InteractiveFly: GeneBrief

CIN85 and CD2AP orthologue: Biological Overview | References

BIOLOGICAL OVERVIEW

Developing tissues require cells to undergo intricate processes to shift into appropriate niches. This requires a functional connection between adhesion-mediating events at the cell surface and a cytoskeletal reorganization to permit directed movement. A small number of proteins are proposed to link these processes. This study identifies one candidate, Cindr, the sole Drosophila melanogaster member of the CD2AP/CIN85 family (this family has been previously implicated in a variety of processes). Using Drosophila retina, it was demonstrated that Cindr links cell surface junctions (E-cadherin) and adhesion (Roughest) with multiple components of the actin cytoskeleton. Reducing cindr activity leads to defects in local cell movement and, consequently, tissue patterning and cell death. Cindr activity is required for normal localization of Drosophila E-cadherin and Roughest, and this study shows additional physical and functional links to multiple components of the actin cytoskeleton, including the actin-capping proteins capping protein alpha and capping protein beta. Together, these data demonstrate that Cindr is involved in dynamic cell rearrangement in an emerging epithelium (Johnson, 2008).

By recruiting proteins into complexes, adaptor proteins create nodes of regulation and activity. The founding member of the CD2AP/CIN85 family of adaptor proteins was initially isolated in a yeast interaction screen as a binding partner of the T cell receptor CD2 (Dustin, 1998), independently from the kidney (MET-1; Lehtonen, 2000) and as a ligand for p130Cas (Kirsch, 1999). The homologue CIN85 was identified as a partner of the E3 ubiquitin ligase Cbl (Take, 2000) and separately as SETA and Ruk (Bogler, 2000; Gout, 2000). Many roles have been ascribed to the CD2AP/CIN85 family but its function in situ remains poorly understood. This study examine the sole Drosophila melanogaster CD2AP/CIN85 orthologue, cindr (Johnson, 2008).

The phenotype of CD2AP knockout mice is chiefly one of tissue degeneration: cardiac hypertrophy, splenic and thymic atrophy, glomerular sclerosis, and a loss of podocyte foot processes (Shih, 1999). The CD2AP/CIN85 family is primarily proposed to function in endocytosis to down-regulate receptor tyrosine kinase activity. This model arises from coimmunoprecipitation and interaction experiments that have identified a wealth of CD2AP/CIN85 interactors, colocalization studies performed in culture or tissue, and in vitro assays. CIN85 constitutively associates with endophilin and, on growth factor stimulation, complexes with Cbl to mediate receptor down-regulation. Furthermore, interactions between CD2AP/CIN85 and other trafficking proteins have been described including AP-2, Dab2, ALIX, and ESCRT-1 (Johnson, 2008 and references therein).

Recent work has also suggested a relationship between CD2AP/CIN85 proteins and actin. They have been detected in actin-rich regions of podocytes and cultured cells and have been found to bind actin in vitro to promote actin bundling. CD2AP has also been reported to bind the actin-capping protein CPα/β dimer and inhibit its function in vitro and anillin (see Drosophila Scraps) at the actin-rich cleavage furrow. CD2AP activity is required for migration of rat gastric mucosal cells and polarization of the cytoskeleton during T cell receptor activation. The role and mechanism by which CD2AP/CIN85 regulates cytoskeletal dynamics within an epithelium in situ remains unclear (Johnson, 2008).

Furthermore, the CD2AP/CIN85 family has also been reported to bind the adhesion molecules E-cadherin and nephrin. Nephrin and NEPH-1 form the backbone of the slit diaphragm, a specialized junction that traverses podocyte foot processes in the mammalian kidney. Direct interactions between CD2AP, nephrin, and podocin and between CD2AP and the podocyte-specific actin-bundling protein synaptopodin are essential for slit diaphragm integrity. In addition, a protein complex containing nephrin, cadherin, p120-catenin, ZO-1, and CD2AP has been isolated from Madin-Darby canine kidney cells and mouse glomerular lysates. Collectively, these data suggest that CD2AP may have a role in anchoring junctions to the cytoskeleton or in regulating actin dynamics at this important intersection (Johnson, 2008).

