discs large 1

Troponin I and Tropomyosin regulate chromosomal stability and cell polarity

The Troponin-Tropomyosin (Tn-Tm) complex regulates muscle contraction through a series of Ca(2+)-dependent conformational changes that control actin-myosin interactions (see video). Pre-cellular embryos of Troponin I, Tm1 and Tm2 mutants exhibit abnormal nuclear divisions with frequent loss of chromosome fragments. During cellularization, apico-basal polarity is also disrupted as revealed by the defective location of Discs large (Dlg) and its ligand Rapsynoid (Raps; also known as Partner of Inscuteable, Pins). In agreement with these phenotypes in early development, on the basis of RT-PCR assays of unfertilized eggs and germ line mosaics of TnI mutants, it was also shown that TnI is part of the maternal deposit during oogenesis. In cultures of the S2 cell line, native TnI is immunodetected within the nucleus and immunoprecipitated from nuclear extracts. SUMOylation at an identified site is required for the nuclear translocation. These data illustrate, for the first time, a role for TnI in the nucleus and/or the cytoskeleton of non-muscle cells. It is proposed that the Tn-Tm complex plays a novel function as regulator of motor systems required to maintain nuclear integrity and apico-basal polarity during early Drosophila embryogenesis (Sahota, 2009).

Troponin I (TnI) and Tropomyosin (Tm) are actin-binding proteins that regulate muscle sarcomere contraction. The Tn-Tm complex contains three different Troponin polypeptides, C, T and I, and it regulates acto-myosin interactions in response to the rise of free calcium. Mammals have three genes expressing TnI known as slow twitch (TNNI1), fast twitch (TNNI2) and cardiac (TNNI3). In humans, mutations in TNNI2 and TNNI3 cause distal arthrogryposis type 2B and familial hypertrophic cardiomyopathy, respectively. In Drosophila, viable mutations in the single gene expressing TnI, wings up A (wupA) [also known as held up (hdp)], result in hypercontraction and degeneration of the indirect flight muscles of the thorax due to recessive hypomorphic point mutations. However, studies on lack of function mutations for this gene have been hampered by the fact that null alleles are dominant lethals. Mammals contain four tropomyosin genes, TPM1-4, while Drosophila has two, Tm1 and Tm2. In humans, mutant TPM1 is thought to be responsible for type 3 familial hypertrophic cardiomyopathy, whereas TPM2 is involved in nemaline myopathy and TPM3 has been linked to dominant nemaline myopathy. TPM1 has also been identified as a suppressor of malignant transformation as it is downregulated in mammalian transformed cells, and its expression is abolished in human breast tumors. Indeed, it is widely accepted that actin regulation plays a crucial role in cell motility, which is a key feature in metastatic cancers (Sahota, 2009 and references therein).

Although some of these pathological phenotypes appear unrelated to muscle biology, several lines of evidence indicate that these muscle-specific proteins could have a role in other cell types and processes. For instance, Tm1 is part of the maternal deposit during Drosophila oogenesis, it is required to localize the oskar mRNA at the posterior pole of the oocyte, and later in development it localizes to various cell types including the gut, brain and epidermis. Also, this study demonstrates that TnI RNA is detected in mature unfertilized eggs, which suggests a role in early embryogenesis. Thus, this study set out to analyze early development phenotypes and their mechanisms in TnI and Tm mutants (Sahota, 2009).

This study shows a novel function for the Tn-Tm complex in regulating nuclear divisions during early embryogenesis in Drosophila. Evidence is provided that TnI is required for maintaining stable chromosomal integrity, which was also show for Tm1 and Tm2. Importantly, the three genes seem required for correct epithelial apico-basal polarity; mutant phenotypes include cellularization defects that mislocalize the polarity markers Discs large (Dlg) and its ligand Rapsynoid (Raps) [also known as Partner of Inscuteable (Pins)]. Consistent with the function of these genes in cellularization and spindle integrity, defects in mitosis and chromosome segregation are observed. In a stable cell line, S2, TnI can be detected within the nucleus. Furthermore, the translocation of TnI to the nucleus is dependent upon a mechanism involving SUMOylation. Taken together, these data implicate the Tn-Tm complex in regulating nuclear functions. Moreover, the results suggest that the Tn-Tm complex is required to maintain correct segregation of chromosomes, as disruption of this complex leads to aberrations including chromosome fragment losses. This is the first evidence that the Tn-Tm complex can regulate both nuclear divisions and cell polarity in Drosophila. This is likely to have important implications in cancer progression since chromosomal instability and the generation of aneuploidies are characteristic hallmarks of many cancers (Sahota, 2009).

This study has immunolocalized TnI to the nucleus and shown nuclear phenotypes in the mutants. It should be noted, however, that the nuclear localization, either in the syncitial embryo or the regular S2 cells, seems dependent on the physiological state of the cell and nucleus. Also, with the techniques used in this study, it cannot be determined whether TnI is bound directly to the chromosomes or through intervening proteins. Because the repertoire of HeLa metaphase chromosome-associated proteins does not include TnI, nor other muscle proteins, the observed effects on chromosome integrity might be produced through indirect links. Nevertheless, one should realize that the referred repertoire is also subject to the technical constrains of the purification methods used in the study of HeLa cells (Sahota, 2009).

This study has also shown that the required nuclear translocation is achieved by SUMOylation, at least in the case of TnI. The putative SUMOylation sequence in exon 10 is required for nuclear import. This site, VKEE, is found in the C-termini of all TnI isoforms because it can be incorporated into the protein sequence, either from exon 9 or exon 10. Thus, all TnI isoforms could be tagged for their function. Other putative SUMOylation sites, if actually used for SUMOylation, could provide further functional diversity for TnI. This mechanism for tagging TnI in Drosophila is likely to be conserved in mammals since the VKEE motif is present in the three TnI gene types (slow twitch, fast twitch and cardiac). Although not addressed in this study, it is possible that a similar mechanism might be used to import Tm1 and Tm2 into the nucleus since they contain suitable motifs in the three isoforms of Tm2 and in one of the two isoforms of Tm1 (Sahota, 2009).

This work on the Tn-Tm complex provides an insight into how DNA aberrations and cellularization defects can be linked, and how this complex is crucially required for both DNA and cellular stability. Given that the Tn-Tm complex is also involved in muscle contraction, it appears likely that there may be other processes where disruption of this complex may be detrimental to the development of the organism. In support of this, it has been shown that mutant TnI allele 23437 displays severe defects in axon guidance and fasciculation and that the TnI L9/wupRA isoform rescues these defects. Considering the role of the Tn-Tm complex in sarcomere contraction and the range of phenotypes described in this study, it seems reasonable to propose that TnI, Tm1 and Tm2 are components of a force-generating complex within the nucleus and in the cytoplasm. However, this remains to be determined since the TnI-associated partners have not being investigated in this study (Sahota, 2009).

Being an actin-binding protein, TnI should perform its nuclear functions in association with actin. This protein is known to help RNA polymerase to move during gene transcription. It is currently a matter of debate whether this function requires actin in a globular or a filament structure. However, a recent study reports the interaction of vertebrate fast skeletal TnI with the estrogen receptor during transcription. By analogy to the role that TnI plays in the sarcomere, where the Tn-Tm complex interacts with the actin filaments, it seems likely that during transcription actin has a filament structure, as in the sarcomere thin filament. Actin is also important for morphogenesis of cells and organs in the early embryo, ranging from nuclear divisions and chromosomal segregation in conjunction with myosin, to the regulation of cell shape and movements. All these processes are also relevant to the formation and progression of tumors. In addition, chromosomal instability, mitotic defects and cell polarity defects are characteristic features of many cancers. The fact that TnI, Tm1 and Tm2 all regulate actin strengthens the argument that they execute this regulation as a complex. Defects in all three genes give rise to similar DNA defects, and also to similar defects in apico-basal cell polarity. These common features provide the basis for a mechanism leading to aneuploidy and aberrant cell signaling. That is, molecules that ensure proper actin function during nuclear divisions also ensure that actin correctly regulates cell polarity, which, in turn, is important in proliferation and growth. The tubulin spindle was also affected in the three mutants, indicating that the integrity of the cytoskeletal network may be compromised when any of these molecules are depleted (Sahota, 2009).

