par-1


REGULATION

Drosophila anterior-posterior polarity requires actin-dependent PAR-1 recruitment to the oocyte posterior

The Drosophila anterior-posterior axis is established at stage 7 of oogenesis when the posterior follicle cells signal to polarize the oocyte microtubule cytoskeleton. This requires the conserved PAR-1 kinase, which can be detected at the posterior of the oocyte in immunostainings from stage 9. However, this localization depends on Oskar localization, which requires the earlier PAR-1-dependent microtubule reorganization, indicating that Oskar-associated PAR-1 cannot establish oocyte polarity. This study analyzed the function of the different PAR-1 isoforms; only PAR-1 N1 isoforms can completely rescue the oocyte polarity phenotype. Furthermore, PAR-1 N1 is recruited to the posterior cortex of the oocyte at stage 7 in response to the polarizing follicle cell signal, and this requires actin, but not microtubules. This suggests that posterior PAR-1 N1 polarizes the microtubule cytoskeleton. PAR-1 N1 localization is mediated by a cortical targeting domain and a conserved anterior-lateral exclusion signal in its C-terminal linker domain. PAR-1 is also required for the polarization of the C. elegans zygote and is recruited to the posterior cortex in an actin-dependent manner. These results therefore identify a molecular parallel between axis formation in Drosophila and C. elegans and make Drosophila PAR-1 N1 the earliest known marker for the polarization of the oocyte (Doerflinger, 2006).

The par-1 gene encodes multiple isoforms that belong to three families (N1, N2, and N3) that differ in their N-terminal domains, raising the possibility that an isoform that is not detected in antibody stainings polarizes the microtubule cytoskeleton. To test this possibility, transgenic flies were generated expressing N-terminally tagged GFP fusions of the major PAR-1 isoforms under the control of the maternally expressed alpha4-tubulin promoter. Their ability was analyzed to rescue the polarity defect of a viable transheterozygous combination of par-1 alleles, in which oskar mRNA is partially or completely mislocalized to the center of the oocyte in 99% of stage 9-10 egg chambers. Expression of either GFP-PAR-1 N1S or N1L fully rescues the posterior localization of oskar mRNA, whereas GFP-PAR-1 N2S and GFP-PAR-1 N3L rescue only partially (30% and 40% mislocalization, respectively). PAR-1 N1S is the major isoform expressed during oogenesis and is strongly reduced in the par-1 hypomorphs that give a polarity phenotype 2 and 3. These results suggest that PAR-1 N1S plays the principal role in the repolarization of the microtubule cytoskeleton, although the N2 and N3 isoforms can partially compensate for a partial loss of N1 when overexpressed (Doerflinger, 2006).

In contrast to the other PAR-1 isoforms, PAR-1 N1S and N1L localize to the posterior of the oocyte at stage 7, as an early response to the polarizing signal from the follicle cells. This localization is microtubule independent and begins before the reorganization of the oocyte microtubule-cytoskeleton that defines the anterior-posterior axis. Furthermore, only the N1 isoforms fully rescue the anterior-posterior polarity defects of par-1 mutants, whereas mutant forms of PAR-1 that localize all around the cortex disrupt oocyte polarity. Thus, anterior-posterior polarity depends on the localization of PAR-1 N1 to the posterior cortex of the oocyte and its exclusion from other regions of the cortex. These results indicate that the actin-dependent posterior recruitment of PAR-1 N1 is essential for the reorganization of the oocyte cytoskeleton (Doerflinger, 2006).

One question raised by the results is why the localization of the PAR-1 N1 isoforms to the posterior cortex has not been detected by antibody staining, given that the antibody was raised against the linker domain and should recognize all forms of PAR-1. This may reflect the higher sensitivity of GFP tagging compared to antibody staining and the low levels of endogenous protein at this site. It seems more likely, however, that the epitopes recognized by this antibody are masked at the cortex, since a new antibody against a different region of PAR-1 shows a very similar posterior cortical localization of endogenous PAR-1 to that seen with GFP-PAR-1 N1S. Conversely, none of the GFP-PAR-1 constructs localize to the pole plasm like the PAR-1 detected in immunostainings. Thus, either the fusion of GFP to the N termini of these isoforms inhibits their recruitment to the pole plasm, or this staining reflects the localization of another splice variant that has not yet been analyzed (Doerflinger, 2006).

The PAR-1 N1 isoforms are targeted to the posterior by a cortical localization signal at the C terminus of the protein and an adjacent antero-lateral exclusion motif (AEM), and these two signals are also necessary and sufficient to target PAR-1 to the lateral cortex in epithelial cells. Mammalian PAR-1b is restricted in a similar way to the lateral domain of polarized MDCK cells, and this is mediated by the phosphorylation of a conserved threonine in the AEM by apically localized atypical Protein kinase C (aPKC), which excludes PAR-1 from the apical cortex and inhibits its activity. Drosophila aPKC shows a similar localization to the subapical region of epithelial cells, suggesting that apical exclusion by aPKC phosphorylation of the AEM domain may be a conserved mechanism for restricting PAR-1 to the lateral cortex. It is more difficult to account for the posterior localization of PAR-1 in the oocyte by this mechanism, however, since DaPKC null mutant germline clones block the specification of the oocyte in the germarium, but a proportion of the mutant egg chambers escape this early defect and go on to develop a normal anterior-posterior axis at stage 9. This suggests that aPKC is not required for the repolarization of the oocyte at stage 7, although it is possible that residual aPKC perdures in these escaper clones and can still function to restrict PAR-1 to the posterior. Alternatively, PAR-1 may be excluded from the anterior and lateral cortex by a different mechanism or by another kinase that phosphorylates the same site in the AEM (Doerflinger, 2006).

The polarization of the anterior-posterior axis in C. elegans also depends on the localization of PAR-1 to the posterior cortex of the one-cell zygote . However, the upstream steps that led to the posterior recruitment of PAR-1 appeared to be quite different in the two systems, because the posterior localization of PAR-1 in C. elegans requires the actin cytoskeleton, whereas the polarization of the Drosophila oocyte is microtubule dependent. The results indicate that the mechanisms are more similar than previously thought, since the posterior recruitment of the PAR-1 N1 isoforms is upstream of microtubule polarity and depends on actin. The localization of C. elegans PAR-1 requires the cortical myosin, NMY2, which associates directly with PAR-1. It will therefore be important to determine whether a myosin is also required for the cortical recruitment of Drosophila PAR-1 (Doerflinger, 2006).

An Oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte

The localization of oskar mRNA to the posterior of the Drosophila oocyte defines the site of assembly of the pole plasm, which contains the abdominal and germline determinants. oskar mRNA localization requires the polarization of the microtubule cytoskeleton, which depends on the recruitment of PAR-1 to the posterior cortex in response to a signal from the follicle cells, where it induces an enrichment of microtubule plus ends. This study shows that overexpressed oskar mRNA localizes to the middle of the oocyte, as well as the posterior. This ectopic localization depends on the premature translation of Oskar protein, which recruits PAR-1 and microtubule-plus-end markers to the oocyte center instead of the posterior pole, indicating that Oskar regulates the polarity of the cytoskeleton. Oskar also plays a role in the normal polarization of the oocyte; mutants that disrupt oskar mRNA localization or translation strongly reduce the posterior recruitment of microtubule plus ends. Thus, oskar mRNA localization is required to stabilize and amplify microtubule polarity, generating a positive feedback loop in which Oskar recruits PAR-1 to the posterior to increase the microtubule cytoskeleton's polarization, which in turn directs the localization of more oskar mRNA (Zimyanin, 2007).

On the basis of these results, a revised model is suggested for how the polarity of the oocyte is established. The polarization of the oocyte is initiated by the posterior-follicle-cell signal, which induces the localization of the PAR-1N1 short and long isoforms to the posterior cortex. This localized PAR-1 then recruits or stabilizes some microtubule plus ends at the posterior cortex, leading to the kinesin-dependent transport of a small amount of oskar mRNA from the center of the oocyte to the posterior pole. Once oskar mRNA reaches the posterior, its translational repression is relieved, and the resulting Oskar protein recruits another population of PAR-1 to the posterior. This PAR-1 can then recruit further microtubule plus ends, which in turn direct the posterior localization of more oskar mRNA. Thus, Oskar protein initiates a positive feedback loop that amplifies the polarization of the microtubule cytoskeleton, leading to an increase in the localization of its own mRNA. The microtubule polarity is not reinforced in oskar protein-null mutants or in mutants that disrupt the localization of translation of oskar mRNA, and this results in only a partial polarization of the microtubule cytoskeleton. On the other hand, oskar mRNA overexpression and premature translation in the middle of the oocyte initiates the positive feedback loop in the wrong place. As a consequence, PAR-1 recruits some of the microtubule plus ends to the center of the oocyte and away from the posterior pole. PAR-1 has also been shown to stabilize Oskar protein through direct phosphorylation. This therefore generates a second positive feedback loop that further reinforces the posterior localization of PAR-1, Oskar protein, and microtubule plus ends (Zimyanin, 2007).

Amplification through positive feedback loops appears to be an emerging theme in cell polarity. For example, the polarization of migrating neutrophils depends on the local enrichment of phosphatidylinositol 3,4,5 triphosphate (PtdInsP3) at the leading edge of the cell, and this is amplified by a Rho-dependent positive feedback loop, in which PtdInsP3 recruits PtdIns-3-OH kinase to the leading edge, which in turn generates more PtdInsP3. A similar mechanism operates during bud-site selection in S. cerevisiae. The bud site is defined by the localization of activated Cdc42-GTP, and this localization induces the assembly of actin cables that extend into the cytoplasm. This signal is then reinforced by the transport of more Cdc42-GTP along the actin cables to the bud site, where it can induce the assembly of further actin cables. This mechanism is enhanced by a second feedback loop, in which Cdc42-GTP recruits the adaptor protein, Bem1, which binds Cdc24, which activates Cdc42. The current results add a third example of the use of multiple feedback loops to reinforce an initially weak cell polarity, and they provide the first case where this amplification involves the microtubule cytoskeleton rather than actin (Zimyanin, 2007).

lethal giant larvae is required with the par genes for the early polarization of the Drosophila oocyte

Most cell types in an organism show some degree of polarization, which relies on a surprisingly limited number of proteins. The underlying molecular mechanisms depend, however, on the cellular context. Mutual inhibitions between members of the Par genes are proposed to be sufficient to polarize the C. elegans one-cell zygote and the Drosophila oocyte during mid-oogenesis. By contrast, the Par genes interact with cellular junctions and associated complexes to polarize epithelial cells. The Par genes are also required at an early step of Drosophila oogenesis for the maintenance of the oocyte fate and its early polarization. This study shows that the Par genes are not sufficient to polarize the oocyte early and that the activity of the tumor-suppressor gene lethal giant larvae (lgl) is required for the posterior translocation of oocyte-specific proteins, including germline determinants. Lgl localizes asymmetrically within the oocyte and is excluded from the posterior pole. Phosphorylation of Par-1, Par-3 (Bazooka) and Lgl is crucial to regulate their activity and localization in vivo. Adherens junctions locate around the ring canals, which link the oocyte to the other cells of the germline cyst. However, null mutations in the DE-cadherin gene, which encodes the main component of the zonula adherens, do not affect the early polarization of the oocyte. It is concluded that, despite sharing many similarities with other model systems at the genetic and cellular levels, the polarization of the early oocyte relies on a specific subset of polarity proteins (Fichelson, 2010).

One general strategy to establish polarity within a cell is to create non-overlapping membrane domains along one specific axis. In most cell types, four complexes are involved in the formation of these domains: (1) Par-3-Par-6-aPKC, (2) Crb-Sdt-dPatj, (3) Scrib-Lgl-Dlg and (4) Par-1. However, the activities and interactions of these complexes depend on the cellular context. In single-cell systems that lack intercellular junctions, such as the C. elegans embryo and vertebrate hippocampal neurons, mutual inhibitions between the Par-3-Par-6-aPKC complex and Par-1 (PAR-2) seem sufficient to establish polarity. By contrast, in the follicular epithelium, the Crb-Sdt-dPatj complex acts redundantly with Par-3-Par-6-aPKC to define the apical side, whereas the Scrib-Lgl-Dlg complex cooperates with Par-1 on the lateral cortex. Consistent with this redundancy, expression of non-phosphorylatable forms of either Par-3 or Par-1 is not able to disrupt the apical-basal polarity of the follicle cells, although they both localize ectopically. This study shows that the early polarization of the oocyte is an intermediate case. The results suggest that the Crb-Sdt-dPatj complex does not act redundantly with the Par-3-Par-6-aPKC complex as it is not required, whereas Lgl could function with Par-1. Consistent with this hypothesis, this study showed that the expression of a non-phosphorylatable form of Par-1 is able to disrupt the early polarization of the oocyte, whereas a non-phosphorylatable form of Par-3 is not able to counter the activities of both Lgl and Par-1. In addition, it was found that Par-1 localizes on the fusome independently of Lgl further suggesting that both could act in parallel pathways (Fichelson, 2010).

The results show that phosphorylation plays a crucial role to regulate the activity and localization of Par-1, Par-3 and Lgl during early oogenesis, but to different extents in each case. Par-1 phosphorylation might not be crucial for its localization within the germarium, since it was found that the non-phosphorylatable and wild-type forms of Par-1 had a similar localization, although Par-1-AEM (apical-lateral exclusion motif (AEM), in which a conserved threonine is replaced by an alanine) appears a bit more cytoplasmic. Its overexpression, however, induces very strong and penetrant polarity defects in the germline. These results contrast with the follicular epithelium, where Par-1-AEM-GFP localizes ectopically to the apical membrane but does not affect the polarization of those cells. Thus, the main function of Par-1 phosphorylation in the germarium might be to downregulate its kinase activity. In addition, it was shown that the microtubule cytoskeleton is a probable target of Par-1 activity, since a strong reduction in microtubules was observed in oocytes expressing Par-1-AEM (Fichelson, 2010).

The localization of endogenous Par-3, wild-type Par-3-GFP and non-phosphorylatable Par-3 (Baz-S151A,S1085A-GFP) also appear identical. However, Baz-S151A,S1085A was unable to localize properly in the absence of the endogenous Par-3. This failure is probably due to the inability of this mutant form of Par-3 to homodimerize. Phosphorylation thus plays an important role for Par-3 localization. This non-phosphorylatable form of Par-3 is, however, still active, as it is able to rescue par-3-null homozygous clones in the follicular epithelium and is also sufficient to induce polarity defects in the oocyte when expressed at later stages of oogenesis. In this latter case, Baz-S151A,S1085A was expressed in the presence of the endogenous Par-3 and was able to reach the cortex of the oocyte and localize ectopically to the posterior pole. The results suggest that this ectopic Par-3 is probably made of heterodimers of endogenous and non-phosphorylatable forms of Par-3. The data further show clear differences with the follicular epithelium, where expression of the non-phosphorylatable form of Par-3 in par-3 mutant clones not only rescues the absence of endogenous protein, but also localizes properly at the apical side only. This could be a consequence of redundant mechanisms in the follicle cells (Fichelson, 2010).

It was found that Lgl localization is strikingly asymmetric in the early oocyte as it is completely absent from the posterior cortex from stage 1 to stage 5-6 of oogenesis. By contrast, it becomes specifically enriched at the posterior cortex from stage 7 onward. This is the first time that such an asymmetry is described within the germline so early during oogenesis for any protein. It was further demonstrated that this asymmetric localization depends on Lgl phosphorylation, as Lgl-3A localizes around the entire oocyte cortex. Phosphorylation thus plays an important role for Lgl localization. Surprisingly, the ectopic Lgl-3A localization is not sufficient to disrupt the early polarization of the oocyte. By contrast, the same Lgl-3A construct induces much stronger polarity defects than wild-type Lgl when overexpressed in embryonic neuroblasts or in the oocyte at later stages of oogenesis. One possible explanation is that at least one of the mutated Serine in Lgl-3A is also required for Lgl activity during the early stages of oogenesis. Another possibility is that an unknown redundant pathway is able to counteract Lgl activity in the germarium (Fichelson, 2010).

One question remaining from this work is the relationship between the localization of Par-1, Par-3 and Lgl, and their function. It is difficult to relate Par-1 localization on the fusome in region 1 of the germarium and the polarization defects induced by its absence in region 3. Furthermore, this study shows that Par-3 localizes around the ring canals with DE-Cadherin and Armadillo on genuine AJs, which are structures playing key roles in the polarization of many epithelia. However, the absence of Par-3 on these junctions in DE-Cadherin mutant clones does not perturb the polarization of the oocyte. The relevant localization of Par-3 for its polarizing activity in the germarium thus remains unknown. Finally, although Lgl localization is clearly asymmetric, excessive or ectopic localization only affect oogenesis after the oocyte becomes polarized (Fichelson, 2010).

Several arguments strongly point to the microtubule cytoskeleton and associated proteins as key targets of the polarity complexes in the germarium: (1) short treatments of microtubules depolymerizing drugs allow the restriction of cytoplasmic proteins into the oocyte in region 2 of the germarium but disrupt their localization to the posterior of the oocyte in region 3; (2) the Orb protein localizes into the oocyte in hypomorphic combinations of dhc64C, which encodes the heavy chain of the minus-end-directed molecular motor Dynein, but fails to translocate to the posterior pole of the oocyte; (3) In strong allelic combinations of Bicaudal D, a binding partner of the dynein-dynactin complex, Orb and centrosomes also fail to migrate to the posterior of the oocyte. Furthermore, in all three cases, the oocyte becomes polyploid later on and reverts to the nurse cell fate. This study shows that overexpression of Par-1-AEM strongly reduces the level of microtubules and induces identical phenotypes. This confirms that the earliest step of oocyte polarization depends on microtubules and is consistent with the well-established function of Par-1 mammalian homologues, the MARKs, in destabilizing microtubules. It is, however, more difficult to explain why the oocyte nucleus becomes polyploid and why the oocyte loses its identity. Although the Par proteins could have a separate function within the nucleus, depolymerizing the microtubules leads to an identical phenotype, which rather suggests that polyploidization of the oocyte nucleus is a direct consequence of the absence of microtubules. A failure to polarize is, however, not sufficient to induce polyploidization of the oocyte nucleus. Indeed, several mutants were found to retain Orb at the anterior of the oocyte but did not produce egg chambers with 16 nurse cells. The link between the cytoplasmic polarization of the oocyte and its nuclear identity thus remains unclear and is an exciting line for future investigations (Fichelson, 2010).

Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction

Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by Protein kinase C (PKC). The Drosophila adducin, Hu-li tai shao (Hts), has been shown to play a role in larval neuromuscular junction (NMJ) growth. This study finds that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), this study shows that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. Evidence is provided that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. It was also shown that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014: PubMed ID).

The Drosophila neuromuscular junction (NMJ) is the site of contact between motor neuron and muscle, and is stably maintained but remodelled during the growth and development of the fly. To permit these differing functions, the NMJ uses an actin- and spectrin-based cytoskeleton both pre- and post-synaptically, where a number of synaptic proteins modify the cytoskeleton dynamically. One such protein involved in the dynamic responses of the synapse to stimuli in vertebrates is the actin- and spectrin-binding protein adducin, a heteromeric protein composed of α, β and γ subunits that is widely expressed in many cell types including neurons and myocytes. The adducins are composed of a globular N-terminal head domain, a neck domain and a C-terminal myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain containing an RTPS-serine residue which is a major phosphorylation site for protein kinase C (PKC), as well as cAMP-dependent protein kinase (PKA). Phosphorylation of adducin in the MARCKS-homology domain inhibits adducin-mediated promotion of actin-spectrin interactions, resulting in cytoskeletal reorganization (Wang, 2014).

Multiple studies have demonstrated that the mammalian MARCKS protein, or more specifically its MARCKS effector domain, can bind to and sequester the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), in artificial lipid vesicles. This interaction has been linked to the regulation of the actin cytoskeleton during the growth and branching of dendrites in rat brains, as well as the directed migration of bovine aortic endothelial cells in wound healing assays. Notably, it has been proposed that aberrant MARCKS regulation of PIP2 signaling may be implicated in the formation of amyloid plaques in Alzheimer's disease. A recent study has also provided evidence that reduced hippocampal levels of MARCKS, and thus PIP2, in mice contributes to age-related cognitive loss (Wang, 2014).

MARCKS binds to PIP2 as the MARCKS effector domain carries basic residue clusters that can interact with acidic lipids in the inner leaflet of the cell membrane. By analogy to other MARCKS-homology domain-containing proteins, it is hypothesized that phosphorylation of adducin at the RTPS-serine may alter the electrostatic interaction between adducin and phosphoinositides, thus reversing the binding between them and causing translocation of adducin from the membrane to the cytosol. In this way, adducin might act as a molecular switch in its regulation of synaptic plasticity, with its localization at the synapse controlled by phosphorylation (Wang, 2014).

In Drosophila, orthologs of adducin are encoded by the hu-li tai shao (hts) locus, and the Hts protein is present at both the pre- and post-synaptic sides of the larval NMJ where it regulates synaptic development. Previous studies have shown that Hts interacts with the scaffolding protein Discs large (Dlg), and regulates Dlg localization at the postsynaptic membrane by promoting its phosphorylation through Partitioning-defective 1 (PAR-1) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), two known regulators of Dlg postsynaptic targeting. Dlg is an important regulator of synaptic plasticity, and likely constitutes a major route by which Hts controls NMJ development. This study found that the main isoforms of Hts at the NMJ are the MARCKS-homology domain-containing isoforms, Add1 and/or Add2. There, the adducin-like isoforms form complexes with Dlg and PIP2, interactions that were identified through Proximity Ligation Assay (PLA). Evidence is provided that Hts promotes the phosphorylation, and thus delocalization, of Dlg at the postsynaptic membrane by regulating the re-distribution of par-1 and camkII transcripts from the muscle nucleus to the cytoplasm. It was also shown that these Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain, further establishing that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014).

Through the use of PLA, this study has shown that Hts forms complexes with Dlg and PIP2 at the postsynaptic region of the larval NMJ, with its ability to associate with these proteins being negatively regulated through phosphorylation of the MARCKS-homology domain. Studies on mammalian adducin have demonstrated that phosphorylation of the MARCKS-homology domain impedes its actin-binding and spectrin-recruiting functions, reduces its affinity for these cytoskeletal components and the membrane, and targets it for proteolysis. It is proposed that phosphorylation of the MARCKS-homology domain in the Add1/Add2 isoforms of Hts in response to upstream signaling events at the synapse reduces their affinity for spectrin-actin junctions and Dlg at the NMJ, but may also hinder their interactions with PIP2 and other phosphoinositides in line with the electrostatic switch model for phosphoinositide binding by the MARCKS-homology domain (Wang, 2014).

It was proposed previously that Hts regulates Dlg localization at the NMJ by controlling the protein levels of PAR-1 and CaMKII, which phosphorylate Dlg and disrupt its postsynaptic targeting. This study now shows that regulation of these kinases appears to occur at the level of transcript processing, with Hts promoting the accumulation of par-1 and camkII transcripts in the muscle cytoplasm. Cytoplasmic accumulation of the transcripts would then presumably lead to higher levels of PAR-1 and CaMKII protein. How is Hts achieving this mode of regulation when it is residing with Dlg at the postsynaptic membrane? One possibility is that Hts at the NMJ is activating a signaling pathway that promotes the transcription and/or stability of par-1 and camkII transcripts, as well as their transport out of the nucleus. Another possibility is that Hts itself, which contains predicted NLS and NES sequences, translocates to the nucleus in response to events at the NMJ, similar to the way that mammalian α-adducin translocates to the nucleus upon loss of cell-cell adhesion in epithelia. This study was unable to detect endogenous Hts in muscle nuclei, however, nuclear Hts levels might be tightly restricted and undetectable under wild-type conditions. Over-expressed wild-type Hts, on the other hand, is readily observable in the nucleus, though not its phosphorylated form - a result also seen with α-adducin. Whatever the mechanism may be, the presence of Hts in a complex with Dlg may allow it to evaluate the status of Dlg and the synapse, and execute a response in the form of regulating Dlg localization through PAR-1 and CaMKII mediated phosphorylation (Wang, 2014).

A recent study has uncovered a novel nuclear envelope budding mechanism that can export select transcripts from muscle nuclei during larval NMJ development, and involves Lamin C (LamC) and the Wnt receptor, DFrizzled2 (DFzz2) (Speese, 2012). Interestingly, camkII, but not dlg, transcripts are regulated by this process, which is consistent with the findings that CaMKII, but not Dlg, expression is regulated by Hts. Future work will determine whether Hts is involved in this LamC/DFzz2-dependent mechanism (Wang, 2014).

Two papers have underscored the importance of phosphoinositides in synaptic development at the Drosophila NMJ (Forrest, 2013; Khuong, 2010). Binding of Hts to PIP2 and probably other phosphoinositides at the NMJ, as seen with other MARCKS-homology domain-containing proteins, may affect the availability of these lipids for processes such as signal transduction, thus affecting synaptic development. Conversely, the localization of Hts at the NMJ may be regulated by the distribution of phosphoinositides. In line with this, postsynaptic knockdown of the phosphoinositide phosphatase Sac1 via transgenic RNAi expression disrupts Hts localization at the NMJ (Wang, 2014).

The observations reported in this study may have important implications for understanding diseases that affect synaptic function in humans and other mammals. Many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a disorder characterized by the progressive loss of motor neurons, have been assumed until recently to be a consequence of neuronal death within the central nervous system. However, there is substantial recent evidence indicating that neuron pathology in ALS and other neurodegenerative diseases is due to a degenerative process that begins in the presynaptic terminal, NMJ or distal axon. This may also be the case in normal aging (Wang, 2014).

Initial interested in adducin arose when elevated levels of phospho-adducin protein was found in the spinal cord tissue of patients who died with ALS, compared to individuals who died without neurological disease. Similar observations were also made in mSOD-expressing mice, a transgenic animal model of ALS. Multiple studies have shown that adducin plays important roles in synaptic plasticity, and that mice mutant for β-adducin display defects in memory, learning and motor coordination. It is clear that modulation of Hts expression and phosphorylation can affect synaptic development. This study provides evidence here that phosphorylation of Hts impedes its function at the larval NMJ, a result that is consistent with the mammalian adducins. In addition, overexpression of phospho-mimetic Hts has dominant negative effects over endogenous Hts. Thus, loss of adducin function through aberrant phosphorylation of the MARCKS-homology domain may be a contributing factor for human neurodegenerative diseases (Wang, 2014).

Protein Interactions

Wnt signaling regulates ß-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by ß-catenin and the other by Jun kinase (JNK). A Dsh-associated kinase has been purified from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the ß-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signaling through ß-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the ß-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-ß-catenin signaling, indicating a link between two important developmental pathways (Sun, 2001).

Dsh is progressively phosphorylated in Drosophila during embryonic development. To identify the kinase that phosphorylates Dsh, various domains of Dsh were expressed as glutathione S-transferase (GST)-fusion proteins and tested for their ability to bind the kinase from Drosophila embryos. Only the GST fusion protein containing the middle domain, DM, but not the N-terminal or C-terminal domain of Dsh, has a high affinity for the kinase. The Dsh-associated kinase activity that is precipitated with GST-DM increases as Dsh becomes progressively phosphorylated during development. Similarly, there is a kinase activity present in Dsh immunoprecipitates. An in-gel kinase experiment has shown that the kinase activity, eluted from Dsh immunoprecipitates, is associated with two major bands and a minor band on a polyacrylamide gel. These bands of 110 kDa, 64 kDa and 130 kDa (minor band) are the kinase and its major fragment. The region on Dsh that interacts with the kinase was mapped more precisely to a 36 amino acid segment, DM5, which is N-terminal to the PDZ domain. Importantly, this region in Dsh is well conserved among Drosophila, C. elegans, Xenopus and mammals (Sun, 2001).

Using GST-DM5 precipitation and in vitro phosphorylation as an assay to monitor the Dsh-associated kinase activity, the kinase was purified 60,000-fold from Drosophila embryos and several peptide sequences were then used to clone the cDNA of this kinase. The cDNA clones encoded a protein kinase that is highly homologous to the C. elegans protein PAR-1 (85% identity in the kinase domain and 42% identity overall), which is known to regulate embryo polarity but whose function in Wnt signaling has not been explored (Sun, 2001).