The challenge remains to understand how different roles of CD2AP/CIN85 are integrated in the organism, which interactors are recruited into CD2AP/CIN85 complexes, and how these are regulated. This study shows that targeted reduction of cindr in the pupal fly eye resulted in defects in overall patterning due to aberrant local cell movements. These defects were linked to misregulation of actin dynamics and mislocalization of Drosophila E-Cadherin (DE-Cad) and the fly NEPH-1 orthologue Roughest (Rst) which, with its binding partner Hibris (Hbs; a Drosophila Nephrin orthologue), is a central mediator of cell-cell adhesion in the pupal retina. Cindr was lined functionally and physically to orthologues of capping protein alpha (Cpa) and capping protein beta (Cpb), and this study further explored the role of Cindr in modulating the actin cytoskeleton during eye maturation. The data support a primary role for cindr in linking junction and actin regulation and help account for many of the phenotypes ascribed to mutations in mammalian CD2AP/CIN85 (Johnson, 2008).

The evidence indicates that Cindr provides a functional link between dynamically regulated surface adhesion and the cytoskeletal changes required for normal pupal eye patterning. Loss of cindr activity leads to misplacement of retinal support cells, which adopt shapes uncharacteristic for their niche in the retinal field. The reasons for this became apparent when cell behavior was examined in live tissue. Reducing cindr prevents 1° cells from maintaining enwrapment of the cone cells; instead, cindr 1° cells are unable to firmly establish this niche and frequently retract, allowing neighboring interommatidial precursor cells (IPCs) to have direct contact with the cone cells. Similar instability was observed in the remaining IPCs fated to establish the 2°/3° hexagonal lattice. Histology demonstrated further changes both to AJ components and the actin cytoskeleton. Although additional roles for Cindr such as regulation of endocytosis cannot be ruled out, no evidence was observed for such a role during cell rearrangements (Johnson, 2008).

In wild-type tissue, Hbs and Rst are localized exclusively to 1°-IPC interfaces during IPC patterning; heterophilic interactions between these molecules are thought to direct the rearrangement of IPCs into single rows around each ommatidium. In cindr-IR tissue (an RNAi inverted repeat knockout), a mislocalization was observed of Rst to the entire IPC circumference. Such mislocalization would impede the generation of a preferential adhesive force, disrupting the direction or flow of cell movement and subsequent patterning. Irregularities were detected in the localization of DE-Cad around the circumference of retinal cells when Cindr was reduced. This presumably resulted in uneven or unreliable junctional stability that further destabilized the dynamic switching of cell positions. Genetic interactions with the loci for rst, hbs, and shg confirmed that these loci cooperate with cindr during patterning (Johnson, 2008).

It was also demonstrated that the actin cytoskeleton is dynamically remodeled during pupal eye patterning and that reducing cindr activity leads to a change in the details of cytoskeletal dynamics. In wild-type tissue, polymerized actin was initially detected almost exclusively at AJs of pre-1° cells and IPCs. These actin rings intensify as cells became rearranged and then, remarkably, once patterning is established, membrane-associated F-actin strongly diminishes. The functional significance of these changes is likely linked to concurrent modification of Rst- and AJ-mediated adhesion. For example, the levels of both DE-Cad and Armadillo (β-catenin) decrease between IPCs as they are rearranged, which would serve to facilitate Rst-mediated IPC movements toward 1° cells. Data indicate that the actin cytoskeleton is coordinately reinforced at AJs as adhesion is weakened. This may serve to maintain the surface integrity of the retinal cells while junctional strength decreases and the tissue is remodeled. Once patterning is achieved, the BMP receptor Thickvein then acts to restrengthen AJs; the reduction of F-actin may reflect the reduced need for a dense actin ring. Throughout this process, Cindr acts as a pivotal regulator to coordinate AJ modification and actin polymerization (Johnson, 2008).

Several data presented in this paper suggest that Cindr acts directly to regulate actin. First, the intensity and distribution of cytoplasmic Cindr puncta in retinal cells tracks that of F-actin in IPCs during development. Second, the striking dynamics of F-actin polymerization are lost, presumably helping account for their abnormal cell movements. Third, strong genetic interactions were observed between cindr-IR and multiple components of the actin regulatory machinery. Fourth, the two capping protein subunits Cpa and Cpb coimmunoprecipitated together with Cindr from Drosophila embryos. And, finally, several phenotypes are shared between tissue mutant for cindr-IR and tissue mutant for cpa or cpb: pupal eye mispatterning, gaps in the distribution of DE-Cad around the circumference of retinal cells, bristle malformation, and tissue degeneration (Johnson, 2008).