In addition to the cytoskeletal network, the localization of Dlg and Pins were also shown to be disrupted in TnI-Tm mutants. Dlg has been described as a neoplastic tumor suppressor and disruption of polarity is a hallmark of cancer progression. The Pins protein is involved in orientation of asymmetric cell divisions, which is important for specifying cell fate. Consistent with the altered Pins expression, spindle orientation defects are observed in the three mutants. Also, spindle orientation is particularly important for specifying neuronal identity in Drosophila neuroblasts. The recycling of molecules for distinct processes is a recurrent theme in development. Indeed, many actin-binding proteins were first identified for their effects on axon guidance and growth, and were subsequently shown to play important roles during cellularization. Also, Dlg was associated with synaptogenesis before its role in cellularization was determined. The novel function for the Tn-Tm complex uncovered in this study might have opened the way to reveal requirements in other actin-associated events. It was observed that TnI, as well as Tm1 and Tm2, are crucial for the correct development of the central nervous system. Further studies on the role of the Tn-Tm complex during nuclear divisions seem appropriate towards understanding how these proteins affect cell proliferation, and might provide novel targets for controlling cell divisions (Sahota, 2009).

Nak regulates Dlg basal localization in Drosophila salivary gland cells

Protein trafficking is highly regulated in polarized cells. During development, how the trafficking of cell junctional proteins is regulated for cell specialization is largely unknown. In the maturation of Drosophila larval salivary glands (SGs), the Dlg protein is essential for septate junction formation. It was shown that Dlg is enriched in the apical membrane domain of proximal cells and localized basolaterally in distal mature cells. The transition of Dlg distribution is disrupted in Numb-associated kinase (nak) mutants. Nak associates with the AP-2 subunit alpha-Adaptin and the AP-1 subunit AP-1gamma. In SG cells disrupting AP-1 and AP-2 activities, Dlg is enriched in the apical membrane. Therefore, Nak regulates the transition of Dlg distribution likely through endocytosis of Dlg from the apical membrane domain and transcytosis of Dlg to the basolateral membrane domain during the maturation of SGs development (Peng, 2009).

This study describes the re-localization of the Dlg protein during Drosophila SG specialization. From this analyses, Dlg underwent a transition from the apical to the basal membrane domain. In addition, Nak and components of AP-1 or AP-2 complexes were found to be required for the Dlg transition. Two possible models could explain the transition of Dlg re-localization in SG cells. First, in the proximal SG cells, newly synthesized Dlg is transported to and maintained in the apical membrane. In the distal cells, however, the newly synthesized Dlg is directed toward the basal membrane. Apically deposited Dlg has to be depleted in the transition zone cells, which could be mediated through the endocytic and lysosomal degradative pathway. An alternatively model is that newly synthesized Dlg is targeted to the apical membrane in all SG cells. However, the apical membrane-associated Dlg is re-routed to the basolateral membrane domains, starting in the transition zone cells and continuing in distal cells, via endocytosis and transcytosis. This model could explicitly explain the Dlg mislocalization in the mutants that were analyzed. Due to defects in endocytosis in the AP-2 complex mutants, the Dlg protein is retained in the apical membrane. In AP-1 mutants such as the AP47^SHE11/EP1112 transheterozygotes, large Dlg-enriched tubular structures were observed. Similar structures have been reported as specific stages, types, or structures of endosome. Therefore, in the absence of AP-1 activity, failure in vesicle budding from TGN or endosomes may lead to the disruption of targeting Dlg to the basal membrane and Dlg retaining in these organelles. Enrichment of Dlg in apical membrane might be an result inadvertently caused by abnormal intracellular transportation or a pool of Dlg being recycled back to the apical membrane through an AP-1-independent route (Peng, 2009).

In nak mutant SG cells, only the transition of Dlg is defective, but not other polarized proteins, such as aPKC, Syx1A, and Arm. In the severe AP47 and α-ada mutants when Dlg was retained at the apical membrane, the ruffled apical membrane might result from the enriched Dlg protein, leading to excess membrane addition. Such phenotype has been described when Dlg was overexpressed during embryonic cellularization. Therefore, the Dlg transition potentially accounts for the establishment of new septate junctions or basolateral membrane addition during SG cell maturation (Peng, 2009).

Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors

When cells undergo apoptosis, they can stimulate the proliferation of nearby cells, a process referred to as compensatory cell proliferation. The stimulation of proliferation in response to tissue damage or removal is also central to epimorphic regeneration. The Hippo signaling pathway has emerged as an important regulator of growth during normal development and oncogenesis from Drosophila to humans. This study shows that induction of apoptosis in the Drosophila wing imaginal disc stimulates activation of the Hippo pathway transcription factor Yorkie in surviving and nearby cells, and that Yorkie is required for the ability of the wing to regenerate after genetic ablation of the wing primordia. Induction of apoptosis activates Yorkie through the Jun kinase pathway, and direct activation of Jun kinase signaling also promotes Yorkie activation in the wing disc. It was also shown that depletion of neoplastic tumor suppressor genes, including lethal giant larvae and discs large, or activation of aPKC, activates Yorkie through Jun kinase signaling, and that Jun kinase activation is necessary, but not sufficient, for the disruption of apical-basal polarity associated with loss of lethal giant larvae. These observations identify Jnk signaling as a modulator of Hippo pathway activity in wing imaginal discs, and implicate Yorkie activation in compensatory cell proliferation and disc regeneration (Sun, 2011).

Many tissues have the capacity respond to the removal or death of cells by increasing proliferation of the remaining cells. In Drosophila, this phenomenon has been characterized both in the context of imaginal disc regeneration and compensatory cell proliferation. These studies implicate the Hippo signaling pathway as a key player in these proliferative responses to tissue damage. After genetically ablating the wing primordia by inducing apoptosis, it was observed that Yki becomes activated to high levels in surrounding cells, based on its nuclear abundance and induction of a downstream target of Yki transcriptional activity. Moreover, high level Yki activation is crucial for wing disc regeneration, as even modest reduction of Yki levels, to a degree that has only minor effects on normal wing development, severely impaired wing disc regeneration. While it was known that Yki is required for wing growth during development, the current observations establish that Yki is also required for wing growth during regeneration, and moreover that regeneration requires higher levels of Yki activation than during normal development (Sun, 2011).

These studies identify Jnk activation as a promoter of Yki activity in the wing disc. Most aspects of imaginal disc development, including imaginal disc growth, normally do not require Jnk signaling. By contrast, Jnk signaling is both necessary and sufficient for Yki activation in response to wing damage. Jnk signaling has previously been linked to compensatory cell proliferation and regeneration in imaginal discs, and it is now possible to ascribe at least part of that requirement to activation of Yki. However, Jnk signaling also promotes the expression of other mitogens, including Wg, which were linked to regeneration and proliferative responses to apoptosis. Wg and Yki are not required for each other's expression, suggesting that they are regulated and act in parallel to influence cell proliferation after tissue damage. The mechanism by which Jnk activation induces Yki activation is not yet known. The observation that it could be suppressed by over-expression of Wts or Hpo suggests that it might impinge on Hippo signaling at or upstream of Hpo and Wts, but the possibility that Jnk-dependent Yki regulation occurs in parallel to these Hippo pathway components cannot be excluded. The high level of nuclear Yki localization is striking by contrast with the more modest effects of upstream tumor suppressors in the Hippo pathway, which suggests that Jnk might regulate Yki through a distinct mechanism, or simultaneously affect multiple upstream regulators (Sun, 2011).