To determine whether dPAR-1 and Dsh form a complex in vivo, endogenous Dsh was immunoprecipitated from Drosophila cells with an affinity purified anti-Dsh antibody and dPAR-1 was detected in the immunocomplex by Western blot using anti-dPAR-1 antibody. These data indicate that dPAR-1 is the kinase responsible for the activity that co-purified with Dsh in the GST pull-down experiment. The PAR-1 protein, prepared by in vitro translation, strongly phosphorylates the middle region of Dsh in vitro. Co-expression of Dsh with wild-type dPAR-1, but not kinase-negative dPAR-1 (dPAR-1 KN), in NIH3T3 cells results in a reduction in Dsh mobility on SDS-PAGE. These data indicate that dPAR-1 directly phosphorylates Dsh in vitro and in vivo (Sun, 2001).

If PAR-1 acts in Wnt signaling, PAR-1 activity might be expected to change in response to Wnt. Drosophila clone-8 cells were stimulated with conditioned medium containing Wingless and it was confirmed that Dsh becomes phosphorylated, and Armadillo is stabilized. The kinase activity of dPAR-1 was also measured under the same conditions. Several experiments have shown that soluble Wg stimulates dPAR-1-specific activity. Wg treatment does not increase the amount of dPAR-1 protein that interacts with GST-DM5. The increased dPAR-1 activity correlates with enhanced Dsh phosphorylation and elevation of Arm levels in Clone-8 cells (Sun, 2001).

To examine whether PAR-1 is required in the Wnt pathway, endogenous PAR-1 activity was suppressed by expressing a kinase-negative PAR-1 (hPAR-1Balpha KN). Chinese hamster ovary (CHO) cells were used because good expression from transfected DNA can be achieved and these cells have a well-characterized response to Wnt. Three hallmarks of Wnt activity were measured: Dsh phosphorylation, ß-catenin stabilization and transcriptional activation. Wnt treatment of CHO cells retards the mobility of Dvl proteins (mammalian homologs of Dsh) on SDS-PAGE, and phosphatase treatment increases the mobility of the Dvl band, thereby confirming that Dvl is phosphorylated in response to Wnt. The hPAR-1Balpha KN suppresses Wnt-mediated phosphorylation of endogenous Dvl proteins, as shown by the reduced amount of a retarded Dvl band. This result is consistent with the data that PAR-1 phosphorylates Dsh in vitro and in cells. Furthermore, both human and Drosophila PAR-1 KN strongly suppress Wnt-induced ß-catenin stabilization. The kinase-negative forms of hPAR-1A, hPAR-1B and hPAR-1C all strongly suppress Wnt-mediated transcriptional activation (measured by LEF1/TCF reporters) in a dose-dependent manner. Importantly, co-expression of wild-type hPAR1 can override the suppression mediated by hPAR-1 KN, indicating that hPAR-1 KN affects Wnt signaling by specifically blocking the effects of endogenous PAR-1 in cells. However, hPAR-1 KN is unable to inhibit the transcriptional activation induced by overexpression of ß-catenin, consistent with PAR-1's role in regulation of Dsh function upstream of ß-catenin (Sun, 2001).

All three human PAR-1 homologs strongly potentiated the responses to Wnt or Dvl3 in CHO cells. The hPAR-1 proteins alone do not activate the signaling pathway but require co-expression of either Wnt or Dvl, indicating that there is synergy between hPAR-1 and other components of the Wnt pathway. The specificity of these responses was verified by their dependency on the co-expression of the LEF1 transcription factor, which is required for Wnt signaling. Furthermore, the effects of hPAR-1 were suppressed by Axin, a negative regulator of the Wnt pathway that acts downstream of Dsh. As predicted, hPAR-1 overexpression does not alter the gene response induced by overexpression of ß-catenin, consistent with the idea that PAR-1 regulates Wnt signaling at a step upstream of Axin and ß-catenin (Sun, 2001).

Dsh also activates an alternative pathway that culminates in the stimulation of JNK in mammalian cells and controls of cell polarity in Drosophila and in vertebrates. Expression of Dvl in NIH3T3 cells induces JNK activation. Interestingly, hPAR-1 strongly suppresses the ability of Dvl3 to activate JNK in NIH3T3 cells. This inhibitory effect depends on the kinase activity of hPAR-1, because a mutation in hPAR-1, which is expected to inactivate its kinase activity (hPAR-1 KN), leads to a loss of its inhibitory effect on JNK activation. Together, these data indicate that PAR-1 promotes the participation of Dsh in the Wnt-ß-catenin pathway but suppresses its function in the JNK pathway, thereby acting as a switch for the downstream responses mediated by Dsh protein (Sun, 2001).

Several studies have shown that Wnt signaling controls axis specification during Xenopus embryogenesis. Ectopic ventral expression of Wnt before the onset of zygotic transcription results in duplication of the dorsal axis: this duplication can be blocked by dominant-negative forms of Frizzled, Dsh and CKIepsilon. Likewise, interference with negative regulators of the Wnt pathway, such as GSK3ß or Axin, by ventral expression of dominant negatives also induces axis duplication. In order to further the understanding of the function of PAR-1 in vertebrates, whether PAR-1 KN can alter Wnt signaling was investigated by injecting PAR-1 RNA into Xenopus embryos. Ventral blastomere injection of XWnt8 RNA results in significant axis duplication, as expected. Co-injection of human or Drosophila kinase-negative PAR-1 RNA significantly inhibits XWnt8-induced axis duplication in injected embryos. Both human and Drosophila kinase-negative PAR-1 can exert functional effects on the Wnt pathway in Xenopus axis duplication experiments (Sun, 2001).

To examine the function of dPAR-1 in Wg signaling in Drosophila embryos, double-stranded RNA-mediated interference (RNAi) was used to generate a dpar-1 loss-of-function phenotype. The ventral epidermis of a wild-type embryo is covered with alternating smooth cuticle and denticle belts. The Wg pathway is required to specify the fate of the epidermal cells that secrete smooth cuticle. RNAi of dpar-1 causes localized ectopic denticle formation and fusion of denticle belts, resembling the wg RNAi phenotype previously described. A similar wg phenotype is also generated with RNAi using double-stranded RNAs derived from different regions of the dpar-1 transcript (Sun, 2001).

Whether Dsh itself or perhaps other proteins bound to Dsh are the most important substrates of PAR-1 remains to be determined. Previous reports have shown that CKIepsilon, a positive regulator of the Wnt pathway, also interacts with Dsh. However, CKIpsilon seems to exert its major effect on more downstream components of the pathway. GBP/Frat (GSK3-binding protein) also interacts with Dsh and has a positive role in the Wnt pathway. Detailed biochemical studies will be required to elucidate the precise function(s) of PAR-1 and its functional interactions with Dsh and other regulators of Dsh in Wnt signaling (Sun, 2001).

Par-1 kinase is critical for polarization of the Drosophila oocyte and the one-cell C. elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Drosophila Par-1 is shown to phosphorylate the posterior determinant Oskar (Osk). It has been demonstrated genetically that Par-1 is required for accumulation of Osk protein. In cell-free extracts Osk protein is intrinsically unstable and Osk is stabilized after phosphorylation by Par-1. The data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk (Riechmann, 2002).

14-3-3 proteins mediate PAR-1 function in axis formation

PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. Drosophila 14-3-3 proteins, 14-3-3epsilon and 14-3-3zeta/Leonardo (Leo) represent the Drosophila homologs of C. elegans PAR-5. 14-3-3 proteins have been identified as Drosophila PAR-1 interactors; PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display phenotypes identical to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries (Benton, 2002).

PAR-1 contains three conserved domains: centrally-located kinase and ubiquitin-associated (UBA) domains, and a C-terminal domain of unknown function. Since the C-terminal domain is dispensable for PAR-1 function in the germline, a yeast two-hybrid screen was performed using a bait containing the kinase and UBA domains. The largest class of preys, representing over 25% of the recovered clones, corresponded to the two Drosophila 14-3-3 proteins, 14-3-3epsilon and 14-3-3zeta/Leonardo (Leo). These interactors represent the Drosophila homologs of C. elegans PAR-5, and this interaction appears to be conserved, since PAR-5 can bind to a fragment of C. elegans PAR-1 (Benton, 2002).

To confirm this interaction by an independent assay, in vitro-synthesized, labeled full-length Drosophila PAR-1 was incubated with bacterially expressed maltose binding protein (MBP)-tagged 14-3-3 proteins bound to amylose beads. Beads containing MBP:14-3-3 fusion proteins, but not MBP alone, efficiently precipitate PAR-1, indicating that this interaction is direct (Benton, 2002).

14-3-3 proteins regulate the activity or subcellular localization of a diverse set of proteins, including several protein kinases, by binding in a phosphorylation-dependent manner to conserved motifs (RSXpSXP or RX1-2pSX2-3pS). Using the yeast two-hybrid system, it was found that 14-3-3 appears to associate with the kinase domain of PAR-1. This contrasts with the interaction of 14-3-3 with other kinases, such as Raf and Wee1, in which 14-3-3 recognizes a phosphoserine-containing motif lying outside the catalytic domain. This interaction with PAR-1 is kinase specific, since 14-3-3 does not bind to the catalytic domains of PKA or aPKC (Benton, 2002).

The region of 14-3-3epsilon that interacts with PAR-1 was determined using the molecular information of three missense alleles of 14-3-3epsilon (Chang, 1997). These alleles were isolated as suppressors of activated Ras or Raf and impair the function of 14-3-3 in Ras/Raf/MAPK signaling. One mutation, E183K, lies within the phosphoserine binding pocket and affects a residue that directly contacts phosphoserine-peptide ligands. The others, F199Y and Y214F, are both located outside this pocket in a hydrophobic region of unknown function. Each of these three mutations were introduced into a 14-3-3epsilon prey clone and their effects on the intermolecular interactions of 14-3-3epsilon were tested (Benton, 2002).

Since 14-3-3 proteins function as dimers, whether these mutations influenced the dimerization property of 14-3-3epsilon was tested. 14-3-3epsilon can form both homodimers and heterodimers with Leo, and none of the three mutations significantly affects these interactions. This is consistent with the location of these mutations in regions distinct from the dimerization interface and indicates that global protein structure and stability are not affected. Interactions were tested of these mutant proteins with a domain of Drosophila Raf that contains a conserved 14-3-3-recognition motif (R740SApSEP745). Raf binds to both Drosophila 14-3-3 isoforms, and the interaction with 14-3-3epsilon is completely abolished by the E183K mutation, but not by the F199Y and Y214F mutations, as expected for an association via the phosphoserine binding pocket (Benton, 2002).

These mutations have opposite effects upon the interaction with PAR-1: E183K does not impair binding, whereas the other mutations either result in a severe (F199Y) or a more modest (Y214F) reduction in the strength of this interaction. These results indicate that 14-3-3epsilon does not bind PAR-1 via its phosphoserine binding pocket, consistent with the lack of canonical binding motifs in PAR-1. The interaction instead appears to be mediated by a novel interface on the external surface of the 14-3-3 molecule. Since the F199 and Y214 residues are conserved in 99% of 14-3-3 sequences, this interface is likely to exist in all isoforms (Benton, 2002).

Since binding of PAR-1 to 14-3-3 should leave the phosphoserine binding pocket vacant, and PAR-1 is a serine/threonine kinase, it was reasoned that PAR-1 might be involved in regulating the phosphorylation-dependent interactions of 14-3-3 with other proteins. Whether PAR-1 can phosphorylate proteins to generate the phosphoserine epitope recognized by 14-3-3 was tested. Using either immunoprecipitated or bacterially expressed PAR-1, efficient phosphorylation was observed of the 14-3-3-interacting portion of Raf. Phosphorylation of Ser743, or the equivalent residue in Raf homologs, is essential for 14-3-3 binding and for Raf function in vivo. This residue was mutated to alanine and this was found to completely abolish the phosphorylation by PAR-1, indicating that PAR-1 specifically phosphorylates Raf to generate a 14-3-3 binding site. This activity of PAR-1 does not require the presence of 14-3-3, and addition of 14-3-3 to this assay does not detectably affect Raf phosphorylation. A mammalian PAR-1 homolog, C-TAK1, is able to phosphorylate proteins such as KSR and Cdc25C within 14-3-3 binding sites, suggesting that this specificity is a conserved property of this kinase family (Benton, 2002).

To test whether 14-3-3 proteins are involved in par-1-dependent processes in vivo, loss-of-function mutations in 14-3-3epsilon and leo were analyzed. Surprisingly, flies homozygous for a protein null allele of 14-3-3epsilon (14-3-3epsilonj2B10) are viable. However, females lay very few eggs, which fail to hatch. Most egg chambers from these females lack differentiated oocytes, as revealed by DNA staining, which distinguishes the oocyte karyosome from the 15 polyploid nurse cells. An identical phenotype is observed in ovaries from flies containing this allele over a deficiency, indicating that the phenotype is specific for this locus. To determine where 14-3-3epsilon is required, clones of this allele were generated in either the germline or somatic follicle cells. Defects in oocyte differentiation were observed only in germline clones; thus, like PAR-1, 14-3-3epsilon is required in the germline for oocyte differentiation (Benton, 2002).

Oocyte determination depends on the MT-dependent transport of specific factors, such as Orb and the germ cell centrosomes, to one cell in the cyst. These factors initially concentrate at the anterior of this cell but subsequently translocate around the nucleus and concentrate along the posterior cortex. This second step appears to require the establishment of a diffuse MTOC along the posterior of the cell and is essential for its stable determination as the oocyte. The formation of this MTOC can be visualized using an antibody to Minispindles (MSPS), a MAP that localizes to sites of MT nucleation. In wild-type egg chambers, MSPS accumulates along the posterior cortex. This accumulation is undetectable in 14-3-3epsilon mutants, indicating that the MTOC has failed to form. Orb and the centrosomes therefore do not undergo the anterior-to-posterior movement and eventually diffuse away as this cell exits meiosis and adopts a nurse cell fate. These phenotypes are indistinguishable from those of par-1 null mutant cysts, indicating that 14-3-3epsilon and PAR-1 function together in this specific step of oocyte determination (Benton, 2002).

In contrast to 14-3-3epsilon mutants, germline clones of a strong lethal allele of leo (leoP1188) display no defects in this process. Since the 14-3-3epsilon phenotype is incompletely penetrant, whether 14-3-3 proteins have partially redundant functions in the germline was tested. Removal of one copy of leo in 14-3-3epsilon mutant clones results in a fully penetrant defect in oocyte determination. Furthermore, removal of one copy of 14-3-3epsilon in leo mutant cysts uncovers an important contribution of leo in this process, since 84% of these cysts display defects in Orb localization. Thus, although 14-3-3epsilon has the predominant function in oocyte determination, Leo can partially compensate in its absence (Benton, 2002).

Polyclonal antibodies against 14-3-3epsilon and Leo were used to examine their localization in the germline. These antibodies are specific, since they do not stain tissue mutant for the corresponding isoform. 14-3-3epsilon is highly expressed in the dividing germline cells in the germarium, and colocalizes with PAR-1 on the fusome, a membranous structure that branches into each germ cell during the early germ cell divisions. The asymmetric partitioning of the fusome during these divisions results in one cell always inheriting more fusome material, which may provide an initial cue to specify this cell as the oocyte. The colocalization of PAR-1 and 14-3-3epsilon on the fusome may therefore represent a mechanism to concentrate these proteins in the future oocyte (Benton, 2002).

At later stages, 14-3-3epsilon colocalizes with PAR-1 at the ring canals, which interconnect the germline cells in each cyst. 14-3-3epsilon can be detected in the cytoplasm and around the cortex of the oocyte but, unlike PAR-1, does not accumulate at the posterior pole. Leo is also expressed in the germline and displays a similar localization to ring canals, but is expressed at very low levels in the germarium (Benton, 2002).

While the fusome and ring canals may represent sites of physical and functional association of PAR-1 with 14-3-3, its localization to these sites is not affected in 14-3-3 mutants, indicating that 14-3-3 binding does not simply act to target PAR-1 to these subcellular destinations. Mutations in the Drosophila PAR-3 homolog Baz cause similar phenotypes in oocyte determination as par-1 and 14-3-3 mutants. However, Baz concentrates at distinct sites in the germarium, in circles around each ring canal that also contain components of adherens junctions, and this localization is not detectably affected in 14-3-3 mutants (Benton, 2002).

The high early cytoplasmic concentration of 14-3-3epsilon has prevented an conclusive determination of whether the fusome localization of 14-3-3epsilon is PAR-1 dependent. However, 14-3-3epsilon is detectable at ring canals in cysts homozygous for a par-1 null allele (Benton, 2002).

To determine if 14-3-3 proteins function with PAR-1 in the repolarization of the oocyte to define the A-P axis, the distribution of osk mRNA and Stau was examined in late-stage egg chambers recovered from homozygous and hemizygous 14-3-3epsilonj2B10 females, and in germline clones of this allele. These mutants display a partially penetrant phenotype, in which osk mRNA and Stau accumulate in dots in the middle of the oocyte. Twenty seven percent of egg chambers display both ectopic and posterior accumulation of osk mRNA and Stau protein, and four percent contain only mislocalized dots. These defects are very similar to those of hypomorphic par-1 mutants and can be strongly enhanced by removal of one copy of par-1 (Benton, 2002).

Although most bcd mRNA localizes normally to the anterior cortex in 14-3-3epsilon mutants, a small proportion is mislocalized along the lateral cortex, and occasionally at the posterior. In contrast to previous observations, such defects in bcd mRNA distribution are also observed in par-1 mutants. These are more pronounced at stages 8-9 than at stage 10, which might reflect a partial recovery in bcd mRNA localization to the anterior between these stages or the diffusion of the mRNA away from the lateral and posterior cortices due to a failure in anchoring. Other mutants that affect the localization of bcd and osk mRNAs, such as gurken, also disrupt the migration of the oocyte nucleus to the dorsal-anterior corner. As in par-1 mutants, however, oocyte nucleus migration appears to be unaffected in 14-3-3epsilonj2B10 mutants (Benton, 2002).

Oocytes that are homozygous for leoP1188 do not display polarity defects. However, strong dominant genetic interactions are observed between 14-3-3 mutants. Thus, these isoforms also function partially redundantly in this process (Benton, 2002).

To gain insights into the basis for the defects in mRNA localization, the organization of the MT cytoskeleton was examined, using a MT plus end marker, Kin:ß-gal. This marker accumulates at the posterior pole in wild-type oocytes, suggesting that the majority of MT plus ends are focused on this site. In contrast, Kin:ß-gal concentrates in the center of 14-3-3epsilon mutant oocytes, indicating that MT plus ends are focused incorrectly, and providing an explanation for the defects in osk mRNA/Stau distribution. The organization of oocyte MTs was directly analyzed using both a FITC-conjugated anti-alpha-tubulin antibody and a Tau:GFP reporter of MT distribution in living egg chambers . In contrast to the wild-type anterior-to-posterior gradient of MTs, 14-3-3epsilon mutants show a uniform distribution of MTs around the oocyte cortex, with the lowest density of MTs in the center. These defects in MT organization are indistinguishable from those of par-1 mutants (Benton, 2002).

The combination of phenotypes in osk and bcd mRNA localization and MT organization is, thus far, unique to par-1 and 14-3-3 mutants, and strongly suggests that they function together in the polarization of the A-P axis (Benton, 2002).

To determine the importance of the 14-3-3 protein interaction domains in vivo, the phenotypes of the 14-3-3epsilon missense alleles, 14-3-3epsilonF199Y and 14-3-3epsilonE183K, were characterized. Neither mutation significantly affects the level or localization of the protein, as assessed by immunostainings. 14-3-3epsilonE183K displays penetrant defects in both oocyte determination and polarization. The penetrance of the latter is almost three times that observed with the protein null allele, indicating that the E183K mutant protein functions as a dominant negative, presumably through the formation of nonfunctional heterodimers with Leo. Thus, the interaction of 14-3-3 dimers with phosphorylated targets is critical for its function in the germline (Benton, 2002).

14-3-3epsilonF199Y mutant egg chambers do not exhibit significant defects in oocyte determination or polarization, consistent with previous reports that this allele only displays phenotypes under genetically sensitized conditions (Chang, 1997). In the absence of leo, however, this allele has a dominant phenotype, with 11% of leoP1188;14-3-3epsilonF199Y/+ egg chambers displaying defects in oocyte determination. Thus, the PAR-1 interaction interface is also important for 14-3-3 function (Benton, 2002).

Thus loss-of-function mutations in 14-3-3 cause phenotypes identical to par-1 mutants in both the initial polarization of the oocyte and the repolarization that defines the A-P axis. These results indicate that 14-3-3 functions as an essential cofactor for PAR-1 in the generation of polarity (Benton, 2002).

Given the diverse roles of 14-3-3 proteins, it is very surprising that the only essential requirement for 14-3-3epsilon is in PAR-1-dependent polarization events in the Drosophila germline. A similar dedication of 14-3-3 function may exist in C. elegans, where animals homozygous for hypomorphic mutations in the 14-3-3 isoform encoded by par-5 are viable but give rise to progeny with highly penetrant defects in the polarization of the A-P axis. Indeed, the discovery that 14-3-3 is required for the initial polarization of the oocyte in the germarium reveals a remarkable homology between the generation of the first A-P asymmetries in flies and worms. Mutations in 14-3-3epsilon give a very specific defect in oocyte determination, in which the oocyte is initially specified correctly but fails to establish a posterior MTOC and to translocate oocyte-specific factors from the anterior to the posterior cortex. This phenotype is identical to that of par-1 null mutants, and the colocalization of PAR-1 and 14-3-3 on the fusome supports the idea that they function together in this process (Benton, 2002).

The Baz/PAR-6/aPKC complex is also required for this step of oocyte determination but localizes to a distinct site in the germarium. Furthermore, it has recently been shown that mutants in the Drosophila homolog of PAR-4 display this phenotype (S. Martin and D.S.J., unpublished data reported in Benton, 2002). Thus, this early polarization of the oocyte requires the Drosophila homologs of five of the six par genes that mediate the A-P polarization of the C. elegans zygote. The final gene, par-2, has no obvious homologs in other organisms and may perform some function that is unique to C. elegans (Benton, 2002).

Although the full complement of PAR proteins is necessary for the initial polarization of the Drosophila oocyte in the germarium, the Baz/PAR-6/aPKC complex does not appear to be required for the repolarization of the oocyte at stage 7. In baz and par-6 null germline clones, a few egg chambers escape the block in oocyte determination, and these complete oogenesis normally, displaying no defects in the localization of Stau to the posterior. Thus, the PAR-1/14-3-3 complex can function to polarize the oocyte independently of these other PAR proteins. PAR-1 also is required for the apical-basal polarity of the follicular epithelium, and localizes to the basolateral domain in these cells. It is interesting to note that 14-3-3epsilon concentrates basolaterally in follicle cells, raising the possibility that it functions with PAR-1 in this process as well. The PAR-1/14-3-3 complex may therefore represent a conserved polarity 'cassette' that plays an analogous role to the Baz/PAR-6/aPKC complex. This requirement is not universal, however, because PAR-1 does not appear to be necessary for the apical-basal polarization of the neuroblasts (J. Kaltschmidt and R. B., unpublished data reported in Benton, 2002), which depends upon Baz, PAR-6, and aPKC. Thus, the two PAR protein complexes may comprise distinct modules that can function either together or separately to generate polarity in different contexts (Benton, 2002 and references therein).

While the common requirement for the PAR proteins strongly suggests that the mechanisms that generate the first A-P asymmetries are conserved between flies and worms, the regulatory relationships between these proteins are not conserved. The hierarchy of PAR protein function in C. elegans has been inferred from the effects of mutants in each par gene on the localization of the other PAR proteins. This analysis places PAR-5 at the top of the hierarchy because it is required for the anterior localization of the PAR-3/PAR-6/PKC-3 complex and the posterior localization of PAR-2 and PAR-1, whereas PAR-1 lies at the bottom because par-1 mutants have little effect on the asymmetric localization of other PAR proteins. In contrast, in Drosophila, Baz and PAR-1 are localized normally in 14-3-3 mutants. Furthermore, although the localization of members of the PAR-3/PAR-6/PKC-3 complex are codependent in the C. elegans zygote, this is not the case in the Drosophila oocyte, nor are they required for the localization of PAR-1 to the fusome. The different positions of PAR-5 and PAR-1 in the C. elegans hierarchy indicate that PAR-5 functions independently of PAR-1 in the localization of the other PAR proteins, but this early requirement makes it difficult to assess whether it is also necessary at other stages in the pathway. The results in Drosophila and the observation that C. elegans PAR-5 and PAR-1 interact in yeast raise the possibility that PAR-5 also functions downstream of PAR-1 (Benton, 2002).

Although the results indicate that PAR-1 and 14-3-3 function together to polarize the oocyte at two stages of oogenesis, the mechanisms by which they generate these polarities are unknown. The repolarization of the oocyte at stage 7 principally affects the organization of the MTs. The original posterior MTOC is disassembled, and the MTs are reorganized to form an A-P gradient, in which most MTs appear to be nucleated from the anterior cortex, with their plus ends extending toward the posterior pole. In 14-3-3 and par-1 mutants the MTs are evenly distributed around the cortex, and a MT plus end marker and osk mRNA/Stau localize to the center of the oocyte. These observations led to the proposal that PAR-1 functions to recruit the plus ends to the posterior. This study shows that par-1 and 14-3-3 mutants also display mislocalization of bcd mRNA around the cortex. Since this mRNA is believed to be transported to the minus ends of MTs, this suggests that MTs are abnormally nucleated from all regions of the oocyte. Thus, PAR-1 and 14-3-3 may also contribute to the generation of the MT gradient by specifically inhibiting MT nucleation along the posterior and lateral cortices. The role of PAR-1 and 14-3-3 in the initial polarization of the oocyte in the germarium is also likely to involve MTs since their loss results in a failure in the formation of an MTOC at the posterior of the cell. The mechanisms that control the formation of this MTOC are not known, however, and it is unclear whether PAR-1 and 14-3-3 function in the same way to polarize the oocyte at both stages (Benton, 2002).

A model for 14-3-3 function with PAR-1 is presented. 14-3-3 proteins regulate the activity of numerous cellular proteins in a phosphorylation-dependent manner by binding as dimers to phosphoserine/threonine-containing motifs. In many cases, this regulation involves sequestration of the target protein in the cytoplasm. For example, 14-3-3 binding to the proapoptotic factor Bad blocks its translocation to mitochondria. 14-3-3 can also directly regulate the activity of its targets: the association of 14-3-3 with serotonin N-acetyltransferase, for example, enhances its ability to bind substrates. The interaction of 14-3-3 with PAR-1 differs from these canonical 14-3-3/target interactions in several respects: (1) the binding of 14-3-3 does not appear to regulate PAR-1 activity, since 14-3-3 mutants have no effect on PAR-1 localization or stability in vivo, or on kinase activity in vitro; (2) the PAR-1 kinase domain lacks both of the well-defined 14-3-3 binding motifs, and interacts with a novel hydrophobic region that is distinct from the phosphoserine binding pocket, which should therefore still be available to bind to other proteins. Thus, 14-3-3 may act as a cofactor for PAR-1 by binding to proteins that are phosphorylated by the kinase. In support of this, it has been demonstrated that PAR-1 can specifically phosphorylate a 14-3-3 binding site in Raf (Benton, 2002).

These observations suggest a model in which PAR-1 has a dual role in regulating 14-3-3/target interactions, first by generating the 14-3-3 binding phosphoepitope, and second by directly delivering 14-3-3 to these sites. Once 14-3-3 is bound to target proteins, its continued association with PAR-1 would maintain the kinase in close proximity to its substrate, which might ensure the stable maintenance of the phosphorylated state (Benton, 2002).

In addition to their role in establishing cell polarity, PAR-1 kinases have been implicated in a diverse range of other cellular processes. The closest mammalian homolog of PAR-1, C-TAK1, was initially purified as an activity that phosphorylates Cdc25C on Ser216. The in vivo significance of this regulation is unknown, but phosphorylation of this site by a distinct kinase, Chk1, induces 14-3-3 binding, and this inhibits Cdc25C as part of the DNA damage checkpoint. C-TAK1 also phosphorylates KSR to promote 14-3-3 binding, which sequesters KSR in the cytoplasm and inhibits EGF signaling. These biochemical activities of C-TAK1 are consistent with the data in Drosophila showing that PAR-1 phosphorylates a 14-3-3 binding site in Raf, and that 14-3-3 mutants give identical phenotypes to par-1 mutants in the germline. The ability to phosphorylate 14-3-3 binding sites may be a general property of PAR-1 kinases, which accounts for the diversity of their functions (Benton, 2002).