These results emphasize the important role of the actin cytoskeleton in regulating or maintaining AJ integrity. However, the data also argue that Cindr regulates the localization of transmembrane adhesion proteins at least in part independently of the cytoskeleton: reducing the genetic component of actin regulators enhances the patterning defects of cindr-IR but not the disruption to DE-Cad, and ectopic Arp66B rescues cindr-IR mispatterning but does not rescue aberrant localization of Rst. In the absence of Cindr, miscoordination of the actin machinery together with aberrant localization of junctional complexes is likely the underlying cause of tissue mispatterning during development. Similarly, deregulation of actin and junction instability is apparently cell lethal in more mature tissue, eventually leading to degeneration of mutant tissue. This may be analogous to the degeneration of mammalian podocytes that has been associated with mutations in CD2AP (Schiffer, 2004; Peters, 2006; Woroniecki, 2006). How Cindr itself is regulated during development remains an open question (Johnson, 2008).

Cindr interacts with anillin to control cytokinesis in Drosophila

Cytokinesis, the final step of cell division, conventionally proceeds to cell separation by abscission, or complete cytokinesis, but may in certain tissues be incomplete, yielding daughter cells that are interconnected in syncytia by stable intercellular bridges. The mechanisms that determine complete versus incomplete cytokinesis are not known. This study reports a novel in vivo role of the Drosophila CD2AP/CIN85 ortholog Cindr in both complete and incomplete cytokinesis. Evidence is shown for the presence of persistent intercellular bridges in the major larval imaginal disc epithelia. During conventional division of both cultured and embryonic cells, Cindr localizes to cleavage furrows, intercellular bridges, and midbodies. Moreover, in cells undergoing incomplete cytokinesis in the female germline and the somatic ovarian follicle cell and larval imaginal disc epithelia, Cindr localizes to arrested cleavage furrows and stable intercellular bridges, respectively. In these structures, Cindr colocalizes with the essential cytokinesis regulator Anillin. Cindr interacts with Anillin, and depletion of either Cindr or Anillin gives rise to binucleate cells and fewer intercellular bridges in vivo. It is proposed that Cindr and Anillin cooperate to promote intercellular bridge stability during incomplete cytokinesis in Drosophila (Haglund, 2010).

Because the in vivo functions of the evolutionarily conserved CD2AP (CD2-associated protein) and SH3KBP1 (SH3-domain kinase binding protein 1)/CIN85 (Cbl-interacting protein of 85 kDa) family of multiadaptor proteins remain incompletely understood, this study investigated the sole Drosophila CD2AP/CIN85 ortholog, Cindr (CG31012). Cindr expression and subcellular localization were systematically analyzed at various stages of Drosophila development and in cultured Drosophila S2 cells by using a Cindr antibody. One or both of the two largest Cindr isoforms were detected during oogenesis, embryogenesis, larval development, in adult flies, and in S2 cells by western blot and immunocytochemistry (Haglund, 2010).

In cultured Drosophila cells undergoing cytokinesis, Cindr localized to cleavage furrows, intercellular bridges, and the midbody. After abscission, Cindr could further be detected in midbody remnants. The essential cytokinesis regulator Anillin has previously been shown to localize in a similar manner during cell division, so its putative colocalization with Cindr was analyzed, and indeed the two proteins colocalize throughout cytokinesis. Cindr also colocalizes with actin at the inner rim of the intercellular bridge. To evaluate the functional importance of Cindr during cytokinesis, the cellular phenotypes were studied after Cindr depletion or overexpression. Effective reduction of cindr by RNA interference-mediated gene silencing (RNAi) resulted in an about 3-fold increase in the number of binucleate S2 cells, as compared to cells treated with control dsRNA. During live imaging experiments, it was observed that Cindr-depleted S2 cells displayed a delay in abscission and cleavage furrow regression. Consistently, overexpression of Cindr in human cells caused a dominant-negative effect, with a clear increase in the fraction of binucleate cells and cells in late cytokinesis (Haglund, 2010).

Given the high expression of Cindr in Drosophila embryos, whether Cindr may be involved in conventional cell division during embryogenesis was investigated. In Drosophila embryonic mitotic domains, GFP-tagged endogenous Cindr was found to colocalize with Anillin at contractile rings during early cytokinesis, as well as at midbodies during late cytokinesis. Taken together, these data implicate a role for Cindr in cytokinesis during conventional cell divisions in cultured Drosophila cells and in the embryo (Haglund, 2010).