Strong Yki activation was detected within the wing and haltere discs in response to Jnk activation, but weaker or non-existent effects in leg or eye discs. Jnk activation has previously been linked to oncogenic effects of neoplastic tumor suppressors in eye discs, and it is possible that Yki activation might be induced in eye discs if a distinct Jnk activation regime were employed. Nonetheless, since identical conditions were employed in both wing and eye discs, isolating them from the same animals, these studies emphasize the importance of context-dependence for Yki activation by Jnk. A link between Jnk activation and Yki activation is not limited to the wing however, as a connection between these pathways was recently discovered in the adult intestine, where damage to intestinal epithelial cells, and activation of Jnk, can activate also Yki (Sun, 2011).

There was a general correspondence between activation of Jnk and activation of Yki under multiple experimental conditions, including expression of Rpr, direct activation of Jnk signaling by Egr or Hep.CA (an activated form of the Jnk kinase Hemipterous), and depletion of lgl. Some experiments, most notably direct activation of Jnk by Hep.CA, revealed a non-autonomous effect on Yki, which could imply that the influence of Jnk on Yki activity is indirect. Although the basis for this non-autonomous effect is not yet known, the hypothesis that it is actually also mediated through Jnk signaling is favored, since it has been reported that Jnk activation can propagate from cell to cell in the wing disc. Consistent with this possibility, a non-autonomous activation of Jnk adjacent to lgl depleted cells was seen to be blocked by depletion of bsk solely within the lgl RNAi cells. Conversely, alternative signals previously implicated in compensatory cell proliferation do not appear to be good candidates for mediating Yki activation, since it was found that Wg is not required for Yki activation in regenerating discs, and prior studies did not detect a direct influence of Dpp pathway activity on Yki activation (Sun, 2011).

Activation of Yki adjacent to Egr- or Rpr-expressing cells was also reduced by over-expression of Wts. This might reflect an influence of Yki on signaling from these cells, but because expression of Wts inhibits Yki activity, and activated Yki promotes expression of an inhibitor of apoptosis (Diap1), it is also possible that this effect could be explained simply by Wts over-expression resulting in reduction or more rapid elimination of Egr- or Rpr-expressing cells; the reduced survival of these cells would then limit their ability to signal to neighbors (Sun, 2011).

Although Jnk has been implicated in compensatory cell proliferation and regeneration, it is better known for its ability to promote apoptosis. The dual, opposing roles of Jnk signaling as a promoter of apoptosis and a promoter of cell proliferation raise the question of how one of these distinct downstream outcomes becomes favored in cells with Jnk activation (see Diverse inputs and outputs of Jnk signaling). Given the links between Jnk activation and human diseases, including cancer, defining mechanisms that influence this is an important question, and the identification of the role of Yki activation in Jnk-mediated proliferation and wing regeneration should facilitate future investigations into how the balance between proliferation or apoptosis downstream of Jnk is regulated (Sun, 2011).

Hippo signaling is regulated by proteins that exhibit discrete localization at the subapical membrane, e.g., Fat, Ex, and Merlin. The observation that disruption of apical-basal polarity is associated with disruption of Hippo signaling underscores the importance of this localization to normal pathway regulation. These observations establish that Hippo signaling is inhibited by neoplastic tumor suppressor mutations, resulting in Yki activation, and that this activation of Yki is required for the tumorous overgrowths associated with these mutations (Sun, 2011).

Although these results agree with these recent studies in linking lgl to Hippo signaling (Grzeschik, 2010), there are some notable differences. A previous study examined lgl mutant clones in the eye imaginal disc, under conditions where cells retained apical-basal polarity, whereas this study examined wing imaginal discs, where apical-basal polarity was lost. Intriguingly this study found that conditions associated with activation of Yki by Jnk in the wing disc were not sufficient to activate Yki in the eye disc. This observation, together with the discovery that loss of polarity in lgl depleted wing cells requires Jnk activation, suggests as a possible explanation for why lgl null mutant clones retain apical-basal polarity in eye discs, that eye disc cells have a distinct, and apparently reduced, sensitivity to Jnk activation as compared to wing disc cells (Sun, 2011).

This study also identified distinct processes linked to Yki activation in the absence of lgl. A previous study reported an effect of lgl on Hpo protein localization (Grzeschik, 2010). In wing discs, the discrete apical localization of Hpo was observed in studies of eye discs. Thus, the proposed mechanism, involving activation of Yki via mis-localization of Hpo and dRassf, might not be relevant to the wing. By contrast, this study identified an essential role for Jnk signaling in regulating Yki activation in lgl-depleted cells in the wing. Because this study did not detect an effect of direct Jnk activation on Yki in eye discs, it is possible that Lgl can act through multiple pathways to influence Yki, including a Jnk-dependent pathway that is crucial in the wing disc, and a Jnk-independent pathway that is crucial in the eye disc. Grzeschik (2010) also linked the influence of lgl in the eye disc to its antagonistic relationship with aPKC. The observation that the influence of aPKC in the wing depends on Jnk activation is consistent with an Lgl-aPKC link, and identifies a role for Jnk activation in the oncogenic effects of aPKC (Sun, 2011).

The observation that the loss of polarity in lgl RNAi discs is dependent upon Jnk signaling was unexpected, but a related observation was recently reported by Zhu; 2010). These results suggest that the established role of the Lgl-Dlg-Scrib complex in maintaining epithelial polarity depends in part on repressing Jnk activity. However, since Jnk activation on its own was not sufficient to disrupt polarity, multiple polarity complexes might need to be disturbed in order for wing cells to lose apical-basal polarity, including both Lgl and additional, Jnk-regulated polarity complexes (Sun, 2011).

The discovery of the role of Jnk signaling in Yki activation provides a common molecular mechanism for the overgrowths observed in conjunction with mutations of neoplastic tumor suppressors, and those associated with compensatory cell proliferation, because in both cases a proliferative response is mediated through Jnk-dependent activation of Yki. Although the molecular basis for the linkage of these two pathways is not understood yet, it operates in multiple Drosophila organs, and thus appears to establish a novel regulatory input into Hippo signaling that is of particular importance in abnormal or damaged tissues. Moreover, Jnk activation has also been observed in conjunction with regeneration of disc fragments after surgical wounding, and thus its participation in regeneration is not limited to paradigms involving induction of apoptosis. It is also noteworthy that under conditions of widespread lgl depletion (i.e., lgl mutant or lgl RNAi), and consequent Jnk activation, the balance between induction of apoptosis and induction of cell proliferation is shifted towards a proliferative response. By contrast, in the wing disc clones of cells mutant for lgl fail to survive, unless oncogenic co-factors are co-expressed. The loss of lgl mutant clones in wing discs was recently attributed to cell competition. Together, these observations suggest that the choice between proliferative versus apoptotic responses to Jnk activation can be influenced by the Jnk activation status of neighboring cells (Sun, 2011).

Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. A genetic screen was performed to identify regulators of neuroblast self-renewal, and a mutation was identified in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of 'neuroblast tumors.' Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1 (PAR-4), or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. It is proposed that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors (Anderson, 2012).