Consistent with this, other PAR-1 substrates have been shown to associate with 14-3-3 or contain conserved potential 14-3-3 recognition motifs. The vertebrate PAR-1 homologs, MARK1 and MARK2, were identified as kinases that phosphorylate Tau to inhibit its MT binding ability. 14-3-3 interacts with the MT binding domain of Tau and appears to compete with tubulin for Tau binding. MARK kinase regulation of Tau may therefore be mediated through 14-3-3, which physically blocks the association of Tau with MTs. PAR-1 also phosphorylates the Wingless pathway component Dishevelled. This phosphorylation has been mapped to a 30 amino acid region of the protein, which contains a putative 14-3-3 recognition motif (amino acids 234-242: RTSSYSS) that is essential for its function in planar polarity (Benton, 2002).

The intimate functional relationship between PAR-1 and 14-3-3 raises the possibility that this kinase might be involved in regulating other processes involving 14-3-3 proteins. For example, the observation that PAR-1 phosphorylates Raf to generate a 14-3-3 binding site makes it a candidate for the unidentified kinase that regulates Raf in vivo. In support of this, this study shows that the F199Y and Y214F mutations in 14-3-3epsilon that affect signaling through Raf, impair the interaction of 14-3-3epsilon with PAR-1 (Benton, 2002).

Although many of the activities of PAR-1 kinases may be mediated by inducing 14-3-3 binding, this is probably not the only mechanism by which they act. Drosophila PAR-1 has recently been proposed to have a third function in the germline, in which it phosphorylates, and so stabilizes, OSK protein at the posterior pole of the oocyte to ensure its levels are high enough to specify the germ cells. Unlike PAR-1, 14-3-3 is not detectably enriched at the posterior, suggesting that this function of the kinase might operate via a 14-3-3-independent mechanism. C. elegans PAR-1 may have a similar function in germline specification, through the regulation of P-granule stability, which does not require PAR-5 (Benton, 2002).

A major question is the nature of the target(s) of PAR-1/14-3-3 that mediate their effects on cell polarity. These are unlikely to be any of the known PAR-1 substrates, such as Dishevelled or Tau, since these are not required for axis formation in Drosophila (R. B. and D. S. J., unpublished data reported in Benton, 2002), but the results lead to the clear prediction that they will bind to 14-3-3 in a PAR-1-dependent manner (Benton, 2002).

A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity; LKB1 is targeted by PAR-1 and PKA

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans. PAR-1 is also required for A-P axis determination in Drosophila. The Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome and is regulated by prenylation and by phosphorylation by protein kinase A. Protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome (Martin, 2003).

The A-P axis of Drosophila is specified during oogenesis when a signal from the posterior follicle cells induces the formation of a polarized oocyte microtubule cytoskeleton, in which most minus ends are nucleated from the anterior cortex, with the plus ends extending towards the posterior pole. These polarized microtubules direct both the localization of bicoid messenger RNA to the anterior of the oocyte to specify where the head and thorax will develop, and the transport of oskar mRNA to the posterior, where it induces the formation of polar granules that contain the abdominal and germline determinants (Martin, 2003).

To identify other genes required for formation of the A-P axis, a genetic screen was carried out in germline clones for mutants that disrupt the localization of Staufen tagged with green fluorescent protein (GFP), which colocalizes with bicoid and oskar mRNAs. Mutants in one lethal complementation group of two alleles abolish the posterior localization of both Staufen and oskar mRNA in 80%-90% of oocytes. In over 40% of mutant oocytes, both bicoid and K10 mRNAs are detected around the whole oocyte cortex, rather than only at the anterior pole. In contrast to wild-type oocytes, which have an anterior-to-posterior gradient of microtubules, mutant oocytes have a high density of microtubules all around the cortex, with the lowest concentration in the centre. Kinesin-ß-galactosidase, a microtubule plus-end marker, also fails to concentrate at the posterior of the oocyte and accumulates instead in the centre. Thus, these mutants disrupt bicoid and oskar mRNA localization by preventing the A-P polarization of the microtubules (Martin, 2003).

Hypomorphic mutants in Drosophila par-1 show defects in the polarization of the oocyte microtubule cytoskeleton and in the localization of bicoid and oskar mRNA that are very similar to the defects of lkb1 mutants. The PAR-1 kinase is also required much earlier in oogenesis for the determination of the oocyte. The oocyte is selected from a cyst of 16 germline cells in the germarium and forms a microtubule-organizing centre, which directs the microtubule-dependent localization of oocyte-specific factors, such as ORB, to this cell. The microtubule-organizing centre then moves from the anterior to the posterior of the oocyte in region 3, the most posterior region of the germarium, and oocyte-specific factors accumulate posteriorly. This anterior-to-posterior switch is disrupted in par-1 null mutants, and the oocyte exits meiosis and becomes a nurse cell (Martin, 2003).

To study the role of lkb1 in this process, mutant germline clones marked by the loss of GFP were induced. ORB still accumulates in the presumptive oocyte in lkb1 cysts but often fails to move to the posterior in region 3; instead, it disperses throughout the cyst as the oocyte exits meiosis and adopts the nurse cell fate. Thus, lkb1 and par-1 share very similar phenotypes in both oocyte determination and polarization, suggesting that they function together. Because the penetrance of the early lkb1 phenotype increases with age (67% of mutant clones in 5-day-old females, rising to 85% after 12 d), wild-type LKB1 activity seems to perdure for several days after the clones are induced, and this presumably accounts for the escapers that show the later oocyte polarity phenotype (Martin, 2003).

Since PAR-1 and LKB1 seem to act in a common pathway, whether either kinase could phosphorylate the other was tested. Although recombinant PAR-1 is not a substrate for LKB1, immunoprecipitated GFP-PAR-1 phosphorylates recombinant LKB1 in vitro. It was found that the phosphorylation site or sites are located in the amino-terminal half of the protein, although it has not been possible to map them precisely. Also a strong genetic interaction is observed between the two genes. The oskar mRNA localization defects in hypomorphic combinations of par-1 alleles cause loss of abdominal segments in the embryo, which can be strongly enhanced by removing one copy of lkb1. In addition, overexpression of GFP-LKB1 partially rescues the A-P polarity phenotype of par-16323/par-1W3, the strongest allelic combination that produces late-stage oocytes. Whereas Staufen localizes to the posterior normally in only 12% of these oocytes, 80% have wild-type amounts of Staufen at the posterior when LKB1 is overexpressed. This phenotype is not rescued by a kinase-dead form of LKB1, indicating that this suppression requires kinase activity. By contrast, overexpression of PAR-1 does not rescue the phenotype of lkb1 mutant germline clones. These results are consistent with a model in which LKB1 is a direct target of PAR-1 regulation in vivo and functions as a downstream effector in the polarization of the oocyte microtubule cytoskeleton (Martin, 2003).

A GFP-LKB1 fusion construct under the control of the endogenous promoter rescues both the lethality and oogenesis phenotypes of lkb1 mutants and is expressed in very low amounts in both the germline and somatic follicle cells of the ovary. The highest expression was observed in the germarium, where GFP-LKB1 colocalizes with PAR-1 on the fusome, a branched membranous organelle that connects the germ cells in a cyst. This localization presumably reflects their common function in early oocyte polarity and determination, because the cell that inherits most fusome is selected to become the oocyte. During the rest of oogenesis, GFP-LKB1 shows a uniform cortical localization in both the germline and the follicle cells. It is enriched in the oocyte from stage 7, when the A-P axis is polarized, and colocalizes with cortical actin, but not with pole plasm components (Martin, 2003).

Follicle cell clones mutant for lkb1 also show a defect in polarity. In severely affected clones, the follicular monolayer is disorganized, with mutant cells rounding up and sorting out of the epithelium. Morphologically wild-type clones show defects in the apical localization of atypical protein kinase C (aPKC) and Armadillo, which become either diffuse or ectopically localized along lateral membranes. In less severely affected cells, the apical localization is discontinuous. These phenotypes are penetrant in large stem-cell clones but not in small clones, indicating that LKB1 activity perdures. Expression of the wild-type or S535E transgenes with arm-GAL4 in lkb1 mutants rescues these follicle cell phenotypes, whereas expression of S535A rescues lethality but gives rise to a completely disorganized follicular epithelium, in which most cells appear unpolarized. Thus, LKB1 is required for cell polarity in the germ line and the follicle cells, and is probably regulated by phosphorylation on the conserved C-terminal serine in both processes (Martin, 2003).

In Drosophila, par-1 and lkb1, the homologue of C. elegans par-4, show very similar phenotypes. In addition, LKB1 is an in vitro substrate for PAR-1 and can suppress the polarity phenotype of par-1 mutants when overexpressed. These results suggest that LKB1 functions downstream of PAR-1. This conclusion is consistent with genetic data in C. elegans that show that par-4 mutants display only a subset of the par-1 A-P polarity phenotypes. Notably, mutants in par-4 and par-1, but not in other par genes, show a disappearance of P granules in the one-cell zygote. Thus, LKB1 and PAR-1 function in a conserved pathway that is required for the polarization of the A-P axis in both worms and flies (Martin, 2003).

Drosophila LKB1 is closely related to human LKB1, and conserved prenylation and PKA phosphorylation sites are essential for the in vivo function of both proteins, indicating that the two are likely to be functional homologues. Mutants in LKB1 cause Peutz-Jeghers syndrome, which is characterized by the formation of intestinal polyps and a high incidence of adeno-carcinomas (tumours of epithelial origin). In addition, mutations in lkb1 have been identified in several sporadic epithelial cancers. The role of LKB1 as a tumour suppressor is not well understood, however, and LKB1 has been proposed to regulate apoptosis, the cell cycle or angiogenesis. In addition, LKB1 seems to function in a context-dependent manner that is different from classical tumour-suppressor genes such as ras or p53. Given that Drosophila lkb1 is required to polarize the epithelial follicle cells, an alternative model is proposed, that loss of LKB1 leads to polyp and tumour formation by disrupting epithelial polarity (Martin, 2003).

PAR-1 kinase plays an initiator role in a temporally ordered phosphorylation process that confers Tau toxicity in Drosophila

Multisite hyperphosphorylation of tau has been implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). However, the phosphorylation events critical for tau toxicity and mechanisms regulating these events are largely unknown. Drosophila PAR-1 kinase is shown to initiate tau toxicity by triggering a temporally ordered phosphorylation process. PAR-1 directly phosphorylates tau at S262 and S356. This phosphorylation event is a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase-5 (Cdk5), to phosphorylate several other sites and generate disease-associated phospho-epitopes. The initiator role of PAR-1 is further underscored by the fact that mutating PAR-1 phosphorylation sites causes a much greater reduction of overall tau phosphorylation and toxicity than mutating S202, one of the downstream sites whose phosphorylation depends on prior PAR-1 action. These findings begin to differentiate the effects of various phosphorylation events on tau toxicity and provide potential therapeutic targets (Nishimura, 2004).

Drosophila has established itself as a model system for studying human neurodegenerative disorders. Fly models of tauopathy have been created by expressing wild-type or FTDP-linked mutant forms of h-tau. Using such models and based largely on overexpression experiments, it has been shown that Shaggy (GSK-3) can promote neurofibrillary tangle (NFT) pathology in photoreceptor neurons (Jackson, 2002). Whether GSK-3 and NFT are necessary for tau-mediated neurodegeneration, however, remains uncertain. Other studies have shown that tau-mediated neurodegeneration could occur without NFT and that GSK-3ß-induced tau hyperphosphorylation in mice could correlate inversely with neuropathology (Nishimura, 2004 and references therein).

Critical testing for a functional role of phosphorylation in tau-mediated neuropathology will require identifying the physiological tau kinase and assessing the consequence of removing this kinase activity on the disease process. Through loss-of-function and overexpression genetic studies and biochemical analysis, it has been shown that PAR-1 is a physiological tau kinase that plays a central role in regulating tau phosphorylation and toxicity in Drosophila. PAR-1 is a Ser/Thr kinase originally identified in C. elegans for its role in regulating cell polarity and asymmetric cell division. PAR-1 homologs have been found in eukaryotes ranging from yeast to mammals and exert essential cellular and developmental functions. MARK kinase, the mammalian homolog of PAR-1, regulates MT dynamics, epithelial cell polarity, and neuronal differentiation. Drosophila PAR-1 plays important roles in MT organization, oocyte differentiation, anterior-posterior axis formation, and Wingless signaling. While analyzing the neuronal function of PAR-1, it was found that Drosophila PAR-1 is a physiological kinase for fly Tau and h-tau. Overexpression of PAR-1 leads to elevated tau phosphorylation and enhanced toxicity, whereas removing PAR-1 function or mutating PAR-1 phosphorylation sites in tau abolishes tau toxicity. Furthermore, an initiator role for PAR-1 has been uncovered in a multisite phosphorylation process that generates pathogenic forms of tau. In this process, phosphorylation by PAR-1 precedes and is obligatory for downstream phosphorylation events, including those carried out by GSK-3 and Cdk5, to generate toxic tau. Consistent with PAR-1 playing an initiator role in the process, mutating PAR-1 phosphorylation sites causes a much more dramatic reduction of overall tau phosphorylation and toxicity than mutating one of the downstream Cdk5/GSK-3 phosphorylation sites. These findings have important implications for understanding the biogenesis of pathogenic tau in neurons and for developing mechanism-based therapeutic strategies (Nishimura, 2004).

To investigate the function of Drosophila PAR-1 in the nervous system, the longest isoform of PAR-1 was overexpressed in the eye using the UAS-GAL4 system. Targeted overexpression of PAR-1 to photoreceptor neurons using the sevenless-GAL4 line resulted in eye degeneration in newly emerged adult flies. Strong PAR-1 expression lines exhibited severely reduced eyes, with fused ommatidia and missing inter-ommatidial bristles, as revealed by scanning electron microscopy (SEM) analysis. Weak or mild expression lines had slightly reduced eyes, which appeared rough and had disordered ommatidia and occasional missing bristles. However, in flies that express two copies of the weak PAR-1 transgene, the eye degeneration phenotype became more severe, similar to that observed in the strong expression line. Thus, overexpression of PAR-1 using the sevenless-GAL4 driver causes eye degeneration in a dosage-dependent manner (Nishimura, 2004).

The degeneration phenotype induced by PAR-1 overexpression could be mediated by abnormal phosphorylation of its substrate(s). MARK kinase was previously shown to phosphorylate tau in vitro. This, together with the fact that expression of h-tau in Drosophila photoreceptor neurons causes similar eye degeneration, led to an investigation of the relationship between tau and PAR-1 in the process. A fly tau homolog was recently shown to be expressed in the nervous system (Heidary, 2001), but no information on its loss-of-function or overexpression phenotype is available. A chromosomal deficiency associated with Tp(3;Y)R97 was used to test possible genetic interaction between endogenous fly tau and PAR-1. Through single embryo PCR analysis, it was confirmed that this deficiency deletes fly tau. In flies overexpressing the strong PAR-1 transgene and heterozygous for the chromosomal deficiency, the eye degeneration phenotype was partially suppressed. This suggests that endogenous fly tau may mediate PAR-1-induced eye degeneration phenotype (Nishimura, 2004).

Possible genetic interaction between fly PAR-1 and h-tau were tested. Overexpression of the four C-terminal tandem repeats (4R) isoform of h-tau containing the R406W mutation (henceforth referred to as h-tauM), which is associated with a familial form of FTDP-17, caused a moderate eye degeneration phenotype. Coexpression of the weak PAR-1 transgene, which by itself had a small effect on eye morphology, enhanced h-tauM toxicity and resulted in smaller sized eyes. The effect of the genetic interaction is also reflected by the extent of photoreceptor neuron loss. PAR-1 and h-tauM coexpressing flies showed a greater neuronal loss than files expressing h-tauM or PAR-1 alone. Interaction between PAR-1 and h-tauM was also observed in cholinergic neurons in the CNS, where coexpression of PAR-1 and h-tauM resulted in profound vacuole formation in both the cell bodies and neuronal processes, whereas expression of h-tauM alone caused only mild vacuole formation in cell bodies and PAR-1 alone had little effect. Thus, elevated expression of fly PAR-1 enhances the toxic effects of h-tauM. Whether PAR-1 and wild-type h-tau genetically interact in the eye was tested. Overexpression of wild-type h-tau also caused a rough eye phenotype. Coexpression of wild-type h-tau and a PAR-1 transgene resulted in an exacerbated phenotype compared to expression of either gene alone. Thus, increased expression of fly PAR-1 enhances the toxic effects of both wild-type and mutant forms of h-tau (Nishimura, 2004).

Recent transgenic animal studies have implicated two kinases, GSK-3 and Cdk5, in the phosphorylation of tau in vivo. Analyses of tau phosphorylation status in transgenic mice overexpressing GSK-3 or Cdk5 have detected increased phosphorylation at certain sites previously identified as their in vitro phosphorylation sites. For example, S202 and PHF-1 sites (S396 and S404) have been shown to be prominent Cdk5 and GSK-3 phosphorylation sites, respectively, and the two kinases may have overlapping specificity at these sites. Tests were performed to see whether these sites in h-tauM were also phosphorylated by the corresponding fly kinases. The activity of Cdk5 is regulated by its binding with neuron-specific activators. Overexpression of Drosophila P35 activator has been shown to elevate endogenous Cdk5 activity. In P35 and h-tauM coexpression flies, the level of phosphorylation at S202 recognized by CP13 antibody is elevated. In addition, phosphorylation at AT270 sites was also significantly increased. Phosphorylation at AT100, AT180, and PHF-1 sites was relatively unchanged. Thus, phosphorylation at S202 and T181 responds to changes in Cdk5 levels. The eye morphology of P35 and h-tauM coexpressing flies appearssimilar to that of flies expressing h-tauM alone, suggesting that elevated Cdk5 activity does not significantly enhance tau toxicity. Shaggy and h-tauM coexpression flies were analyzed next. Coexpression of Shaggy and h-tau results in enhanced eye degeneration phenotypes. In the coexpression flies, significantly increased tau phosphorylation was observed at PHF-1, CP13, AT180, and AT100 sites. It is concluded that these phospho-epitopes contain GSK-3 phosphorylation sites and that elevated phosphorylation at these sites enhances tau toxicity (Nishimura, 2004).

The fact that many of the above-tested phosphorylation sites for GSK-3 and Cdk5 kinases are affected in S2A suggests that phosphorylation by the two kinases is regulated by prior PAR-1 action. To test this idea further, the phosphorylation status of GSK-3 and Cdk5 phosphorylation sites was analyzed in PAR-1 and h-tauM coexpression flies. In addition to 12E8 sites, significant increase of phosphorylation was observed at CP13 and PHF-1 sites in these flies. In contrast, phosphorylation at other sites such as AT100 sites was little changed, suggesting that PAR-1 is not a rate-limiting factor for these phosphorylation events. Since in vitro kinase assays showed that PAR-1 is incapable of directly phosphorylating the CP13 and PHF-1 sites, the elevated phosphorylation at these sites in PAR-1 coexpressing flies are likely mediated by downstream kinases such as Cdk5 and GSK-3 (Nishimura, 2004).

Whether coexpression of PAR-1, GSK-3, or Cdk5 has any modulating effect on S2A toxicity was further tested in vivo. PAR-1 and S2A coexpression flies showed a mild rough eye phenotype similar to PAR-1 overexpression alone, indicating that PAR-1 overexpression does not confer additional toxicity to S2A. Co-overexpression of GSK-3 or Cdk-5 also did not change S2A toxicity. These results further support the notion that phosphorylation by PAR-1 at S262 and S356 is a prerequisite for the subsequent phosphorylation by downstream kinases such as GSK-3 and Cdk5 to generate toxic tau species (Nishimura, 2004).

Since the S2A mutation disrupts tau phosphorylation at multiple downstream sites, it does not allow distinguishing the contribution of individual phosphorylation sites to tau toxicity. This issue was addressed by making point mutations in the downstream phosphorylation sites. Focus was placed on the S202 site because it is phosphorylated by Cdk5 and GSK-3 in vivo and because AT8 antibody, which is sensitive to phosphorylation at this site, was considered an Alzheimer-diagnostic antibody. Transgenic flies were generated that express h-tauM containing an Ala substitution at S202 (S202A). Western blot analysis demonstrated that, as predicted, S202A protein was no longer recognized by CP13 or AT8 antibodies. Significantly, phosphorylation at 12E8, AT100, PHF-1, AT180, and AT270 sites was unaffected by S202A mutation. This suggests that unlike S262 and S356 sites, the phosphorylation state of S202 does not influence that of other sites. Examination of external eye morphology by SEM and photoreceptor staining of eye sections has shown that, unlike S2A, S202A is as toxic as h-tauM. This suggests that phosphorylation by GSK-3 and Cdk5 at S202 site plays a rather limited role in conferring tau toxicity. This result supports the notion that PAR-1 plays an initiator role in the pathogenic phosphorylation process and further suggests that phosphorylation at downstream sites other than S202 or a combination of those downstream phosphorylation events makes a major contribution to tau toxicity (Nishimura, 2004).

Thus PAR-1, the fly homolog of mammalian MARK kinase, plays a central role in conferring tau toxicity in vivo. This study reveals PAR-1 function in triggering a temporally ordered phosphorylation process that is responsible for generating toxic forms of tau. This multisite phosphorylation process involves downstream kinases such as Cdk5 and GSK-3, whose action depends on prior phosphorylation of h-tau by PAR-1. A nonphosphorylatable mutation at S202, one of the downstream GSK-3/Cdk5 target sites whose phosphorylation depends on prior PAR-1 action, has a much smaller impact on overall tau phosphorylation and toxicity than mutations at PAR-1 phosphorylating sites. This strongly supports the initiator role of PAR-1 in generating toxic species of tau and further implies that the toxic form of tau may be phosphorylated at a subset or all of the other downstream sites (Nishimura, 2004).

These results indicate that tau phosphorylation by PAR-1/MARK represents one of the earliest events in the pathological process. Consistent with this notion, the 12E8 epitope sites are phosphorylated in all the isoforms of h-tau found in AD brain. Relatively little is known about the upstream events that act through PAR-1/MARK to regulate h-tau phosphorylation in the disease process. Understanding how PAR-1/MARK kinases are regulated during normal development may provide some clues. PAR-1 belongs to a group of evolutionarily conserved PAR proteins that control cell polarity. Genetics studies have shown that PAR-1 is regulated by a PAR-3/PAR-6/aPKC protein complex. Recently, the mammalian PAR-3 and PAR-6 proteins have been shown to regulate hippocampal neuronal polarity through interaction with the phosphatidylinositol 3-kinase signaling pathway (Shi, 2003). Furthermore, recent biochemical studies have identified a protein kinase capable of activating MARK/PAR-1. It would be interesting to test whether these factors may regulate h-tau toxicity through PAR-1 (Nishimura, 2004).

It was previously shown that PAR-1 regulates the Wingless/Wnt pathway in Drosophila and Xenopus by phosphorylating the core component Dishevelled. It is thus interesting that GSK-3, another core component of Wingless pathway, acts downstream of PAR-1 to phosphorylate h-tau. These results are consistent with the notion that the Wingless pathway may be involved in regulating tau phosphorylation. It has been proposed that the pathway components are utilized differently in tau phosphorylation than in canonical Wnt signaling. The data indicate that PAR-1 and GSK-3 directly phosphorylate tau in an ordered fashion, with PAR-1 action preceding that of GSK-3. One parsimonious explanation for the requirement of prior phosphorylation by PAR-1 is that PAR-1 phosphorylation reduces the affinity of tau for MT and releases it from the MT network, therefore allowing easy access by other kinases. If that is the case, the mechanism may operate in a region-specific manner since certain phosphorylation sites do not depend on prior PAR-1 action. The data are also consistent with the idea that PAR-1 phosphorylation at 12E8 sites provides docking sites for intermediary kinase(s) and/or adaptor molecule(s), which facilitate subsequent phosphorylation by GSK-3 and Cdk5. It appears that the phosphorylation at certain downstream sites is achieved through a complex process. For example, phosphorylation at AT100 sites depends on prior PAR-1 action, but PAR-1 co-overexpression does not increase phosphorylation at these sites. Instead, co-overexpression of GSK-3 can lead to increased phosphorylation at AT100 sites. Previous in vitro studies have shown that the generation of AT100 epitope requires a PHF-like conformation of tau and the sequential phosphorylation by GSK-3 and PKA. It remains to be determined whether GSK-3 and PKA act downstream of PAR-1 to phosphorylate AT100 sites in flies (Nishimura, 2004).

Recent studies suggest that synaptic dysfunction may be one of the earliest events in the pathogenesis of AD. It is reasonable to speculate that abnormally phosphorylated tau may contribute to synaptic dysfunction in AD. MT is known to be essential in synaptic vesicle transport during neurotransmission and in the formation and maintenance of synaptic structures. Given the known function of tau in MT binding, it is conceivable that phosphorylation may induce the detachment of tau from MT, thereby affecting MT dynamics and disrupting synaptic functions. Alternatively, phospho-tau could acquire certain new activities unrelated to MT binding, such as gaining affinity for some unidentified factors at the synapse and interfering with their function. While estimates of the levels of tau phosphorylation at S262/S356 sites (in h-tauM only and h-tauM and PAR-1 coexpression transgenic flies) do not distinguish between these two possibilities, they nevertheless indicate that phospho-tau constitutes a substantial fraction of the total protein. Future genetic studies in Drosopila could help reveal the exact mechanisms of phospho-tau toxicity (Nishimura, 2004).

Rapid progress in an understanding of the biogenesis of amyloid plaques have led to efforts to develop AD therapy based on inhibition of the processing and aggregation of Aß or clearance of mature amyloid plaques. In view of the role of tau in the disease process, it might be more rational to target both amyloid and tau pathologies in therapeutic approaches. This study is beginning to dissect the relative contribution to tau toxicity of individual phosphorylation events. Further analysis will identify the kinases acting downstream of PAR-1 and playing dominant roles in conferring tau toxicity. Future studies will also test whether deregulation of the PAR-1-initiated multisite phosphorylation process may underlie certain idiopathic tauopathies and whether PAR-1 and the downstream kinases could serve as therapeutic targets (Nishimura, 2004).

Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia

The Ser/Thr kinase Par-1 is required for cell polarisation in diverse organisms such as yeast, worms, flies and mammals. During Drosophila oogenesis, Par-1 is required for several polarisation events, including localisation of the anterior determinant bicoid. To elucidate the molecular pathways triggered by Par-1, a genome-wide, high-throughput screen for Par-1 targets was carried out. Among the targets identified in this screen was Exuperantia (Exu), a mediator of bicoid mRNA localisation. Exu is a phosphoprotein whose phosphorylation is dependent on Par-1 in vitro and in vivo. Two motifs were identifed in Exu that are phosphorylated by Par-1; their mutation abolishes bicoid mRNA localisation during mid-oogenesis. Interestingly, exu mutants in which Exu phosphorylation is specifically affected can to some extent recover from these bicoid mRNA localisation defects during late oogenesis. These results demonstrate that Par-1 establishes polarity in the oocyte by activating a mediator of bicoid mRNA localisation. Furthermore, this analysis reveals two phases of Exu-dependent bicoid mRNA localisation: an early phase that is strictly dependent on Exu phosphorylation and a late phase that is less phosphorylation dependent (Riechmann, 2004).

Par-1 has two distinct functions in bicoid mRNA localisation. Par-1 is necessary for the release of bicoid mRNA from the oocyte cortex. Genetic epistasis experiments indicate that the exu independent function of par-1 acts at a step upstream of exu in bicoid mRNA localisation. By generating mutants that abolish Exu phosphorylation, two phases of Exu dependent bicoid mRNA localisation could be further distinguished; an early phase, in which bicoid mRNA localisation is abolished when Exu is unphosphorylated and a late phase, in which the requirement for Exu phosphorylation is less stringent. Thus, these results show that bicoid mRNA localisation is a multi-step process, and that redundant mechanisms are used to ensure the anterior accumulation of bicoid mRNA (Riechmann, 2004).