During oogenesis, Cindr was found expressed in both germ cells and the surrounding somatic follicle cell epithelium. In germ cells, Cindr localizes strongly to small ring-shaped structures in the anterior part of the germarium, which is organized as follows. In region 1, germ stem cell daughter cells, called cystoblasts, undergo four mitotic divisions by incomplete cytokinesis, leading to the formation of a cluster of 16 interconnected germ cells. The 16-cell cluster moves into region 2a, where the arrested cleavage furrows begin maturing into intercellular bridges called ring canals. In order to determine the identity of the Cindr-postive rings, costainings were performed with known cleavage furrow and ring canal components. It was found that Cindr colocalizes with Anillin (CG2092) and Pav-Klp (CG1258), two constituents of arrested cleavage furrows, in region 1 of the germarium. In this area, Cindr localizes strongly to cleavage furrows surrounding fusomes, organelles that branch out in the germ cell cysts during the mitotic divisions. Interestingly, in region 2a, upon fusome breakdown and recruitment of the ring canal markers hts-RC (CG9325) and phospho-tyrosine (pTyr), Cindr disappeared from cleavage furrows. After this point Cindr could not be detected at growing ring canals during the rest of oogenesis, which is interesting, because other known components remain for longer time (e.g., Anillin until stage 3; Pav-Klp, pTyr, and hts-RC throughout oogenesis). It is therefore concluded that Cindr marks arrested cleavage furrows in mitotically active and newly formed germ cell clusters, but disappears from growing ring canals, suggesting a role in cleavage furrow formation and/or arrest, but not in ring canal growth (Haglund, 2010).

In Drosophila, each individual egg chamber is formed through encapsulation of the germ cells by an epithelium of somatic follicle cells, starting in region 2a of the germarium. Interestingly, whereas Cindr disappears from arrested cleavage furrows in the female germline, it abruptly appears in dot-like structures in the forming egg chamber follicle cell epithelium in region 2a and remains in such structures throughout oogenesis. These are present throughout the follicle cell epithelium and consistently localize apically or at the level of the nuclei and at the borders between adjacent follicle cells. They are localized basally of E-cadherin-containing adherens junctions and do not overlap with markers for early (Rab5), late (Rab7), or recycling (Rab11 or Rab4) endosomes. In light of the implication for Cindr in cytokinesis, reports were found that describe incomplete cytokinesis in the follicle cell epithelium, giving rise to follicle cell clusters with apically localized stable intercellular bridges. Indeed, Cindr clearly colocalizes with both Anillin and Pav-Klp, two known stable intercellular bridge components, at intercellular bridges throughout oogenesis. Consistently, ultrastructural investigation by immunoelectron microscopy also demonstrated the presence of Cindr at follicle cell intercellular bridges, where it localizes mainly at their inner rim in transverse, longitudinal, and oblique sections (Haglund, 2010).

Having identified Cindr at stable intercellular bridges during oogenesis, it was next asked whether Cindr would be present at stable intercellular bridges during other stages of Drosophila development. For this purpose, larval imaginal disc epithelia, out of which the wing and leg discs have previously been shown to contain stable intercellular bridges, were examined. Indeed, Cindr localizes to distinct structures present at cell borders that colocalize with Anillin throughout wing and leg discs and is also detected in similar structures in eye and antennal discs. These were confirmed to be intercellular bridges by electron microscopy and were ultrastructurally similar to follicle cell intercellular bridges. Cindr localizes mainly to the inner rim also in wing disc intercellular bridges. These data indicate that all larval imaginal discs examined contain persistent intercellular bridges and that Cindr represents a stable intercellular bridge component during Drosophila development (Haglund, 2010).

Additionally, also in the larval brain, it was found that Cindr localizes at structures present at cell borders that colocalize with Anillin and bridges displaying the characteristic intercellular bridge ultrastructure. Indeed, the larval brain has been suggested to contain stable intercellular bridges, but this remains an area of investigation. Interestingly, intercellular bridges of dividing neuroblasts display a more variable morphology. Consistent with the data in, Cindr moreover localizes to contractile rings in the larval imaginal discs and brain (Haglund, 2010).

The role of Cindr at stable intercellular bridges was examined in vivo. RNAi-mediated cindr depletion in somatic follicle cells results in a significant increase in the number of binucleate cells, as compared to control egg chambers, which contain essentially only mononucleate cells. Similarly, RNAi-mediated knockdown of anillin dramatically increases the number of binucleate follicle cells. Interestingly, the increase in binucleate cells is accompanied by a significant decrease in the average number of follicle cell intercellular bridges per nucleus, whereas the number of bridges compared to the number of cells was essentially unchanged. The higher levels of binucleate cells in Anillin- compared to Cindr-depleted epithelia may be accounted for by a faster depletion of Anillin compared to Cindr protein levels. In Anillin-depleted epithelia, binucleate follicle cells were indeed detected in early egg chamber stages arising from defective cell divisions, whereas binucleate Cindr-depleted cells were detected only at late egg chamber stages after the cease of follicle cell divisions in stage 6. These findings indicate a role for Cindr in stabilizing intercellular bridges in the follicle epithelium during oocyte growth (Haglund, 2010).