This study presents evidence that the evolutionary-conserved protein Sgt1 acts with Hsp90, LKB1 and AMPK to promote apical localization of the Par and Pins complexes in prophase neuroblasts. It is proposed that Sgt1/Hsp90 proteins function together based on multiple lines of evidence: (1) they show conserved binding from plants to humans; (2) the sgt1^s2383 mutant results in a five amino acid deletion within the CS domain, which is the Hsp90 binding domain; (3) sgt1 and hsp83 have similar cell cycle phenotypes; and (4) sgt1 and hsp83 have similar neuroblast polarity phenotypes. The Sgt1/Hsp90 complex either stabilizes or activates client proteins (Zuehlke, 2010); it is suggested that Sgt1 activates LKB1, rather than stabilizing it, because it was not possible to rescue the sgt1 mutant phenotype by simply overexpressing wild type LKB1 protein. No tests were performed for direct interactions between Sgt1 and LKB1 proteins, and thus the mechanism by which Sgt1 activates LKB1 remains unknown (Anderson, 2012).

LKB1 is a 'master kinase' that activates at least 13 kinases in the AMPK family. It is suggested that LKB1 activates AMPK to promote neuroblast polarity because overexpression of phosphomimetic, activated AMPKα can rescue the lkb1 and sgt1 mutant phenotype. It remains unclear how AMPK activity promotes apical protein localization. An antibody to activated AMPKα (anti-phosphoT385-AMPKα shows spindle and cytoplasmic staining that is absent in ampkα mutants, and centrosomal staining that persists in AMPKα null mutants, but no sign of asymmetric localization in neuroblasts. AMPK activity is thought to directly or indirectly activate myosin regulatory light chain to promote epithelial polarity. AMPK is activated by a rise in AMP/ATP levels that occur under energy stress or high metabolism; AMP binds to the γ regulatory subunit of the heterotrimeric complex and results in allosteric activation of the α subunit. ampkα mutants grown under energy stress have defects in apical/basal epithelial cell polarity in follicle cells within the ovary. In contrast, AMPKα mutants grown on nutrient rich food still show defects in embryonic epithelial polarity, neuroblast apical polarity, and visceral muscle contractio. Larval neuroblasts, embryonic ectoderm, and visceral muscle may have a high metabolic rate, require low basal AMPK activity, or use a different mechanism to activate AMPK than epithelial cells. What are the targets of AMPK signaling for establishing apical cortical polarity in larval neuroblasts? AMPK could directly phosphorylate Baz to destabilize the entire pool of apical proteins, but currently there is no evidence supporting such a direct model. AMPK may act via regulating cortical myosin activity: clear defects have been seen in cortical motility, ectopic patchy activated myosin at the cortex, and failure of cytokinesis in sgt1, lkb1, and ampkα mutants. This strongly suggests defects in the regulation of myosin activity, but how or if gain/loss/mispositioning of myosin activity leads to failure to establish apical cortical polarity remains unknown. Lastly, the defects in apical cell polarity seen at prophase could be due to the prometaphase cell cycle delays (Anderson, 2012).

What activates the Sgt1-LKB1-AMPK pathway to promote cell polarity during prophase? In budding yeast, Sgt1 requires phosphorylation on Serine 361 (which is conserved in Drosophila Sgt1) for dimerization and function (Bansal, 2009); this residue is conserved in Drosophila Sgt1 but its functional significance is unknown (Anderson, 2012).

Sgt1/Hsp90/LKB1/AMPK are all required for apical Par/Pins complex localization, but Sgt1 must act via a different pathway to promote Dlg/Scrib cortical localization, because only the sgt1 mutant affects Dlg/Scrib localization, and overexpression of activated AMPKα is unable to restore cortical Scrib in sgt1 mutants. The mechanism by which Sgt1 promotes Dlg/Scrib cortical localization is unknown (Anderson, 2012).

This study has shown that sgt1 mutants lack Par/Pins apical polarity in prophase neuroblasts, but these proteins are fairly well polarized in metaphase neuroblasts. The rescue of cortical polarity is microtubule dependent, probably occurring via the previously described microtubule-dependent cortical polarity pathway containing Pins, Dlg and Khc-73. The weak polarity defects still observed in sgt1 metaphase neuroblasts may be due to the poor spindle morphology. The lack of microtubule-induced polarity at prophase, despite a robust microtubule array in prophase neuroblasts, suggests that the microtubule-induced cortical polarity pathway is activated at metaphase. Activation of the pathway could be via expression of the microtubule-binding protein Khc-73; via phosphorylation of Pins, Dlg or Khc-73 by a mitotic kinase like Aurora A; or via a yet unknown pathway (Anderson, 2012).

It was somewhat surprising that the sgt1 pins double mutants had increased numbers of brain neuroblasts, because each single mutant had reduced neuroblast numbers. The double mutant phenotype may be due to loss of both Pins and cortical Dlg/Scrib, as the sgt1 pins double mutant phenotype is similar to the dlg pins double mutant phenotype. It could also be due to a change in an unknown downstream effector of both Sgt1 and Pins. A not mutually exclusive possibility is that the sgt1 pins double mutant phenotype is due to loss of all Par/Pins cortical polarity. This model is consistent with the observation that sgt1 or pins single mutants retain some neuroblast cortical polarity, whereas the sgt1 pins double mutants lack all known neuroblast cortical polarity. It is proposed that the apolar double mutant neuroblasts partition cell fate determinants equally to both siblings, and that both siblings frequently assume a neuroblast identity. This is supported by the recent finding that when the neuroblast spindle is aligned orthogonal to a normal apical/basal polarity axis, such that both siblings inherit equal amounts of apical cortical proteins, the siblings always acquire a neuroblast identity. Thus, equal partitioning of apical/basal cell fate determinants (in spindle orientation mutants) or failure to establish any cortical polarity (sgt1 pins mutants) may result in neuroblast/neuroblast siblings and an expansion of the neuroblast population (Anderson, 2012).

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).

Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97

Stardust (Sdt) and Discs-Large (Dlg) are membrane-associated guanylate kinases (MAGUKs) involved in the organization of supramolecular protein complexes at distinct epithelial membrane compartments in Drosophila. Loss of either Sdt or Dlg affects epithelial development with severe effects on apico-basal polarity. Moreover, Dlg is required for the structural and functional integrity of synaptic junctions. Recent biochemical and cell culture studies have revealed that various mammalian MAGUKs can interact with mLin-7/Veli/MALS, a small PDZ-domain protein. To substantiate these findings for their in vivo significance with regard to Sdt- and Dlg-based protein complexes, the subcellular distribution of Drosophila Lin-7 (DLin-7) was analyzed, and genetic and biochemical assays were performed to characterize its interaction with either of the two MAGUKs. In epithelia, Sdt mediates the recruitment of DLin-7 to the subapical region, while at larval neuromuscular junctions, a particular isoform of Dlg, Dlg-S97, is required for postsynaptic localization of DLin-7. Ectopic expression of Dlg-S97 in epithelia, however, was not sufficient to induce a redistribution of DLin-7. These results imply that the recruitment of DLin-7 to MAGUK-based protein complexes is defined by cell-type specific mechanisms and that DLin-7 acts downstream of Sdt in epithelia and downstream of Dlg at synapses (Bachmann, 2004).

This study has shown that the single fly homologue of Lin-7 is a component of different MAGUK-based protein complexes in epithelia and at synaptic junctions. This finding is in line with the previously reported association of LIN-7 and mLin-7 with various membrane specializations in worms or mammals, respectively. Nonetheless, these results deviate from these earlier reports in several regards and thereby imply novel roles for Sdt and Dlg-S97. Most notably, the requirement for either MAGUK to recruit DLin-7 to distinct membrane domains was not simply predictable from studies on their homologues in other species (Bachmann, 2004).

