discs large 1

Protein Interactions

The cell adhesion molecule Fasciclin II (FASII) is involved in synapse development and plasticity. Proper localization of FASII at type I glutamatergic synapses of the Drosophila neuromuscular junction is mediated by binding between the intracellular tSXV bearing C-terminal tail of FASII and the PDZ1-2 domains of Discs-Large (DLG). Moreover, mutations in fasII and/or dlg have similar effects on presynaptic ultrastructure, suggesting their functional involvement in a common developmental pathway. DLG can directly mediate a biochemical complex and a macroscopic cluster of FASII and Shaker K+ channels in heterologous cells. These results indicate a central role for DLG in the structural organization and downstream signaling mechanisms of cell adhesion molecules and ion channels at synapses (Thomas, 1997b).

Both the Fasciclin II (Fas II) cell adhesion molecule and the Shaker potassium channel are localized at the Drosophila neuromuscular junction, where they function in the growth and plasticity of the synapse. Both proteins contain -S/T-X-V sequences at their C termini, identifying them as proteins that could interact with PDZ domains. The GAL4-UAS system was used to drive expression of the chimeric proteins CD8-Fas II and CD8-Shaker. The C-terminal sequences of both Fas II and Shaker are necessary and sufficient to drive the synaptic localization of a heterologous protein. The PDZ-containing protein Discs-Large (Dlg) controls the localization of Fas II and Shaker, most likely through a direct interaction with their C-terminal amino acids. Transient expression studies show that the pathway these proteins take to the synapse involves either an active clustering or a selective stabilization in the synaptic membrane. Following a pulse of protein expression, the CD8-Shaker protein is initially distibuted uniformly on the muscle membrane, followed by concentration at the synapse. Thus the results are consistent with a model of uniform membrane targeting followed by active clustering or selective retention at the synapse. If interactions with Dlg stabilize FasII and Shaker at the synapse, one mechanism to regulate the levels of Fas II and Shaker could be through regulation of these interactions. Such a regulation has been demonstrated for the mammalian inwardly rectifying potassium channel Kir2.3, in which phosphorylation of its PDZ-interaction motif by cAMP-dependent protein kinase (PKA) inhibits its binding to PSD-95. A similar mechanism to regulate interaction between Dlg and FasII or Shaker could provide an activity-dependent mechanism for regulation of the levels of these proteins at the synapse (Zito, 1997).

Human adenomatous polyposis coli gene (APC), for which there is no known Drosophila homolog, binds to the adherens junction protein ß-catenin, a mammalian homolog of Armadillo  (one component of the Wingless signaling pathway). Overexpression of APC blocks cell cycle progression. The mammalian APC-ß-catenin complex has been shown to bind to mammalian DLG, suggesting the possibility of a similar interaction in Drosophila. This requires the carboxyl-terminal region of APC and the DLG homology repeat region of mammalian DLG. APC is a good substrate for GSK3ß, the mammalian homolog of Shaggy/Zeste white 3 in vitro, and the phosphorylation sites map to the central region of APC. Binding of ß-catenin to this region is dependent on phosphorylation of GSK3ß. These interactions, some but not all of which are known to occur in Drosophila, may prove to be important aspects of segment polarity in the fly. It is already known that Dishevelled, another DLG homolog in the fly interacts with Armadillo (Matsumine, 1996 and Rubinfeld, 1996, Yanagawa, 1995).

Another interaction may occur in Drosophila, an interaction of DLG with protein 4.1 homologs. Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. Synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Thus protein 4.1, known to interact with the cytoskeleton, also interacts with DLG family members (Marfatia, 1995). A protein in Drosophila with protein 4.1 homology is the recently discovered Inscuteable (Kraut, 1996).

Distribution of two family 4.1 proteins, Expanded and Coracle, are disrupted in dlg mutants. Loss of Discs large also affects the distribution of Fasciclin III and neuroglian, two transmembrane proteins thought to be involved in cell adhesion (see in DLG part 1: Biological overview). These results suggest that DLG serves as a binding protein linking cell surface receptors with the cytoskeleton via family 4.1 proteins (Woods, 1996)

Coracle protein is a component associated with septate junctions of epithelial cells. In tall columnar epithelia, such as in the hindgut, there is an obvious polarity of Coracle expression toward the apicolateral epithelial surface. Imaginal disc cells show intense staining in regions of cell contact just below the apical epithelial surface. This region appears to coincide with the septate junction. Mutations in cor show defects in dorsal closure, leaving a small scab in the dorsal side of embryos, or a much larger dorsal opening and abnormalities in head involution. coracle mutations dominantly suppress Ellipse, a hypermorph allele of the Egf-receptor (Fehon, 1994).

Drosophila Neurexin is required for septate junction and blood-nerve barrier formation and function. NRX is localized apicolaterally, adjacent to Crumbs, which delimits the zonula adherens. These two proteins are not coexpressed, placing NRX apicolaterally. Both Fasciclin3 and NRX colocalize at salivary gland synaptic junctions. NRX precisely colocalized with D4.1/Coracle except in the PNS and CNS where D4.1/Coracle is only expressed in a few cells.

No defects in the localization of Discs large protein is detected in nrx mutants. However, D4.1/Coracle is not restricted to septate junctions in nrx mutants. These results suggest that the short cytoplasmic portion of NRX that shows homology to glycophorin C is required to localize D4.1/Coracle to septate junctions, creating a parallel with red blood cell cytoskeletal anchoring proteins (Baumgartner, 1996).

Discs large (DLG) mediates the clustering of synaptic molecules. Synaptic localization of Dlg itself is regulated by CaMKII. Dlg and CaMKII colocalize at synapses and exist in the same protein complex. Constitutively activated CaMKII phenocopies structural abnormalities of dlg mutant synapses and dramatically increases extrajunctional Dlg. Decreased CaMKII activity causes opposite alterations. This was most clearly demonstrated by examining the size of the postsynaptic junctional membrane -- the SSR. Constitutive CaMKII activation results in poorly developed SSR, while CaMKII inhibition results in an overdeveloped SSR. These alterations phenocopy previously described effects of changing Dlg levels at the synaptic membrane. Mutations in dlg result in a poorly developed SSR, while overexpressing wild-type Dlg results in an overdeveloped SSR. The similarity between the phenotypes elicited by constitutive activation of CaMKII and those observed in dlg mutants is not solely restricted to the morphology of the SSR. An increase in the number of active zones, an enlargement of the boutons, and an abnormal Fas II clustering around these boutons is observed. Thus, these results strongly suggest that the changes in synaptic structure and composition elicited by changing CaMKII activity are the result of changes in DLG distribution. In vitro, CaMKII phosphorylates a Dlg fragment with a stoichiometry close to one. Moreover, expression of site-directed dlg mutants that block or mimic phosphorylation has effects similar to those observed upon inhibiting or constitutively activating CaMKII. It is proposed that CaMKII-dependent Dlg phosphorylation regulates the association of Dlg with the synaptic complex during development and plasticity, thus providing a link between synaptic activity and structure (Koh, 1999).

Antibodies against Drosophila CaMKII were used to determine its distribution at the larval NMJ. CaMKII is localized at wild-type NMJs around type I synaptic boutons in a pattern similar to Dlg. Double labeling with anti-Dlg reveals that Dlg and CaMKII are colocalized at bouton borders. In addition, transgenic CaMKII is targeted to synaptic boutons, as visualized by increased immunoreactivity levels at NMJs, upon expression of a CaMKII transgene in both motor neurons and muscles, by using muscle specific and motor neuron specific GAL4 activators. CaMKII and DLG colocalization is supported by immunoprecipitation experiments. Both proteins exist in the same complex in body wall muscles. When anti-CaMKII is used for immunoprecipitations of body wall muscle extracts, Dlg coimmunoprecipitates from wild-type, CaMKII+, or CaMKII-T287D overexpressing extracts. Moreover, transgenic Dlg expressed in dlg^X1-2 mutants is also coimmunoprecipitated by CaMKII. Thus, Dlg and CaMKII are closely associated at type I NMJs (Koh, 1999).

It is proposed that the anchoring of Dlg at the synapse is optimal when Dlg is in the dephosphorylated state. Upon phosphorylation, Dlg is less restricted to the synaptic complex. Because Dlg is essential for proper synaptic localization of Fas II and Shaker, these binding partners are similarly free to move away from the synaptic complex upon CaMKII-dependent phosphorylation. An alternative possibility however, and one that is not mutually exclusive, is that CaMKII-dependent phosphorylation of Dlg affects its targeting to the synapse during development. However, CaMKII and DLG colocalize at synapses. Therefore, it is likely that CaMKII-dependent phosphorylation of Dlg occurs at the synapse. It is known that synaptic Fas II is downregulated by increased cAMP levels, suggesting that PKA is also involved in coupling synaptic activity to structural plasticity at NMJs. Both PKA and CaMKII can be activated by various synaptic stimuli. Whether both signal transduction pathways act together or in parallel to regulate Dlg-dependent localization of Fas II remains to be determined (Koh, 1999 and references).