To address the molecular mechanisms by which Cindr may act at intercellular bridges, it was finally asked whether Cindr and Anillin may interact, given their similar localization pattern throughout Drosophila development and the presence of a Px(P/A)xxR motif (PLARLR, amino acids 145-150) in Anillin. Such motifs interact with the SH3 domains of mammalian CD2AP/CIN85 family members (Kowanetz, 2003; Kurakin, 2003) and in fact CD2AP interacts with such a motif in human Anillin (Monzo, 2005). The SH3 domains of Cindr show high sequence homology with the SH3 domains of CD2AP/CIN85, and in pull-down experiments via four GST-tagged parts of Anillin, Cindr indeed associated with the most N-terminal (GST-A1) PLARLR-containing region of Anillin. Importantly, mutation of the consensus arginine to alanine (R150A) in the motif abolished the interaction with Cindr. An interaction was detected between Cindr and the C-terminal (GST-A4) region of Anillin, which is interesting because CD2AP and human Anillin were reported to associate only via an N-terminal Px(P/A)xxR motif (Monzo, 2005). Taken together, these data indicate that Cindr interacts with Drosophila Anillin via two distinct sites: the PLARLR motif via its SH3 domains and an additional site in the C terminus of Anillin (Haglund, 2010).

In summary, this study identified a novel in vivo role of Cindr as a general component of stable intercellular bridges during Drosophila development. Cindr localizes to arrested cleavage furrows in the female germline and to somatic stable intercellular bridges in the follicle cell and larval imaginal disc epithelia. Only a limited number of such components have previously been described, including Anillin, Pav-Klp, and Mucin-D, and interestingly Cindr interacts with Anillin and colocalizes with it at intercellular bridges throughout Drosophila development. Given the increase in binucleate cells and decrease in intercellular bridge numbers upon Cindr and Anillin depletions in follicle cells, it is proposed that Cindr and Anillin interact to promote intercellular bridge stability in tissues that undergo incomplete cytokinesis. One mechanism may be via stabilization of actin at the inner rim of the bridge, as indicated by the fact that Cindr/CD2AP/CIN85 and Anillin are well-established actin regulators and Cindr indeed localizes to the inner rim of stable follicle cell intercellular bridges that is lined with actin filaments. During conventional cell divisions, Cindr and Anillin may play a similar role, as shown by the fact that Cindr colocalizes with actin in the bridge, and Cindr was also found participating in the final abscission step, like CD2AP (Monzo, 2005). It is noted that the CD2AP/CIN85/Cindr family of proteins may function as scaffold proteins during cytokinesis because of their ability to oligomerize and to associate with Anillin, and possibly septins, MgcRacGAP/RacGAP50C, and MKLP1/Pav-Klp (Monzo, 2005; Havrylov, 2009). Finally, the presence was shown of persistent intercellular bridges in the major larval imaginal disc epithelia, suggesting that their development may require coordinated intercellular communication within cellular syncytia. A better understanding of complete and incomplete cytokinesis in vivo, to which these data contribute, not only expands the knowledge about these processes during development, but may also provide clues to how accurate cytokinesis regulation is achieved to prevent cell division defects associated with cancer development (Haglund, 2010).

I-BAR protein antagonism of endocytosis mediates directional sensing during guided cell migration; Cindr/Dcortactin complex acts to promote endocytosis in opposition to DMIM

Although directed cellular migration facilitates the coordinated movement of cells during development and repair, the mechanisms regulating such migration remain poorly understood. Missing-in-metastasis (MIM) is a defining member of the inverse Bin/Amphiphysin/Rvs domain (I-BAR) subfamily of lipid binding, cytoskeletal regulators whose levels are altered in a number of cancers. This study provides the first genetic evidence that an I-BAR protein regulates directed cell migration in vivo. Drosophila MIM (dmim) is involved in Drosophila border cell migration, with loss of dmim function resulting in a lack of directional movement by the border cell cluster. In vivo endocytosis assays combined with genetic analyses demonstrate that the dmim product regulates directed cell movement by inhibiting endocytosis and antagonizing the activities of the CD2-associated protein/Cortactin complex in these cells. These studies demonstrate that DMIM antagonizes pro-endocytic components to facilitate polarity and localized guidance cue sensing during directional cell migration (Quinones, 2010).