Pals1, a putative mammalian homologue of Sdt, binds mLin-7 in vitro (Kamberov, 2000). The physiological significance of this finding remains unclear since Pals1 localizes to tight junctions of epithelial cells, whereas a basolateral localization for mLin-7 was emphasized in several other reports. In Madin-Darby canine kidney cells, however, mLin-7 has also been detected at tight junctions. The current results now indicate that the interaction between the fly orthologues of Pals1 and mLin-7 is employed in epithelia for the recruitment of DLin-7 to the Crb-Sdt complex within the SAR (Bachmann, 2004).

The virtual absence of DLin-7 from basolateral plasma membrane compartments in Drosophila imaginal disc epithelia is in striking contrast to the situation in both mammals and nematodes. Differences in the expression, subcellular localization and binding capacity of potential interaction partners may account for this discrepancy. Two types of evolutionary conserved proteins have been implicated in the basolateral membrane recruitment of LIN-7 and mLin-7: the MAGUKs LIN-2/mLin-2 (CASK) and β-catenin. The latter was found to recruit mLin-7 to cadherin-based epithelial junctions via its C-terminal PDZ-binding motif (Perego, 2000). Although this motif (tDTDL) is conserved in C. elegans β-catenin, it is aberrant in the fly orthologue, Armadillo (tDTDC). In fact, a direct interaction between DLin-7 and Armadillo was not detectable in a yeast two-hybrid assay. Hence it appears unlikely that DLin-7 and Armadillo exhibit a mode of interaction similar to that of their counterparts in mammals. In contrast, DLin-2 (Caki/CamGUK) and DLin-7 displayed strong interaction in the yeast two-hybrid assay. Therefore DLin-2 would be expected to compete with Sdt for binding to the L27 domain of DLin-7 when expressed in epithelia. An epithelial expression of DLin-2, however, has not yet been documented and, instead, both immunostainings and mRNA analyses revealed that DLin-2 is predominantly expressed in the CNS (Bachmann, 2004).

Sdt is not expressed at detectable levels at larval NMJs and thus cannot contribute to the postsynaptic enrichment of DLin-7 at these junctions. Instead it was demonstrated that Dlg-S97 is required for the recruitment of DLin-7 to scaffolding complexes within the subsynaptic reticulum (SSR) around type I boutons. Severe mutations in dlg cause a decrease in the length of the SSR to about 40%. In immunofluorescence analyses, however, the reduction of both endogeneous or Flag-tagged DLin-7 at dlg^XI-2 mutant NMJs appeared clearly more dramatic, suggesting that impaired recruitment of DLin-7 is not simply due to reduced SSR complexity. This reasoning is supported by co-immunoprecipitation experiments that revealed a physical linkage between DLin-7 and Dlg-S97. This linkage is most likely indirect, as implied by the failure of EGFP-Dlg-S97 to recruit cytosolic DLin-7 in epithelia. Although an interaction between DLin-7 and the N-terminal domain of Dlg-S97 was monitored in yeast, it was also noted that this interaction is much weaker compared with those displayed by DLin-7 in combination with DLin-2 or Sdt. In accordance with recent biochemical studies and cell culture assays, which imply the coupling of SAP97 and mLin-7 via MAGUKs such as mLin-2 or MPP3, it is therefore proposed that Dlg-S97 and DLin-7 are linked via an intermediate protein factor. In fact, both the N-terminal domain of Dlg-S97 and DLin-7 can bind to L27 domains of DLin-2 in vitro . The presence of DLin-2 at larval NMJs, however, remains questionable. Unfortunately it was not possible to employ an antibody against DLin-2 to address this issue in further detail. Third instar larvae that are homo- or hemizygous for the DLin-2 mutant allele caki^x-307 exhibit normal levels of both DLin7- and Dlg-S97-specific immunofluorescence. This allele has been characterized as a deletion that removes large portions of the gene including the region encoding the PDZ-, SH3- and GUK domains of DLin-2. Nonetheless some residual function might be displayed by a truncated DLin-2^x-307 mutant isoform. Thus, the observations strongly argue against, but do not completely rule out, an involvement of DLin-2 in the recruitment of DLin-7 to NMJs. It should be noted, however, that Dlg-S97, DLin-2 and DLin-7 could co-assemble into synaptic protein complexes in the CNS where they are found equally enriched within the neuropil regions (Bachmann, 2004).

In light of recent work, which has revealed complex intramolecular interactions displayed by SAP97, one should also consider the possibility that the SAP97-type N-terminus is only accessible upon binding of tissue- or compartment-specific factors to other domains within Dlg-S97. This mode of regulation, however, would not apply to Dlg-S97N-EGFP and thus cannot explain its inability to induce nuclear targeting of DLin-7 in epithelia (Bachmann, 2004).

It has been proposed that the targeting of SAP97 to epithelial membranes depends on mLin-2. This hypothesis was based on the finding that the expression of truncated mLin-2 exerts a dominant-negative effect on the localization of SAP97 in cultured epithelial cells. It is stressed that this hierarchical mode does not apply to the respective fly homologues, since Dlg-A, which lacks the SAP97-type N-terminus, is efficiently targeted to epithelial septate junctions and to NMJs. Moreover, the recruitment of Dlg-S97 by a DLin-7 binding MAGUK could hardly explain the Dlg-S97-dependent recruitment of DLin-7 (Bachmann, 2004).

These analyses strongly suggest that the interactions between DLin-7 and Sdt or Dlg-S97 take place within the respective submembraneous target regions. In addition, these interactions could play a role during the trafficking of DLin-7. In mammalian neurons mLin-7 was found in a complex with mLin-2, mLin-10 and the NMDA-type glutamate receptor subunit NR2B on dendritic vesicles, which are transported along microtubules. Likewise, the subcellular targeting of Dlg-like MAGUKs involves the association with vesicles and/or intracellular membrane compartments and depends on microtubular transport (Bachmann, 2004).

In vertebrates, mLin-7 isoforms have been detected in axonal and dendritic compartments. The postsynaptic colocalization of DLin-7 and Dlg-S97 is reminiscent of the association of mLin-7 with PSD-95/SAP90, a prominent Dlg-like MAGUK present in postsynaptic densities of vertebrate neurons. Interestingly, a recently discovered isoform of PSD-95 (PSD-95β) exhibits a SAP97-type N-terminus with conserved binding properties. In light of the current findings it is speculated that PSD-95β, as opposed to conventional PSD-95, is involved in the postsynaptic recruitment of mLin-7. SAP97 could also serve this role, although a physical association of SAP97 and mLin-7 at synaptic junctions has not yet been reported. A possible association of DLin-7 with the presynaptic membrane of synaptic boutons can hardly be resolved by confocal microscopy in the presence of strong postsynaptic immunoreactivity. Targeted expression of Flag-DLin-7 in motorneurons did not yield considerable immunofluorescence signals at NMJs, suggesting that DLin-7 is barely targeted to presynaptic nerve terminals. It should be noted, however, that the relative pre-versus postsynaptic abundance of a protein does not necessarily reflect its functional impact on either side of the synaptic cleft. For instance, while Dlg, Scrib and D-VAP-33A are clearly enriched postsynaptically at larval NMJs, genetic rescue and gain-of-function experiments have highlighted the importance of the minor presynaptic component in all three cases (Bachmann, 2004).

The roles of DLin-7 within the SAR and at synapses remain elusive. Overexpression of Flag-DLin-7 did not result in easily detectable phenotypes within epithelia or at NMJs. This analyses led to the prediction that DLin-7 acts downstream of Sdt or Dlg-S97. Accordingly, loss-of-function alleles of DLin-7 are expected to mimic previously described or yet concealed phenotypical aspects of sdt and dlg mutants. The partial reduction of DLin-7 as achieved by transgenic expression of dsRNA had no obvious effect on the shape of boutons or on epithelial polarity. Current studies are therefore aimed at both the generation of complete loss-of-function alleles and monitoring more subtle phenotypes. In accordance with previous studies in other species, DLin-7 can be expected to bind at least one ligand via its single PDZ domain. Thereby it may help to retain this ligand within the respective compartment and/or to regulate its endosomal sorting (Bachmann, 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).