Amphiphysin family members are implicated in synaptic vesicle endocytosis, actin localization and one isoform is an autoantigen in neurological autoimmune disorder; however, there has been no genetic analysis of Amphiphysin function in higher eukaryotes. Drosophila Amphiphysin is localized to actin-rich membrane domains in many cell types, including apical epithelial membranes, the intricately folded apical rhabdomere membranes of photoreceptor neurons and the postsynaptic density of glutamatergic neuromuscular junctions. Flies that lack all Amphiphysin function are viable, lack any observable endocytic defects, but have abnormal localization of the postsynaptic proteins Discs large, Lethal giant larvae and Scribbled, altered synaptic physiology, and behavioral defects. Misexpression of Amphiphysin outside its normal membrane domain in photoreceptor neurons results in striking morphological defects. The strong misexpression phenotype coupled with the mild mutant and the lack of phenotypes suggest that Amphiphysin acts redundantly with other proteins to organize specialized membrane domains within a diverse array of cell types. In other words, Drosophila Amphyphysin functions in membrane morphogenesis, but an additional role in endocytosis cannot yet be dismissed (Zelhof, 2001).

Recruitment of Scribble to the synaptic scaffolding complex requires GUK-holder, a novel dlg binding protein

Membrane-associated guanylate kinases (MAGUKs), such as Discs-large (Dlg), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between Dlg and another PDZ scaffolding protein, Scribble (Scrib), during the establishment of cell polarity in developing epithelia. The biochemical nature of this interaction has remained elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. This study reports a new Dlg-interacting protein, GUK-holder (GUKh), that interacts with the GUK domain of Dlg and that is dynamically expressed during synaptic bouton budding. At Drosophila synapses Dlg colocalizes with Scrib and this colocalization is likely to be mediated by direct interactions between GUKh and the PDZ2 domain of Scrib. Dlg, GUKh, and Scrib form a tripartite complex at synapses, in which Dlg and GUKh are required for the proper synaptic localization of Scrib. These results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse (Mathew, 2002).

In Drosophila, dlg mutants in which the GUK domain is absent exhibit abnormalities in synapse structure. Moreover, transgenic Dlg lacking the GUK domain fails to localize at synapses when expressed in a dlg mutant background. These findings imply that the GUK domain is required for a synaptic function and targeting of Dlg. To gain further insight on how the GUK domain of DLG exerts its various functions, proteins interacting with this domain were sought. GUK-holder, a novel synaptic protein contains a WH1/EVH1-like domain in its N-terminal half and a PDZ binding motif at its C terminus; the PDZ binding motiif has been identified as a GUK interactor. GUKh is expressed in a dynamic fashion during synaptic bouton formation. In addition, it also binds to a PDZ domain of Scribble (Scrib; a tumor suppressor protein interacts genetically with Dlg in developing epithelia) thus physically linking Dlg to Scrib. Indeed, coimmunoprecipitation analyses together with immunocytochemical studies on wild-type and mutant larvae provide strong evidence that Dlg, GUKh, and Scrib exist in a tripartite complex at the NMJ. Most notably, normal GUKh function is required for the synaptic localization of Scrib (Mathew, 2002).

To understand the functional significance of the GUK domain of Dlg, binding partners of this domain were sought using a yeast two-hybrid screen. The GUK domain of Dlg (amino acids 765-960 was used as bait to screen a late embryonic stage Drosophila cDNA library. Thirty-eight interacting clones were recovered from this screen, and from these, nine were overlapping cDNAs representing a single novel gene, GUK-holder (Mathew, 2002).

To determine the precise regions of interaction between Dlg and GUKh, deletion constructs of the Dlg GUK domain and of the GUKh C terminus were generated and assayed for binding using the yeast two-hybrid assay. Nearly the entire GUK domain is necessary for an interaction with GUKh, since deletion of more than 15 residues from either end results in a loss of binding. A construct encompassing the last 156 amino acids of GUKh (amino acids 888-1044) is sufficient to mediate binding to the GUK domain of Dlg, defining this region as the GUK-holding domain (Mathew, 2002).

An affinity-purified polyclonal antiserum directed against the last 238 C-terminal amino acids of GUKh (GUKh-C) was prepared. In Western blots from body wall muscle extracts, the antibody detects a single band of ~110 kDa, consistent with the predicted size of the L isoform of GUKh. Moreover, anti-GUKh immunoreactivity is reduced at the NMJs or CNS of the hypomorphic gukh mutants gukh^J3E1and gukh², eliminated in gukh^2EM9 embryos, and enhanced upon overexpression of a gukh transgene, confirming the specificity of the antibody (Mathew, 2002).

To establish that Dlg and GUKh interact in vivo, a coimmunoprecipitation of body wall muscle extracts was performed using GUKh antibody. In wild-type, Dlg-specific bands at 97 and 116 kDa coprecipitate with the 110 kDa GUKh band, suggesting that GUKh exists in the same complex as Dlg. In contrast, Dlg is not coimmunoprecipitated from dlg^XI-2 mutants that lack the GUK domain. Together, these results strongly suggest that GUKh binds to the GUK domain of Dlg in vivo (Mathew, 2002).

The interaction of Dlg and GUKh is reminiscent of the interaction between the GUK domain of mammalian MAGUKs and GKAP (Kim, 1997; Takeuchi, 1997). Moreover, while GUKh and GKAP do not share significant sequence homology, both proteins terminate in a similar tS/TXV/L/I PDZ binding motif (i.e., tETAL versus tQTRL. In fact, GKAP proteins link the GUK domain of PSD-95/SAP90 to the PDZ domain of Shank/ProSAP (Boeckers, 1999; Tu, 1999). By analogy, it is inferred that GUKh might link Dlg to other PDZ domain-containing proteins. Recent studies have revealed that at epithelia Dlg exists in a complex with Scribble (Scrib), a protein comprising 16 leucine-rich repeats followed by four PDZ domains. However, the molecular nature of this interaction remained elusive. In this study, a coimmunoprecipitation assay was performed on body wall muscle extracts using a Scrib-specific antibody. Anti-Scrib efficiently coimmunoprecipitates Dlg from wild-type but not from a severe hypomorphic scrib allele. This indicates that, similar to the case in epithelia, Dlg and Scrib may exist in a complex at the NMJ. In line with this finding, Scrib exhibits striking colocalization with Dlg at type I boutons (Mathew, 2002).

Whether GUKh might provide a physical link between Dlg and Scrib was assessed. Indeed, GUKh is detected in anti-Scrib immunoprecipitates from wild-type but not from scrib mutant extracts. Moreover, immunoprecipitation of Dlg by anti-Scrib antibodies from a hypomorphic gukh allele is dramatically reduced (Mathew, 2002).

To investigate the possibility that the interaction between GUKh and Scrib might be direct, a yeast two-hybrid assay was used; this shows that GUKh specifically interacts with the PDZ2 domain of Scrib but not with either its PDZ3-4 or its LRR motifs. The interaction between GUKh and the PDZ2 domain of Scrib is mediated by the C terminus of GUKh, since just the ten last amino acids of GUKh are sufficient for this interaction. Deletion of the last 23 amino acids of GUKh (GUKh-DeltaC) prevents the interaction with the PDZ2 domain of Scrib. Moreover, when the ten amino acid peptide contains a mutation (L->A) at the C-terminal residue, it fails to interact with PDZ2. In addition, the last ten amino acids of Shaker K⁺ channel, which strongly binds to PDZ1-2 of Dlg, failed to bind PDZ2 of Scrib, demonstrating a degree of ligand specificity. In contrast, constructs encompassing PDZ1-2 or PDZ3 of Dlg failed to bind GUKh. Together, the localization, immunoprecipitation, and yeast two-hybrid studies strongly suggest that Dlg, GUKh, and Scrib may form a tripartite complex in which GUKh serves as a physical link between Dlg and Scrib (Mathew, 2002).

Together, these yeast two-hybrid, coimmunoprecipitation, and colocalization studies provide compelling evidence that GUKh interacts with Dlg in vivo. This interaction is mediated by a region near the C terminus of GUKh. However, as revealed by genetic analysis, the synaptic localization of GUKh does not depend on Dlg. This suggests that domains other than the Dlg interacting motif may mediate its synaptic localization. For instance, the single WH1-like domain of GUKh might interact directly or indirectly with the synaptic cytoskeleton. WH1 domains in other proteins bind F-actin, actin-associated proteins such as zyxin, vinculin, and profilin, or the spectrin-bound scaffolding protein Shank/ProSAP. Association of GUKh with cytoskeletal elements might also be mediated by those sequences that exhibit moderate similarity to the actin binding protein Kelch (Mathew, 2002).