Although MIM is one of the founding members of the I-BAR subfamily of lipid-binding BAR domain proteins, MIM functions have not yet been delineated in vivo, although studies have been conducted with IRSp53 knockout mice. Because the BAR subfamilies are represented in Dipterans, Drosophila was used to better understand the in vivo roles of these important proteins. Drosophila MIM (DMIM, GenBank/EMBL/DDBJ accession no. CG33558) shares strong identity in the I-BAR and WH2 domains with its vertebrate counterpart and ABBA, and less similarity with the I-BAR family member IRS53. Previous structural studies have demonstrated that MIM binds and bends phospholipid-containing membranes (Mattila, 2007). To determine whether DMIM retains similar properties, the lipid-binding properties of purified fly and human MIM I-BAR domains were compared. Both human and DMIM proteins bound to PI(4,5)P2-containing vesicles, whereas the DMIM mutant I-BAR domain failed to bind to these vesicles. The DMIM I-BAR domain was also able to bind to PI(3,4,5)P3-containing vesicles, whereas the human I-BAR was not. This binding was quite specific as mutation of the conserved lipid-binding motifs completely abrogated lipid binding (Mattila, 2007). Further, fly or human MIM overexpression in Drosophila S2 cells results in the same dramatic cytoskeletal reorientation and extensions, indicating that DMIM functions similarly to its vertebrate orthologue (Quinones, 2010).

BAR domain proteins function through regulating the assembly of protein complexes at membrane surfaces. To understand mechanistically how dmim regulates border cell guidance sensing, attempts were made to discover which proteins, previously known to regulate border cell migration, interact with the scaffolding portion of DMIM. In GST pull-downs and coimmunoprecipitation assays, DMIM formed protein complexes with Drosophila Cortactin (Dcortactin), but not with Rac1, Cdc42, several RPTPs, and the BAR domain protein amphiphysin. Dcortactin is a major Src phosphorylation target downstream of growth factor signaling and is part of the pro-endocytic complex comprised of the BAR domain protein endophilin and its binding partner CD2AP. The endophilin/CD2AP/cortactin complex helps to provide force for endocytosis and scission of the early endosome by inducing local actin polymerization. Like their vertebrate counterparts, DMIM interacts with Dcortactin through the proline-rich domain. Both MIM and cortactin also affect the directional migration of vertebrate cells in culture. Cells treated with siRNA against either MIM or cortactin display a reduction in cell motility in response of EGF. Simultaneous knockdown of both MIM and cortactin results in normal cell motility, suggesting that these two proteins work antagonistically. The loss of both proteins resulting in a wild-type phenotype also suggests that there is redundancy with another set of proteins with a similar function (Quinones, 2010).

The functional relationship between DMIM and Dcortactin was determined through the examination of border cell phenotypes in double mutants. Previous analysis of dcortactin mutants identified a mild border cell migration phenotype. Surprisingly, it was found that dmim; dcortactin double mutant border cells do not enhance the mutant phenotype, but rather display a wild-type phenotype. This suggests that DMIM acts antagonistically to Dcortactin to regulate directional cell migration in border cells. Because cortactin is recruited to endophilin via the adapter CD2AP as shown in mammalian cells, if the CD2AP/cortactin complex functions antagonistically to MIM in guided migration, then CD2AP mutant border cells should phenocopy dcortactin mutants, and also rescue the dmim mutant border cell cluster migration phenotype. The effects of mutations in the single fly CD2AP gene, cindr, on border cell migration were assayed. Previous studies have indicated that two cindrⁱ RNAi lines have strong effects on photoreceptor cell morphology (Johnson, 2008). These two independent cindrⁱ lines were expressed in border cells, where they demonstrated a moderate border cell migration phenotype, mimicking that of dcortactin mutants. Like dcortactin, cindrⁱ expression in a dmim mutant background partially restores the ability of border cell clusters to migrate, increasing the fraction of clusters that have migrated more than half the distance to the oocyte. It was also found that the loss of dcortactin or cindr results in a mild reduction in the uptake of the FM4-64 dye in border cells. As predicted, knockdown of dmim in combination with either dcortactin or cindr results in a partial rescue of the dmim increase in dye uptake. From these data, it is concluded that the Cindr/Dcortactin complex acts to promote endocytosis in opposition to that of DMIM (Quinones, 2010).