Identification of an Aurora-A/Pins^LINKER/ Dlg spindle orientation pathway using induced cell polarity in S2 cells

Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. A method has been developed to construct cortical polarity in a normally unpolarized cell line and this method was used to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. A previously unrecognized evolutionarily conserved Pins domain (Pins^LINKER) was identified that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized Pins^TPR domain has no function alone, but placing the Pins^TPR in cis to the Pins^LINKER gives dynein-dependent precise spindle orientation. This 'induced cortical polarity' assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity (Johnston, 2009).

A surprising result of these studies is the importance of the Pins^LINKER domain for spindle orientation in the S2 assay and within neuroblasts in vivo. Only this domain is sufficient for spindle orientation, and a single point mutation in the linker domain (S436A) results in spindle orientation defects in larval neuroblasts that closely mimic the pins null mutant phenotype. On the basis of domain mapping and epistasis analysis, a linear pathway has been identified from cortical Pins^LINKER to the plus ends of astral microtubules: (1) Aurora-A phosphorylates Pins^LINKER on a single amino acid, serine 436, (2) the phosphorylated Pins^LINKER binds and recruits Dlg, (3) the kinesin Khc-73 moves to astral microtubule plus ends using its motor domain and may be anchored at the plus ends by its Cap-Gly domain (Siegrist, 2005), and (4) the Khc-73^MBS domain binds the cortical Dlg^GK domain, thereby linking Khc-73⁺ astral microtubule plus ends to the Dlg cortical domain. Interestingly, this pathway is active in both directions during mitosis. Cortical Pins acts through Dlg and Khc-73 to regulate spindle orientation, and spindle-associated Khc-73 acts through Dlg and Pins to induce Pins/Galphai functional cortical polarity in neuroblasts (Johnston, 2009).

Why does the Pins^LINKER pathway provide only partial spindle orientation function? Live imaging rules out several possible explanations, such as Pins^LINKER-induced spindle rocking variability, or that Pins^LINKER functions during only a narrow window during mitosis. Live imaging shows that in Pins^LINKER cells, the spindle drifts until it is immobilized at the edge of the crescent. This is consistent with the fact that Khc-73 is a plus end-directed motor protein, and thus unable to generate pulling forces to bring the centrosome closer to the cell cortex; at best, it would provide a static link between astral microtubules and the cell cortex (Johnston, 2009).

The Pins^TPR domain can improve the Pins^LINKER spindle orientation to a level matching wild-type neuroblasts. It is proposed that the Pins^TPR domain directly binds Mud and that Mud interacts with the dynein/dynactin/Lis1 complex to enhance Pins^LINKER spindle orientation. This model is based on five observations. First, the Pins^TPR domain binds Mud in vitro and the two proteins coimmunoprecipitate from in vivo lysates; this interaction is conserved in the related C. elegans and mammalian proteins. Second, the Pins^TPR and Pins^TPR+LINKER but not the Pins^LINKER can recruit Mud to the cortex of S2 cells. Third, Pins^TPR+LINKER-mediated spindle orientation requires the dynein complex proteins Dlc and Lis1. Fourth, Pins^TPR+LINKER-mediated spindle orientation exhibits rapid, directional spindle movement toward the center of the Pins cortical crescent, similar to dynein-dependent spindle orientation in Drosophila neuroblasts. Fifth, Pins^TPR+LINKER-mediated spindle orientation leads to dynein-dependent movement of the spindle pole close to the cell cortex, consistent with dynein minus end-directed pulling of astral microtubules, as observed in other cell types (Johnston, 2009).

If Pins^TPR recruits Mud, and Mud recruits the dynein complex, then why doesn't Pins^TPR have spindle-orienting function on its own? The simplest model is that Pins^TPR/Mud alone is unable to recruit or activate the dynein complex. Alternatively, the Pins^LINKER pathway could be required for 'presenting' microtubule plus ends to an active Pins^TPR/Mud/Dynein complex, which fits with the requirement for Pins^TPR and Pins^LINKER acting in cis. In summary, these data show that the Pins^TPR and Pins^LINKER domains provide distinct functions, both of which are required for optimal spindle orientation. Interestingly, spindle orientation in S2 cells does not show 'telophase rescue'—a phenomenon whereby spindles that are partially oriented in metaphase/anaphase neuroblasts become aligned with the cell polarity axis by telophase -- consistent with the absence of redundant spindle orientation pathways in this assay (Johnston, 2009).

The Pins^TPR pathway is regulated by Galphai binding to the GoLoco domain, relieving intramolecular TPR-GoLoco interactions, and making the TPR domain accessible for intermolecular interactions. In addition, Galphai is required to recruit Pins to the cell cortex, where it can interact with regulator and effector proteins. In the S2 spindle orientation assay, a requirement for Galphai can be bypassed by simply deleting the GoLoco domain (thereby freeing the TPR for intermolecular interactions) and tethering the Pins^TPR+LINKER protein to the cortex by fusion with the Ed transmembrane protein. Thus, Galphai is important to activate and localize full-length Pins, but not as an effector of Pins-mediated spindle orientation (Johnston, 2009).

In contrast, the Pins^LINKER pathway is not regulated by Galphai, because full-length Pins in the absence of Galphai provides equal spindle orientation to Pins^LINKER, suggesting that the Pins^LINKER is active when Pins is in the 'closed' form. The Khc-73 mammalian ortholog GAKIN transports hDlg to the cell cortex, but there are several reasons to think that this mechanism does not activate the Pins^LINKER pathway. First, cortically tethered Dlg^GK domain requires Khc-73 for spindle orientation, which rules out a role for Khc-73 in merely transporting Dlg to the cortex; second, khc-73 RNAi does not block the ability of Pins^LINKER to recruit Dlg to the cortex (Johnston, 2009).

This study has shown that Aurora-A kinase activates the Pins^LINKER spindle orientation pathway by phosphorylating S436 in the linker domain and that this pathway is required for accurate spindle orientation in vivo for larval neuroblast asymmetric cell division. Neuroblasts expressing the nonphosphorylatable form of Pins (S436A) have a weaker spindle orientation phenotype than aurora-A null mutants, as expected because of Aurora-A regulation of multiple Pins-independent processes required for spindle orientation, such as centrosome maturation, cell-cycle progression, and cell polarity in flies. However, this study shows that a Pins phosphomimetic mutant (S436D) allows spindle orientation even after RNAi depletion of Aurora-A levels, suggesting that Aurora-A phosphorylation of PinsS436 is essential for Pins-dependent spindle orientation in the S2 cell assay. Furthermore, the finding that the PinsS436A protein has no spindle orientation activity in pins mutant larval neuroblasts, and has dominant-negative activity in the presence of endogenous Pins, shows that the Aurora-A/Pins^LINKER pathway is required for spindle orientation in larval neuroblasts as well (Johnston, 2009).

The Pins spindle orientation pathway is cell-cycle regulated: interphase S2 cells that have polarized Pins^TPR+LINKER do not capture centriole/centrosomal microtubules. There are at least two reasons for the lack of Pins interphase activity. First, the level of the Aurora-A kinase is low during interphase, and Aurora-A phosphorylation of Pins S436 has been shown to be is essential for Pins-mediated spindle orientation. Second, interphase centrosomes are immature, lacking Cnn and nucleating few microtubules. Expression of the Pins S436D protein, which is fully functional during mitosis even after Aurora-A depletion, still has no ability to capture centrioles during interphase. Thus, both centrosome maturation and Aurora-A activation are required for Pins-mediated spindle orientation in S2 cells (Johnston, 2009).