Exu protein is an essential mediator of bicoid mRNA localisation. Par-1 kinase phosphorylates Exu, and this phosphorylation is necessary for anterior localisation of bicoid mRNA during mid-oogenesis. Exu phosphorylation does not affect Exu localisation, its ability to form mobile particles, or its colocalisation with bicoid mRNA. How then might Par-1 phosphorylation enable Exu to mediate bicoid mRNA localisation? Experiments in which fluorescently labelled bicoid mRNA was microinjected into living egg chambers have revealed that Exu is required in the nurse cells for anterior localisation of bicoid mRNA within the oocyte. These experiments have led to a model whereby Exu associates in the nurse cells with bicoid mRNA and mediates the recruitment of additional nurse cell factors required for targeting of bicoid mRNA to the anterior of the oocyte. The finding that mutation of Exu phosphorylation sites results in a phenotype that is, during mid-oogenesis, indistinguishable from that of exu-null mutants suggests that Exu phosphorylation is involved in the recruitment of these anterior-targeting factors in the nurse cells. Phosphorylation might increase the binding affinity of Exu for these nurse cell factors, promoting their association with bicoid mRNA. The colocalisation of Exu-GFP, Par-1 and bicoid mRNA in patches in the nurse cells suggests that this is where the bicoid RNP complexes assemble (Riechmann, 2004).

The consequences of exu and par-1 mutations on bicoid mRNA localisation are distinct. Although loss of exu function results in diffuse bicoid mRNA distribution in the ooplasm, a reduction in par-1 function causes cortical localisation of the mRNA. An Exu protein has been generated that localises bicoid mRNA independent of phosphorylation by Par-1 and rescues exu mutants, but that is unable to rescue bicoid mRNA localisation in par-1 mutants. Therefore, the cortical mislocalisation of bicoid mRNA in par-1 mutant oocytes is independent of Exu function. What might be the other function of Par-1 in localisation of bicoid mRNA? The fact that bicoid localisation requires the microtubule cytoskeleton, together with the report that oocyte microtubules are improperly polarised in par-1 mutants, suggests that cortical localisation in the mutants is caused by a microtubule defect. It has been proposed that microtubules of different qualities may nucleate from different regions of the oocyte cortex. A simple explanation for the aberrant localisation of bicoid mRNA in par-1 oocytes would be that the subset of microtubules nucleating from the anterior corners of the oocyte and serving as tracks for anterior transport of bicoid mRNA are not restricted to the anterior corners, but spread along the cortex, resulting in the lateral cortical localisation of bicoid mRNA. However, this model is not supported by the genetic epistasis experiments, which indicate that the exu independent function of par-1 acts at a step upstream of exu in bicoid mRNA localisation. Therefore, a different model is favored, in which in wild-type oocytes bicoid mRNA first localises cortically preceding its targeted transport along microtubules. In this model, most of the bicoid mRNA entering the oocyte moves in a nonpolar fashion, either passively or by active transport, to the oocyte cortex. Only after this cortical localisation does the targeted transport of bicoid mRNA to the anterior corners of the oocyte commence. In par-1 mutants, the improperly organised microtubule cytoskeleton prevents release of the mRNA from the cortex to the (anterior-targeting) microtubules and the mRNA remains cortically localised. In exu mutants, the polarity of the microtubules is normal and bicoid mRNA is released from the cortex. However, its targeted transport to the anterior is impaired and the mRNA is diffusely distributed in the ooplasm (Riechmann, 2004).

The requirement for Exu phosphorylation in bicoid mRNA localisation decreases during the later stages of oogenesis. This is revealed by the partial recovery of bicoid mRNA localisation in exu mutants that abolish phosphorylation. These mutants are indistinguishable from exu-null mutants through stage 10b of oogenesis, but during early embryogenesis two-thirds of the mutants localise enough bicoid mRNA at the anterior to support formation of a Bicoid protein. This indicates that the mechanism of bicoid mRNA localisation changes after stage 10b of oogenesis, from an early phase that is strictly dependent on Exu phosphorylation, to a late phase that is less dependent on phosphorylation. Stage 10b is the stage at which ooplasmic streaming commences, providing a possible mechanism for localisation of bicoid mRNA in mutants in which Exu phosphorylation cannot occur. Before stage 10b, anterior targeting of bicoid mRNA could be mediated solely by directed transport of bicoid mRNA complexes along microtubules, a process that is strictly dependent on Exu phosphorylation. After stage 10b, this directed transport might be complemented or replaced by a passive trapping mechanism, which has also been postulated for the localisation of oskar and nanos mRNAs during late oogenesis. This mechanism relies on the movements generated by ooplasmic streaming, which could bring bicoid mRNA complexes into contact with the anterior cortex of the oocyte, where the mRNA could be trapped by localised anchoring molecules. This change in the mechanism of bicoid mRNA localisation would occur at the time of assembly of the anterior MTOC that is essential in the late phase of bicoid mRNA localisation, suggesting that the MTOC might be involved in the trapping mechanism. Such a trapping mechanism would be differentially affected in Exu-null mutants and in mutants that specifically abolish Exu phosphorylation. It is possible that Exu provides bicoid mRNA not only with factors required for anterior targeting, but also with factors required for anchoring of bicoid mRNA. Unphosphorylated Exu might be inactive in recruiting the factors for anterior targeting, but be competent for binding of factors required for anchoring (Riechmann, 2004).

It is proposed that in the first phase of bicoid mRNA localisation, the mRNA is transported to the anterior corners of the oocyte, resulting in a ring-like distribution. This targeted transport requires the formation of RNP complexes that contain bicoid mRNA and specific anterior-targeting factors that allow the RNPs to identify those microtubules that nucleate from the anterior corners of the oocyte. Assembly of this complex takes place in the nurse cells and requires the phosphorylation of Exu by Par-1. Upon entry of the complex into the oocyte, a specific proportion of the RNP complexes encounter the microtubules that nucleate from the anterior corners, and these complexes are directly transported to their final destination. However, a large proportion of the complexes does not find these microtubules directly, and moves first to the oocyte cortex. The transfer of these cortically localised complexes to microtubules nucleating from the anterior corners solely requires a properly polarised microtubule network. Only at this stage can the nurse cell factors assembled on the mRNA act to transport the cortically localised complexes to the anterior corners of the oocyte. During the second phase of bicoid mRNA localisation, the ring-shaped distribution changes to a disc-shaped distribution and a MTOC forms at the anterior of the oocyte. The third phase of bicoid mRNA localisation begins after the onset of ooplasmic streaming. In this late phase, the mechanism of bicoid mRNA localisation changes from targeted transport to passive trapping, mediated by ooplasmic streaming, and the mRNA is anchored at the anterior margin. The generation of exu mutants that abolish phosphorylation allows distinguishing between early and the late mechanisms of bicoid mRNA localisation, since the two mechanisms differ in their sensitivity to Exu phosphorylation (Riechmann, 2004).

It has also been shown that Par-1 controls posterior patterning by phosphorylating Oskar. In addition, Par-1 regulates anterior patterning by phosphorylating Exu. Although Oskar is an intrinsically unstable protein whose stability is increased by Par-1 phosphorylation, Par-1 phosphorylation does not affect Exu stability but does affect its ability to mediate bicoid mRNA localisation. Thus, Par-1 uses at least two different mechanisms to generate polarity within the same cell. Interestingly, these two Par-1 substrates, Oskar and Exu, are unique to Diptera, showing that during evolution Par-1 gained fly-specific mediators of cell polarisation as substrates. Par-1 is therefore flexible in the mechanisms and in the targets by which it mediates cell polarisation. This is in striking contrast to the PDZ-containing proteins Par-3 and Par-6, which appear to establish polarity by the assembly of a conserved protein complex (Riechmann, 2004).

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. This study found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and this study show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and evidence is presented that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation (Wang, 2011).

Dlg is a Drosophila member of a family of MAGUK scaffolding proteins that has four mammalian members, SAP97/hDlg, PSD-93/Chapsyn-110, PSD-95/SAP90 and SAP102/NE-Dlg, and which has important developmental and regulatory roles in the nervous system and in epithelia. Phosphorylation is emerging as a mechanism for the regulation of the localization and function of the Dlg family. The post-synaptic targeting of Dlg to the Drosophila NMJ is inhibited by phosphorylation of the first PDZ domain by CaMKII and of the GUK domain by PAR-1. In mammalian neurons, CaMKII phosphorylation of PDZ1 of PSD-95 terminates long term potentiation-induced spine growth by inducing translocation of PSD-95 out of the active spine. Furthermore, phosphorylation of PDZ1 of either PSD-95 or SAP97/hDlg by CaMKII regulates their interaction with NMDA subunits, and additional cell culture studies in epithelial cells demonstrate effects of SAP97/hDlg phosphorylation by various kinases (Wang, 2011).

An important function of Dlg at the NMJ is involvement in synaptic plasticity during muscle growth, at least in part by controlling the localization at the pre-synaptic and post-synaptic membranes of Fas2, a homophilic cell adhesion molecule of the immunoglobulin superfamily that binds Dlg. The breaking and restoration of Dlg/Fas2-mediated adhesion between the pre- and post-synaptic membranes is likely critical to synaptic growth during development. This study has shown that Hts exists in a complex with Dlg, probably at both the post-synaptic membrane of the NMJ and at the lateral membrane in epithelia, where the two proteins are likely brought into close proximity by a shared association with the spectrin-actin junction. Hts is therefore positioned to locally regulate Dlg and participate in synaptic plasticity at sites of association with the membrane cytoskeleton, and it positively contributes to phosphorylation of Dlg on its GUK domain. This site of phosphorylation is a target for PAR-1, and consistent with this it was found that Hts can regulate the levels of PAR-1. Phosphorylation of Dlg impedes its targeting to the post-synaptic membrane and it is proposed that Hts regulates this targeting at the synapse by controlling the levels of PAR-1 at the post-synaptic membrane. This could occur through Hts acting as a scaffold participating in localized stabilization or translation of PAR-1 at the post-synaptic membrane and, consistent with the latter mechanism, localized post-synaptic translation of NMJ proteins has been reported in Drosophila. Over-expression of Hts permits synaptic overgrowth, which may in part be due to effects on Dlg targeting to the post-synaptic membrane, but unpublished results indicating additional routes of action. Furthermore, if Hts were acting mainly by elevating PAR-1 levels, synaptic undergrowth would be expected rather than overgrowth with Hts over-expression, as the post-synaptic over-expression of PAR-1 leads to decreased bouton number and an oversimplified synapse. CaMKII also regulates synaptic growth and Dlg targeting at the NMJ through phosphorylation. There is currently no antibody available to examine phosphorylation of Dlg by CaMKII in vivo, but it was determined that Hts controls the levels of this kinase similarly to PAR-1, suggesting that Hts may also be regulating Dlg targeting via CaMKII-dependent phosphorylation (Wang, 2011).

Two other studies address Hts function in neurons, one describing a role in photoreceptor axon guidance and the other characterizing pre-synaptic Hts function at the NMJ (Ohler, 2011; Pielage, 2011). The latter study reported a phenotype of synaptic retraction at the hts mutant NMJ, revealed by the absence of the pre-synaptic marker Bruchpilot from extensive stretches of the NMJ, which is consistent with the observation of small synapses in hts mutants stained for the synaptic vesicle marker CSP-2 (Pielage, 2011). However, in addition to retraction, this group observed overgrowth of the hts mutant NMJ, visible with anti-HRP staining. Although no quantification was performed using anti-HRP, qualitative examination of hts mutant muscle 6/7 NMJs in the current study did not indicate an overgrowth phenotype. A possible explanation for this discrepancy is the choice of the muscle 6/7 NMJ for analysis in contrast to Pielage, who focused on muscle 4. Unlike most other larval muscles, muscles 6 and 7 are not innervated by type II boutons. A component of the synaptic overgrowth reported by Pielage is the extension of thin, actin-rich extensions somewhat similar in appearance to type II and type III processes and, accordingly, growth of these processes is impaired with pre-synaptic over-expression of Hts (Pielage, 2011). Pielage proposes that pre-synaptic Hts restricts synaptic growth through its function as an actin-capping protein. At the muscle 6/7 NMJ this role may not be so prevalent and the post-synaptic growth-promoting function of Hts prevails. By examining NMJs other than muscle 6/7 in hts mutant larvae it was possible to confirm the overgrowth phenotype observed by Pielage (Wang, 2011).

Synaptic plasticity at the Drosophila NMJ has parallels with the structural changes seen at synapses in cellular models of learning such as long-term facilitation (LTF) in Aplysia and long-term potentiation in the mammalian hippocampus, and several studies indicate that the current results with hts at the NMJ may be relevant to these other systems. Phosphorylation of the conserved serine residue in the MARCKS domain of adducin is increased during LTF in Aplysia, and mice lacking β-adducin exhibit impaired synaptic plasticity and learning (Bednarek, 2011; Gruenbaum, 2003; Porro, 2010; Rabenstein, 2005; Ruediger, 2011; Wang, 2011 and references therein).

Similar to the plasticity exhibited by the NMJ during development, the morphogenesis of epithelia involves what has been referred to as 'epithelial plasticity' in which cell-cell adhesions are disassembled and cells become motile, for example in epithelial-mesenchymal transitions. The acquisition of motility by epithelial cells is also involved in tumor cell invasion and metastasis. The Drosophila follicular epithelium is a model for studying both developmental and pathological epithelial plasticity, and recent observations indicate that Dlg and Fas2 collaborate to prevent inappropriate invasion of follicle cells between neighboring germ cells. Furthermore, border cell migration, an invasion of a subset of follicle cells between germ cells occurring during normal oogenesis, requires downregulation of Fas2 expression in the border cells. PAR-1 is required for detachment of border cells from the follicular epithelium and it is interesting to speculate that this might involve regulation of Dlg localization. These various results indicate that regulation of the Dlg/Fas2 complex is important for the epithelial plasticity exhibited by follicle cells and this mechanism likely applies to epithelia in the developing embryo. As in muscle, Hts over-expression causes elevated levels of PAR-1 and CaMKII in epithelial cells. While Hts over-expression does not cause discernible Dlg delocalization in the epidermis, it disrupts the membrane localization of Dlg in amnioserosa cells. Dlg in the amnioserosa might be particularly susceptible to Hts function, as it not incorporated into septate junctions in this tissue, unlike in the epidermis. Furthermore, if the delocalization of Dlg by Hts is CaMKII-dependent, it would be more pronounced in the amnioserosa than the epidermis as Hts can only weakly promote CaMKII accumulation in the epidermis. In addition to showing that Hts can regulate Dlg localization in the amnioserosa, this study has determined that proper cortical localization of Hts in amnioserosa cells in the early stages of dorsal closure is dependent on Dlg. This result suggests that where Dlg and Hts are together in a complex, Dlg stabilizes the membrane localization of Hts. This stabilization may occur in cells other than the amnioserosa but might not be readily detectable. Hts localizes all around the epithelial membrane, with much of it not co-localizing with Dlg. In the very flat, squamous cells of the amnioserosa early in dorsal closure, the proportion of lateral membrane Hts dependent on Dlg for stabilization may be greater than in more columnar epithelial cells such as those in the epidermis (Wang, 2011).

Consistent with the effects that they have on each other's localization, the frequency of cuticle defects seen in dlg hts zygotic double mutant embryos suggests that Hts and Dlg co-operate in epithelial development, and it is of interest that mammalian adducin has recently been implicated in the stabilization and remodeling of epithelial junctions (Naydenov, 2010). Embryos deficient in Hts likely have a diminished ability to delocalize Dlg, effectively reducing the pool of Dlg available for de novo junction formation, and this situation would be worsened by reducing Dlg levels with a dlg allele. Conversely, reducing Dlg in an embryo deficient in Hts may further compromise Hts function through effects on Hts membrane localization. One of the phenotypes seen in hts mutants, the frequency of which is increased by additionally reducing Dlg, is that of embryos which secrete only small pieces of cuticle. This phenotype is characteristic of maternal zygotic dlg mutant embryos (Wang, 2011).

The interaction of Hts with Dlg suggests that adducin could be a regulator of the septate junction and it will be of interest to examine adducin effects on septate junction morphology in the nervous system. In the Drosophila nervous system septate junctions are formed between glial cells, whereas in mammals they are found between glial and neuronal membranes at the paranodal junction. Interestingly, β-adducin was recently identified as a paranodal junction protein localized to the neuronal membrane where it co-localizes with NCP1, the vertebrate homolog of the Drosophila septate junction protein Neurexin IV (Ogawa and Rasband, 2009) (Wang, 2011).

Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, the effects of Par-1 on Baz localization were studied in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. This study also shows that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, it is proposed that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis (Nam, 2007).

Clonal analysis suggests that Baz is crucial for targeting or maintenance of Par-6 and aPKC. In contrast, Baz protein expression was not significantly reduced in par-6 or apkc null clones, although Baz protein distribution was mislocalized to the basolateral region. This implies that Baz plays a nodal role among the Par-6 complex proteins. A caveat in this analysis is that par-6 and apkc mutant clones are very small (1-2 ommatidia) compared to the relatively large baz mutant clones, raising the possibility that par-6 and apkc mutant clones analyzed may represent rare escaper cells that survive with weaker phenotypes. However, this may not be the case because nearly identical phenotypes were seen in more than 50 par-6 or apkc mutant clones. The data suggesting the central role for Baz in Par-6 and PKC localization are also consistent with studies on embryonic epithelia, in which Baz localization to the membrane precedes localization of Par-6, implying Par-6-independent membrane localization of Baz. The requirement of Baz for the localization of Par-6/aPKC but not vice versa suggests that initial Baz localization may be independent of Par-6 and aPKC, as reported in embryonic epithelia (Nam, 2007).

Similar relationships among Par-6 complex proteins have also been observed during asymmetric cell division in C. elegans. PAR-3, the homolog of Baz, is properly localized in the absence of either PKC-3 (aPKC) or PAR-6, whereas it is indispensable for localization of both PKC-3 and PAR-6. These studies suggest that Baz/Par-3 is a major component in the control of cell polarity in diverse systems, including the photoreceptors in Drosophila. However, the Par-6 complex may exist in different compositions with unique functions, depending on various developmental contexts. For instance, Baz/Par-3 and Par-6 colocalize to the apical cortex of dividing neuroblasts in the Drosophila CNS and in dividing cells in C. elegans embryos, whereas Baz is distinctly localized to the AJ basal to the Par-6 domain in the apical membrane of photoreceptors. It is also striking that whereas Baz is not critically required for photoreceptor differentiation in the larval eye imaginal disc, it becomes crucial during pupal eye development. These data suggest that Baz is required for specific developmental events such as junctional reorganization and rhabdomere formation, although it is expressed in photoreceptors from the time of neuronal fate specification as well as in undifferentiated cells prior to retinal development (Nam, 2007).

Par-1 is a key regulator of Baz localization in ovarian follicle cells. In this system, Par-1 is localized to the basolateral membrane and is essential for exclusion of Baz expression from the basolateral membrane. During early embryogenesis, Par-1 is transiently restricted to the lateral membrane, but at mid-gastrulation it is localized near the apical domain immediately below the region of spot adherens junction (SAJ). Par-1 is required for the restriction of SAJ, preventing the expansion of the E-Cad SAJ marker into the lateral membrane, but not affecting Crb-Dpatj apical markers (Nam, 2007).

In contrast to follicle and embryonic epithelia, in eye imaginal discs, par-1 LOF clones show no significant apical basal polarity defects, suggesting that Par-1 is not required for cell polarity in eye imaginal disc epithelia. This raises a question of whether Par-1 function is dispensable for photoreceptor morphogenesis. In this study, analysis was focused on pupal eye development, since some cell polarity genes such as crb are not required in larval imaginal discs, although they become essential later during the pupal stage when the retina undergoes dramatic reorganization of cell junctions and the apical basal pattern in the photoreceptor cells. Analysis of pupal eyes suggests that Par-1 is required for the distal-proximal growth or maintenance of apical and AJ domains of photoreceptor cells, as Baz and apical markers often fail to form continuous AJ and rhabdomeres along the distal-proximal axis of the retina in par-1 null mutant clones. These phenotypes are similar to the defects shown previously in the eyes of Crb complex mutants. Like par-1 mutations, loss of these gene functions also affects the extension/maintenance of AJ and rhabdomeres but not apical basal cell polarity. Since Baz is essential for proper targeting of Par-6 and Crb complex proteins, loss of Par-1 function may result in mislocalization of Crb complex through affecting Baz localization, although it is possible that Par-1 may also be directly involved in localization of Crb complex proteins independent of Baz. In the eye imaginal disc, it has been reported that Par-1 is localized to the apical-marginal zone and AJ. In the pupal eye, it was also found that Par-1 is enriched in the apical region of photoreceptor clusters, although a low level of Par-1 is also detected broadly along the basolateral membrane. Thus, Par-1 localization is not restricted to the basolateral membrane but appears to be regulated in a complex pattern in different cell types (Nam, 2007).

In ovarian follicle cells, phosphorylation of Baz by Par-1 is required for proper localization of Baz to the apical region of the cells, and BazSA mutated proteins are abnormally localized to the basolateral membrane. In contrast, the current data show that under conditions of overexpression, Baz protein mutated at Par-1 phosphorylation sites is targeted to the AJ whereas wild-type Baz is ectopically localized. Overexpressed wild-type Baz may be abnormally targeted to non-AJ sites, but it is also possible that ectopic Baz may recruit AJ proteins to form ectopic AJs. Nonetheless, the data suggest that, in photoreceptor cells, Par-1-dependent phosphorylation is not essential for initial localization of Baz to AJ. Instead, dephosphorylation of Baz may be a key for the localization of Baz to AJ. This explanation is consistent with the data that the pattern of AJ and apical markers is severely disrupted by overexpression of Par-1 but not by loss of Par-1, suggesting that Baz phosphorylation by Par-1 must be suppressed to maintain photoreceptor cell polarity. The data provide genetic evidence to support that Mts is a major enzyme responsible for antagonizing the effects of Par-1-dependent Baz phosphorylation (Nam, 2007).

PP2A has been implicated in the regulation of tight junction formation in MDCK epithelial cells by interacting with aPKC. However, it is unlikely that the Mts role in Baz localization is mediated through aPKC. First, wild-type and mutant BazSA are localized to completely different sites even though both have an intact phosphorylation site for aPKC. Second, the phenotype of Par-1 overexpression is mimicked by inhibition of Mts but not by loss of aPKC. The idea of specific antagonism between Par-1 and Mts is also supported by the enhancement of the Par-1 overexpression phenotype by reduction of mts gene dosage but not apkc. Thus, a model is proposed in which the localization of Baz to AJ in photoreceptors cells during early pupal eye development depends on the removal of Par-1-mediated phosphorylation by Mts PP2A activity. In this model, Mts plays a pivotal role in regulation of Baz localization and consequent maintenance of photoreceptor cell polarity. On the contrary, Par-1 does not play an essential role for the initial targeting of Baz to AJ, but it is required for growth or stability of AJs and rhabdomeres during photoreceptor morphogenesis. It will be interesting to see whether the antagonistic interaction of Par-1 and PP2A plays an important role in regulation of Baz localization and function in various developmental contexts in Drosophila and other animal species (Nam, 2007).

PP2A antagonizes phosphorylation of Bazooka by PAR-1 to control apical-basal polarity in dividing embryonic neuroblasts

Bazooka/Par-3 (Baz) is a key regulator of cell polarity in epithelial cells and neuroblasts (NBs). Phosphorylation of Baz by PAR-1 and aPKC is required for its function in epithelia, but little is known about the dephosphorylation mechanisms that antagonize the activities of these kinases or about the relevance of Baz phosphorylation for NB polarity. This study found that protein phosphatase 2A (PP2A) binds to Baz via its structural A subunit. By using phospho-specific antibodies, it was shown that PP2A dephosphorylates Baz at the conserved serine residue 1085 and thereby antagonizes the kinase activity of PAR-1. Loss of PP2A function leads to complete reversal of polarity in NBs, giving rise to an 'upside-down' polarity phenotype. Overexpression of PAR-1 or Baz, or mutation of 14-3-3 proteins that bind phosphorylated Baz, causes essentially the same phenotype, indicating that the balance of PAR-1 and PP2A effects on Baz phosphorylation determines NB polarity (Krahn, 2009).

Apical-basal polarity of NBs is controlled by a relatively small number of proteins which assemble into protein complexes localized to the NB cortex in an asymmetric fashion. These cortical proteins interact with each other in a functional hierarchy. At the top of the hierarchy is Baz, because it can localize to the apical NB cortex in loss-of-function mutants for any of the other factors, including PAR-6, aPKC, Insc, Pins, and others (Krahn, 2009).

This study shows that Baz gets frequently mislocalized to the basal NB cortex when it is moderately overexpressed or when it is excessively phosphorylated at S1085, either by overexpression of PAR-1 or by loss-of-function of PP2A. It is expected that similar antagonistic activities of kinases and phosphatases regulate the phosphorylation state of additional sites of Baz/PAR-3 that are relevant in different cellular contexts. Loss of function of 14-3-3ζ and to a lesser extent of 14-3-3epsilon causes mislocalization of endogenous Baz in NBs, whereas overexpression of 14-3-3ζ and 14-3-3epsilon suppresses the mislocalization of overexpressed Baz. It is therefore suggested that the ratio of Baz phosphorylated at S1085 to the amount of available 14-3-3 determines whether Baz gets mislocalized to the basal cortex. In this model, the 14-3-3 proteins function as a buffer to inactivate mislocalized, phosphorylated Baz. This inactivation could be explained by the inhibition of aPKC binding to Baz upon association of 14-3-3 with Baz. If the amount of overexpressed Baz exceeds the buffering capacity of 14-3-3, this would lead to the formation of active Baz/aPKC complexes at the basal cortex. These basally localized, active Baz/aPKC complexes may in turn affect the localization of PAR-1. The mammalian aPKC homolog PKCζ can phosphorylate PAR-1 at a conserved serine residue, and this phosphorylation causes a strong reduction of PAR-1 kinase activity and the release of PAR-1 from the plasma membrane. If the same was true in Drosophila, it would explain the total reversal of NB polarity, because the now basally localized aPKC would phosphorylate PAR-1, which would cause its release from the membrane and the establishment of a new apical cortical domain at the previously basal cortex (Krahn, 2009).

PAR-1, 14-3-3 proteins, and PP2A are strongly expressed during oogenesis, and maternal contributions may account for difficulties identifying requirements during early embryogenesis. In contrast, eliminating maternal expression of these genes results in phenotypes too severe to allow the study of neurogenesis. However, overexpression of a dominant-negative form of Mts from early neurogenesis onward also caused polarity reversal only in late-stage NBs. While this experiment does not exclude the possibility that the late onset of polarity reversal in NBs is due to the perdurance of the maternal gene products, it points to a fundamental difference in the mechanism of how NB polarity is controlled immediately after delamination as opposed to subsequent asymmetric divisions. The majority of late-stage NBs showing polarity reversal were not in direct contact with the overlying epithelium and thus may rely exclusively on intrinsic polarity cues, in contrast to NBs that have just delaminated and maintain contact to the overlying epithelium. Late-stage NBs lacking contact to the overlying epithelium show a higher variability of spindle orientation as compared to early-stage NBs in close contact to the epithelium. Thus, late-stage NBs may be particularly sensitive to changes in the phosphorylation state and general activity level of Baz, because they rely on Baz as the main cue for orienting their polarity axis (Krahn, 2009).

It is interesting to note that mutations uncoupling spindle orientation from the localization of cell fate determinants commonly show fully random spindle orientation, including a variety of oblique orientations. In contrast, hyperphosphorylation of Baz at S1085 resulted very rarely in oblique orientations, and spindles were always aligned with the asymmetric crescents of cell fate determinants. Although a good explanation for why there is a strong bias for either total reversal of polarity or misorientation of the spindle by 90° is not available, the findings point to the existence of a spatial cue functioning upstream of Baz that defines a polarity axis perpendicular to the plane of the epithelium (Krahn, 2009).

Regulation of cyclin A localization downstream of Par-1 function is critical for the centrosome orientation checkpoint in Drosophila male germline stem cells.

Male germline stem cells (GSCs) in Drosophila melanogaster divide asymmetrically by orienting the mitotic spindle with respect to the niche, a microenvironment that specifies stem cell identity. The spindle orientation is prepared during interphase through stereotypical positioning of the centrosomes. It has been demonstrated that GSCs possess a checkpoint ('the centrosome orientation checkpoint') that monitors correct centrosome orientation prior to mitosis to ensure an oriented spindle and thus asymmetric outcome of the division. This study shows that Par-1, a serine/threonine kinase that regulates polarity in many systems, is involved in this checkpoint. Par-1 shows a cell cycle-dependent localization to the spectrosome, a germline-specific, endoplasmic reticulum-like organelle. Furthermore, the localization of cyclin A, which is normally localized to the spectrosome, is perturbed in par-1 mutant GSCs. Interestingly, overexpression of mutant cyclin A that does not localize to the spectrosome and mutation in hts, a core component of the spectrosome, both lead to defects in the centrosome orientation checkpoint. It is proposed that the regulation of cyclin A localization via Par-1 function plays a critical role in the centrosome orientation checkpoint (Yuan, 2012).