The genetic data suggest guided cell migration is regulated in part from competition for cortactin between the pro-endocytic BAR domain complex endophilin/CD2AP, and the anti-endocytic BAR MIM. To confirm this model, MIM antagonism of cortactin and other endocytic regulators was examined in cultured vertebrate cells to determine whether double mutants would also restore normal receptor uptake in vitro. As predicted, whereas treatment with siRNA against cortactin decreases the levels of EGF receptor endocytosis, siRNA-mediated knockdown of both MIM and cortactin restores receptor uptake levels back to those in the control-treated cells. Consistent with the genetic data, combination knockdowns of MIM with CD2AP also restore EGF uptake levels to control values. Whether MIM antagonism extended to other endocytic regulators was further tested. Knockdown of endophilin leads to a reduction in endocytosis with the combined knockdown of endophilin and MIM demonstrating a reproducible trend in elevation of endocytosis over endophilin alone. Knockdown of Cbl, clathrin, or dynamin results in decreased endocytosis and is not rescued in combination knockdowns with MIM, suggesting that MIM acts uniquely through CD2AP/cortactin. These results, along with previous studies, suggest that endophilin could be interacting with additional proteins when CD2AP and cortactin are unavailable during EGFR endocytosis (Quinones, 2010).

Because circular dorsal ruffles (CDRs) and cortactin have been shown to be an alternative endocytosis pathway for the internalization of growth factor receptors, MIM and cortactin’s role in CDR formation was investigated. In vivo, no evidence was found for CDR formation in migrating border cells. In cultured cells, it was observed that the loss of MIM resulted in a reduction of the CDR formation in response to both PDGF-BB and EGF. However, in contrast to border cells, EGF endocytosis in MIM and cortactin double mutant cells failed to rescue CDR formation. These results suggest that the effects seen in border cell migration are not a result of increased internalization of the growth factor receptors due to dorsal ruffle formation (Quinones, 2010).

The kinetics of cortactin association with guidance cue addition was examined by performing cortactin immunoprecipitations in mouse embryonic fibroblasts upon the addition of EGF ligand. Under serum-free conditions, cortactin was found to associate with both endophilin/CD2AP and MIM complexes, but within 5 min after EGF addition, cortactin begins to disassociate from the CD2AP/endophilin complex. In the absence of MIM, cortactin fails to dissociate from CD2AP after EGF addition, resulting in an increase of the amount and duration of endophilin/CD2AP in complex with cortactin. The duration of cortactin association mirrors that of the persistent pERK1/2 immunoreactivity. This work and other studies indicate that both CD2AP and MIM associate through their proline-rich domains with cortactin’s SH3 domain. Indeed, in vitro-translated CD2AP readily associates with the purified SH3 domain of cortactin. Increasing concentrations of purified MIM abrogate the binding of cortactin to CD2AP, supporting a direct competition between MIM and CD2AP for cortactin binding. MIM lacking its proline-rich domain (MIM 1-277) fails to compete with CD2AP, providing additional support for the idea that MIM antagonizes the ability of cortactin to associate with the pro-endocytic CD2AP/endophilin complex. Stable GFP-tagged MIM and endophilin lines were used to examine cortactin association with each of the proteins at different EGF ligand concentrations over time. The antagonism between MIM and CD2AP/endophilin for cortactin binding could be explained by the prolonged association between cortactin and MIM, which persists longer after stimulation with EGF ligand than the association between endophilin and cortactin, even at different ligand concentrations (Quinones, 2010).

cindr, the Drosophila homolog of the CD2AP Alzheimer's disease risk gene, is required for synaptic transmission and proteostasis

The Alzheimer's disease (AD) susceptibility gene, CD2-associated protein (CD2AP), encodes an actin binding adaptor protein, but its function in the nervous system is largely unknown. Loss of the Drosophila ortholog cindr enhances neurotoxicity of human Tau, which forms neurofibrillary tangle pathology in AD. Cindr is expressed in neurons and present at synaptic terminals. cindr mutants show impairments in synapse maturation and both synaptic vesicle recycling and release. Cindr associates and genetically interacts with 14-3-3zeta, regulates the ubiquitin-proteasome system, and affects turnover of Synapsin and the plasma membrane calcium ATPase (PMCA). Loss of cindr elevates PMCA levels and reduces cytosolic calcium. Studies of Cd2ap null mice support a conserved role in synaptic proteostasis, and CD2AP protein levels are inversely related to Synapsin abundance in human postmortem brains. These results reveal CD2AP neuronal requirements with relevance to AD susceptibility, including for proteostasis, calcium handling, and synaptic structure and function (Ojelade, 2019).