While an association of GUKh with the actin-based synaptic cytoskeleton currently remains hypothetical, the C-terminal tETAL motif specifically binds to the second PDZ domain of Scrib. Anatomical and biochemical experiments suggest that in vivo, Dlg, Scrib, and GUKh may exist in the same complex at the NMJ. Alternatively, the three proteins could interact pairwise, forming separate heterodimers. Since GUKh was found to still localize normally at dlg mutant NMJs, it is proposed that Dlg and GUKh act in concert rather than in a hierarchical manner to recruit Scrib. As a possible mechanism, binding to the GUK domain of Dlg could cause sterical changes in GUKh, such that the tETAL motif becomes available for interaction with Scrib. A caveat to this study is that hypomorphic gukh mutants were used, and therefore, a requirement of GUKh in Dlg localization cannot be ruled out (Mathew, 2002).

While Dlg and Scrib are colocalized along the rims of synaptic boutons, which, as has been demonstrated for Dlg, comprise both the presynaptic membrane and the postsynaptic junctional region (SSR), GUKh intersects that region only in a narrow strip. Yet, in budding boutons, GUKh displays a complementary pattern to Dlg. These observations suggest that GUKh may not be continuously bound to Dlg but rather may be involved in transient interactions. The process of bouton budding is a dynamic process that is characterized by equally dynamic changes in both GUKh and Dlg distribution. The accumulation of GUKh at the core of budding boutons and the disappearance of Dlg at the border of buds suggest that both proteins serve different roles during this process. Interestingly, FasII, a molecule that mediates synapse stabilization but that also imposes an adhesive constraint on synaptic growth, faithfully resembles the changes in distribution of Dlg during budding, consistent with a role for Dlg in synaptic localization. The presence of GUKh at budding regions may represent a role for this protein in destabilizing regions of the synaptic bouton, thereby allowing for bud formation (Mathew, 2002).

In contrast to GUKh, Scrib is expressed throughout the SSR in exact colocalization with Dlg. Nonetheless, Scrib localization at distal regions of the SSR is also affected in gukh mutants. In fact, considering the hypomorphic character of the gukh alleles that were used in this study, the effect on Scrib localization appears remarkably strong. This observation might indicate that GUKh activity is required only temporarily and/or in a locally restricted fashion to prime a secondary mechanism by which Scrib becomes associated with the SSR, e.g., through a more direct interaction with Dlg. Interestingly, presynaptic expression of GUKh-C is largely sufficient to restore postsynaptic Scrib localization at gukh mutant NMJs. Together, these observations suggest a second, more indirect mechanism by which GUKh contributes to the recruitment of Scrib to the postsynaptic SSR; such a mechanism may involve trans-synaptic signaling (Mathew, 2002).

These studies provide evidence for one mechanism by which scaffolding proteins with different interaction domains may be linked to form a network of multiprotein complexes. GUKh, in physically linking Dlg and Scrib, can therefore bring together these complexes and their associated proteins. Since a single protein forms this link, it would be a straightforward point at which to also separate the complexes, along with their actions, to regulate different aspects of synapse formation. Examples would be during synapse stabilization and during synapse growth through bouton budding. Thus, this work provides a means by which macromolecular complexes can mediate and finely tune various structural changes at the highly dynamic structure of the synapse (Mathew, 2002).

Partner of Inscuteable interacts directly with Discs-large in the establishment planar polarity during asymmetric cell division in Drosophila

In the dorsal thorax (notum) of the Drosophila pupa, the pI cell divides unequally with its spindle axis aligned with the anterior-posterior (a-p) axis of the fly body. It produces two different daughter cells, pIIa and pIIb. During this division, Numb and its adaptor protein Partner of Numb (Pon) form an anterior crescent and segregate unequally into the anterior pIIb cell. In fz mutant pupae, the division of the pI cell is oriented randomly relative to the a-p axis and the Numb crescent does form, but at a random position. Thus, Fz is not required to establish planar asymmetry per se, but is necessary to orient the axis of the asymmetric cell division. This indicates that additional genes may be required for establishing, rather than orienting, planar asymmetry in the pI cell (Bellaïche, 2001 and references therein).

Fz organizes the actin cytoskeleton at the site of hair formation. Planar polarity in the pI cell is established by a mechanism that involves a remodeling of the previously established apical-basal polarity. During the pI cell division, Baz and DaPKC relocalize from the apical cortex to the posterior lateral cortex, while Dlg and Partner of Inscuteable (Pins) accumulate asymmetrically at the anterior lateral cortex. This redistribution along the a-p axis leads to the formation of two complementary planar domains at the cell cortex. This mechanism of polarity establishment is distinct from the one described in Drosophila neuroblasts. In these cells, Pins is recruited via Insc by Baz to the apical cortex, and acts in a Dlg-independent manner to maintain the Baz/DmPAR-6/DaPKC/Insc complex at the apical cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell (Bellaïche, 2001).

In the dividing pI cell, Numb and Pon colocalize at the anterior pole of the lateral cortex, marked with Fasciclin3 (Fas3), below the adherins junction (AJ), marked with DE-Cadherin (Shotgun). In epithelial cells in interphase, Baz colocalizes with Shotgun at the AJ around the apical cortex. In the pI cell, Baz accumulates at the posterior cortex during mitosis. Prior to chromosome condensation, this accumulation is seen at the level of the AJ. Then, during prophase and metaphase, Baz forms a posterior crescent below AJ and opposite to Numb. At telophase, the pIIa cell inherits a higher level of Baz than its sister cell. DaPKC shows a similar distribution to Baz in the pI cell (Bellaïche, 2001).

In neuroblasts, a key function of the Baz/DaPKC/DmPAR-6 complex is to recruit the Insc and the Pins proteins. However, in the pI cell, Insc is not expressed and Pins does not colocalize with Baz at the posterior cortex. Rather, it localizes to the anterior pole in early prophase and colocalizes with Numb at the anterior lateral cortex at metaphase (Bellaïche, 2001).

Because DaPKC and Baz have a dual function in epithelial polarity and asymmetric neuroblast division, it was hypothesized that genes required for epithelial polarity might also regulate planar polarity in the pI cell. To test this hypothesis, the planar distribution of various proteins known to be distributed asymmetrically along the apical-basal axis of epithelial cells was examined. Of these, only Dlg was identified as a protein localizing asymmetrically along the planar axis in the pI cell. Dlg overlaps with Fas3 below the AJ in interphase cells. In dividing pI cells, Dlg redistributes in part along the planar a-p axis. From late prophase onward, Dlg becomes enriched at the anterior cortex, where it colocalizes with Numb and Pins. During this time, Dlg does remain detectable at the posterior lateral cortex. At telophase, a higher level of Dlg segregates into the pIIb cell. Thus, the accumulation of Dlg/Pins and Baz at opposite poles of the cell defines two complementary cortical domains oriented along the a-p planar axis of the pI cell. The position of the mitotic spindle at metaphase correlates with the localization of these two cortical domains. The posterior spindle pole is positioned near the accumulation of Baz, and the anterior spindle pole lies near the accumulation of Dlg. In both pI and epidermal cell, the mitotic spindle poles are found below the AJ, which appear to remain functional since they retain their ability to recruit Arm (Bellaïche, 2001).

To determine the possible function of Baz in the planar polarization of the pI cell, clones of baz mutant cells were studied in the notum. Loss of baz activity does not affect the localization of Shotgun and Dlg, indicating that apical-basal polarity in the notal epithelium is maintained in the absence of Baz. In the dividing pI cell, Numb either does not localize asymmetrically or forms a weak crescent at the anterior cortex at prometaphase. In contrast, Pins localizes asymmetrically at the cortex of the pI cell during division. Moreover, baz mutant pI cells divide within the plane of the epithelium with a normal a-p orientation with Pins localizing at the anterior cortex. This shows that baz is required for the asymmetric localization of Numb but is not essential to establish asymmetry nor to orient polarity along the a-p axis (Bellaïche, 2001).

The function of Pins during the asymmetric division of the pI cell was analyzed using a viable null allele of pins^Delta1-50 that does not affect epithelial cell polarity. To study the function of Dlg, two hypomorphic alleles, dlg^SW and dlg^1P20 were used that were predicted to encode truncated proteins lacking the C-terminal 14 and 43 amino acids, respectively and which do not perturb apical-basal polarity. The GUK domain of Dlg is partly deleted in the mutant Dlg^1P20 protein, but should be unaffected in the mutant Dlg^SW protein. In the pI cell, the Dlg^SW protein accumulates normally at the anterior cortex, whereas the mutant Dlg1P20 protein is cortical, but fails to accumulate anteriorly (Bellaïche, 2001).