Thus Par-1 acts as a component of the centrosome orientation checkpoint, probably through its ability to influence cyclin A localization. This checkpoint ensures the asymmetric outcome of GSC division by delaying cell cycle progression when centrosomes are not correctly oriented. Such a checkpoint would provide an additional layer of accuracy in oriented stem cell division. This study highlights the importance of cyclin A localization in the centrosome orientation checkpoint. Intriguingly, it was reported that in cultured mammalian cells, cyclin A is confined to the endoplasmic reticulum (ER) via its interaction with a protein called SCAPER (Tsang, 2007). The spectrosome/fusome has been shown to be a part of the ER (Snapp, 2004), therefore, regulation of cyclin A through its localization is likely evolutionarily conserved (Yuan, 2012).

The fact that the wild type misoriented GSCs tend to have lower/non-detectable cyclin A levels suggests that GSCs degrade cyclin A or that the arrest point of the centrosome orientation checkpoint is before cyclin A accumulation. It is possible that distinct mechanisms stall the cell cycle, depending on when the centrosome misorientation is sensed. For example, when the centrosome misorientation is detected earlier in the cell cycle (i.e., before cyclin A accumulation), the cell cycle would be stalled before cyclin A protein synthesis/accumulation. In contrast, when the centrosome misorientation is detected later in the cell cycle (i.e., after cyclin A accumulation), the cell cycle would be stalled by preventing translocation of cyclin A from the spectrosome to the cytoplasm/nucleus. Further studies are required to dissect the detailed mechanisms that monitor centrosome orientation, possibly depending on the cell cycle stage (Yuan, 2012).

It is currently unclear how Par-1 might regulate cyclin A localization in response to centrosome misorientation. Direct interaction between Par-1 and cyclin A was not detected in immunoprecipitation experiments, thus the molecular mechanism by which Par-1 regulates cyclin A localization to the spectrosome/fusome remains to be determined. It is formally possible that cyclin A mislocalization and the defective checkpoint response are two unrelated consequences of par-1 mutation. However, considering that the expression of cyclin A mutant proteins defective in spectrosome localization is sufficient to perturb the centrosome orientation checkpoint, the possibility is favored that cyclin A is indeed part of a Par-1-dependent checkpoint response to centrosome misorientation. Future identification of proteins that recruit/anchor cyclin A to the spectrosome will provide further insight into this process (Yuan, 2012).

This study has shown that the mother centrosome is consistently located at the hub–GSC interface, while the daughter centrosome migrates to the opposite side. Whether the centrosome orientation checkpoint monitors the correct positioning of the mother centrosome or any centrosome is currently unknown. However, given that dedifferentiated GSCs, which must have lost their 'original' mother centrosome (generated earlier during development) when they committed to differentiation, still retain the centrosome orientation checkpoint, the centrosome orientation checkpoint does not appear to monitor the presence of 'original' mother centrosomes. It is still possible that the centrosome orientation checkpoint monitors the presence of 'mature' centrosomes (not necessarily from earlier in development, but > 2 cell cycle-old centrosomes) at the hub–GSC interface. Interestingly, it was recently shown that the daughter centrosome is consistently inherited by stem cells during the divisions of Drosophila neuroblast. Given the precise inheritance of mother or daughter centrosomes depending on the context/stem cell system, it is tempting to speculate that the centrosome orientation checkpoint monitors the presence of the mother centrosome in male GSCs, and possibly an equivalent mechanism monitors the daughter centrosome inheritance in neuroblasts (Yuan, 2012).

In developing embryos, cyclin A localization was reported to be dispensable for its activity. Even the plasma membrane-bound form of cyclin A was shown to be able to fulfill its function to promote mitosis. Indeed, the mutant forms of cyclin A protein used in this study (NLS-CycA and Cyclin AγC) are 'functional' in that they can promote the cell cycle progression into mitosis. Instead, it is proposed that these cyclin A mutant proteins cannot be subjected to a negative regulation by Par-1. It is possible that the embryonic cell cycle has minimal negative regulation as in embryonic stem cells, while male GSCs have an additional regulatory step (i.e., the centrosome orientation checkpoint) that negatively regulates mitotic entry (Yuan, 2012).

The lack of spindle misorientation in Dsas-4 mutant male GSCs is intriguing. In the complete absence of the centrosome, the spindle was correctly oriented in dividing GSCs, while defective centrosome function in cnn mutant leads to abrogation of the centrosome orientation checkpoint. Dsas-4 mutant male GSCs apparently orient the mitotic spindle via anchorage of spindle pole to the apically-localized spectrosome, which is highly reminiscent to the spindle orientation mechanism in female GSCs. The prediction would be that the spindle orientation is randomized in Dsas-4 hts double mutant male GSCs, which lacks both the centrosome and spectrosome. Unfortunately, the analysis of the double mutant was technically very challenging; Dsas-4 single mutant flies die as pharate adult, and the survival of the double mutant was worse. Furthermore, it was never possible to observe any mitotic GSCs from those pharate adult double mutants that were recovered and analyzed. Thus, future studies will be required to test this prediction (Yuan, 2012).

This study illuminates the importance of stem cell-specific regulators of the general cell cycle machinery such as cyclin A. We propose that stem cells have developed elaborate mechanisms to ensure an asymmetric outcome of the stem cell division, the failure of which can lead to tumorigenesis or tissue degeneration (Yuan, 2012).

Par-1 regulates tissue growth by influencing hippo phosphorylation status and hippo-salvador association

The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. This study reports the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevates Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminishes Hpo target gene expression and reduces organ size. par-1 functions downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, it was also found that Par-1 physically interacts with Hpo and Sav and regulates the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibits the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, evidence is provided that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, these findings identified Par-1 as a novel component of the Hpo signaling network (Huang, 2013).

The Hpo signaling pathway has emerged as a conserved pathway that controls tissue growth and balances tissue homeostasis via the regulation of the downstream Sd-Yki transcription complex. Despite the importance of this pathway in development and carcinogenesis, many unknown regulators of the Hpo pathway remain to be identified. This study identified Par-1 as one such Hpo pathway regulator via a genetic overexpression screen using Drosophila EP lines. This study demonstrated that Par-1 was essential for the restriction of Hpo signaling. It was also demonstrated that overexpression of Par-1 promotes tissue growth via the inhibition of the Hpo pathway, whereas loss of Par-1 promotes Hpo signaling to suppress growth and induce apoptosis. Using the Drosophila eye and wing imaginal discs as well as cultured cells, this study provides the first genetic and biochemical evidence for a function of Par-1 in the Hpo pathway (Huang, 2013).

Although the conserved function of Hpo has been well studied, the regulatory mechanism of its kinase activity is still largely obscure. Currently, the regulatory mechanism of Hpo kinase activity is believed to mainly be dependent on autophosphorylation by altering the phosphorylation status of the Thr195. However, whether the uncharacterized phosphorylation events of Hpo, which have been identified in several recent proteome-wide phosphorylation studies, contribute to the regulation of Hpo activity is still unknown. By studying the mechanism underlying Par-1 function in Hpo signaling, this study demonstrated that Par-1 induces Hpo phosphorylation at Ser30 and this leads to the regulation of Hpo kinase activity (Huang, 2013).

In recent proteome-wide phosphorylation studies using Drosophila embryos, it was suggested that Hpo was phosphorylated at Ser30 in vivo, indicating an important role for the Ser30 site in the regulation of Hpo activity. To determine the biological significance of Hpo phosphorylation at Ser30 induced by Par-1, whether Ser30 phosphorylation state affects Hpo phosphorylation at Thr195, which is important for Hpo activation, was tested. Par-1, but not Par-1-KD, was shown to significantly inhibit Hpo phosphorylation levels at Thr195, whereas this inhibitory effect was abolished when the Ser30 site was mutated. More importantly, phosphorylation at Thr195 was slightly elevated when Ser30 was mutated into an alanine. These findings suggested that Par-1 regulates Hpo activity via antagonizing phosphorylation at the Thr195 site by regulating Ser30 phosphorylation. It has been reported that the Hpo Thr195 site is not only auto-phosphorylated but also phosphorylated by Tao-1, a partner of Par-1 in the regulation of microtubule dynamics. Thus, it was asked whether Par-1-induced phosphorylation at Ser30 also affects Tao-1-mediated phosphorylation at Thr195. Par-1 was shown to suppress Tao-1-mediated phosphorylation at Thr195. The antagonistic effect of Par-1 and Tao-1 on Hpo phosphorylation at Thr195 motivated the examination of the interrelationship of Par-1 and Tao-1 in the Hpo pathway. It was found that Tao-1 disrupted Par-1-induced phosphorylation mobility shift of Hpo-KD, suggesting that the function of Par-1 in the Hpo pathway was modulated by upstream signaling (Huang, 2013).

Several unresolved questions remain. The interaction between Par-1 and Hpo/Sav may be tightly regulated because full-length Par-1 only weakly interacts with Hpo/Sav, unlike the interaction with the N-terminal fragment of Par-1. However, the triggering signal for Par-1 to interact with Hpo/Sav is still unknown. It has been reported that Par-1 is activated by Tao-1 and LKB1. This study established that Par-1 antagonized Tao-1 in Hpo signaling: in Drosophila, the antagonistic relationship between Par-1 and Tao-1 in microtubule regulation has been previously reported. Thus, it is unlikely that Tao-1 functions as the trigger. Whether LKB1 functions as an activator of Par-1 in Hpo signaling was investigated by expressing the LKB1 transgene in different organs. Unlike Par-1, ectopic LKB1 expression limits both wing and eye growth, indicating that LKB1 is also not the trigger (Huang, 2013).

This study has shown that Par-1 and Tao-1 exhibit opposing effects on Hpo signaling. Given that Tao-1 and Par-1 are partners that regulated microtubule dynamics via the phosphorylation of Tau, Tau may have a function in Hpo signaling. To investigate this hypothesis, genetic and biochemical studies were employed, and it was found that Tau RNAi failed to suppress the expression of Hpo pathway-responsive genes. In addition, Tau did not trigger Hpo phosphorylation and Sav dissociation in vitro, indicating that Par-1 regulates Hpo signaling independent of Tau. Interestingly, it has been previously suggested that Par-1 does not regulate Tau activity in Drosophila, indicating an evolutionary difference between Par-1 and Tau-1 function (Huang, 2013).

This study has provided evidence that Par-1 regulates Hpo signaling via the phosphorylation of Hpo or the destruction of the Hpo/Sav complex. Because Par-1 is a well-known polarity regulator and polarity components, such as Crumb and Lgl, have been shown to be involved in the Hpo signaling pathway, it is possible that Par-1 may regulate Hpo signaling via a polarity complex, or its activity might be regulated via a polarity complex. Indeed, the localization of Crumb and Patj were affected by Par-1 expression. Thus, further studies on polarity complexes and Hpo signaling will help elucidate this problem (Huang, 2013).

A Par-1-Par-3-centrosome cell polarity pathway and its tuning for isotropic cell adhesion

To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy, but AJs are dynamic during epithelial development and remodeling, and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation. This study shows that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer, and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization. Par-1 was found to promote the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference (Jiang, 2015).

The identification of Par-1 as an inducer of Baz-centrosome co-recruitment is surprising given its well-established role in inhibiting Baz complex formation in Drosophila, C. elegans, and mammalian systems. It is proposed that Par-1 contributes to both global inhibition and local promotion of Baz complex assembly, providing a simple and potent Baz polarization mechanism (Jiang, 2015).

The Baz-centrosome positive feedback loop is evident from the specific accumulation of Baz next to cortical centrosomes, the MT requirement for Baz accumulation, the Baz requirement for centrosome recruitment, and the dynein role for drawing Baz and centrosomes together. Significantly, Par-1 is also necessary and sufficient for the loop and seems to have two direct roles. One is promotion of astral microtubules around the centrosome, an effect consistent with known effects of Par-1 on MT regulators, but requiring further elucidation in the Drosophila embryo. The other is the phosphorylation of Baz at Ser-151 and Ser-1085. These modifications have well-characterized inhibitory effects on Baz cortical association, but strikingly, they are also enriched where the Baz-centrosome positive feedback loop occurs and appear necessary for Baz entry into the loop. It is speculated that phospho-regulated Baz-14-3-3 protein interactions mediate further protein interactions, or induce conformational changes, important for Baz-MT association. Indeed, 14-3-3 proteins can bridge MT motors, a Par-3 conformational change induces direct MT binding, Par-3 directly binds a dynein subunit, and other links to MTs are known (Jiang, 2015).

Although the Par-1-Par-3-centrosome pathway can be a potent Baz polarization mechanism, it is normally attenuated within a homeostatic system. During early cellularization, Par-1 localizes over the entire PM and presumably phosphorylates Baz and MT regulators. In response, it is proposed that Baz is continually displaced and diffuses over the PM but is additionally primed for MT interactions. Simultaneously, the two centrosomes found atop each nucleus would provide the positional information for localizing Baz around the apical circumference through dynein-mediated MT associations. As Baz accumulates, it recruits aPKC to the apical domain, from where aPKC then displaces Par-1. Normally, this Baz-aPKC-Par-1 negative feedback loop seems to keep the Par-1-Baz-centrosome pathway in check. In the absence of aPKC, the Par-1- Baz-centrosome pathway continues unabated, leading to excessive Baz and centrosome polarization, loss of AJ isotropy, and later epithelial dissociation (Jiang, 2015).

Intriguingly, focused accumulations of Par-3 and AJs colocalize with cortical centrosomes during C. elegans intestinal development and during zebrafish collective cell migration. Moreover, Par-1 induces centrosomal MT interactions with AJs during human liver lumen formation in vitro and is needed for Baz-centrosome associations during the asymmetric division of Drosophila germline stem cells. Thus, induction of the Par- 1-Par-3-centrosome pathway, with regulated shifts to aPKC or Par-1 activities, may be generally relevant to developmental transitions of animal tissues (Jiang, 2015).


DEVELOPMENTAL BIOLOGY

Par-1 transcripts are expressed in both the germline and somatic follicle cells throughout oogenesis, but appear to be uniformly distributed at all stages. However, stainings with the Par-1 antisera reveal that the protein localizes to a number of specific sites in both the germ cells and the follicle cells. Par-1 localizes cortically in the follicle cells at early stages, and is restricted to the basolateral membrane domain of the columnar epithelium in stage 10 egg chambers, similar to the distribution of mammalian Par-1 homologs in cultured epithelial cells (Bohm, 1997). The earliest Par-1 staining in the germline is localized to the fusome, a branched structure extending through the ring canals that functions to orient mitotic spindles during the cell divisions in the germarium. As the cysts develop further, Par-1 localizes to the ring canals and the cortical cytoskeleton of the nurse cells. No asymmetric localization has been detected within the oocyte during stages 1-8, but in early stage 9 egg chambers, Par-1 is transiently enriched at the anterior of the oocyte. It then starts to accumulate at the posterior of the oocyte, and becomes progressively more concentrated at the posterior pole during stages 9-10. The posterior localization of Par-1 is very similar to that of the first components of the pole plasm, OSK mRNA and Staufen (Stau) protein, which also show a transient anterior localization before moving to the posterior cortex during stage 9. Indeed, Par-1 colocalizes with a GFP:Stau fusion protein at the posterior of the oocyte. Thus, like its counterpart in C. elegans, Drosophila Par-1 is one of the earliest markers for the posterior pole. The localization of Par-1 cannot be reliably followed at later stages in oogenesis because the chorion blocks antibody penetration, but the protein is not enriched at the posterior of early embryos (Shulman, 2000).

Par-1 and the polarization of the oocyte

After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Par-1 is shown to become restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. baz is required for Par-1 relocalization within the oocyte and the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization (Vaccari, 2002).

In Drosophila, early oogenesis involves formation of a germline cyst consisting of an oocyte surrounded by 15 nurse cells. The cyst develops in region 1 of the germarium, where a germline stem cell-derived cystoblast divides four times with incomplete cytokinesis and produces a cyst of 16 cells interconnected by the fusome, a continuous membrane-skeletal organelle. The fusome is asymmetrically partitioned into the sibling cells and acts at multiple steps in early oogenesis. During the cyst divisions, it is connected to one spindle pole and guides the cells through an invariant division pattern. Later on, it acts as a scaffold for the migration of the cyst centrosomes into the oocyte, resulting in the organization of a polarized microtubule (MT) network in the cyst. During oocyte determination, the fusome progressively disassembles. In region 2a, the presumptive oocyte, which contains the most fusomal remnants, accumulates determinants such as Bicaudal D (BicD), Egalitarian (Egl), and oo18 RNA binding protein (Orb). Between germarial region 2b and region 3 (also referred to as stage 1 of oogenesis), the oocyte itself becomes polarized, as revealed by the relocalization of Orb from the anterior to the posterior of the oocyte (Vaccari, 2002 and references therein).

Genetic analysis of Drosophila par-1 function has revealed a requirement for the kinase during oocyte polarization; in par-1 null alleles, Orb fails to relocate to the posterior of the oocyte and eventually disappears, after which the oocyte loses its fate and becomes a nurse cell. In addition to Orb, two other oocyte determinants, BicD and Egl, also fail to relocalize within par-1 null oocytes. It has been unclear how Par-1, reported to localize exclusively to the fusome during early oogenesis, could affect the polarization process, which takes place several hours after disappearance of the fusome. D. melanogaster par-1 encodes several isoforms, which are composed of three alternative N termini (N-ter 1–3), a constant kinase, and a spacer domain; they also may include or exclude a C-terminal (C-ter) domain. It was reasoned that the reported Par-1 localization, as revealed by a pan-Par-1 antibody raised against the spacer domain (Par-1 spacer antibody), might only partially reflect the distribution of the kinase. Germline expression of an isoform (N1S) consisting of N-ter 1, the kinase, and the spacer domains and lacking the C-ter allows normal development of par-1 null oocytes. A peptide antibody was raised specifically against N-ter 1 (N1 antibody) and compared the Par-1 signals detected by this antibody and those detected by the original Par-1 spacer antibody. In contrast to the spacer antibody, which detects Par-1 on the fusome, the N1-specific antibody first detects Par-1 in the cyst as the fusome disappears, with the signal becoming restricted to the cytoplasm of the oocyte during its determination in region 2b. This novel Par-1 signal colocalizes with the oocyte marker Orb. The fact that the N1 antibody does not detect Par-1 on the fusome might be explained by a selective accessibility of different domains of the protein. In fact, the N1-specific antibody, which detects Par-1 in the early oocyte, does not recognize fusome-associated Par-1, even in ovaries in which the protein is overexpressed and visible as a GFP fusion on the fusome (Vaccari, 2002).

During oocyte specification, localization of the determinants BicD, Egl, and Orb to the early oocyte relies on the asymmetric distribution of microtubules in the cyst, evident as a dense focus of MT in the oocyte. Depolymerization of the MT by colchicine abolishes the localization of BicD, Egl, and Orb and results in egg chambers with 16 nurse cells and no oocyte. It was therefore asked if the restriction of Par-1 to the oocyte during the transition from region 2a to region 2b is also MT dependent. Ovaries of flies fed with colchicine for 12 hr fail to localize Par-1 and Orb to the oocyte, indicating that Par-1 restriction to the oocyte is indeed MT dependent. This is in contrast to the localization of Par-1 to the fusome, which occurs independently of MT (Vaccari, 2002).

The distribution of Par-1 within the oocyte was further examined by focusing on the transition between regions 2b and 3, when par-1-dependent polarization of the oocyte occurs. In germarial region 2b, Par-1 is enriched anterior to the oocyte nucleus. In region 3, the protein is mainly detected at the posterior of the oocyte, where it remains. During this relocalization, Par-1 colocalizes completely with BicD in the germline. Because par-1 is required for BicD relocalization within the oocyte, the distribution of Par-1 was examined in BicD hypomorphs that allow differentiation of an oocyte. Par-1 is detected but mislocalized in an anterior dot within the BicD mutant oocytes. Hence, Par-1 and BicD are interdependent for their relocalization to the posterior of the oocyte region 3b (Vaccari, 2002).

To assess whether the MT cytoskeleton mediates relocalization of Par-1 and BicD within the oocyte, wild-type ovaries were dissected a short time after treatment with colchicine. A screen was carried out for region 3 egg chambers in which the focus of oocyte MT was destroyed. In these, BicD and Par-1 remain anterior to the oocyte nucleus, indicating that MTs are required for oocyte polarization (Vaccari, 2002).

The MT motor Dynein has been reported to influence development of the germline cyst. Loss-of-function mutants in dhc64C, encoding the heavy chain of the minus end-directed molecular motor Dynein, fail to develop an egg chamber because of mitotic failure in the germarium. However, hypomorphic dhc64C mutants develop an oocyte and 15 nurse cells. In a high percentage of such egg chambers, both Par-1 and BicD remain at the anterior of the oocyte in region 3. Hence, after its initial requirement in cyst formation, the minus end-directed motor Dynein is involved in the relocalization of Par-1 and BicD to the posterior of the oocyte (Vaccari, 2002).

It has been reported that, like Par-1, the D. melanogaster homolog of Par-3 Bazooka (Baz) is necessary for oocyte maintenance. Germline clones of a baz null allele are defective in oocyte polarization, establishing a functional parallel with C. elegans, in which the PAR genes polarize the early embryo. In the worm, restriction of Par-1 activity to the posterior cortex of the embryo crucially depends on Par-3, but the reverse is not the case. In the Drosophila germarium, Par-1 localization to the fusome appears to be independent of Baz. To assess whether the subsequent localization of Par-1 in the oocyte requires Baz, Par-1 distribution was further evaluated in baz germline clones. Par-1 is initially detected at the anterior of the oocyte in region 2b, but the protein disappears in region 3, revealing that Par-1 relocalization is baz dependent (Vaccari, 2002).

The relative distributions of Par-1 and Baz were investigated during later stages of oogenesis in wild-type ovaries. Par-1 is detected at the posterior of the oocyte as of germarial region 3 and becomes tightly associated with the cortex between oogenesis stages 3 and 5. Concomitantly, Baz becomes transiently localized to the anterior of the oocyte. The anterior localization of Baz is specific; it is not observed in germline clones of a baz null allele. Interestingly, maximal expression of Baz at the anterior coincides with the apparent tightening of Par-1 signal at the posterior cortex, suggesting a role for Baz in sharpening Par-1 localization. At this stage, the localization of the two proteins appears to be mutually exclusive. The anterior enrichment of Baz between stages 2 and 5 is absent in germline clones of the par-1 null allele (Vaccari, 2002).

The mutually exclusive distribution of Par-1 and Baz in the Drosophila oocyte is strikingly reminiscent of that observed in the C. elegans embryo. The ability to visualize the two proteins has allowed their respective roles in achieving this distribution to be genetically evaluated. The seeming dependence of Baz localization on par-1 is in contrast to results in C. elegans, in which localization of Par-3 is independent of Par-1. However, the absence of Baz in par-1 germline clones may well reflect the loss of oocyte fate and the onset of its degeneration that occurs at stage 1. Nonetheless, the anterior localization of Baz during stages 2–5 suggests that, after acting in oocyte polarization in region 2b/3, baz may be required again during oogenesis. The existence and nature of such a second requirement for baz after oocyte polarization is not yet clear (Vaccari, 2002).

Research has shown that the fusomal localization of Par-1 is unaffected in baz mutants, suggesting a difference between the Drosophila oocyte and the C. elegans embryo, in which Par-1 localization depends on par-3. Remarkably, at the time when oocyte polarization takes place, baz is in fact required for Par-1 localization within the oocyte. Hence, it appears that a similar relationship exists between Par-1 and Baz at the time when their activities are critical in the two organisms (Vaccari, 2002).

Impairment of the MT cytoskeleton and mutations in BicD and dhc64C also affect Par-1 relocalization within the oocyte. Conversely, in par-1 mutants, the MT cytoskeleton is not focused in the oocyte, BicD fails to relocalize, and Dynein is not enriched in the oocyte. The mutual interdependence of these genes and the MT suggests that all these components cooperate to form a polarization complex in the oocyte. Interestingly, the N1 antibody begins to detect Par-1 only when its function is genetically required, suggesting that, in region 2, the kinase may undergo a change in conformation or in its association with other factors (Vaccari, 2002).

The presence and localization of Par-1 in the oocyte at the time of its determination and polarization complements the previously reported localization of Par-1 on the fusome prior to oocyte determination and establishes Par-1 as a unique oocyte marker, for at least two reasons. (1) Absence of any one of the oocyte determinants, BicD, Egl, or Orb, prevents the concentration of the two other determinants in this cell. In contrast, in the absence of Par-1, it is the relocalization of the determinants within the oocyte that is specifically affected. (2) BicD, Egl, and Orb are not present on the fusome, and the observed enrichment of these determining factors in the oocyte is the result of the enrichment of their RNAs in this cell during its specification. In contrast, no par-1 RNA is detected in the germline at such early stages. The idea that Par-1 is initially loaded on the fusome, where it perdures during the cyst divisions, and that it is later preferentially inherited by the oocyte, is favored. Taken together, the facts that par-1 mutants show no fusomal defects and that accumulation of Par-1 itself in the oocyte requires MT suggest that Par-1 does not affect the oocyte MT cytoskeleton from its fusomal location. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where it acts in conjunction with BicD, Egl, and Dynein to effect polarization (Vaccari, 2002).

Genetic interaction screens identify a role for hedgehog signaling in Drosophila border cell migration

Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration. Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par-1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E-Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh-Rac and Hh-Par-1 signaling was illustrated in the wing, in which Hh-dependent phenotypes were enhanced by loss of Rac or par-1. This study has identified a pathway by which Hh signaling connects to Rac and Par-1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways (Geisbrecht, 2013).

A role for the Hh signaling pathway in collective migration of the border cells was uncovered in two independent genetic screens. Previous genetic mosaic screens in border cells identified a role for cos2 in polar cell differentiation, but had yet to reveal a role for Hh signaling components in border cell migration. It has long been recognized that alternative screening methods are advantageous in uncovering genes that may be required earlier in development and/or for those genes with redundant functions. Both of these explanations are supported by published literature and the data presented in this study. First, ectopic Hh signaling, either by overexpression of hh itself or loss of the downstream components ptc, cos2, or Pka-C1, produces early ovarian phenotypes that include oocyte mis-positioning and excess polar cells. These events occur prior to border cell recruitment and migration and thus may complicate analyses of Hh signaling in subsequent oogenic processes. This potential issue was bypassed by inducing downregulation of the Hh pathway specifically in border cells just prior to their migration. Second, the migration defects due to loss of Hh pathway components appear to be incompletely penetrant. Despite considerable reduction of Hh signaling due to overexpression of ptc, most border cells were able to complete their migration. However, the significant suppression of RacN17 motility defects by overexpression of Hh particularly indicates an important functional role for this pathway in border cells. The data are thus consistent with other, at present unknown, signaling pathways functioning in concert with Hh for proper cell migration (Geisbrecht, 2013).

The Hh pathway is capable of regulating a wide variety of cellular responses through transcriptional regulation of downstream target genes. In most tissues, Hh is secreted from a local source, but the downstream effects occur only in ptc-receiving cells that may reside up to ten cell diameters away. In migrating border cells, the results presented in this study suggest an autocrine mechanism where Hh is both produced and received by the same cells. Both the hh-lacZ and multiple ptc-lacZ enhancer traps/reporters reveal transcriptional activity in the outer, migratory cells of the cluster. Furthermore, this study has shown that hh expression is regulated by JAK/STAT signaling and is independent of Slbo regulation. It remains a distinct possibility that the Hh signal is relayed between border cells within the cluster. Nonetheless, the data favor a role for Hh specifically in border cells rather than receiving Hh signal from other cells in the ovary. This idea is supported by findings that migration was impaired when Hh and proteins required for its signal reception and transduction were knocked down by RNAi selectively in the border cells using slbo-GAL4. Furthermore, border cells mutant for disp did not complete their migration. As Disp is required in Hh-secreting cells for release of lipid-modified active Hh this further indicates that border cells produce Hh signal. It is still unclear whether paracrine versus autocrine Hh signaling is biologically important. However, a number of studies have reported roles for autocrine Hh activity in the Drosophila wing disc and optic primordium in the embryo and the salivary gland in the larva, as well as autocrine Sonic hedgehog (Shh), a vertebrate Hh homolog, in neural stem cells, B-cell lymphoma, and interferon-stimulated cerebellar dysplasia during brain development. This type of Hh signaling mechanism also occurs in a variety of human tumors, where abnormal Hh pathway activation in an autocrine fashion increases cell proliferation and invasion (Geisbrecht, 2013).