Cell polarity and spindle orientation has been induced in a cultured cell line in this study. This system was used to identify two pathways regulating spindle orientation, to establish molecular epistasis within each pathway, and to identify the target of the mitotic kinase Aurora-A that coordinates cell-cycle progression with spindle orientation. In the future, this system should be useful for characterizing spindle orientation pathways from other Drosophila cell types or from other organisms, identifying the mechanisms that control centrosome or spindle asymmetry, and characterizing the establishment and maintenance of cortical polarity. In each of these cases, the induced polarity system should be useful for rapid protein structure/function studies and high-throughput drug or RNAi loss-of-function studies (Johnston, 2009).

A perisynaptic ménage à trois between Dlg, DLin-7, and Metro controls proper organization of Drosophila synaptic junctions

Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. This study has identified Metro (Menage a trois), a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging it was further demonstrated that the complex controls the size of postsynaptic glutamate receptor fields. These findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization (Bachmann, 2010).

The establishment of neural networks involves mechanisms that coordinate the assembly and selective stabilization of synapses. Multivalent scaffold molecules that link transmembrane proteins to the cytoskeleton are candidate determinants of synapse stability. Recent studies imply a stabilizing role for Postsynaptic density protein-95 (PSD-95), a principal vertebrate synaptic scaffold protein, during activity-dependent maturation of glutamatergic synapses. The linkage of PSD-95 to ionotropic glutamate receptors (GluRs), however, makes it difficult to assess a direct involvement in synaptic structural integrity independent from activity-related effects. Moreover, the existence of additional PSD-95/Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) accounts for partial redundancy and functional diversification including perisynaptic and extrasynaptic activities (Bachmann, 2010 and references therein).

In Drosophila a single gene, discs-large (dlg), encodes DlgA and DlgS97. The latter is specified by an N-terminal L27 domain and thus corresponds to the predominant isoform of vertebrate SAP97. Both isoforms, collectively referred to as Dlg, are present at glutamatergic larval neuromuscular junctions (NMJs). Strikingly, Dlg omits GluR-containing PSDs but is enriched within the subsynaptic reticulum (SSR), a postsynaptic membrane specialization commonly categorized as perisynaptic. Strong dlg mutants display aberrant motornerve terminal morphology and severely reduced SSR complexity. Dlg further controls the size of synaptic contacts (i.e., active zones and PSDs), possibly involving the perisynaptic cell adhesion molecule Fasciclin II (FasII) as binding partner. Similar to various dlg alleles, strong fasII alleles display enlarged active zones. Mutations that specifically abolish DlgS97, however, result in a similar phenotype while leaving junctional FasII largely unaffected, suggesting that DlgS97 acts in a FasII-independent pathway to restrict synaptic contacts (Bachmann, 2010).

To specify the role of DlgS97 isoform-specific interactions have been analyzed and it has been shown that DlgS97 is crucial for proper NMJ localization of the PDZ domain protein DLin-7. It is further predicted that this interaction relies on a linker protein expressed in muscles but not in epithelia. Proteins bearing a tandem of L27 domains such as the MAGUK CASK/mLin-2 or members of the p55 subfamily of MAGUKs have emerged as primary candidates to serve the linkage between vertebrate Lin-7 (Veli) and SAP97 in epithelial cells. This study introduces Metro, a novel Drosophila MAGUK, as the missing link between DlgS97 and DLin-7 at NMJs. Genetic analyses reveal that the three scaffold proteins control each other. NMJs lacking Metro display reduced growth and are predestined to structural abnormalities. Notably, Metro and DLin-7 are involved in the dimensioning of glutamate receptor fields. These findings show that Metro and DLin-7 augment the complexity of the perisynaptic scaffold system and thereby control the synaptic organization of the NMJ (Bachmann, 2010).

This study has focused on a complex formed by Drosophila SAP97β alias DlgS97, the MPP-like MAGUK Metro, and the Veli/MALS homolog DLin-7. Related scaffold complexes exist at vertebrate epithelial junctions and at presynaptic photoreceptor terminals. The existence of the respective complexes in vertebrate CNS neurons and their synaptic or extrasynaptic roles therein remain elusive. Using larval NMJs as an in vivo model, this study now shows that DlgS97-Metro-DLin-7-type complexes indeed control the proper organization of a synaptic junction (Bachmann, 2010).

Genetic analyses implied that Metro constitutes the exclusive link between DlgS97 and DLin-7 at NMJs. In vitro binding assays, however, revealed that the interaction between DlgS97 and Metro is fairly weak. This issue was eventually clarified by two observations: (1) DLin-7 is absolutely required for DlgS97-dependent localization of Metro-B at NMJs, and (2) biochemical studies revealed that binding of the Metro homolog MPP7 to Veli promotes its binding to hDlg. This allosteric mechanism is thus evolutionary conserved and distinguishes Metro and its closest mammalian homologs from other MPP-like MAGUKs and Cmg/Cask. In this way a one-to-one ratio of Metro and DLin-7 can be maintained, possibly translating into a balance of yet-to-be-identified junctional binding partners of the PDZ domains of either protein. Moreover, it was found that the spectrin-based cytoskeleton to some extent assures the junctional anchorage of Metro and DLin-7 in the absence of DlgS97. This link might contribute to a regular positioning of DlgS97-based scaffold complexes dictated by a spectrin-defined network (Bachmann, 2010).

The knock-out of metro leads to a considerable reduction of DlgS97. Such chronic downregulation of DlgS97 may be partially compensated by recruitment of DlgA. Stabilization of DlgS97 by Metro and DLin-7, however, involves the formation of higher-order complexes driven by (1) the ability of L27 domains to form dimers of dimers and (2) the tandem arrangement of L27 domains in Metro. Formally, such complexes are unlimited. In addition to the reduced abundance of junctional DlgS97, the breakdown of its multimeric context is consider as a crucial consequence of lacking Metro and DLin-7 (Bachmann, 2010).

The formation of new boutons strongly correlates with a temporally restricted downregulation of the Dlg-based scaffold at the respective site. Normally, Dlg reassociates with the nascent bouton shortly thereafter and recent studies suggest that GluRs and the actin regulators dPix and Pak act upstream of Dlg in this process. The occurrence of relatively large boutons with only a few GluR clusters and very little Dlg in both metro and DLin-7 mutants suggests that the formation of the postsynaptic scaffold is disturbed. Interestingly, a knockdown of MPP7 was found to cause a significant delay in the establishment of epithelial tight junctions. Considering the temporal aspect, it is proposed that Metro and DLin-7 are required to synchronize junctional expansion and scaffold assembly. Indeed various other abnormalities were observed that are consistent with a role for Metro and DLin-7 in balancing NMJ growth and stability. Reduced proliferation of boutons was accompanied by an overall enlargement of boutons, a reciprocal correlation, observed in several instances, possibly indicative of a disturbed linkage between submembranous scaffold and cytoskeletal elements. Notably, enlarged boutons may harbor more active zones, explaining the virtual invariance in the overall number of synaptic contacts at mutant versus wild-type NMJs (Bachmann, 2010).

The data indicate a stabilizing role for Metro and DLin-7, possibly as part of a back-up system to cope with operational demands such as junctional plasticity. It remains elusive, however, whether both proteins are regulated to weaken or strengthen the Dlg-based scaffold. Weakening may be involved in defining sites for bouton formation, but might also be a prerequisite for the de novo formation of synaptic contact sites on preexisting boutons. The striking protein instability of Metro in the absence of DLin-7 is suggestive in this regard, as it implies that upon breakdown, the complex can only be reassembled based on newly synthesized Metro. This would result in latency, which in turn could contribute to the temporal fidelity of the processes (Bachmann, 2010).