The possible role of Dlg and Pins in regulating the position of the mitotic spindle was investigated. Spindle movements were analyzed in living pupae using Tau-GFP. It was found that the a-p orientation of the pI division does not depend on the activity of pins and is not affected in the dlg^1P20mutant. In wild-type and pins mutant pI cells, the spindle lines up with the planar polarity axis 3-4 min prior to the metaphase-anaphase transition. In contrast, the spindle often rotates throughout metaphase in dlg mutant pI cells. It is concluded that Dlg regulates the localization or the activity of factors responsible for spindle rotation (Bellaïche, 2001).

The roles of Dlg and Pins in the asymmetric localization of Numb and Pon were examined. The interphase localization of Numb at the cortex and of Pon around the nucleus does not depend on the function of the dlg or pins genes. At metaphase, however, the anterior localization of both proteins requires the activity of both dlg and pins. Thus, in pins mutant cells at prometaphase, the crescent of Numb and Pon is either not detected or weak. Nevertheless, both proteins segregate into the anterior cell at anaphase and telophase. In dlg^1P20 mutant pI cells, Numb does not accumulate at the anterior cortex and Pon remains cytoplasmic at metaphase. At telophase, Numb and Pon segregate equally into both daughter cells. These results show that Dlg and Pins are required to localize Numb and Pon at the anterior cortex in the pI cell. Consistently, nonsensory cells are transformed into neurons leading to a bristle loss phenotype in adult flies. Furthermore, the genetic interaction seen between dlg^sw and pins suggests that dlg and pins act in the same process to specify the fate of the pI daughter cells (Bellaïche, 2001).

Pins colocalizes with the anterior accumulation of Dlg and dlg and pins mutations genetically interact. This raises the possibility that the two proteins interact directly. Indeed, in a yeast two-hybrid screen using full-length Dlg as bait, one Pins clone (encoding amino-acid residues 235 to 658) was isolated. To further test for a direct interaction between Dlg and Pins and to identify the Pins interaction domain of Dlg, blot overlay experiments were performed using GST-fusion proteins. A biotinylated Pins protein has been found to interact with the SH3 domain but not with the PDZ1, PDZ2, PDZ3, HOOK, or GUK domains of Dlg. The Dlg-Pins complex is also detected in brains and imaginal discs by coimmunoprecipitation experiments. This interaction is abolished by a single amino-acid substitution (L556P) in the SH3 domain, which does not noticeably affect Dlg stability in dlg^m30 mutant larvae but does result in disc overgrowth and late larval lethality (Bellaïche, 2001).

Consistent with this direct interaction, Pins and Dlg are mutually dependent for their accumulation at the anterior cortex. A very weak crescent of Pins is seen at the anterior cortex in dlg^1P20 mutant pI cells at metaphase, suggesting that the GUK domain might facilitate the interaction between the SH3 domain of Dlg and Pins. Conversely, Dlg does not become enriched at the anterior cortex of pins mutant pI cells at metaphase. It is concluded that Dlg directly interacts with Pins via its SH3 domain, and that this interaction is important for the anterior accumulation of both Dlg and Pins (Bellaïche, 2001).

The role of Pins and Dlg in localizing Baz asymmetrically was examined. In pins mutant pI cells, Baz accumulates at the posterior cortex at metaphase, but the asymmetry is less pronounced than in wild-type cells. This raises the possibility that Pins participates in the asymmetric localization of Baz. In dlg^1P20 mutant pupae, Baz is correctly localized to the apical posterior cortex prior to chromosome condensation, but does not form a cortical crescent below the AJ during late prophase and prometaphase. Instead, Baz accumulates in the cytoplasm and remains cortical only at the level of the AJ. Thus, the initial posterior localization of Baz at the level of the AJ does not depend on the activity of the GUK domain of Dlg, but its cortical localization below the AJ does require dlg activity. It is concluded that planar polarization of the pI cell cannot be maintained without Dlg activity (Bellaïche, 2001).

To test whether the initial Dlg-independent localization of Baz at the posterior cortex depends on Fz signaling, the distribution of Baz was studied in fz mutant pupae. In wild-type pupae, a clear accumulation of Baz is seen at the level of the AJ in 61% of the interphase pI cells. By contrast, an asymmetric distribution of Baz at the apical cortex is detected in only 19% of the interphase pI cells in fz mutant pupae. In the remaining 81% of the cells, the asymmetric accumulation of Baz is either weak or similar to that seen in the surrounding epithelial cells. This indicates that Fz signaling regulates the initiation of the asymmetric localization of Baz at the posterior cortex. At metaphase, however, Baz and Pins form misoriented crescents relative to the a-p axis that localize at opposite poles in fz mutant pI cells. It is concluded that the formation of the two opposite Baz and Pins domains does not depend on fz activity, and that planar asymmetry can be established in the absence of Fz signaling. However, as previously seen for pins, the asymmetric distribution of Baz is less pronounced in fz mutant pI cells than in wild-type cells. Moreover, Dlg is distributed around the entire cell cortex, indicating that Fz signaling is required for the anterior accumulation of Dlg (Bellaïche, 2001).

Since Pins localizes asymmetrically in a Fz-independent manner, it was asked whether Pins is necessary to localize Baz at one pole of the pI cell in the absence of Fz. It was found that Baz localizes uniformly around the basal-lateral cortex in 82% of the fz;pins double mutant pI cells at metaphase. Moreover, although Numb forms a crescent at anaphase in pI cells mutant for pins or fz, no Numb crescent is seen at either metaphase or anaphase in fz;pins double mutant pI cells. Consistently, loss of fz activity enhances the pins bristle loss phenotype. These data show that Pins and Fz act in a redundant manner to exclude Baz from the anterior cortex and to establish planar asymmetry in the pI cell (Bellaïche, 2001).

These results show that Pins localizes to the anterior cortex in a Baz-independent manner, in an orientation opposite that of Baz, as does Numb. Pins cooperates with Fz to exclude Baz from the anterior cortex of the pI cell. In contrast, in neuroblasts, Pins localizes in a Baz-dependent manner to the apical pole, opposite Numb, and stabilizes the Insc/Baz/DmPAR-6/DaPKC complex. Nevertheless, Pins promotes the localization of Numb in both cell types (Bellaïche, 2001).

One important difference between pI cells and neuroblasts is the lack of insc expression in pI cells. To test the functional significance of this lack of Insc, Insc was expressed in the pI cell. Under these circumstances, Insc and Pins localize at the anterior cortex. Insc triggers the anterior relocalization of Baz, while Numb forms a posterior crescent at anaphase. The pI cell division remains planar. This contrasts with the effect of Insc in epithelial cells. In these cells, Insc localizes apically and orients the spindle along the apical-basal axis. This further indicates that the apical-basal polarity is remodeled in the pI cell. It is concluded that the ectopic expression of Insc is sufficient to reverse the planar polarity axis of the pI cell and to modify the activity of Pins relative to Baz. In the absence of Insc, the Dlg/Pins complex excludes Baz, while expression of Insc leads to the formation of a Pins/Insc/Baz complex. In both cases, Numb localization is opposite that of Baz (Bellaïche, 2001).

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (Dlg), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (Fas2) and the position specific (PS) integrin receptors, including ßPS (Myospheroid) and its alpha partners (alphaPS1, alphaPS2, PS3/alpha Volado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that Dlg and Fas2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. ßPS integrin physically associates with the synaptic complex anchored by the Dlg scaffolding protein, which contains CaMKII and Fas2. This study demonstrates an alteration of the Fas2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory ßPS integrin mutations increase the expression and synaptic localization of Fas2. Synaptic structural defects in ßPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of Fas2 (distal in pathway). These studies demonstrate that ßPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology (Beumer, 2002).

ßPS integrin is a transmembrane receptor present in both pre- and post-synaptic membranes at the larval NMJ. Dlg is a synaptic scaffolding protein associated with both pre-and post-synaptic membranes and is also involved in the regulation of synaptic morphology, through the localization of diverse transmembrane proteins (including Fas2). Studies were undertaken to determine whether ßPS integrin associates with the DLG complex to tie together these disparate receptor components in a common molecular machine. In confocal analyses, ßPS integrin and Dlg co-localize at the larval NMJ. Dlg clearly has less extensive expression, more tightly localized at NMJ boutons, whereas ßPS is more extensive through the subsynaptic reticulum (SSR) and also localized at extrasynaptic sites in the muscle, including the muscle sarcomere and attachment sites. However, both proteins are most intensively expressed at the NMJ presynaptic/postsynaptic interface, where they co-localize. Therefore, tests were performed to determine if ßPS integrins form part of the synaptic complex linked by Dlg. Protein immunoprecipitation assays were performed using rabbit anti-DLG to probe Oregon R head extracts. Dlg antibodies clearly co-immunoprecipitate Dlg and ßPS integrin protein, consistent with co-localization observed in confocal analysis. Inspection of the ratio of both bound proteins and proteins not bound to beads (immunoprecipitation versus flow through lanes) indicates that a large portion of the ßPS integrin protein associates with a complex containing Dlg. The fact that ßPS was found to co-immunoprecipitate with the complex mediated by Dlg provides support for integrins existing in a synaptic complex with Fas2 and CaMKII at the synapse (Beumer, 2002).