A question raised by this study is what role the Hh signaling pathway plays in border cell migration. As depicted in a proposed model, border cells with reduced Hh activation exhibited altered localization of E-cad and depolarized p-Tyr, either of which could affect border cell motility. E-cad is required for border cell migration by promoting proper adhesion with the nurse cell substrate. Importantly, disruption of E-cad localization contributes to migration defects caused by loss of steroid hormone signaling and cell polarity genes. Loss of one of the guidance ligands, pvf1, disrupts E-cad localization in border cells similar to what was observed when Hh activity was impaired. Given that PVF1 signals to the receptor PVR on border cells, this suggests a connection between Hh and RTK signaling. Indeed, wild-type border cells exhibit polarized activation of the RTKs PVR and EGFR prior to migration as assayed by global tyrosine phosphorylation and specific phosphorylation of PVR (Tyr-1428). Disruption of this polarized RTK by several mechanisms, as shown by reduced or mislocalized p-Tyr, impairs border cell migration. The data suggest that Hh signaling restricts polarized p-Tyr to the front of the border cell cluster. Interestingly, overexpression of vertebrate Shh in keratinocytes increased activation of EGFR and invasion through matrix. Moreover, there is evidence for synergism between Hh and EGFR signaling to activate Gli (Ci homolog) transcription targets in human cells. Nonetheless, proteins other than RTKs (and/or their targets) can be phosphorylated on tyrosines and thus recognized by the p-Tyr antibody; thus, the role for Hh signaling in border cells may be independent of the RTK pathways. Regulators of endocytosis are also required for localized, high levels of p-Tyr in border cells. However, in contrast to loss of hh, loss of endocytic pathway members do not significantly impair E-cad levels or localization. Thus, Hh likely regulates border cell migration by a distinct mechanism. Further experiments will be needed to determine if Hh signaling is required for the proper levels or distribution of an unknown protein(s) that affects tyrosine phosphorylation and E-cad localization during border cell migration (Geisbrecht, 2013).

The data presented in this study suggest a link between the Rac, Hh, and Par-1 signaling pathways. However, understanding of how these proteins function together to modulate migration remain a mystery. The results from the suppression screen indicate that overexpression of Hh can overcome Rac-dependent migration defects. In fact, Hh was the strongest suppressor obtained from the screen. A simple explanation for this observation is that Ci induces transcription of one or more as yet unknown downstream target genes required in the migratory process. However, the entire repertoire of Ci targets has yet to be elucidated and specific targets (apart from ptc itself) in border cells are unknown. Interestingly, Rac2 was recently uncovered as a potential target of the Hh signaling pathway in the Drosophila embryo. Both Rac1 and Rac2 are essential for border cell migration. Thus, it is intriguing to speculate that upregulation of Rac2 by Hh is a possible mechanism to overcome the RacN17 migration phenotype. Alternatively, the Hh pathway may directly or indirectly affect regulation of Rac protein activity either by increasing the amount of active Rac-GTP or by regulating the subcellular localization of activated Rac within the border cell cluster. Hek 293 cells exposed to Shh had increased levels of the active form of the small GTPase RhoA. Similarly, Shh stimulated RhoA and Rac via phosphoinositide 3-kinase (PI3K) signaling during chemotaxis of fibroblasts. Because border cells do not rely on PI3K activity, another mechanism is likely involved (Geisbrecht, 2013).

What is the connection between Par-1 and the Hh signaling pathway? One possibility is the emerging requirement for microtubules in Hh signaling. Disruption of microtubules with the drug nocodazole prevents downstream transcriptional responses, possibly due to nuclear translocation of Gli proteins. Furthermore, Cos2-mediated subcellular motility and translocation of its cargo Ci requires microtubules in Drosophila. Par-1 is a central player in mediating microtubule polymerization and dynamics. More specifically, phosphorylation of microtubule-associated proteins (MAPs) by the Par-1 kinase induces detachment of MAPs from microtubules. It is interesting to speculate that the kinase activity of Par-1 is essential in the Hh pathway to regulate Cos2 or MAP proteins for Ci mobility. Another possibility is that Par-1 functions through Rac. Overexpression of Par-1 partially suppressed the RacN17 migration defect, similar to overexpression of Hh. Although it is unclear why Par-1 rescued the Rac phenotype, it is possible that Par-1 acts in parallel to Hh signaling to promote Rac-mediated border cell motility. Notably, mammalian MARK2 (Par-1 homolog) promotes microtubule growth downstream of Rac1 at the leading edge of migrating cells. Further studies, however, are needed to determine the precise molecular relationships amongst the Rac, Hh and Par-1 pathways in collectively migrating border cells (Geisbrecht, 2013).

Cell shape changes are important for most aspects of morphogenic processes, including cell contractility and cell migration. Hh signaling induces cell shape changes in the developing Drosophila eye via regulation of non-muscle myosin II. Thus, the role of Hh in border cells may be to regulate cell shape during migration. Accumulating evidence points to a non-canonical role for Hh in mediating mammalian cell migration. Shh can function as a chemoattractant in migrating cells and guidance of axons independent of Gli-induced gene transcription. In axon guidance, Shh stimulates phosphorylation and activation of Src kinase and thereby facilitates axon turning through regulation of the actin cytoskeleton. Specifically, Shh induced phosphorylation of Src at Tyr-418, an activating site, and polarization of Src family kinases within the axon. This appears to be consistent with the finding that loss of Hh activity depolarized global p-Tyr distribution. In other migratory cell types, Shh also acts as a chemoattractant that induces cytoskeletal rearrangements and migration independent of the canonical transcriptional respons. However, the mechanism of Hh function in border cell migration is likely to be different for several reasons. First, Hh is unlikely to function as a long-range chemoattractant, because border cells are the likely source of Hh signal. Second, a role for Src in border cell migration is unknown at present, so Hh may mediate tyrosine phosphorylation of other substrates in border cells. Third, these is evidence that Ci is involved in border cell migration and therefore canonical Hh-induced transcription is predicted to be important. Nonetheless, the results are consistent with a conserved role for the Hh pathway in regulating cytoskeletal-mediated events in migrating cells, which in border cells likely functions through the Rac GTPase (Geisbrecht, 2013).

Effects of Mutation or Deletion

Given the analogous posterior localization of PAR-1 in the C. elegans zygote and the Drosophila oocyte, an examination of whether Drosophila PAR-1 has a similar role to the nematode gene in the localization of the germline determinants and the polarization of the A/P axis was undertaken. The null allele, par-1W3, is homozygous lethal, and germline clones arrest oogenesis at about stage 5, preventing analysis of its effect on axis formation. The maternal-effect phenotypes were examined of the homozygous viable mutant combinations par-16821/par-16323, par-16323, and par-16323/par-1W3, all of which which reduce the levels of PAR-1 protein without disrupting the function of mei-W68. The progeny of mothers of all three genotypes show typical posterior group phenotypes: the embryos lack abdominal segments, and pole cells fail to form, giving rise to a grandchildless phenotype, in which the adult 'escapers' have agametic gonads. Furthermore, these embryos show little or no Vasa staining at the posterior pole, indicating that the phenotypes arise from a defect in pole plasm formation. The severity of these phenotypes correlates well with the reduction in the levels of Par-1 protein in the mutant ovaries. The strongest mutant combination, par-16323/par-1W3, produces a completely penetrant posterior group phenotype and has the lowest amounts of maternal Par-1, whereas the other mutant combinations express more protein and produce weaker phenotypes. In addition to its requirement for posterior patterning, loss of par-1 causes other less penetrant maternal-effect phenotypes, such as defects in the organization of the head skeleton and telson, and a low frequency of death prior to cuticle deposition, but these appear to be unrelated to its function in A/P axis formation (Shulman, 2000).

A similar ectopic mislocalization of OSK mRNA is observed in mutants such as gurken (grk), Egfr, Pka, and Notch. These mutations disrupt signaling between the posterior follicle cells and the oocyte, resulting in the formation of a symmetric microtubule cytoskeleton that localizes OSK mRNA to the oocyte center and BCD mRNA to the posterior as well as the anterior pole. However, in par-1 oocytes, BCD mRNA localization is indistinguishable from wild type. Furthermore, whereas the oocyte nucleus frequently fails to migrate to the anterior in grk or Notch, it always moves to the correct position in par-1 egg chambers. The localization of GRK mRNA to the dorsal-anterior margin of the oocyte is also unaffected, consistent with the normal dorsal-ventral patterning of par-1 eggs and embryos. Thus, the par-1 mutants cause a novel polarity phenotype, in which the posterior transport of OSK mRNA is redirected to the center of the oocyte, whereas BCD and GRK mRNAs and the oocyte nucleus localize normally at the anterior (Shulman, 2000).

Since the posterior localization of OSK mRNA is dependent on a polarizing signal from the posterior follicle cells, an examination was carried out to see whether these cells are correctly specified in par-1 mutants, and whether Par-1 function is required in the germline or somatic cells of the egg chamber. Unlike grk mutants, in which the 'posterior' follicle cells adopt an anterior fate and express the enhancer-trap line L53b, this line is expressed solely at the anterior in par-16323/par-1W3 egg chambers, indicating that Par-1 is not required for Gurken signaling to the posterior follicle cells. Since germline clones of the par-1 null allele block oogenesis, GFP-marked germline clones of the hypomorphic allele, par-16821, were generated and it was found that Stauffen is mislocalized to the center of the oocyte. In addition, a par-1(N1S) transgene expressed from the germline-specific alpha4-tubulin promoter efficiently rescues the par-16323/par-1W3 OSK mRNA localization and embryonic patterning defects. Thus, par-1 is required autonomously in the germline for the posterior localization of OSK mRNA, and this function must occur after stage 2, when the transgene is first expressed (Shulman, 2000).

Drosophila par-1 acts at an early step in embryonic-axis formation

The Drosophila homolog of C. elegans PAR-1 becomes asymmetrically localized as polarity is established mutations affecting its expression perturb posterior patterning, germ-line development and posterior localization of pole-plasm components. It is proposed that par-1 has an important and conserved function in the establishment of polarity and development of the germ-line lineage in both of these species (Tomancak, 2000).

Analysis of the distribution of Drosophila PAR-1 in ovaries reveals that it is present in the spectrosome and early fusome of the germarium and becomes enriched in the oocyte during early oogenesis. During stages 5-8 of oogenesis, the protein is uniformly distributed along the oocyte cortex, and at stage 9, it is enriched at the posterior pole, although it can be detected at a low level throughout the oocyte cortex. PAR-1 is also expressed in follicle cells. To determine the role of Drosophila PAR-1, P-element insertions were identified within the genomic region encompassing the par-1 locus and the overlapping mei-w68 locus. Two insertions, l(2)k06323 (par-19A) and l(2)k05603 (par-1574) affect expression of PAR-1 isoforms. Both P elements cause reductions in levels of isoforms of Mr ~110K and lead to increased levels of an isoform of Mr ~116K. Excision of the par-19A P element restores the wild-type expression pattern, demonstrating that these changes are caused by insertion of the P element and confirming the identity of the affected proteins as PAR-1 isoforms. In par-1-mutant egg chambers, distribution of PAR-1 was found to be altered. In stage-9 oocytes, PAR-1 enrichment at the posterior pole is reduced in these mutants; instead the protein appears evenly distributed along the cortex. These changes in PAR-1 distribution presumably reflect changes in the amounts of the various PAR-1 isoforms, as detected by Western blotting (Tomancak, 2000).

Females harboring the par-19A/par-19A and par-19A/par-1574 mutations are fertile; however, 20% of embryos produced by these individuals died, exhibiting defects ranging from disturbances in posterior patterning, such as fusion or bifurcation of segments, to complete absence of abdominal segments. Abdominal patterning in Drosophila is initiated by assembly of the posterior pole plasm, which is also required for formation of the germ-line precursors, the pole cells. In the majority of par-1-mutant embryos, the number of pole cells is significantly reduced, indicating that pole-plasm formation may be affected by par-1 mutations (Tomancak, 2000).

Pole-plasm assembly is induced by localization and translational derepression of oskar messenger RNA at the posterior pole of the oocyte, creating a localized source of Oskar protein, which in turn recruits further pole plasm components. In 70% of par-19A-mutant egg chambers at stages 8-10, oskar mRNA is either fully or partially mislocalized to the middle of the oocyte. This aberrant distribution of oskar mRNA was mirrored by similar defects in localization of Staufen, another pole-plasm component. Moreover, in 20% of stage 8-9 egg chambers, some or all of the Oskar protein detected is found in the middle of the oocyte, indicating that pole-plasm mispositioning may be a cause of defects in abdominal patterning and pole-cell formation (Tomancak, 2000).

Thus, Drosophila PAR-1, like its counterpart in C. elegans, has a function in establishing embryonic polarity, and becomes asymmetrically distributed during the period when polarity is specified. Mutations that alter its expression lead to alterations in the distribution of pole-plasm components, reduced numbers of pole cells and defective posterior patterning. Given the nature of the changes in expression, these defects could result from reduced levels of a critical isoform, inappropriate levels or distribution of an isoform, or a combination of the above (Tomancak, 2000).

Drosophila PAR-1 may influence polarity through the cytoskeleton. Localization of Oskar mRNA to the posterior pole requires both microtubule-dependent and microfilament-dependent processes. The idea that Drosophila PAR-1 regulates microtubules is supported by the fact that mammalian PAR-1 homologs exhibit microtubule-destabilizing activities. In C.elegans, however, there is no evidence of a role for microtubules in par-1-dependent processes, and PAR-1 protein has been shown to interact with non-muscle myosin, an actin motor. Furthermore, proper asymmetric localization of P granules is resistant to microtuble-depolymerizing drugs but sensitive to microfilament-depolymerizing agents. It is therefore also possible that Drosophila PAR-1 acts through a microfilament-dependent mechanism, such as anchoring oskar mRNA and/or OSKAR protein to the posterior pole. Indeed, maintenance of pole-plasm components at the posterior pole of the Drosophila embryo depends on an intact actin cytoskeleton. Previous studies have shown fundamental differences in the early development of Drosophila and C. elegans with respect to establishment of embryonic polarity. In spite of these marked differences, PAR-1 and Drosophila PAR-1, members of the PAR-1/MARK/KIN1 family of Ser/Thr kinases, are distributed asymmetrically at the cell periphery in oocytes and in early embryos of Drosophila and C. elegans, respectively. This finding raises the possibility that at the heart of these two widely divergent systems lies a conserved mechanism to initiate cell polarity and germ-line development (Tomancak, 2000).

Drosophila par-1 is required for oocyte differentiation and microtubule organization

Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which a protein null mutation was used, reveal that Drosophila par-1 is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The Par1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, Par1 colocalizes with microtubules along the basolateral membrane. However, in either case Par1 localization is independent of microtubules. It is concluded that Drosophila par-1 plays at least two essential roles during oogenesis: it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian Par1 homologs (Cox, 2001).

To investigate the role of Par1 in oogenesis, a polyclonal antibody was generated against a portion of the linker region that is shared by all predicted protein isoforms. Consistent with previous studies, staining with the Par1 antisera reveals that Par1 is expressed in both the germline and the soma. Par1 germline expression is first detectable in the germarium, where it is localized to the fusome, a highly branched structure rich in membrane skeletal proteins such as the Adducin-like hu-li tai shao (hts) gene product. Par1 colocalizes with the fusome component Hts. Subsequently, during cyst development, Par1 colocalizes with actin to the ring canals. In the soma, Par1 is asymmetrically localized to the basolateral membrane of the follicular epithelium. In apical cross section, Par1 is cortically localized in follicle cells, and in transverse sections Par1 is enriched along the basolateral membranes of follicular epithelia. This pattern is highly similar to that observed for the mammalian Par1 homolog MARK, which is also localized basolaterally in cultured epithelial cells. To control for the specificity of the affinity-purified Par1 antisera, Par1 expression was analyzed in homozygous mutant germline and somatic cell clones of a protein null allele (par-1delta16. Par1 basolateral staining is completely eliminated in the mutant follicle cell clones, and fusome and ring canal expression is similarly lost in mutant germline clones (Cox, 2001).

Previous studies of hypomorphic alleles of par-1 have revealed a role in establishing anterior-posterior polarity in egg chambers and subsequent embryonic axis formation. To investigate whether par-1 function is required earlier in oogenesis, a protein null allele was generated in par-1 (par-1delta16). The par-1delta16 allele originates from the imprecise excision of the l(2)k06323 line and is homozygous embryonic lethal. This allele contains a 16 kb deletion removing most of the coding sequences 3' of the P-element insertion; the deletion includes the entire kinase domain and the majority of the linker region. The fact that this allele is embryonic lethal indicates an additional function for par-1 during zygote development. Therefore, to determine the role of par-1 in early oogenesis, mosaic egg chambers were generated whose germline cyst was homozygous mutant for par-1delta16. The FLP/DFS technique was used to generate both par-1 mutant and wild-type germline clones. The germline phenotype of par-1 mutant clones is largely indistinguishable from that of control ovoD females when analyzed by DAPI staining to reveal nuclear morphology. In either case, there are no vitellogenic oocytes observed, and mutant females fail to produce any eggs. These results indicate that par-1 is required early in the germline for oocyte development (Cox, 2001).

To further characterize the germline requirement for par-1 function during oocyte specification, the mosaic analysis was repeated in the absence of ovoD. The FLP/FRT technique was used to generate par-1 mutant germline clones, which are marked by the absence of nuclear GFP expression. Mosaic egg chambers containing homozygous par-1 germline clones were counterstained with the chromatin marker propidium iodide to determine the number and size of the nuclei. Mosaic egg chambers lacking par-1 in germline cells were compared with control egg chambers, which retain par-1 expression in both germline and follicle cells. Control egg chambers invariably contain 15 nurse cells and a single oocyte. In contrast, par-1 mosaic egg chambers contain the normal number of 16 cells; however, all of these cells develop as nurse cells, as indicated by their polyploid nuclei. Mosaic egg chambers that contain wild-type germline cells surrounded by a monolayer of par-1 mutant follicle cells give rise to normal germline cysts of 15 nurse cells and a single oocyte. Phenotypic analysis of germline mosaic egg chambers further reveals a par-1 dependent block at stage 5 to stage 6 of oogenesis. These results taken together clearly demonstrate that par-1 is required in the germline to regulate oocyte differentiation (Cox, 2001).

To analyze how oocyte determination is blocked in par-1 mutant egg chambers, an examination was carried out to see how removing Par1 affects the formation or structure of the fusome and ring canals to which Par1 localizes within the germline. The morphology of fusomes was examined in par-1 mutant cysts by double labeling germaria with nuclear GFP and monoclonal anti-Hts antibody 1B1, a marker for the fusome. In wild-type germaria, a spherical fusome or spectrosome (Sp) is present in germline stem cells and cystoblasts. Following cystoblast division, the fusome (Fu) undergoes a morphogenesis from a spherical fusome in the cystoblast into a polarized, branched structure in the 2-, 4-, 8-, and 16-cell cysts. Analysis of par-1 mutant clones reveals that par-1 is not required for formation of the fusome and that, furthermore, fusome morphology is indistinguishable from wild-type clones within the same germaria. Therefore, while par-1 is not required for fusome formation, the defects in cyst development and oocyte determination suggest that par-1 may nevertheless play an important role as a component of the fusome in establishing and/or maintaining cyst polarity (Cox, 2001).

The ring canals are stable intercellular bridges that maintain the connection between all cells within the 16-cell germline cyst. In wild-type germline cysts, the pattern of ring canal connections is highly invariant; one of the two cells with four ring canals invariably differentiates to form the oocyte, while the remaining 15 cells become nurse cells. Therefore, the pattern of ring canal connections reflects the spatial pattern of the cystoblast mitotic divisions. To analyze the size, formation, and spatial distribution of ring canals in par-1 mutant clones, wild-type and mutant germline cysts and egg chambers stained with rhodamine-conjugated phalloidin were compared to visualize the filamentous actin component of ring canals. In wild-type germaria, each 16-cell germline cyst displays a stereotypic pattern of ring canal connections. In region 2b, when the germline cyst flattens out across the width of the germarium, the spatial distribution of ring canals is manifest as an orderly, linear arrangement across the width of the germline cyst. Similarly, in region 3 of the germarium, the spatial distribution of ring canals remains highly ordered. However, in par-1 mutant germline cysts, the arrangement of ring canals in both region 2b and region 3 of the germarium is disrupted. In region 2b, the wild-type linear distribution of ring canals is clearly disrupted; likewise, in region 3, the arrangement of ring canals appears less ordered and more unevenly distributed. In mosaic egg chambers, the size and location of the ring canal connections is also disrupted. The most posterior germline cell in the egg chamber did not display the typical cluster of the four largest ring canals characteristic of the oocyte. In contrast, in wild-type egg chambers the oocyte is invariantly positioned at the posterior and is easily identified by its four large ring canals at the anterior margin of the cell. Thus, par-1 function is required early in germline cyst formation to establish cyst polarity. Furthermore, the lack of cyst polarity correlates with the failure in oocyte determination that results in the differentiation of 16 polyploid nurse cells (Cox, 2001).

While the precise mechanism of oocyte determination is unknown, one hallmark of this process is the differential accumulation of specific oocyte differentiation factors within a single cell of the germline cyst. A number of these molecules, including Bic-D, Egl, Orb, and Dhc64C, appear to be directly involved in this process since they are required for determination of the oocyte. To further define the role of par-1 in oocyte determination, the expression pattern of these proteins was examined in par-1 mutant germline clones during early and late stages of oogenesis and that expression was compared to wild-type control germaria and egg chambers. In contrast to wild-type germline cysts, par-1 mutant cysts fail to maintain the accumulation of Bic-D protein in a single cell. In region 2b of the germarium, Bic-D protein appears to localize to a single cell; however, this differential localization is not maintained in region 3 of the germarium. Later in oogenesis, par-1 mosaic egg chambers also fail to accumulate Bic-D in a single cell, but rather they display diffuse expression of Bic-D throughout the egg chamber. Similarly, par-1 mutant cysts and egg chambers fail to accumulate Orb, Dhc64C, and Staufen proteins within a single cell. This failure leads to diffuse expression throughout the germline cyst or egg chamber. To investigate the integrity of the microtubule network, oskar mRNA expression was examined in par-1 mosaic egg chambers by in situ hybridization. oskar localization to the future oocyte has previously been shown to require a stable MTOC and microtubule network. In wild-type ovarioles, oskar mRNA is first detected in region 2b, where it begins to accumulate in a single cell. Subsequently, oskar mRNA is localized posteriorly in the oocyte of each developing egg chamber. In contrast, par-1 mosaic egg chambers fail to accumulate oskar mRNA posteriorly. These results indicate that par-1 is required for the differential accumulation of oocyte differentiation factors and further suggests that par-1 may function in establishing and/or maintaining polarity of the microtubule network (Cox, 2001).

In addition to its role in the establishment of axial polarity and the localization of morphogens within the developing oocyte, the microtubule cytoskeleton has a very important role in the early events of oocyte determination. The microtubule cytoskeleton is critical in many aspects of oocyte determination since microtubule depolymerizing drugs disrupt the accumulation of oocyte differentiation factors, such as Bic-D and oskar mRNA, and lead to the development of cysts with 16 nurse cells. Given that par-1 is also required for the differential accumulation of oocyte factors and for oocyte determination, the organization of the microtubule cytoskeleton was analyzed in par-1 mutant clones. In wild-type germaria, the oocyte is characterized by the presence of an active MTOC which organizes the microtubule network within the developing germline cyst. In region 3 of the germarium, the MTOC is clearly present in a single posterior cell. However, in par-1 mutant cysts there is no apparent posterior MTOC formation; rather, the microtubules appear evenly distributed within region 3 of the germarium. Similarly, in stage 2 mutant egg chambers, there is no apparent posterior accumulation of microtubules, but rather the microtubules remain evenly distributed within the mutant cyst. Finally, in later-stage par-1 mosaic egg chambers there remains no posterior accumulation of microtubules or visible MTOC formation. These results indicate that par-1, like Bic-D and Egl, is required for the formation and/or maintenance of an active MTOC within the future oocyte. The even distribution of microtubules within the mutant germline cysts and egg chambers and the failure to maintain an active posterior MTOC may account in part for the defects observed in cyst polarity and, ultimately, oocyte determination (Cox, 2001).

To better understand the relationship between par-1 function and microtubule organization, a detailed analysis was conducted of microtubule structure in par-1 mutant germline clones. To examine the dependence of Par1 localization on an intact microtubule cytoskeleton, adult females were fed with the microtubule depolymerizing drug colchicine. To assay the effect of the drug on disrupting the microtubule cytoskeleton, the production was monitored of egg chambers containing 16 nurse cells and no oocyte and microtubule structure was analyzed by immunofluorescence. In a wild-type germarium, Par1 is expressed on the spectrosome and the fusome, and the microtubules display a highly organized network originating from the posterior MTOC. Following microtubule depolymerization, no disruption was observed in the localization of Par1 to the spectrosome or fusome. This indicates that Par1 localization within the germline is independent of microtubules (Cox, 2001).

Finally, the orientation and attachment of mitotic spindle poles to the fusome during cystocyte divisions is believed to ensure the proper pattern of cell divisions necessary to generate a polarized cyst. Therefore, spindle orientation was examined in dividing par-1 mutant germline cysts. par-1 mutant germline cysts were stained with antibodies to alpha-tubulin and the Hts-related antigen 1B1 to label microtubules and fusomes, respectively. No apparent defect in spindle orientation or attachment to the fusome was found. These results taken together suggest that par-1 regulates microtubule dynamics during cyst formation and oocyte determination by establishing and/or maintaining polarization of the microtubule network (Cox, 2001).

Given that Par1 functions to regulate microtubule dynamics and cyst polarity in the germline, attempts were made to test whether par-1 plays similar roles in the somatic follicle cells. par-1delta16 follicle cell clones were generated and egg chambers were stained to assay both microtubule and epithelial organization as well as follicle cell polarity. par-1 mutant follicle cell clones were examined by double labeling with anti-alpha-tubulin and anti-alpha-Spectrin antibodies to assay microtubule organization and epithelial integrity, respectively. In wild-type follicle cells, alpha-tubulin is normally localized to the basolateral membrane in a pattern very similar to that observed for Par1. However, in par-1 mutant clones, the basolateral alpha-tubulin expression is completely abolished. The effect on alpha-tubulin expression of removing par-1 function is specific since alpha-Spectrin expression is unaffected in par-1 mutant follicle cell clones. Moreover, anti-Spectrin staining reveals defects in epithelial shape and organization. Wild-type follicle cells at this stage form a columnar monolayer that surrounds the oocyte; however, par-1 mutant follicle cells appear to disrupt this monolayer. By using anti-Spectrin staining as a cortical marker, two defects were observed in monolayer organization. (1) It was found that follicle cells lacking Par1 function appear to lose their characteristic columnar shape and appear irregular in size and shape. (2) A disruption was observed in the monolayer such that two follicle cells appear to be stacked on top of one another rather than in a monolayer. This result suggests that disruption in the monolayer may lead to a reorientation or randomization of the plane of division in par-1 mutant follicle cells. An alternative and equally probable interpretation could be that mutant follicle cell division occurs normally; however, following division there may be a reorientation of the mutant follicle cells perpendicular to the wild-type orientation (Cox, 2001).