Given the strong dependency of Metro on DlgS97 it seemed plausible that loss of Metro would affect the size of receptor fields. A novel method was used that, once established, allowed measurement of the size of a high number of receptor fields. In this way it was found that the receptor fields were indeed enlarged at metro mutant NMJs. The specificity of this phenotype was further confirmed by transgenic rescue, which, however, remained incomplete. Although Metro-B clearly occurred as the principal isoform at NMJs, it is possible that the A- and/or C-variants are required at a low level or just temporarily to cover metro function at NMJs entirely. Compared with the enormous expansion of synaptic contacts associated with simultaneous depletion of DlgA and DlgS97, the effects on receptor field size detected in this study appear moderate. It is conceivable, however, that the reduced spacing between synaptic contacts that were frequently observed in the mutants represents a prestage toward the fusion of neighboring contacts (Bachmann, 2010).

While the size of receptor fields differed markedly between metro mutants and controls, no striking differences occurred in local GluRIID-specific fluorescence intensities. Moreover, despite the structural abnormalities, metro mutants displayed a rather normal profile of electrophysiological parameters. In particular, quantal currents were not significantly altered, consistent with the assumption that the composition and local density of GluRs remained normal and that transmitter release from a single vesicle does not saturate a normal-sized receptor field. Notably, normal mEJC amplitudes have been measured in case of a pronounced enlargement of synaptic contacts and increased mEJC amplitudes in strong dlg alleles have been assorted to enlarged synaptic vesicles rather than the size increment of synaptic contacts. The fact that the frequency of spontaneous release events and the evoked transmission remained unaffected is consistent with the virtual invariance in the number of active zones facing GluR fields at mutant NMJs and further implies that the presynaptic release machinery is largely intact in the metro mutants (Bachmann, 2010).

To date there is little evidence for the enrichment of Metro-like MAGUKs at synapses in the mammalian CNS, whereas mammalian homologs of Dlg and DLin-7 are prominent presynaptic and postsynaptic components of excitatory synapses. Reminiscent of the current observations, depletion of Veli in mice was found to cause a moderate increase in synaptic size, and yet this effect was assigned to its presynaptic interaction with liprin-α via Cask. Notably, there is no previous report on a close link between DLG-MAGUKs and Veli at synapses of CNS neurons, despite the presence of Cask as a potential linker protein. Nevertheless an association of MPP3 with SAP97 and Veli is implied by coimmunoprecipitations from rat brain. Moreover, MPP3 was found to bind to a serotonin receptor and to the CAM Necl-1/SynCAM3 at extrasynaptic sites. The current results thus lead to a proposal that the perisynaptic interplay of Metro, DlgS97, and DLin-7 represents a conserved mechanism that confers structural fidelity and stability onsynaptic systems during development and plasticity (Bachmann, 2010).

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).

Inscuteable regulates the Pins-Mud spindle orientation pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). This study investigated the mechanism of spindle orientation by the Insc-Pins complex. Previously, two Pins spindle orientation pathways were defined: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. The current study examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. It was found that the Insc-Pins complex requires Galphai for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. These results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud) (Mauser, 2012).

Spindle positioning is important in many physiological contexts. At a fundamental level, spindle orientation determines the placement of the resulting daughter cells in the developing tissue, which is important for correct morphogenesis and tissue organization. In other contexts, such as asymmetric cell division, spindle position ensures proper segregation of fate determinants and subsequent differentiation of daughter cells. This study examined the function of a protein thought to provide a 'passive' mark on the cortex for subsequent recruitment of the spindle orientation machinery. During neuroblast asymmetric cell division, Insc has been thought to mark the cortex based on the location of the Par polarity complex (Mauser, 2012).

Ectopic expression of Insc in cells that normally do not express the protein has revealed that it is sufficient to induce cell divisions oriented perpendicular to the tissue layer, reminiscent of neuroblast divisions. Expression of the mammalian ortholog of Inscuteable, mInsc, in epidermal progenitors has shown that this phenotype is not completely penetrant over time. Expression of mInsc leads to a transient re-orientation of mitotic spindles, in which mInsc and NuMA initially co-localize at the apical cortex. After prolonged expression, however, the epidermal progenitors return to dividing along the tissue polarity axis, a scheme in which mInsc and NuMA no longer co-localize. These results indicate that Insc and Mud can be decoupled from one another (Mauser, 2012).

This study examined the effect of Insc-Pins complex formation both in an induced polarity spindle orientation assay and in in vitro binding assays. The results indicate that Insc plays a more active role in spindle positioning than previously appreciated. Rather than passively coupling polarity and spindle positioning systems, Insc acts to regulate the activity of downstream Pins pathways. The Dlg pathway is unaffected by Inscuteable expression while the Mud pathway is inhibited by Insc binding (Mauser, 2012).

Recent work on the mammalian versions of these proteins explains the structural mechanism for competition between the Insc-Pins and Pins-Mud complexes. The binding sites on Pins for these two proteins overlap making binding mutually exclusive because of steric considerations. The observation of Insc dissociation of the Pins-Mud complex in Drosophila (this work) and mammalian proteins (LGN-NuMA) suggests that Insc regulation of Mud-binding is a highly conserved behavior (Mauser, 2012).

This competition between Mud and Insc for Pins binding is consistent with previous work done with a chimeric version of Inscuteable/Pins (Yu, 2000). This protein, in which the Pins TPR domain was replaced with the Inscuteable Ankyrin-repeat domain, bypasses the Insc-Pins recruitment step of apical complex formation. In these cells, the chimeric Insc-Pins protein was able to rescue apical/basal polarity and spindle orientation in metaphase pins mutant neuroblasts. As this protein lacks the Mud-binding TPR domain, Mud binding to Pins is not absolutely necessary for spindle alignment. Importantly, the PinsLINKER domain is still intact in the Insc-Pins fusion, implying that Dlg, not Mud, function is sufficient for partial activity, as observed in the S2 system (Mauser, 2012).

The Mud and Dlg pathways may play distinct roles in spindle positioning. The Dlg pathway, through the activity of the plus-end directed motor Khc73, may function to attach the cortex to the spindle through contacts with astral microtubules. In contrast, the Mud pathway, through the minus-end directed Dynein/Dynactin generates force to draw the centrosome towards the center of the cortical crescent. Fusion of the Pins TPR motifs, which recruit Mud, to Echinoid does not lead to spindle alignment, indicating that the Mud pathway is not sufficient for spindle alignment. The PinsLINKER domain does have partial activity on its own, however, and when placed in cis with the TPRs leads to full alignment. In this framework, the function of Insc may be temporal control, ensuring that microtubule attachment by the Dlg pathway occurs before the force generation pathway is activated (Mauser, 2012).

In the temporal model of Insc function, what might cause the transition from the Insc-Pins-Dlg complex, which mediates astral microtubule attachment, to the Mud-Pins-Dlg complex, which generates spindle pulling forces? By early prophase, Inscuteable recruits Pins and Gαi to the apical cortex. During this phase of the cell cycle, Mud is localized to the nucleus in high concentration. Apically-localized Pins binds Dlg, creating an apical target for astral microtubules. During early phases of mitosis, Inscuteable would serve to inhibit binding of low concentrations of cytoplasmic Mud to the Pins TPRs to prevent spurious activation of microtubule shortening pathways. After nuclear envelope breakdown, Mud enters the cytoplasm in greater concentrations and could then act to compete with Insc for binding to Pins, allowing Pins output to be directed into microtubule-shortening pathways (see Proposed model for Inscuteable regulation of spindle orientation). Future work will be directed towards testing additional aspects of this model (Mauser, 2012).

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