Potential role of Dlg in Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell

Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Gαi complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. It is proposed that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity (Bellaïche, 2004).

Planar polarization of the pI cell occurs prior to division and is required, upon entry into mitosis, to direct the Dlg-Pins-Galphai and Baz-Par6-aPKC complexes at the anterior and posterior cortex, respectively. The localization of Pins at the anterior cortex is regulated positively by the Stbm-Pk complex and negatively by Dsh. (1) Loss of stbm activity results in a delay in the cortical localization of Pins during prophase; (2) concomitant expression of Stbm and Pk leads to a broadening of the cortical crescent of Pins at prophase; (3) loss of dsh PCP activity similarly results in an extended Pins crescent at prophase. Moreover, analysis of the defective partitioning of Pon::GFP suggests that the Stbm-Pk complex acts antagonistically to Dsh to localize at the anterior cortex a centrosome-attracting activity. It is proposed that the Stbm-Pk complex organizes the anterior cortex and recruits the Dlg-Pins-Galphai complex as well as molecules regulating spindle positioning (Bellaïche, 2004).

Cortical localization of Pins is a novel read-out of PCP signaling in the pI cell that is distinct from the ones previously identified in wing and eye cells. In wing epidermal cells, Fz promotes the formation of a polarized actin cytoskeleton via a pathway that possibly involves a direct interaction between Dsh and a Daam1-Rho complex and a Rho Kinase-dependent phosphorylation of cytoplasmic myosin. Whether Dsh also regulates microfilament assembly in pI cells remains to be studied. In photoreceptor cells, the read-out for PCP signaling is the transcriptional regulation of the Delta gene in R3. Thus, the conserved core of PCP signaling molecules have different, cell-type specific read-outs (Bellaïche, 2004).

How does Stbm direct the localization of Pins to the anterior cortex? One hypothesis is that Stbm directs the anterior localization of Pins via the regulated assembly of a Stbm-Dlg-Pins complex. The anterior accumulation of Pins depends on its interaction with Dlg. Evidence is provided that Stbm may bind Dlg. (1) In vitro binding studies indicate that Stbm interacts with Dlg. It is noted, however, that PDZ-containing proteins other than Dlg may also bind Stbm in this assay. (2) The localization of Stbm overlaps with the distribution of Pins and Dlg in dividing pI cells. (3) The PBM motif of Stbm appears to regulate the re-localization of Stbm in pI cells. The data are therefore consistent with a model in which, upon mitosis, the binding of Stbm to Dlg in turn promotes the binding of Pins to Dlg and, hence, localization of Pins at the anterior cortex where Stbm and Dlg accumulations overlap. This model predicts that the PBM of Stbm should be required for the anterior localization of Pins. It was found, however, that StbmDeltaPBM is fully functional and that Pins is properly recruited at the anterior cortex in stbm^6c mutant pI cells expressing StbmDeltaPBM. One interpretation of this result is that Stbm regulates the localization of Pins not only via the PBM-dependent assembly of the Dlg-Pins complex but also via a second PBM-independent mechanism. Since Dsh acts redundantly with Baz to localize Pins asymmetrically, it is suggested that this second mechanism may involve Dsh. Accordingly, in stbm^6c mutant pI cells, uniformly distributed Dsh activity would prevent Pins cortical localization. By contrast, since the PCP function of stbm does not depend on its PBM, the activity of Dsh should be restricted to the posterior cortex in stbm^6c mutant pI cells expressing StbmDeltaPBM. Dsh should therefore restrict Pins localization to the anterior cortex in this mutant background. Another interpretation of the correct localization of Pins in stbm^6c mutant pI cells expressing StbmDeltaPBM is that Stbm recruits Pins via a mechanism that does not involve an interaction with Dlg (or any other PDZ-containing proteins). Future studies will address how the Stbm-Pk complex regulates the localization of Pins in the pI cell (Bellaïche, 2004).

Different mechanisms appear to cooperate to maintain Pins asymmetric localization. baz is required for the asymmetric localization of Pins in the absence of dsh PCP activity. This indicates that Baz can regulate the maintenance of Pins asymmetric localization at prometaphase. The loss of asymmetric localization of Pins in dsh baz mutant pI cells suggests that Dsh may also contribute to maintain Pins asymmetric localization at prometaphase. Dsh does not merely act by excluding Stbm, a positive regulator of Pins localization in prophase, because Pins localizes asymmetrically in baz stbm double mutant pI cells. The mechanisms by which Baz and Dsh regulates Pins localization are not known. However, because Pins regulates its own localization via a Gß13F-dependent positive feedback loop, one hypothesis is that Baz and/or Dsh negatively regulates Gß13F signaling activity (Bellaïche, 2004).

One of the best examples of PCP in mammals is the stereotyped planar orientation of the stereociliary bundles that are located at the apical cortex of each mechanosensory hair cell within the cochlea. In these cells, the first sign of polarization is the stereotyped movement, at the luminal surface of the cell and along the neural-abneural axis, of the kinocilium, the single tubulin-based cilium, from the center towards the abneural pole of the cell. Recently, a mutation in a stbm homolog, Vangl2, has been shown to result in the defective orientation of the stereociliary bundles. This planar cell polarity defect appears to result from the randomly oriented center-to-periphery movement of the kinocilium. Because LGN, a mammalian homolog of Pins, is known to regulate microtubule stability, it is tempting to speculate that Vangl2 may regulate via LGN a microtubule-dependent process regulating kinocilium movement along the neural-abneural axis. Future studies will reveal whether the regulation of Pins/LGN cortical localization is a conserved read-out of PCP (Bellaïche, 2004).

Targeting Dlg to the NMJ

Mutations in rho-type guanine exchange factor (rt/GEF), also called dpix, were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dPix/rtGEF is homologous to mammalian Pix. dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations lead to decreased synaptic levels of the PDZ protein Discs large, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Direct interaction of mammalian Pix with Pak has been detected. Thus, a Rho-type GEF (Pix) and Rho-type effector kinase (Pak) regulate postsynaptic structure (Parnas, 2001).

In mammals, the Pix family contains two members: alphaPix (Cool-2) and ßPix (Cool-1). Pix has an SH3 domain, a DBL-homology GEF domain, and a pleckstrin homology domain. The Cool (for cloned-out of library)/Pix (for PAK-interactive exchange factor) proteins directly bind to members of the PAK family of serine/threonine kinases and regulate their activity. In Drosophila, dPix is localized to the PSD: dpix mutations lead to the loss of synaptic Pak kinase. Paks are a family of Cdc42/Rac1-activated serine/threonine kinases important in regulating actin-containing structures. In the fly NMJ, Pak kinase is localized to the PSD. In mammals, Pak is recruited to focal complexes in a Cdc42-, Rac1-, and Pix- dependent manner (Parnas, 2001).

Since the dpix mutation eliminates Pak kinase from the synapse, and since Pak is a downstream target of the Rac/Cdc42 pathway, which includes Pix, it was reasoned that the elimination of Pak kinase from the synapse may be responsible for the inefficient clustering of Dlg. Thus, dpak mutants were stained with antibodies for Dlg and dPix. Several combinations of dpak alleles were used. dpak¹¹ is a protein null, and dpak⁶ has a stop codon at position 113; however, there is still some synaptic expression in this allele. dpak⁴ has a missense mutation in the Dock binding domain, and Pak kinase protein levels are normal. Finally, dpak⁷ has not been characterized molecularly, but behaves genetically as a null allele. In all allelic combinations of dpak mutants, dPix levels and localization are normal. In the allelic combination dpak¹¹/dpak⁴, Dlg levels at the synapse are also normal. However, in dpak¹¹/dpak⁶, Dlg levels are somewhat lower than wild-type (reduction of 57%), and Pak kinase levels are reduced by 66%. In dpak¹¹/dpak⁷, Dlg levels are reduced to the same extent as in dpix mutants (75%), and Pak kinase is absent. Fas II levels are also reduced to the same extent as in dpix mutants (a reduction of 19.3%). Levels of GluRIIA are also reduced, although less than levels in dpix mutants (reduction of 56%). These results are consistent with Pak kinase acting as a downstream effector of dPix. Nevertheless, there are differences between dpak and dpix mutants. In dpix mutants, the synapse looks abnormal and irregular; whereas in dpak mutants, even when Dlg levels are lowered, the synapse looks normal. Also, in dpak mutants, the muscles are thin and degenerated, and the muscle nuclei are mislocalized. In dpix larvae, the muscle activity appears weaker (as assessed by larval motility) than in wild-type larvae, but they are not as affected as in dpak mutants, and muscle nuclei are localized normally. It should be noted that the ultrastructure of dpix and dpak muscles are completely normal, and muscle differentiation per se does not seem to be affected. No structural correlate could be found that would explain the weaker muscles of dpix larvae (Parnas, 2001).

dpix mutations lead to the decrease in synaptic levels of the PDZ protein Discs-large (Dlg), the cell adhesion molecule Fasciclin II (Fas II), the glutamate receptor (GluR) subunit GluRIIA, and to the elimination of the subsynaptic reticulum (SSR). In Drosophila, the PSD-95 homolog Dlg has been shown to be directly responsible for the clustering of the Shaker potassium channel and to partially control the clustering of the cell adhesion molecule Fas II to the NMJ. Many, but not all, dpix defects are mediated through Pak kinase. Thus, the data suggest a pathway for synaptic clustering from dPix to Pak kinase to Dlg to Shaker and to Fas II (Parnas, 2001).