Since par-1 is required in follicle cells to organize and/or stabilize the microtubule cytoskeleton as well as to maintain the integrity of the epithelial monolayer, the potential role of par-1 in the establishment and/or maintenance of follicle cell polarity was examined. Mutant follicle cell clones were labelled with anti-alpha-tubulin and anti-Armadillo (Arm) antibodies to assay microtubule organization and epithelial polarity, respectively. Armadillo displays a polarized accumulation within follicle cells with intense staining apically in the adherens junctions as well as laterally between cell surfaces. In contrast to the polarized pattern of Arm accumulation in wild-type follicle cells, par-1 mutant follicle cells display an even and slightly weakened distribution of Arm protein. Furthermore, in an apical cross section, it is clear that par-1 mutant follicle cells display a highly disrupted pattern of Arm expression along the cell cortex. These defects in Arm accumulation suggest a loss of polarity within the par-1 mutant follicle cells and further indicate that Par1 may function to organize and restrict the basolateral membrane domain within follicle cells since removing par-1 function leads to a loss of this apical Arm restriction. To further assay the role of par-1 in organizing the basolateral membrane domain, mosaic egg chambers were stained with anti-Neurotactin to assay both apical and lateral membrane integrity. Neurotactin is a transmembrane glycoprotein expressed in epithelial tissues along the apical and lateral membranes. Similar to the effects of par-1 on microtubule stabilization, a dramatic destabilization was observed of both apical and lateral Neurotactin expression in par-1 mutant follicle cell clones. These results taken together strongly suggest that Par1 is required to organize the microtubule cytoskeleton as well as maintain polarity within the follicular epithelium (Cox, 2001).

PAR-1 is required for the maintenance of oocyte fate in Drosophila

The PAR-1 kinase is required for the posterior localization of the germline determinants in C. elegans and Drosophila, and localizes to the posterior of the zygote and the oocyte in each case. Drosophila PAR-1 is also required much earlier in oogenesis for the selection of one cell in a germline cyst to become the oocyte. Although the initial steps in oocyte determination are delayed, three markers for oocyte identity, the synaptonemal complex, the centrosomes and Orb protein, still become restricted to one cell in mutant clones. However, the centrosomes and Orb protein fail to translocate from the anterior to the posterior cortex of the presumptive oocyte in region 3 of the germarium, and the cell exits meiosis and becomes a nurse cell. Furthermore, markers for the minus ends of the microtubules also fail to move from the anterior to the posterior of the oocyte in mutant clones. Thus, PAR-1 is required for the maintenance of oocyte identity, and plays a role in microtubule-dependent localization within the oocyte at two stages of oogenesis. PAR-1 localizes on the fusome, and provides a link between the asymmetry of the fusome and the selection of the oocyte (Huynh, 2001a).

To examine the phenotype caused by the complete lack of PAR-1 activity in the germline, the ovoD technique was used to generate clones of par-1W3, a null mutation that deletes most of the par-1-coding region. This technique provides a powerful selection for homozygous mutant clones in the germline, because the ovoD transgene blocks oogenesis at an early stage, so only the mutant clones that have lost the transgene survive. However, no late stage egg chambers were recovered, suggesting that cysts that lack PAR-1 arrest their development before stage 6. Therefore germline clones were induced that were marked by the loss of nuclear GFP, so that the mutant egg chambers could be identified at all stages of oogenesis. Egg chambers with homozygous mutant germlines do develop, but they remain small, and never reach the stage where the oocyte is larger than the nurse cells. The oocyte can be distinguished from the nurse cells early in oogenesis, because it is arrested in meiosis and condenses its chromosomes into a hollow sphere called the karyosome, whereas the nurse cells endoreplicate their DNA to become polyploid. In the mutant egg chambers, none of the cells forms a karyosome, and instead all 16 germ cells become polyploid. The oocyte can also be identified by staining for Orb protein, which localizes in a crescent at the posterior of the cell. Orb does not localize to any of the cells in par-1 mutant egg chambers after stage 3, indicating that mutant cysts develop 16 nurse cells and no oocyte (Huynh, 2001a).

The 16 nurse cell phenotype of par-1 null germline clones is very similar to that produced by mutations in egl and BicD. These mutants disrupt the earliest known step in the selection the oocyte, which is the formation of the synaptonemal complex (SC) in the two pro-oocytes. In egl mutants, all cells in the cyst enter meiosis and reach the pachytene stage, but then lose the SC and revert to the nurse cell pathway of development. In contrast, none of the cells enters meiosis and forms any SC in BicD null germline clones. To see whether par-1 is also required for these early steps, the behavior of the SC was examined in par-1 mutant clones. In contrast to wild type, mutant cysts contain up to 16 cells with SC in early region 2a, indicating that all of the cells have entered meiosis. Unlike egl mutant cysts, however, the SC does not disappear from all cells. Instead, the SC becomes progressively restricted to two cells, and then to one cell in region 3 (also called stage 1). However, this restriction is delayed compared with wild-type cysts, where the SC is always restricted to the oocyte by region 2b. In addition, the SC disappears prematurely from mutant oocytes. Wild-type oocytes retain a compacted SC until stage 3-4, whereas the SC can no longer be detected in mutant stage 2 egg chambers that have just left the germarium (Huynh, 2001a).

This behavior of the SC suggests that par-1 acts at two stages in the germarium: (1) it plays a role in the selection of the pro-oocytes and the oocyte, since too many cells enter meiosis in early region 2a, and the choice of the oocyte is delayed; (2) par-1 is required to keep the oocyte in meiosis after stage 1, since the SC is not maintained (Huynh, 2001a).

The restriction of the SC to one cell suggests that at least some of the initial steps in oocyte determination still occur in par-1 clones. The behavior of other early oocyte markers was examined: the oocyte-specific cytoplasmic protein Orb and the centrosomes. In wild-type cysts, Orb protein accumulates in the pro-oocytes in region 2a of the germarium, and then concentrates at the anterior of the oocyte in late region 2a. As the cyst enters region 3, Orb moves from the anterior to the posterior of the oocyte, and forms a crescent at the posterior pole. The same movement has been reported for a BicD-GFP fusion protein. In par-1 mutant cysts, Orb protein still accumulates in one cell, but this localization is delayed until region 3 (stage 1), like the restriction of the SC. Furthermore, Orb never translocates to the posterior of the oocyte, and the protein disappears from the oocyte by stage 2 (Huynh, 2001a).

The migration of the centrosomes from the nurse cell to the oocyte can be followed by staining for gamma-tubulin. Like Orb, the centrosomes localize to the anterior of the oocyte in region 2b. They then move from the anterior to the posterior of the oocyte in region 3, and coalesce to form a bright dot at the posterior pole. In par-1 mutant clones, the centrosomes accumulate at the anterior of the oocyte in late region 2b or early region 3, indicating that the first phase of centrosome migration occurs normally. The centrosomes remain at the anterior, however, and never translocate to the posterior (Huynh, 2001a).

These results indicate that the loss of par-1 blocks oocyte determination at a novel step. The initial selection of the oocyte occurs normally, since three independent oocyte markers, the SC, Orb and the centrosomes, still accumulate in one cell, but the identity of this cell is not fixed, and it soon reverts to the nurse cell pathway of development. This reversion to the nurse cell fate correlates with a defect in the migration of Orb and the centrosomes from the anterior to the posterior of the oocyte, suggesting that this PAR-1-dependent A-P movement is required for the maintenance of oocyte identity (Huynh, 2001a).

PAR-1 is required for the correct organization of microtubules in the oocyte at stage 9, whether this early phenotype in oocyte determination is also a consequence of a defect in the microtubule cytoskeleton was also examined. In a wild-type germarium, the microtubule cytoskeleton becomes progressively polarized in region 2 and is clearly focused on one cell in late region 2b. par-1 mutant clones stained for alpha-tubulin show a slight delay in the focusing of the microtubules to one cell in region 2b, which is similar to the delay in Orb restriction. However, the overall organization of the microtubules in region 2 appears essentially normal (Huynh, 2001a).

Since it is difficult to interpret the organization of the microtubules within the oocyte in anti-tubulin stainings, markers for the minus-ends of the microtubules were used to examine the polarity of these microtubules in wild type and par-1 mutant cysts. Although the centrosomes lie at the minus ends of the microtubules in most cells, this is not necessarily the case in the germline, since they appear to be inactivated when the cyst leaves region 1, and a Nod-GFP transgene was therefore used as an alternative marker for the minus ends. Nod-GFP localization in wild-type cysts reveals that the minus ends are first focussed at the anterior of the oocyte, before switching to the posterior as the cyst buds off the germarium. In par-1 mutant cysts, Nod-GFP still becomes restricted to the anterior of one cell, but never re-localizes to the posterior (Huynh, 2001a).

Furthermore, the minus end-directed motor, dynein, shows an identical behavior as that of Nod-GFP: it localizes to the anterior of one cell in mutant cysts, but fails to switch to the posterior in region 3. Thus, par-1 is required for a reorganization of the microtubule cytoskeleton of the oocyte in region 3, and this may account for the failure of Orb and the centrosomes to move from the anterior to the posterior of the cell (Huynh, 2001a).

The par-1 null allele is also mutant for mei-W68, which shares a promoter with par-1 and lies entirely within its first intron. Since Mei-W68 is required for the dsDNA breaks that initiate meiotic recombination, some or even all of the phenotypes of par-1 clones could therefore be due to this meiotic defect rather than the loss of par-1 itself. To distinguish between these possibilities, the Gal4/UAS system and a Nanos-Gal4-VP16 driver were used to express the N1S isoform of par-1 in the germarium as a GFP-fusion protein, to determine whether it could rescue the phenotype of par-1W3 germline clones. The fusion protein localizes to a branched structure in regions 1 and 2 of the germarium, which is presumably the fusome, as the endogenous protein is a component of this structure. More importantly, PAR-1N1S completely rescues the delay in Orb localization to the oocyte in par-1W3 homozygous cysts, and also restores the anterior-posterior movement of Orb within the oocyte in region 3. Furthermore, this construct rescues the posterior movement of the centrosomes, and the maintenance of oocyte identity. Thus, these phenotypes are specific to par-1, and are not due to the reduction in Mei-W68 activity or any other mutations on the par-1W3 chromosome (Huynh, 2001a).

The N1L isoform of PAR-1, which differs only from N1S by the exclusion of five amino acids in an alternative exon in the linker region of the protein and the presence of the conserved C-terminal par-1 domain, was also expressed. The PAR-1N1L fusion protein also localizes to the fusome, but is unable to rescue any of the early defects in par-1W3 germline clones, although it is functional because it rescues the par-1 phenotype at other stages of development. This suggests that the par-1 domain inhibits the early function of the kinase, and provides the first example where different PAR-1 isoforms have different activities (Huynh, 2001a).

The GFP-PAR-1 localization in the germarium is intriguing, since it has been suggested that the fusome provides the initial cue for the determination of the oocyte. To confirm that par-1 is recruited to the fusome, wild-type germaria were stained for PAR-1, using an antibody that recognises all isoforms of the protein, and Hu li tai shao (Hts), an integral component of the fusome. PAR-1 and Hts co-localize on the spectrosome, which is the precursor of the fusome in the germline stem cells and cystoblasts, and on the fusome itself. This co-localization is most apparent in region 1, but some PAR-1 persists on the fusome in region 2. In addition, the PAR-1 antibody labels small particles that do not contain Hts, but this is due to a cross-reacting antigen, because these are not seen with the GFP-PAR-1 fusions, and do not disappear in par-1 null germline clones (Huynh, 2001a).

Mutants in other components of the fusome, such as Hts and alpha-Spectrin, produce cysts with fewer than 16 nurse cells, because the precise pattern of cyst divisions is disrupted. However, all par-1W3 mutant egg chambers contain 16 cells, indicating that par-1 is not required for this function of the fusome. To rule out the possibility that the lack of an effect on the pattern of divisions in the cyst is due to the perdurance of wild-type PAR-1 protein in mutant clones, mutant egg chambers derived from persistent stem cell clones that were induced 10 days earlier, were also analysed and the same result was obtained. To test if par-1 affects the formation or morphogenesis of the fusome, mutant cysts were stained for alpha-Spectrin, which is a component of the spectrosome and the fusome at all stages of its development. The spectrosome is asymmetrically partitioned during stem cell division, so that two-thirds remain in the daughter stem cell, and one-third in the cystoblast, and this asymmetry is unaffected in par-1W3 clones. Furthermore, the fusome is asymmetrically partitioned at each subsequent division, as in wild type: when a mutant cystoblast divides, one cell still inherits more fusome than the other; this asymmetry is maintained throughout the next three mitoses, and can be seen in four-cell cysts and in 16-cell cysts that have stopped dividing. Thus, PAR-1 localizes on the fusome, but is not required for its formation or its asymmetric segregation (Huynh, 2001a).

The re-localization of cytoplasmic markers in region 3 reveals the earliest known A-P polarity within the oocyte. It is very intriguing that PAR-1 is required both for this polarization, and the later polarization of the oocyte at stage 9. This raises the question of whether the two are linked. For example, it is possible that the altered organization of the microtubules and mislocalization of Oskar mRNA at stage 9 in par-1 hypomorphs is a consequence of earlier problems in the polarization of the oocyte in the germarium. This seems unlikely, however, for several reasons. (1) The localization of all known markers for oocyte polarity changes during stage 7, suggesting that the early polarization of the oocyte is erased when it re-polarizes in response to a signal from the posterior follicle cells. (2) In the vast majority of egg chambers that are mutant for par-1 hypomorphs, Orb protein moves normally from the anterior to the posterior of the oocyte, and is maintained there as it is in wild type. Furthermore, the Oskar mRNA localization defect of these mutants can be rescued by a par-1 transgene that is only expressed after the cysts have left the germarium. The view that PAR-1 plays a role in the anterior-posterior polarization of the oocyte at two stages of oogenesis is therefore favored. It will be interesting to determine whether these processes are related in other ways, and whether PAR-1 serves a common function in oocyte determination and Oskar mRNA localization (Huynh, 2001a).

Bazooka and PAR-6 are required with PAR-1 for the maintenance of oocyte fate in Drosophila

The anterior-posterior axis of C. elegans is defined by the asymmetric division of the one-cell zygote, and this is controlled by the PAR proteins, including PAR-3 and PAR-6 (see Drosophila par-6), which form a complex at the anterior of the cell, and PAR-1, which localizes at the posterior. PAR-1 plays a similar role in axis formation in Drosophila: the protein localizes to the posterior of the oocyte and is necessary for the localization of the posterior and germline determinants. PAR-1 has recently been shown to have an earlier function in oogenesis, where it is required for the maintenance of oocyte fate and the posterior localization of oocyte-specific markers. The homologs of PAR-3 (Bazooka) and PAR-6 are also required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that develop 16 nurse cells and no oocyte. Furthermore, oocyte-specific factors, such as Orb protein and the centrosomes, still localize to one cell but fail to move from the anterior to the posterior cortex. Thus, PAR-1, Bazooka, and PAR-6 are required for the earliest polarity in the oocyte, providing the first example in Drosophila where the three homologs function in the same process. Although these PAR proteins therefore seem to play a conserved role in early anterior-posterior polarity in C. elegans and Drosophila, the relationships between them are different, since the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm (Huynh, 2001b).

The determination of the oocyte can be followed by the migration of the centrosomes, which move along the fusome to cluster at the anterior of the oocyte in region 2b, and then translocate to the posterior of the oocyte in region 3. In baz mutant cysts, the centrosomes accumulate at the anterior of the oocyte but never move to the posterior cortex. Furthermore, alpha-tubulin stainings of mutant cysts indicate that microtubules remain focused on the anterior of the oocyte and fail to rearrange. These phenotypes are identical to those produced by par-1 mutants, indicating that Bazooka and PAR-1 act in the same step in oocyte determination. Neither is necessary for the initial selection of the oocyte, because Orb and the centrosomes still become restricted to one cell, but they are required for the maintenance of oocyte fate, since the oocyte soon dedifferentiates and becomes a nurse cell. This failure to maintain oocyte identity correlates with a block in the movement of oocyte-specific factors and the centrosomes from the anterior to the posterior of the cell, suggesting that this early polarization is important for the further development of the oocyte (Huynh, 2001b).

PAR-6 has been shown to localize to the same protein complex as PAR-3 in C. elegans, Drosophila, and mammalian cells and is essential both for the localization and the function of this complex. In Drosophila, Bazooka and PAR-6 colocalize to the apical side of the embryonic ectoderm, where they are necessary for the maintenance of epithelial polarity, and both proteins are also inherited by the neuroblasts when they delaminate and are required for the basal localization of cell fate determinants during their asymmetric divisions. To test if Drosophila PAR-6 also functions with Bazooka during oogenesis, germline clones were generated of the par-6Delta226 allele, which is a deletion of the promoter, the start codon, and the first 121 amino acids of the protein and is therefore a strong loss of function mutation if not a null. The majority of mutant egg chambers appear small, oval-shaped, and contain 16 polyploid nurse cells and no oocyte, indicating that PAR-6 is also required for oocyte determination. Furthermore, Orb and the centrosomes accumulate in one cell at the posterior of the cyst, although with a slight delay compared to wild-type. Both remain at the anterior of the oocyte, however, and fail to translocate to the posterior pole. Thus, the loss of PAR-6 from the germline gives an identical phenotype to Bazooka and PAR-1. As is the case for bazooka germline clones, some of the par-6 mutant egg chambers escape the early arrest and go on to produce normal eggs. When the females are scored 2 days after eclosion, half of the egg chambers form a normal oocyte, and about a quarter still do so after 10 days. This increase in the penetrance of the phenotype with age shows that PAR-6 protein perdures for many days after the clones are produced. Consistent with this, PAR-6 appears to be unusually stable in the embryo; the protein can be detected throughout embryogenesis in zygotic par-6 null embryos, at levels that are only slightly lower than in wild-type. However, the continued presence of escapers after 10 days suggests that PAR-6 may not be essential for oocyte determination in all cases and that there may be redundant pathways that can partially compensate for its absence (Huynh, 2001b).

To investigate the relationships between Bazooka, PAR-6, and PAR-1 during oocyte determination, their localizations were analyzed in both wild-type and mutant germaria. In region 2a to region 3 of the germarium, Bazooka localizes around the ring canals, in a ring that is about twice the diameter of that formed by actin. This localization is very similar to that of the adherens junction components Shotgun (E-cadherin) and Armadillo. A double staining was therefore performed for Arm and Baz. Although Arm localizes to these rings before Bazooka in early region 2a, the two proteins colocalize from the middle of region 2a until region 3, when they both disappear. Bazooka also colocalizes with Shotgun and Armadillo in the zonula adherens of the embryonic epithelium, which provides a boundary between the apical and basolateral membrane domains. This raises the possibility that the Shotgun, Armadillo, and Bazooka rings in the germarium perform a similar function by marking the separation between an anterior and a posterior domain within the oocyte. It is unclear whether PAR-6 also localizes to these rings, since none of the available antibodies give any significant staining that disappears in par-6 null germline clones

These results show that PAR-1, Bazooka, and PAR-6 act in the same step in oocyte determination, providing the first example in Drosophila where these three homologs of C. elegans PAR proteins participate in the same process. Furthermore, mutants in all three genes disrupt the movement of oocyte-specific proteins and the centrosomes from the anterior to the posterior of the oocyte, which is the earliest visible sign of polarity within the oocyte. Given the role of these PAR proteins in other systems, it seems very likely that their primary function in the germarium is in the anterior-posterior polarization of the oocyte, and that the failure to maintain oocyte fate is a consequence of this defect (Huynh, 2001b).

It is intriguing that this very early anterior-posterior polarity of the Drosophila oocyte requires three of the PAR proteins that mediate the anterior-posterior polarization of the first cell division in C. elegans. Although this suggests that these proteins act in a conserved pathway for generating cell polarity in these two systems, the relationships between the localizations of these proteins are quite different in the Drosophila oocyte and C. elegans zygote. Thus, at least some aspects of their function are not conserved, and it will therefore be interesting to determine whether the downstream pathways that generate other cellular asymmetries in response to this polarity are related (Huynh, 2001b).

PAR-1 regulates the polarised microtubule cytoskeleton in the Drosophila follicular epithelium

The PAR-1 kinase plays a conserved role in cell polarity in C. elegans, Drosophila and mammals. The role of PAR-1 in epithelial polarity was investigated by generating null mutant clones in the Drosophila follicular epithelium. Large clones show defects in apicobasal membrane polarity, but small clones induced later in development usually have a normal membrane polarity. However, all cells that lack PAR-1 accumulate spectrin and F-actin laterally, and show a strong increase in the density of microtubules. This is consistent with the observation that the mammalian PAR-1 homologs, the MARKs, dramatically reduce the number of microtubules, when overexpressed in tissue culture cells. The MARKs have been proposed to destabilize microtubules by inhibiting the stabilizing activity of the Tau family of microtubule-associated proteins. This is not the case in Drosophila, however, since null mutations in the single tau family member in the genome have no effect on the microtubule organization in the follicle cells. Furthermore, PAR-1 activity stabilizes microtubules: microtubules in mutant cells depolymerize much more rapidly after cold or colcemid treatments. Loss of PAR-1 also disrupts the basal localization of the microtubule plus ends, which are mislocalized to the center of mutant cells. Thus, Drosophila PAR-1 regulates the density, stability and apicobasal organization of microtubules. Although the direct targets of PAR-1 are unknown, it is suggested that it functions by regulating the plus ends, possibly by capping them at the basal cortex (Doerflinger, 2003).

Although par-1 mutants produce similar polarity phenotypes in the follicle cells to mutants in the components of the Bazooka/PAR-6/aPKC complex, they are not identical. par-1 clones cause a complete disruption of polarity only when induced early in the follicle cell lineage. The smaller clones that arise later in oogenesis often show little or no reduction in the localization of apical and basolateral markers, and usually remain as a single layer of cells. By contrast, even late clones of bazooka or aPKC produce penetrant epithelial defects. This difference is also apparent in the embryo, where loss of zygotic bazooka, PAR-6 or aPKC disrupts epithelial organization. par-1 homozygous embryos, however, display no obvious epithelial polarity phenotype, although it is not possible to remove the maternal PAR-1 completely. The reason for low penetrance of polarity defects in smaller par-1 follicle clones is unclear, but similar differences between early large clones and smaller late clones have been observed for crumbs and lkb1 (the Drosophila par-4 homolog - required for the early A-P polarity of the oocyte and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis). One possibility is that these genes are required for the initial formation of the follicular epithelium, but not for its maintenance. This seems unlikely to be the case for par-1, however, because small clones containing only one or two cells can sometimes show strong apicobasal polarity defects. A more likely explanation is that the low penetrance of this phenotype in small clones is due to the perdurance of the PAR-1 that was present at the time when the clones were induced. In support of this, the penetrance of the polarity defects of par-1 clones increases with clone size and the stage of oogenesis, as one would expect if the protein is gradually degraded over time, and is diluted out by cell division (Doerflinger, 2003).

In contrast to apicobasal membrane polarity, the density, the stability and the organization of MTs are disrupted in all par-1 clones, regardless of their size or the stage of oogenesis. This is likely to represent a distinct function of the kinase from its other roles in cell polarity, because it is much more sensitive to a reduction in activity. The effects of PAR-1 on MT density are consistent with results on the mammalian PAR-1 homologs, MARK1 and MARK2. These experiments show that removal of PAR-1 causes an increase in the density of MTs in each cell, whereas overexpression of MARK1 or MARK2 in unpolarized tissue culture cells causes most MTs to disappear (Doerflinger, 2003).

The MARKs have been proposed to regulate the MT cytoskeleton by phosphorylating Tau family MAPs, thereby inhibiting them from binding to and stabilizing MTs. Although PAR-1 does phosphorylate Drosophila Tau in vitro, tau null mutations are viable and fully fertile and have no effect on the arrangement of MTs in either the follicle cells or the oocyte. Therefore, this mechanism cannot account for its function in organizing the MTs in the follicle cells. The viability of tau mutants is surprising, given the many functions ascribed to Tau in human neurons. It does have a precedent, however, since tau knockout mice are viable, have a morphologically normal nervous system, and display only defects in MT stability and organisation in small-caliber axons. The mild phenotype of tau in mice has been proposed to be due to functional redundancy with the closely related MAP2, but this cannot be the case in Drosophila, which does not contain a MAP2 homolog. It may be redundant with other types of MAP, however, and the best candidate is Futsch, which has significant structural and functional homology to mammalian MAP1B. MAP1B appears to have functional overlap with both Tau and MAP2 in mammals, because mice that are homozygous for null mutations in map1b and tau, or map1b and map2, show defects in axonal elongation, neuronal migration and MT organization that are much more severe than in mice lacking only one of these genes (Doerflinger, 2003).

Another compelling argument that PAR-1 regulates MTs by a different mechanism from that proposed for the MARKs is that it is required to stabilize rather than destabilize MTs, at least in epithelial cells. The MTs in follicle cells are among the most stable in nature, because they are almost completely resistant to cold or to prolonged colchicine treatments. By contrast, the MTs in par-1 mutant cells appear to be highly dynamic because: (1) they disappear after brief colchicine treatments; (2) they depolymerize at 4°C, but re-grow in a few minutes after return to 25°C; (3) they are lost during fixation, if the fixative is too weak (Doerflinger, 2003).

The opposite effects of PAR-1 and the MARKs on MT stability may indicate that these closely related kinases have evolved to fulfil distinct functions in invertebrates and mammals. It is also possible, however, that this reflects the different experimental approaches and cell-types that have been used to examine their activities. The MARKs have been assayed by over-expressing them in CHO cells, which are a transformed line of rapidly dividing, undifferentiated and unpolarized cells. By contrast, the loss-of-function phenotype of PAR-1 was examined in post-mitotic follicle cells, highly polarized and differentiated epithelial cells. The two cell types also have very different microtubule cytoskeletons. In CHO cells, microtubules are nucleated from a central centrosome, and are presumably reasonably dynamic, because they disassemble at each mitosis, whereas the follicle cells lose their centrosomes when they form a columnar epithelium, and nucleate a very stable apicobasal array of microtubules. It would therefore be interesting to test the effects on MT stability of disrupting the function of PAR-1 homologs in more similar mammalian cell-types, such as polarized MDCK cells (Doerflinger, 2003).

In addition to its effect on stability, PAR-1 is required to maintain the normal organization of the MTs. The MT arrangement in the follicle cells is typical of a polarized epithelium, with the minus ends associated with the apical membrane, and the plus ends at the basal side of the cell. The arrangement of minus ends appears to be largely unchanged in par-1 mutant cells, but a marker for the plus ends, Kinesin:ß-gal, accumulates in the center rather than the basal region of the cell. This phenotype is very similar to that observed in par-1 mutant oocytes, in which the plus ends become abnormally focussed in the center of the oocyte, rather than at the posterior, and there is an increase in the density of MTs around the cortex. Thus, PAR-1 may regulate the MTs in the same way in the two cell types, and most probably acts primarily on the plus ends. PAR-1 may also have some effect on the distribution of the minus ends of MT in the oocyte, as bicoid mRNA, which is believed to be transported towards minus ends, is found around the lateral cortex of mutant oocytes, rather exclusively at the anterior. Although the possibility that there is also an effect on the minus ends in mutant follicle cells cannot be ruled out, this is not detectable using Nod:ß-gal (Nod is a kinesin-related protein) as a marker (Doerflinger, 2003).

It seems paradoxical that the loss of PAR-1 should increase the density of MTs in follicle cells, while decreasing their stability, but one possible explanation is suggested by studies in mammalian cells on populations of stable MTs that are marked by detyrosinated alpha-tubulin. These MTs are resistant to nocadazole-induced depolymerization, and fail to incorporate new tubulin subunits, leading to the proposal that they are capped at their plus ends in a way that prevents both the addition and loss of tubulin. Thus, it is possible that PAR-1 stabilises the MTs in the follicle cells by capping plus ends when they reach the basal cortex, thereby preventing them from either growing or shrinking. If the conditions inside the cell favour MT polymerization, the loss of the PAR-1-dependent cap would allow the plus ends to continue to grow once they reach the basal cortex. This could account for both the increase in MT density and the redistribution of plus ends to the center of mutant cells. However, the uncapped MTs would rapidly shrink under conditions that favor MT depolymerization, such as cold or colchicine treatment, explaining why the MTs disappear in mutant cells (Doerflinger, 2003).

par-1 null clones also show fully penetrant and cell-autonomous increases in the recruitment of ß-spectrin and actin to the lateral cortex. Like the microtubule phenotype, these effects appear to be independent of the defect in apicobasal membrane polarity, since the latter is much less penetrant. These phenotypes may therefore reflect a third distinct function of the kinase. It is also possible, however, that the MT defects are a consequence of the changes in actin organization or vice versa. In this context, it is interesting to note that Rho family GTPases, major regulators of the actin cytoskeleton, have also recently been found to control the capping of MT plus ends at the leading edge of migrating cells. The Rac and Cdc42 effector, IQGAP, interacts with the plus end binding protein, CLIP170, to stabilize MTs transiently, whereas the Rho effector, mDia, leads to the formation of more stable MTs, perhaps through the plus-end binding protein EB1. Given that PAR-1 does not appear to function through the obvious candidate, Tau, it would be interesting to test whether this kinase acts through either of these pathways to regulate MT organization in epithelial cells (Doerflinger, 2003).