The dpix phenotype is consistent with at least two functions at the postsynaptic compartment: targeting and stabilization of postsynaptic components. In dpix mutants, Pak kinase is completely missing from the synapse. Since Pix is known to directly interact with Pak in mammals and target it to focal complexes, the data best fit with the model in which dPix targets Pak kinase to the synapse via a direct interaction. Furthermore, overexpressing either Pak kinase or a membrane-tethered gain-of-function form of Pak kinase does not result in any accumulation of Pak kinase at the synapse. Still, it is possible that Pak kinase is targeted to the synapse via a different mechanism and fails to stabilize in dpix mutants (Parnas, 2001).

In contrast to Pak kinase, Dlg and GluRIIA are not completely eliminated from the synapse in dpix mutants, but rather, their levels are reduced. In the case of Dlg, its localization pattern is also disrupted, indicating that dPix controls the postsynaptic targeting of Dlg at least to some extent, as well as its stabilization at the synapse. The localization pattern of GluRIIA (in subsynaptic domains opposite active zones) is intact. Thus, dPix is not necessary for the synaptic targeting of GluRIIA per se, but rather, it is important for maintenance of its levels and/or stabilization (Parnas, 2001).

Discs large functions in the asymmetric distribution of Neuralized

In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). The E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. It is proposed that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling (Le Borgne, 2003).

The mechanisms by which Neur localized at the anterior cortex of the dividing pI cell were investigated. The role of the cytoskeleton was studied by applying drugs to cultured nota. Colcemid, a microtubule-depolymerizing agent, was found to have no significant effect. In contrast, both Latrunculin A, an agent that depolymerizes actin microfilaments, and the myosin motor inhibitor butanedione-2-monoxime (BDM) strongly impaired or completely inhibited the asymmetric localization of Neur. Thus, both myosin motor activity and an intact actin cytoskeleton are required for the formation and/or maintenance of the Neur crescent at the anterior cortex of the dividing pI cell. These requirements for Neur localization are similar to the ones seen earlier for Numb and Pon. Neur also behaves in a manner similar to Numb and Pon in that localization of Neur at the anterior cortex of the pI cell depends on planar polarity genes and on the polarity genes discs-large and pins. Moreover, mispartitioning of Neur in dlg and pins mutant cells correlates with a loss in asymmetric internalization of Dl. These data indicate that Neur and Numb share part of the same molecular machinery to localize asymmetrically in the pI cell (Le Borgne, 2003).

Microtubule-induced Pins/Gαi cortical polarity in Drosophila neuroblasts; Interaction of Dlg with Khc-73

Cortical polarity regulates cell division, migration, and differentiation. Microtubules induce cortical polarity in yeast, but few examples are known in metazoans. Astral microtubules, kinesin Khc-73, and Discs large (Dlg) induce cortical polarization of Pins/Gαi in Drosophila neuroblasts; this cortical domain is functional for generating spindle asymmetry, daughter-cell-size asymmetry, and distinct sibling fates. Khc-73 localizes to astral microtubule plus ends, and Dlg/Khc-73 and Dlg/Pins coimmunoprecipitate, suggesting that microtubules induce Pins/Gαi cortical polarity through Dlg/Khc-73 interactions. The microtubule/Khc-73/Dlg pathway acts in parallel to the well-characterized Inscuteable/Par pathway, but each provides unique spatial and temporal information: The Inscuteable/Par pathway initiates at prophase to coordinate neuroblast cortical polarity with CNS tissue polarity, whereas the microtubule/Khc-73/Dlg pathway functions at metaphase to coordinate neuroblast cortical polarity with the mitotic spindle axis. These results identify a role for microtubules in polarizing the neuroblast cortex, a fundamental step for generating cell diversity through asymmetric cell division (Siegrist, 2005).

A current model for the establishment of neuroblast cortical polarity is that an unknown cue recruits Baz, aPKC, Par-6, and Insc to the apical cortex of the neuroblast just prior to prophase, which is closely followed by the apical recruitment of Pins/Gαi proteins, presumably via Insc-Pins direct interactions. This is termed the cortical “Insc/Par pathway” of Pins/Gαi localization to distinguish it from the Insc-independent “microtubule-based pathway” of Pins/Gαi localization that is the focus of this paper (Siegrist, 2005).

insc²² null mutant embryos (insc mutants) lack apical localization of the Insc/Par complex proteins (Insc, Baz, aPKC, and Par-6), but interestingly that Pins, Gαi, and Dlg still form robust crescents in the majority of insc mutant metaphase neuroblasts. Similar results were observed in mitotic neuroblasts from embryos homozygous for the TE35 deficiency in which insc is not transcribed. Although Pins/Gαi/Dlg crescents form in insc mutants, the timing and position of crescent formation differed from wild-type. First, in wild-type neuroblasts Pins/Gαi/Dlg crescents always formed at the apical surface adjacent to the overlying ectoderm, whereas in insc mutant neuroblasts Pins/Gαi/Dlg crescents were found at all positions around the cortex. Second, in wild-type neuroblasts Pins/Gαi crescents formed by early prophase (94%), whereas in insc mutants Pins/Gαi crescents were not detected at prophase but only at metaphase (78%). These results suggest that there is an Insc/Par-independent pathway that is active at metaphase to induce formation of Pins/Gαi/Dlg cortical crescents (Siegrist, 2005).

A clue to the identity of the Insc/Par-independent pathway was the observation that Pins/Gαi/Dlg crescents were always colocalized over one spindle pole, which can be mispositioned relative to the overlying ectoderm in insc mutants. This observation suggested that either spindle microtubules induced cortical polarity, or cortical polarity formed spontaneously at a nonapical position and induced spindle alignment. To distinguish between these mechanisms, microtubules were depolymerized in insc mutant neuroblasts with Colcemid, and Pins/Gαi/Dlg cortical crescents were scored. Colcemid treatment of insc mutant neuroblasts resulted in a nearly complete loss of Pins/Gαi/Dlg crescents: Pins is mostly cytoplasmic and Gαi/Dlg are uniform cortical. In contrast, Colcemid treatment of wild-type neuroblasts had no effect on Pins/Gαi/Dlg crescent formation, likely due to the association of Pins/Gαi/Dlg with the apical Insc/Par complex. In fact, the Insc/Par pathway of Pins/Gαi/Dlg localization requires only Insc and Baz proteins, because aPKC mutants that lack aPKC/Par-6 protein localization but retain Baz/Insc localization still formed Pins/Gαi/Dlg crescents in the absence of microtubules. It is concluded that spindle microtubules have the ability to induce Pins/Gαi/Dlg cortical crescents over one spindle pole in the absence of an Insc/Par pathway (Siegrist, 2005).

Recent work has shown that microtubules can directly regulate cortical polarity in yeast during C. elegans meiosis and in migrating cells. An important question is the extent to which microtubules regulate cortical cell polarity in other contexts. This study identifies a microtubule/kinesin pathway for inducing cortical polarity in Drosophila neuroblasts. This pathway is sufficient to induce cortical polarization of the evolutionarily conserved Dlg, Pins, and Gαi proteins and is necessary for reliable spindle orientation relative to apical Insc/Par cortical proteins (Siegrist, 2005).

A model is presented for the microtubule/Khc-73/Dlg pathway, in the absence of the Insc/Par function.