PAR-1 kinase regulates epithelial detachment and directional protrusion of migrating border cells

Many cells that migrate during normal embryonic development or in metastatic cancer first detach from an epithelium. However, this step is often difficult to observe directly in vivo, and the mechanisms controlling the ability of cells to leave the epithelium are poorly understood. In addition, once cells detach, they must assume a migratory phenotype, involving changes in cytoskeletal and signaling dynamics. Drosophila border cells provide a model system in which a combination of forward genetics and live-cell imaging can allow researchers to investigate the cellular and molecular mechanisms of epithelial cell detachment and migration in vivo. This study identified the Drosophila homolog of the serine/threonine kinase PAR-1 (MARK/Kin1) in a screen for mutations that disrupt border cell migration. Previous studies identified two proteins, Apontic and Notch, that indirectly affect border cell detachment by regulating transcription of downstream targets. In contrast, PAR-1 directly modulates apical-basal polarity between border cells and epithelial cells to promote detachment. Furthermore, PAR-1, but not the apical polarity complex protein PAR-3, promotes the directionality of transient cell protrusions, which border cells require for sensing the chemoattractant gradient. It is concluded that PAR-1-dependent apical-basal polarity is required for proper detachment of migratory border cells from neighboring epithelial cells. Moreover, polarity controlled by PAR-1 influences the ability of migratory cells to sense direction, a critical feature of migration. Thus, this work reveals new insights into two distinct, but essential, steps of epithelial cell migration (McDonald, 2008).

Although detachment is critical for the separation of migratory cells from epithelia, the molecular and cellular mechanisms underlying this process are poorly understood. This study demonstrates that disrupting apical-basal polarity inhibits the ability of border cells to detach from the follicle cell epithelium. Overexpression of a PAR-3 mutant that cannot be phosphorylated by PAR-1 phenocopied the loss of par-1. Loss of lkb1 affected border cell detachment in a manner similar to what was seen with par-1 mutants. In addition, a specific genetic interaction was observed between the two genes. Although Drosophila LKB1 was initially reported to be downstream of PAR-1, a recent study demonstrated that it phosphorylates and activates PAR-1 in vitro and in vivo at a conserved threonine within the kinase domain. A model is proposed in which a conserved network of LKB1 and basolateral PAR-1 directly phosphorylates and inhibits apical PAR-3 between border cells and follicle cells and thereby promotes the separation of the two cell types (McDonald, 2008).

How does the proper spatial restriction of apical polarity proteins lead to border cell detachment? The apical PAR-3/PAR-6/aPKC complex is required for formation and stabilization of epithelial adherens and tight (subapical) junctions. Overexpression of PAR-3 in mammalian cells promotes tight junction assembly, suggesting a model in which PAR-1-dependent restriction of PAR-3 to the apical junction allows the disassembly and/or remodeling of adhesion between border cells and adjacent epithelial cells. Analysis of specific cell membrane markers revealed that loss of par-1 caused mislocalization of apical PAR-3 and E-cadherin at the interface between border cells and follicle cells. In addition, overexpression of PAR-3 and PAR-6 in border cells disrupts the polarized distribution of membrane proteins, including the adherens junction proteins E-cadherin and Armadillo/β-Catenin. Expansion of the apical PAR-3 domain inhibits border cell detachment, possibly by increasing adhesion and preventing disassembly of junctions between border cells and follicle cells (McDonald, 2008).

The results suggest that the ability to detach is a property of the basolateral domain and requires restriction of the apical domain, which may be a general mechanism for promoting detachment of epithelial cells. During epithelial-to-mesenchymal transitions induced by TGFβ signaling, TGFβ receptors bind to and phosphorylate the apical polarity protein PAR-6 to allow recruitment of the ubiquitin ligase Smurf1; this in turn degrades RhoA, resulting in loss of tight junctions. Furthermore, the oncogene ErbB2 directly blocks the formation of the PAR-3/PAR-6/aPKC complex to inhibit breast epithelial cell polarity and tissue organization, apart from its function in cell proliferation. These studies demonstrate that downregulation of apical-basal polarity, which in turn disrupts cell-cell junctions, remodels epithelia during tissue morphogenesis and tumor progression. It is proposed that PAR-1 is another protein that remodels specific epithelial junctions to promote cell migration. However, other basolateral proteins such as Scribble and Discs large seem to suppress rather than promote detachment of epithelial cells. Therefore, the relationship between apical/basal polarity and epithelial cell invasion and detachment remains to be fully understood (McDonald, 2008).

Border cells are guided to the oocyte by secreted ligands that bind to multiple receptor tyrosine kinases. The secreted proteins PVF1, Spitz, and Keren are synthesized in the oocyte. The receptor for PVF1 is PVR, and the receptor for both Spitz and Keren is the Drosophila EGF Receptor (EGFR); both receptors are expressed on border cells. Border cells, like all migrating cells that undergo chemotaxis, polarize in response to these guidance cues. One of the most obvious manifestations of polarization is the extension of prominent protrusions primarily from the front or leading edge of the cluster. Inhibiting the functions of PVR and EGFR together causes border cells to extend protrusions in all directions, indicating that the activity of guidance receptors limits protrusion to the leading edge. However, guidance signaling not only induces protrusion extension from the front but also suppresses them from the rear of the cluster. Thus, activation of guidance receptors at the front polarizes the cluster. Similarly, loss of par-1 causes more protrusions to extend to the side and rear of the cluster, suggesting that directional polarity is lost (McDonald, 2008).

As border cells initiate their migration, the leading edge coincides with the apical domain, although this orientation changes after the cluster detaches. PAR-1 localizes to the lateral sides of border cells and not to the leading or apical side of the cluster. One possibility is that PAR-1 restricts the localization of an unknown protein to the leading edge and that this in turn promotes the formation of stable protrusions at the front. Although PAR-1 restricted PAR-3 to the apical side, PAR-3 SA overexpression did not disrupt directional protrusion extension. Alternatively, PAR-1 might activate a protein at the side and/or back of the cluster, and this protein might either suppress or destabilize protrusions at the lagging edge. The known role for PAR-1 in regulating microtubule stability could contribute directly to protrusion extension and dynamics. Although no gross changes were observed in the levels or distribution of α-tubulin, PAR-1 could have a subtle role in regulating microtubule dynamics of border cell protrusions. Thus, PAR-1 participates in generating polarity with respect to the leading and lagging edges of the migrating border cells, independently of its effect on PAR-3 (McDonald, 2008).

Whereas border cells detach and migrate as part of normal development, similar mechanisms occur in pathological conditions such as tumor invasion and metastasis. The work presented here establishes PAR-1-dependent apical-basal polarity as an essential mechanism for cell detachment from a polarized epithelium. Furthermore, PAR-1 plays a second role during cell migration by polarizing the border cell cluster, possibly in response to guidance signaling, and is required for normal protrusion morphology. Given the diversity of mechanisms that contribute to polarizing epithelial cells and migrating cells, as well as the high degree of functional conservation of polarity proteins, it will be of great interest to determine whether and in what contexts PAR-1 homologs induce epithelial cell detachment and protrusions during development or in tumor metastasis in vertebrates (McDonald, 2008).

Par-1 kinase establishes cell polarity and functions in Notch signaling in the Drosophila embryo

The Drosophila protein kinase Par-1 is expressed throughout Drosophila development, but its function has not been extensively characterized because of oocyte lethality of null mutants. This report characterizes the function of Par-1 in embryonic and post-embryonic epithelia. Par-1 protein is dynamically localized during embryonic cell polarization, transiently restricted to the lateral membrane domain, followed by apicolateral localization. Maternal and zygotic par-1 was depleated by RNAi and a requirement was revealed for Par-1 in establishing cell polarity. Par-1 restricts the coalescing adherens junction to an apicolateral position and prevents its widespread formation along the lateral domain. Par-1 also promotes the localization of lateral membrane proteins such as Delta. These activities are important for the further development of cell polarity during gastrulation. By contrast, Par-1 is not essential to maintain epithelial polarity once it has been established. However, it still has a maintenance role since overexpression causes severe polarity disruption. Additionally, a novel role is found for Par-1 in Notch signal transduction during embryonic neurogenesis and retina determination. Epistasis analysis indicates that Par-1 functions upstream of Notch and is critical for proper localization of the Notch ligand Delta (Bayraktar, 2006).

The first embryonic epithelium in Drosophila to develop is the blastoderm, which forms by cellularization. Nuclear division without cytokinesis continues for 13 rounds until nearly 5000 nuclei are present in a syncitium. The nuclei migrate peripherally beneath the plasma membrane, divide once more, and become enveloped by invaginating membrane. Membrane growth occurs by insertion of new membrane at distinct sites, first apically and then apicolaterally. The leading edge of each membrane invagination forms a furrow canal, and is associated with basal spot adherens junctions (SAJs) composed of E-cadherin and ß-catenin. Apical SAJs also arise during lateral membrane growth and eventually coalesce to form the apicolateral AJ at mid-gastrulation, while the basal SAJs disappear. The blastoderm epithelium is the direct progenitor of the ectoderm, from which such diverse epithelia as the epidermis and imaginal discs are derived. To explore the role Par-1 plays in Drosophila zygotic development, localization of Par-1 protein was examined in embryos; anti-Par-1 staining was detected throughout cellularization. At early stages of cellularization, staining was detected along the original plasma membrane and in the vicinity of the furrow canal. As cellularization progressed, staining was detected in the vicinity of the furrow canal and along the lateral membrane, while the original plasma membrane staining attenuated. When cellularization was complete, Par-1 staining was associated with the lateral domain of blastoderm cells, as well as punctate staining in the cytoplasm. Apical staining of the original plasma membrane was not apparent, and Par-1 did not overlap with E-cadherin in the AJ. At mid-gastrulation, the apical and lateral membrane regions of ectodermal cells were stained, as was the cytoplasm at a lower level. Low levels of cytoplasmic staining were also detected in neuroblasts and mesodermal cells. Late stage embryos exhibited punctate Par-1 staining, with a conspicuously higher level in the central nervous system relative to other tissues (Bayraktar, 2006).

Anti-Par-1 staining was maintained in epithelia that were derived from the blastoderm. The eye imaginal disc is a fully polarized epithelium that is directly derived from mid-embryonic stage ectoderm epithelium without a mesenchymal intermediate. Specific anti-Par-1 staining was observed in the eye imaginal disc of third-instar larvae in the apical domain, marginal zone, and AJ. This pattern was reminiscent of the pattern observed in the gastrulating embryonic ectoderm, from which the eye disc is derived. At this stage, Par-1 overlaps with E-cadherin in the disc cell AJ (Bayraktar, 2006).

The data presented here indicate that Par-1 is specifically involved in restricting SAJs to a region adjacent to the future apical domain during cellularization. Par-1 also restricts the AJ to the same region in older cells as they undergo gastrulation. Depletion of Par-1 results in expansion of the AJ into the lateral domain. This effect is not unique to par-1(RNAi) embryos. The Dlg/Scrib complex is required to restrict the AJ from the lateral domain. However, three observations indicate that Par-1 is not simply acting through this complex to localize the AJ. First, Par-1 does not regulate the localization of Dlg in blastoderm or ectoderm. Second, the Dlg complex does not begin to restrict the AJ until gastrulation, a time considerably later than Par-1 initially acts. Third, loss of Scrib results in lateral expansion of apical Crb, a phenomenon not observed when Par-1 is depleted. Altogether, these data indicate that Par-1 restricts the AJ by a mechanism independent of the Dlg/Scrib complex, perhaps preceding it. Interestingly, the presence of the Dlg complex in gastrulating ectoderm is not sufficient to re-establish an apical AJ when Par-1 is depleted, suggesting that the Par-1 and Dlg/Scrib pathways are obligatory (Bayraktar, 2006).

It might not be that Par-1 simply defines the limits of the AJ. Rather, it might also act positively to specify the basolateral domain. Par-1 is required for localization of Delta to the basolateral region. This positive effect of Par-1 on Delta is not due to preventing the AJ from inhibiting Delta. Delta co-localizes with the AJ both normally and when Par-1 is depleted. So how does Par-1 guide membrane regionalization during cellularization? Par-1 protein is distributed along the lateral membrane immediately basal to the incipient AJ. Based on this, three models suggest themselves. Par-1 could assemble a diffusion barrier that physically blocks movement of SAJs into the lateral region and limits movement of Delta into the apicolateral region. If par-1(RNAi) disrupts such a barrier, then other mechanisms must maintain apical Crb restriction from the lateral region. An alternative is that Par-1 has a role in the polarized targeting of transport vesicles carrying SAJ and Delta proteins. In this model, Par-1 might interact with the 'exocyst', a secretory targeting apparatus involved in polarized segregation of transmembrane proteins. Data from yeast Par-1 indicate that it directly associates with a t-SNARE, a membrane-bound component of the exocyst. Moreover, Par-1 phosphorylation of the t-SNARE protein triggers its release from the cell membrane. If Drosophila Par-1 also interacts with the exocyst, then it might selectively block the fusion of SAJ exocytic vesicles to the basolateral membrane. In support of this model, punctate intracellular staining of Par-1 can be seen during cellularization and is reminiscent of vesicles. Par-1 might also stimulate targeting of other cargo, such as Delta-loaded vesicles, to the basolateral membrane. Consistent with this notion, Par-1-depleted ectoderm cells accumulate Delta-positive cytoplasmic vesicles. Finally, Par-1 could differentially affect the stability of proteins in the lateral domain, by de-stabilizing some and stabilizing others. This could occur through degradation or rapid recycling via endocytosis (Bayraktar, 2006).

How directly would Par-1 participate in these mechanisms? This is unclear at present. None of the known substrates for Drosophila Par-1 kinase include Delta or AJ components. In ovarian follicle cells, Par-1 phosphorylation of Baz prevents Baz association with Par-6-aPKC. Baz and Par-6 are among the earliest acting proteins in polarization of blastoderm. During cellularization, Baz associates with the apicolateral membrane, whereas Par-6 is localized to the apical cortex. If either Baz or Par-6 is mutated, the apical AJ proteins do not coalesce but disperse along the lateral membrane. Thus, Baz could be a candidate for mediating the effects of Par-1 in the blastoderm. However, several observations indicate that Par-1 phosphorylation of Baz is not necessarily essential to establish AJ localization. First, Par-1 does not have a polarized distribution during early cellularization and is detected in the apicolateral regions where Baz and Par-6 are already localized. A similar co-distribution is seen by the time the ectoderm has reached mid-gastrula stage. Second, although Baz is required for apical localization of Crb and Patj, Par-1 has no significant effect on their apical localization. Third, establishment of the AJ in follicle cells is not dependent upon Par-1 phosphorylation of Baz (Bayraktar, 2006).

Par-1 plays a curious role in maintenance of polarity of imaginal disc epithelia that derive directly from ectoderm. Par-1 is localized to the apical and marginal zones of imaginal disc cells but is not essential for their polarity. Possibly, redundant mechanisms operate in the absence of Par-1. This idea is supported by overexpression experiments. When Par-1 is overexpressed, the AJ and apical domain are disorganized, and cells are compromised for differentiation, growth and death. This result argues that Par-1 normally plays a role in maintaining cell polarity that is sensitive to its activity level. By contrast, Par-1 is essential to maintain polarity in follicle cell epithelia surrounding adult egg chambers, suggesting that redundancy is restricted to imaginal discs (Bayraktar, 2006).

Par-1 also regulates Notch signaling and it acts upstream of Notch as determined by epistasis analysis. Two different Notch signaling decisions regulated by Par-1 are detected. The first was in the embryonic ectoderm where Par-1 depletion disables Notch-mediated lateral inhibition. The second is in the eye imaginal disc where Par-1 overexpression disables Notch-mediated eye cell determination. Since Notch is disabled when Par-1 is missing or overactive, it suggests that Par-1 is not playing an instructive role in Notch signaling. Rather, it is probably a permissive effect that is related to cell polarity regulation. Indeed, localization of Delta is dramatically reduced along the basolateral domains of blastoderm and ectoderm cells of par-1(RNAi) embryos. It is reasonable to think that Par-1 acts in Notch signaling by localizing Delta to a region of the membrane where it can make a productive interaction with Notch. This permissive model of Notch signaling is nevertheless specific; other regulators of ectoderm polarity do not affect Notch signaling. Moreover, other signaling pathways active in the ectoderm are unaffected by Par-1. Interestingly, a synergistic interaction between Par-1 and Notch was found in the eye imaginal disc. Disc growth significantly increased when Par-1 was overexpressed with ligand-independent Notch. The extra eye tissue developed photoreceptors, indicating the ectopic cells are properly specified. Since loss of cell polarity is associated with hyperplasia in the eye disc, this supports the notion that Par-1 exerts this effect through perturbation of eye disc cell polarity. The synergistic interaction with Notch may be useful in the future for screening of genes involved in tumor formation or progression to a cancerous state (Bayraktar, 2006).

Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis

The Drosophila dorsal-ventral (DV) axis is polarized when the oocyte nucleus migrates from the posterior to the anterior margin of the oocyte. Prior work suggested that dynein pulls the nucleus to the anterior side along a polarized microtubule cytoskeleton, but this mechanism has not been tested. By imaging live oocytes, this study found that the nucleus migrates with a posterior indentation that correlates with its direction of movement. Furthermore, both nuclear movement and the indentation depend on microtubule polymerization from centrosomes behind the nucleus. Thus, the nucleus is not pulled to the anterior but is pushed by the force exerted by growing microtubules. Nuclear migration and DV axis formation therefore depend on centrosome positioning early in oogenesis and are independent of anterior-posterior axis formation (Zhao, 2012).

The correct positioning of the nucleus is important for several developmental processes, such as cell migration, formation of the neuromuscular junction, and asymmetric cell divisions, whereas nuclear mislocalization is a feature of neurological disorders, such as lissencephaly. Positioning of the nucleus plays an essential role in Drosophila axis formation, as the movement of the nucleus from the posterior of the oocyte to a point at its anterior circumference breaks radial symmetry to polarize the DV axis. At stage 7 of oogenesis, an unknown signal from the posterior follicle cells induces a major reorganization of the oocyte microtubule cytoskeleton. The posterior microtubule organizing center (MTOC) is disassembled, and microtubules are nucleated from the anterior-lateral cortex, resulting in an anterior-posterior (AP) gradient of microtubules that defines the AP axis. It is believed that dynein subsequently uses this polarized microtubule cytoskeleton to pull the nucleus to the oocyte anterior, making polarization of the DV axis dependent on the prior polarization of the AP axis (Zhao, 2012).

To investigate the mechanism of nuclear positioning directly, the movement of the nucleus in living oocytes was imaged. The nucleus migrates at a speed of 4.0 ± 0.7 μm/hour, and takes 2-3 hours to move across the oocyte. The trajectory of the nucleus is variable: sometimes it moves around the cortex of the oocyte directly to an anterior corner, but it often migrates up the center of the oocyte and then turns to move along the anterior cortex, confirming the random nature of this symmetry-breaking event. This study observed that all migrating nuclei have large posterior indentations, suggesting that they are being pushed rather than pulled toward the anterior. This could reflect an intrinsic reorganization of the nuclear architecture, or a deformation induced by an external force to the nucleus. In support of the latter view, the direction of the indentation correlates with the direction of migration, suggesting that the same force creates the indentation and moves the nucleus. This indentation is not an artefact of long-term imaging in oil, as egg chambers dissected directly into strong fixative have identical indentations (Zhao, 2012).

This idea that the nucleus is pulled to the anterior by dynein is based on the finding that mutations in the dynein accessory factors, Lis1 and Bic-D, as well as disruption of the dynactin complex result in mislocalized nuclei at stage 10. This is not compatible with the pushing model of nuclear movement, as motor proteins can only pull their cargoes. Therefore this study re-examined the role of the dynein complex by imaging the nucleus in Lis1 mutant egg chambers. Lis1 mutant oocytes are much smaller than normal because dynein is required for transport from the nurse cells into the oocyte. Nevertheless, the oocyte nucleus migrates normally with a prominent posterior indentation. Thus, dynein is presumably required for the anchoring of the nucleus once it has reached the anterior, rather than for its migration. Consistent with this, the nuclei are only rarely mispositioned in Lis1 and Bic-D mutant oocytes at stage 9, but are mislocalized much more frequently at later stages (Zhao, 2012).

Both actin and microtubule polymerization can generate pushing forces that lead to cellular or organelle deformations. Two lines of evidence suggest that microtubules are responsible for the nuclear indentation: First, depolymerization of actin with latrunculin A or B does not affect nuclear positioning, whereas the microtubule-depolymerizing drug, colcemid, induces mislocalized nuclei. Secondly, several microtubule-associated proteins become enriched on the posterior nuclear envelope during migration, including the Dynein light intermediate chain (Dlic), Calmodulin (Cam), and the Drosophila NuMA homolog Mushroom body defect, Mud (Zhao, 2012).

To test the role of microtubules in the formation of the indentation, colcemid was added to living egg chambers expressing the +TIP protein, EB1-GFP (end binding-1), which forms a 'comet' on the growing plus ends of microtubules. Colcemid takes 3.5 min to diffuse into the oocyte, as monitored by a decrease in the number of EB1 comets on growing microtubule plus ends. As soon as microtubule growth starts to decrease, the indentation diminishes in size. A focus of EB1-GFP persists posterior to the nucleus for several minutes, and as this disappears, the nucleus relaxes completely and becomes spherical. Thus, the nuclear indentation depends on microtubule polymerization and its size is proportional to the number of growing microtubules (Zhao, 2012).

Using EB1-GFP to track the growing microtubule plus ends in time-lapse movies of nuclear migration revealed several strong foci of EB1-GFP behind the indentation, with growing microtubules emanating from them in all directions. This indicates that microtubules are nucleated from MTOCs behind the nucleus. These MTOCs resemble the centrosomes, which migrate from the nurse cells into the oocyte during early oogenesis in a dynein-dependent manner, and localize to the posterior cortex as a result of the initial oocyte polarity. Indeed, the centriolar markers Sas4 and PACT, as well as a marker for pericentriolar material (PCM), Centrosomin (Cnn), localize to the foci behind the nuclear indentation at the onset of migration. The centrosomes behave rather dynamically during migration and change reversibly from a dense cluster to a more dispersed distribution. Upon completion of nuclear migration, the centrosomes are recruited to the anterior-dorsal cortex of the oocyte, presumably as a consequence of the activation of the dynein-dependent anchoring mechanism that retains the nucleus in this position. Active centrosomes are therefore positioned behind the nucleus before and during migration (Zhao, 2012).

To test the role of the centrosomes in creating the nuclear indentation, they were inactivated by laser ablation. Upon ablation of the entire cluster of centrosomes, the indentation disappears and the nucleus becomes spherical within 1 min. This nuclear relaxation may occur more rapidly, as centrosome ablation causes local bleaching of the nuclear envelope, making it impossible to monitor nuclear shape during the first minute. Local laser ablation of the nuclear envelope at the site of the indentation has no effect, however, excluding the possibility that the disappearance of the indentation is a consequence of bleaching of the nuclear membrane. Furthermore, ablation of the anterior of the nucleus does not affect the indentation, arguing against any pulling force from the anterior. As described above, centrosomes are sometimes scattered behind the nucleus, causing multiple indentations. Ablating one cluster of centrosomes abolishes only the indentation facing them. The non-ablated centrosomes remain active and induce an indentation on the adjacent side of the nucleus. Thus, the nucleus is not a rigid structure and the growing microtubules from the centrosomes exert force on the nuclear envelope to induce its deformation (Zhao, 2012).

The centrosomes are dispensable for oogenesis. Therefore nuclear migration was examined in DSas-4 mutant ovaries that lack centrosomes. Consistent with the previous study, all nuclei migrate to the anterior-dorsal corner and show a posterior indentation during migration. GFP-Cnn is still localized in foci behind the nucleus and EB1-GFP tracks reveal active posterior MTOCs. Thus, acentrosomal MTOCs form in the absence of centrosomes and can provide the pushing force for nuclear migration (Zhao, 2012).

As a further test of the idea that the centrosomal microtubules push the nucleus to the anterior, par-1 hypomorphs, in which some centrosomes fail to migrate to the posterior of the oocyte, were examined. These anterior centrosomes induce anterior nuclear indentations, leading to dumbbell-shaped nuclei, confirming the role of centrosomal microtubules in pushing the nucleus. These ectopic centrosomes eventually fuse with the posterior centrosomes to move the nucleus to the anterior-dorsal corner. This explains why the nucleus migrates normally in par-1 mutants, even though the anterior-posterior axis is not polarized. Consistent with this, the nucleus in wild-type oocytes can migrate to the anterior before the anterior-to-posterior microtubule gradient is established (Zhao, 2012).

Another documented example of nuclear positioning by microtubule pushing comes from S. pombe, where microtubule bundles push against the cell ends to maintain the nucleus in the cell center. The oocyte nucleus moves a much greater distance, however, and appears to be pushed by the force exerted by single growing microtubules. To test the feasibility of this mechanism, Stoke's law (F = 6πηrv) to estimate the drag force (F) exerted on the nucleus. Assuming a cytoplasmic viscosity (η) &asymp 100 Pas and the measured values of the nuclear radius (r) ∼ 5 &muν;m and the velocity of migration (v) ∼ 4 μm/hour yields a drag force ∼ 10 pN. It is expected that the actual drag force is lower, because nuclear migration is so slow (1 nm/s) that the cytoplasmic actin mesh will turn over ahead of the nucleus, decreasing the effective viscosity. The longest microtubules can reach ∼ 10 μm between the posterior of the nucleus and the posterior oocyte cortex, resulting in a critical buckling force Fc ≈ 5 pN. This value is probably an underestimate, as microtubules embedded within an elastic cytoplasm in vivo have been reported to bear compressive loads a hundred times higher than in vitro. Each microtubule can therefore generate a pushing force of at least 5 pN. Thus, the force of only two microtubules pushing at any time should be sufficient to move the nucleus to the oocyte anterior (Zhao, 2012).

The number of microtubules pushing the nucleus was measured using EB1-GFP. 15.3 ± 1.6 microtubules hit the nuclear indentation per minute in one z-plane (n = 10, 2 oocytes), and they continue growing and presumably exerting force on the nucleus for 2.77 ±. 0.14 s. Given the thickness of a confocal section (0.8 μm) and the radius of the indentation [4.3 ± 0.2 μm (n = 10)], an average of 5.9 ± 0.7 microtubules are pushing the nucleus at any given time. Microtubule polymerization can therefore provide sufficient pushing force to drive nuclear migration (Zhao, 2012).

The migration of the nucleus is triggered by an unknown signal from the posterior follicle cells, which could act either by activating the centrosomes or by releasing the nucleus from a posterior tether. To address this question, when the indentation appears during oogenesis was examined. Active centrosomes are already localized behind the nucleus at stage 5 of oogenesis and induce a posterior indentation. This suggests that the centrosomes continually exert a pushing force on the nucleus, which is tethered to the posterior until it receives a signal for migration. The nucleus remains at the posterior in gurken (grk) mutants, which block follicle cell signaling to the oocyte (39% penetrance). These posterior nuclei still maintain a posterior indentation later in oogenesis, suggesting that they fail to migrate because they are not released from the posterior tether. Indeed, microtubules growing from active centrosomes probably always exert a pushing force on the nucleus that must be countered by an opposing pulling force or anchor to keep the nucleus in place. For example, a clear nuclear indentation is still visible adjacent to the centrosomes after the nucleus is anchored at the anterior (Zhao, 2012).

The results lead to a revised model for how the oocyte nucleus moves to break radial symmetry and polarize the Drosophila dorsal-ventral axis. This model explains the failure to recover mutants that specifically disrupt nuclear migration, since the driving force is provided solely by microtubule polymerization. Furthermore, the results imply that migration is triggered by the release of the nucleus from a posterior anchor, rather than by microtubule reorganization. Thus, polarization of the dorsal-ventral axis is independent of the formation of the microtubule array that defines the anterior-posterior axis, as previously proposed (Zhao, 2012).


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par-1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 August 2018

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