• (1) At prophase, the microtubule/Khc-73/Dlg pathway is unable to polarize. Dlg/Gαi are uniform cortical, and Pins is predominantly cytoplasmic. It is not clear when Khc-73 is localized to microtubule plus ends because Taxol-treated neuroblasts metaphase arrest (Siegrist, 2005).
• (2) At metaphase, Khc-73 is localized at microtubule plus ends where it can contact cortical Dlg protein. It is proposed that Khc-73 first contacts Dlg at the cortex because no Dlg or Dlg:eGFP is detected on astral microtubules or colocalized with Khc-73 at microtubule plus ends. Association of Khc-73 with microtubule plus ends may be mediated by its CAP-Gly domain, similar to the Clip-170 and APC plus-end binding proteins (reviewed in Galjart, 2003). The Khc-73/Dlg interaction could occur between the Khc-73 MBS stalk domain and the Dlg GK domain because the related GAKIN/hDlg domains directly interact (Asaba, 2003; Hanada, 2000). While Dlg can be precipitated with the Khc-73 MBS domain from embryonic lysate, these direct protein–protein interactions between Khc-73 and Dlg were not detected in vitro, suggesting that this interaction may be highly regulated (Siegrist, 2005).
• (3) Dlg clustering occurs over one spindle pole, although low levels persist around the cortex. An attractive model for Dlg clustering is that Khc-73/Dlg interaction blocks Dlg SH3-GK intramolecular interactions to favor Dlg intermolecular oligomerization. In support of this model, Dlg:eGFP can be expressed specifically in neuroblasts and an anti-GFP antibody can be used to immunoprecipitate endogenous Dlg proteins. It is not clear why clustering occurs over just one spindle pole; perhaps the spindle poles are intrinsically different, or perhaps cortical heterogeneity (e.g., residual Par proteins) favors crescent formation over one spindle pole (Siegrist, 2005).
• (4) Pins/Gαi cortical clustering occurs. Pins/Gαi clustering requires Dlg and may be mediated by direct Dlg/Pins interactions and Pins/Gαi interactions. However, the Dlg/Pins interaction must be highly regulated or indirect, since it has been possible to coimmunoprecipitate Dlg/Pins from in vivo lysates but no binding was seen by in vitro assays (Siegrist, 2005).
• (5) Khc-73/Dlg/Pins/Gαi signals to the mitotic spindle. Loss of any of these proteins results in spindle orientation and varying morphology defects, even in the presence of the Insc/Par pathway. It is not clear which protein most directly mediates the cortex to microtubule signal, but it is intriguing to note that the Pins ortholog LGN can bind the microtubule-associated protein NuMA and regulate spindle biology in mammals. Gα subunits can bind tubulin to regulate microtubule dynamics, and finally Khc-73/Dlg microtubule binding could also directly regulate spindle behavior (Siegrist, 2005).

Asymmetric localization of Pins/Gαi proteins can be induced by two distinct pathways in embryonic neuroblasts: a well-characterized cortical pathway involving the Insc/Par proteins and a microtubule-dependent Khc-73/Dlg pathway. Each pathway is regulated differently and has unique features that provide different temporal and spatial information for generating cortical polarity (Siegrist, 2005).

First, each pathway is initiated by a different mechanism and provides unique information for the timing of Pins/Gαi polarization. The Insc/Par pathway is initiated at late interphase in response to an unknown extrinsic cue and is required for the early prophase cortical polarization of Pins/Gαi. In contrast, the Khc-73/Dlg pathway is initiated later at prometaphase/metaphase by astral microtubules and is required for cortical polarization of Pins/Gαi only in the absence of Insc/Par complex proteins. Consistent with this timeline, asymmetric enrichment of Dlg normally occurs well after polarization of Insc/Par/Pins/Gαi during the prometaphase/metaphase transition, and this temporal progression of Dlg cortical enrichment is not affected in insc mutants. The temporal polarization of Dlg coincides precisely with the onset of Pins/Gαi cortical polarity at prometaphase/metaphase that occurs in the absence of the Insc/Par pathway (Siegrist, 2005).

Next, each pathway provides different spatial information for the cortical polarization of Pins/Gαi. The Insc/Par pathway recruits Pins/Gαi to the apical cortex of the neuroblast at a position just below the overlaying epithelium, thus coordinating neuroblast cortical polarity with the neuroblast environment. In the absence of this pathway (e.g., insc mutant neuroblasts), cortical polarity can be generated but is not linked to tissue polarity, resulting in mispositioning of neuroblast progeny. In contrast, the microtubule/Khc-73/Dlg pathway induces Pins/Gαi crescent formation over one spindle pole, thus coordinating the neuroblast cortical polarity with the spindle axis. In the absence of this pathway (e.g., dlg mutant or Khc-73 RNAi neuroblasts), Insc/Baz can still recruit Pins/Gαi to the apical cortex, yet the spindle is not always properly aligned with this cortical polarity. Together these two pathways ensure the correct temporal and spatial positioning of apical complex proteins relative to extrinsic and intrinsic landmarks (Siegrist, 2005).

Drosophila sense organ precursors (SOPs) divide asymmetrically to generate an anterior pIIb cell and a posterior pIIa cell. During this division, Pins, Gαi, Dlg, and Numb form cortical crescents over the anterior spindle pole, and Baz localizes over the posterior spindle pole. Cell division orientation is fixed along the anterior/posterior axis by planar polarity cues mediated by the seven pass transmembrane receptor Frizzled. However, Frizzled signaling is required only for the position of Dlg/Pins crescents, not for their formation. When both frizzled and microtubules were remove together, it was found about 10% of the mitotic SOPs lack Pins crescents. This mild phenotype suggests that while astral microtubules may contribute to Dlg/Pins polarization in SOPs, there must be an additional mechanism involved. The best candidates for this third mechanism are the Par proteins because Par crescents still form in frizzled mutant SOPs at metaphase (Siegrist, 2005).

There are many similarities between asymmetric division of fly neuroblasts and the C. elegans zygote, but there are also striking differences. One of the most noteworthy differences is that C. elegans par mutants undergo symmetrically sized embryonic cell divisions, whereas in Drosophila, par or insc mutants maintain sibling cell size asymmetry. This work provides an explanation for this discrepancy. It is shown that astral microtubules are capable of generating Pins/Gαi cortical polarity in the absence of localized Par proteins and that this microtubule-induced Pins/Gαi cortical polarity is fully functional for generating an asymmetric spindle, cell size, and unique daughter cell fates. It is likely that C. elegans lacks this “microtubule-based pathway” for inducing GPR1/2 (Pins) and Gα cortical polarity, at least during the first embryonic cell division, because posterior cortical localization of GPR1/2 is absent in par mutants and the daughter cells are equal in size. Interestingly, an increase is observed in symmetrically dividing neuroblasts in neuroblasts lacking both Insc/Par and microtubule pathways, compared to loss of single pathways alone. It appears that either the Insc/Par or microtubule/Khc-73/Dlg pathway is sufficient to induce Pins/Gαi cortical polarity, which generates daughter cells of different sizes and fates (Siegrist, 2005).

The microtubule/kinesin-induced Dlg clustering pathway described in this study may be evolutionarily conserved. In mammals, hDlg and the Khc-73 ortholog GAKIN are coexpressed in T cells and coimmunoprecipitate, and T cell activation leads to recruitment of hDlg to the immunological synapse (Hanada, 2000). Interestingly, GAKIN targets hDlg into ectopic cellular projections in MDCK cells, and this targeting depends on microtubules (Asaba, 2003). This has lead to the hypothesis that GAKIN may use a microtubule-based mechanism to target hDlg to the T cell immune synapse, similar to the microtubule/Khc-73 pathway described in this paper (Siegrist, 2005).

PAR-1 kinase phosphorylates Dlg and regulates its postsynaptic targeting at the Drosophila neuromuscular junction

Targeting of synaptic molecules to their proper location is essential for synaptic differentiation and plasticity. PSD-95/Dlg proteins have been established as key components of the postsynapse. However, the molecular mechanisms regulating the synaptic targeting, assembly, and disassembly of PSD-95/Dlg are not well understood. This study shows that PAR-1 kinase, a conserved cell polarity regulator, is critically involved in controlling the postsynaptic localization of Dlg. PAR-1 is prominently localized at the Drosophila neuromuscular junction (NMJ). Loss of PAR-1 function leads to increased synapse formation and synaptic transmission, whereas overexpression of PAR-1 has the opposite effects. PAR-1 directly phosphorylates Dlg at a conserved site and negatively regulates its mobility and targeting to the postsynapse. The ability of a nonphosphorylatable Dlg to largely rescue PAR-1-induced synaptic defects supports the idea that Dlg is a major synaptic substrate of PAR-1. Control of Dlg synaptic targeting by PAR-1-mediated phosphorylation thus constitutes a critical event in synaptogenesis (Zhang, 2007).

Rearrangement of synaptic protein composition and structure is a fundamental mechanism governing synaptic plasticity. As organizers of the postsynapse, PSD-95/Dlg proteins have been intensively studied as substrates mediating synaptic plasticity. The signaling pathways that couple internal or external cues to the localization and function of PSD-95/Dlg are not well defined. This study has found that PAR-1 kinase plays a critical role in regulating the postsynaptic targeting of Dlg at the Drosophila NMJ. PAR-1 does so by phosphorylating Dlg at a Ser residue in the GUK domain. The conservation of this Ser residue in all members of the MAGUK proteins suggests that this phosphorylation event may represent a general mechanism by which the MAGUK proteins are regulated. This is the first time the PAR-1 family of Ser/Thr kinase has been shown to play an important role in synaptic development and function (Zhang, 2007).

PAR-1 directly phosphorylates Dlg, and overactivation of PAR-1 disrupts Dlg's postsynaptic targeting. The physiological function of PAR-1 in regulating Dlg synaptic targeting is supported by loss-of-function analysis, which indicates that phosphorylation by PAR-1 negatively regulates Dlg synaptic targeting. Consistent with this, in vivo FRAP analysis shows that the nonphosphorylatable DlgSA-GFP recovers much faster than DlgWT-GFP, and that the recovery of DlgWT-GFP is facilitated by PAR-1 loss-of-function, but impeded by PAR-1 overexpression. At first glance, it may seem somewhat counterintuitive that DlgSA-GFP, which accumulates to a greater degree at the synapse than DlgWT-GFP does, is replaced more quickly and to a greater extent that DlgWT-GFP. Since FRAP analysis suggested that the recovered Dlg comes primarily from Dlg protein reserved or newly synthesized in the muscle cytoplasm rather than from diffusion of Dlg protein from the neighboring synapses, the most likely explanation is that PAR-1-mediated phosphorylation regulates the transport of Dlg from the extrasynaptic to the synaptic regions. DlgSA-GFP may be transported more efficiently from the extrasynaptic region to the postsynapse. Upon reaching the postsynapse, DlgSA-GFP may also associate with the synaptic membrane more tightly (Zhang, 2007).

Previous studies have demonstrated that the GUK domain, in which the S797 residue is located, plays an important role in the trafficking and synaptic targeting of Dlg. The importance of the GUK domain in mediating Dlg function is also highlighted by the fact that many of the identified dlg mutations are clustered in this domain. Two types of protein-protein interactions involving the GUK domain have been detected: (1) intramolecular interaction with the SH3 domain, and (2) protein-protein interactions with GUK binding proteins, including an MT binding protein and a kinesin motor. Since MT and MT-based motor proteins provide a major driving force for protein and mRNA trafficking, it is possible that PAR-1-mediated phosphorylation may regulate Dlg interaction with the MT-based transport system (Zhang, 2007).

Morphological and electrophysiological rescue experiments strongly support that Dlg is a critical downstream target through which PAR-1 impacts synapse differentiation and function. However, the rescue of PAR-1 overexpression-induced defects by DlgSA-GFP is not complete, raising the possibility that other synaptic substrates are affected by PAR-1. It is also possible that some of the PAR-1 overexpression phenotypes are neomorphic. A possible neomorphic effect caused by the synaptic upregulation of a kinase has recently been described. None of the known postsynaptic markers, such as CaMKII, FasII, or GluRIIA has the KXGS motif, suggesting that they may not be PAR-1 targets. In other developmental contexts, PAR-1/MARK kinases phosphorylate a number of substrates. Whether any of these PAR-1 substrates function at the synapse awaits further investigation. The existence of other synaptic targets of PAR-1 could also explain why it was not possilbe to effectively rescue par-1 mutant phenotypes with the Dlg-GFP variants, although there are other possible explanations for this result. For example, phosphorylated Dlg may possess certain biological activity that cannot be provided by DlgSD-GFP. Even if some of the phenotypes caused by altered PAR-1 activities are mediated by other substrates, several lines of evidence indicate that the mislocalization of Dlg is a primary effect of PAR-1 phosphorylation of Dlg, rather than a secondary consequence of synaptic damages caused by PAR-1 action on some unknown target(s): (1) the phospho-mimetic DlgSD-GFP is mislocalized in a wild-type background, in the presence of normal synaptic structures; (2) another postsynaptic marker, GluRIIA, retains its predominant postsynaptic localization in a PAR-1 overexpression situation; (3) the subsynaptic reticulum loss and synaptic transmission defects caused by PAR-1 overexpression using DlgSA-GFP could largely be rescued; (4) in a condition where postsynaptic structure was maintained with exogenous DlgSA-GFP, endogenous Dlg was still mislocalized in the presence of overexpressed PAR-1 (Zhang, 2007).

Recent studies suggest that posttranslational modification plays a role in regulating the trafficking of PSD-95/Dlg. In mammalian central synapses, N-terminal palmitoylation is critical for the intracellular sorting, postsynaptic targeting, and surface expression of PSD-95. Cyclin-dependent kinase 5 (Cdk5) phosphorylates the N-terminal region of PSD-95, inhibiting its oligomerization, channel clustering activity, and possibly, synaptic localization. This study establishes PAR-1-mediated phosphorylation at the C-terminal GUK domain as a regulatory mechanism in the synaptic targeting of Dlg. In addition, two independent studies have been conducted to study the function of CaMKII at the Drosophila NMJ. However, divergent results were obtained on the effect of CaMKII on synaptic development and function, and it appears that further studies are needed to clarify the function of CaMKII at the Drosophila NMJ. Future studies of upstream signaling events in the regulation of PAR-1 at the synapse, especially those which potentially regulate the PAR-1-Dlg phosphorylation cascade, will provide new insights on molecular mechanisms that regulate synaptic differentiation and plasticity (Zhang, 2007).

It is interesting to note that in addition to postsynaptic defects, altering PAR-1 activity leads to profound defects in presynaptic development and function. This indicates that PAR-1 regulates the coordinated maturation of pre- and postsynaptic structures. PAR-1 could regulate the adhesion between the pre- and postsynaptic membranes or trans-synaptic signaling. Intriguingly, a previous study has revealed a presynaptic localization and function for Dlg in regulating neurotransmission. Since a fraction of PAR-1 is localized at the presynapse, it raises the possibility that PAR-1 may also play a role there. Further studies are needed to test whether PAR-1 may act through Dlg or other substrates at the presynapse to affect neurotransmission. Previous studies have also implicated BMP as a retrograde signal that modulates presynaptic development and function in response to postsynaptic alterations. It would be interesting to explore the relationship between PAR-1 and Dlg-mediated synaptic effects and BMP-mediated retrograde signaling (Zhang, 2007).

The current model predicts that PAR-1 overactivation causes Dlg hyperphosphorylation and delocalization from the synapse, producing certain Dlg loss-of-function effects. In contrast, loss of PAR-1 function has the opposite effect, causing Dlg overactivation phenotypes. Most of the phenotypes observed are consistent with this model. For example, at the morphological level, PAR-1 overexpression and Dlg inactivation both lead to SSR loss, whereas loss of PAR-1 and overexpression of Dlg promote SSR growth. At the electrophysiological level, PAR-1 overexpression or Dlg loss of function leads to reduced EJC amplitude, whereas loss of PAR-1 has the opposite effect. The genetic interaction between PAR-1 and Dlg is also consistent with an antagonistic effect of PAR-1 on Dlg. Some inconsistencies of certain PAR-1 overexpression phenotypes and previously published dlg mutant phenotypes are noted. For example, overexpression of PAR-1 in the postsynapse causes reductions in both active zone number and synaptic vesicle density, whereas quite variable phenotypes, ranging from no obvious structural alteration in the presynapse to reduction in synaptic vesicle density or increase in active zone number, have been described for different dlg mutant alleles. Similarly, in electrophysiological studies, a decrease was found in both mEJC and EJC amplitudes, but no significant change in quantal content, in both Mhc>PAR-1 animals and dlgX1-2 mutants. These neurotransmission phenotypes are different from those previously reported for dlg mutant alleles dlgm52 and dlgv59, in which EJC was increased, whereas mEJC was not changed. However, a recent study also reported features of reduced neurotransmission in dlgX1-2 mutant. It is therefore possible that different dlg mutant alleles may differentially affect synaptic function (Zhang, 2007).

Recent studies have revealed a tight correlation between synaptic dysfunction and the pathogenesis of neurodegenerative diseases and other neurological disorders. In AD in particular, synaptic dysfunction occurs decades before the onset of amyloid plaque and neurofibrillary tangle formation and discernable neuronal loss. Intriguingly, loss of PSD-95 protein has been observed in AD patients. It is conceivable that under disease conditions, an increase of PAR-1/MARK activity might occur in response to certain neurotoxic insults, leading to abnormal phosphorylation and delocalization of PSD-95 from the postsynapse, eventually leading to neuronal dysfunction and death. Further studies in human AD postmortem tissues and mouse AD models will test the potential role of PAR-1/MARK kinases in regulating PSD-95 function and disease pathogenesis (Zhang, 2007).

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