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disorder that is estimated to affect 13 million people in the United States by 2050 (Alzheimer's Association). At autopsy, AD is characterized by extracellular neuritic plaques and intraneuronal neurofibrillary tangles, predominantly composed of misfolded and aggregated β-amyloid (Aβ) peptide and the microtubule-associated protein tau (MAPT or Tau), respectively. Substantial evidence indicates that AD pathology disrupts synaptic function at an early stage, and synapse loss is strongly associated with clinical progression. Human genome-wide association studies (GWASs) have discovered nearly 30 common variants associated with AD risk; however, most of these newly implicated genes remain poorly studied, especially in the nervous system. Prior work leveraged a human MAPT transgenic model in the fruit fly, Drosophila melanogaster, to identify AD susceptibility gene candidates that interact with Tau-mediated mechanisms. Among the findings, knockdown of cindr, the ortholog of the AD risk gene CD2-associated protein (CD2AP), enhanced Tau-induced neurodegeneration (Ojelade, 2019).

Before its identification from AD GWASs, loss-of-function mutations in CD2AP were identified as a rare cause of familial, autosomal dominant, focal segmental glomerulosclerosis, and progressive renal dysfunction has been recapitulated in mice hetero- or homozygous for a Cd2ap knockout allele. CD2AP encodes an adaptor protein that contains three SH3 motifs, a proline-rich region, and an actin binding domain. In the kidney, CD2AP is required to maintain structural integrity of the slit diaphragm, the specialized junctions between the foot processes of glomerular podocytes. Additional studies support a role for CD2AP in cytoskeletal anchoring of adhesion complexes and regulation of membrane localization for cell surface receptors via endocytosis. Several other AD susceptibility loci harbor genes with links to cell adhesion (CASS4, FERMT2, and PTK2B) and endocytosis (BIN1, PICALM, and RIN3). Nevertheless, the function of CD2AP in the adult nervous system, or its mechanism in neurological disorders, remains largely unknown. Studies in mice have connected CD2AP to both axonal growth (Harrison, 2016) and maintenance of blood-brain barrier integrity (Cochran, 2015). CD2AP has also been implicated in amyloid precursor protein trafficking and processing in cultured neurons (Ubelmann, 2017), although Cd2ap loss of function did not affect total Aβ levels or amyloid plaque burden in AD mouse models (Ojelade, 2019).

Besides suggesting potential interactions with Tau (Shulman, 2014), investigations in the fruit fly have implicated cindr with conserved roles in nephrocytes, retinal development, and oogenesis. This study has used Drosophila to elucidate the role of cindr in the nervous system, confirming a requirement for synaptic vesicle endocytosis and revealing requirements for ubiquitin-proteasome-mediated protein turnover and presynaptic Ca2+ homeostasis. Moreover, evidence is presented that the role of CD2AP in synaptic proteostasis is conserved in the mammalian brain and may contribute to vulnerability for Tau-mediated neurotoxic mechanisms in AD (Ojelade, 2019).

CD2AP is an AD susceptibility gene, but to date, its function in the adult nervous system is poorly defined. A cross-species experimental strategy implicates Cindr in synaptic biology and defines potential links to AD pathogenic mechanisms. First, Cindr was shown to be present at the synapse and interacts with other presynaptic proteins. Second, cindr loss of function causes impairments in synapse maturation and synaptic vesicle recycling and release. Third, the synaptic structural and functional defects are directly linked to requirements for Cindr in UPS-mediated protein turnover and Ca2+ homeostasis. Lastly, this study shows that Cindr functions with 14-3-3ζ to regulate proteasome activity, and this role is likely conserved in mouse and human brains. The results therefore enhance understanding of cindr/CD2AP and suggest potential mechanisms for its contribution to AD susceptibility (Ojelade, 2019).

Synaptic loss is strongly associated with cognitive decline in AD, and both Aβ and Tau have been linked to synaptic dysfunction. In Drosophila, loss of cindr causes a combination of endocytic and exocytic synaptic defects. Prior studies have implicated cindr/CD2AP in endocytosis in other contexts. Consistent with this, loss of cindr caused a reduced synaptic vesicle density at the NMJ and depression of synaptic responses during high-frequency stimulation, reminiscent of other genes implicated in endocytosis. Notable among these is like-AP180, which is homologous to another AD susceptibility gene PICALM. Loss of cindr caused reduced evoked potentials and enhanced facilitation, which both result from diminished presynaptic Ca2+, possibly because of increased PMCA levels. Altered Ca2+ handling has also been linked to AD pathogenesis, in which PMCA has been implicated. PMCA is a Ca2+ efflux pump with an established requirement for neuronal Ca2+ homeostasis. It was possible to rescue cindr phenotypes either directly through increasing extracellular [Ca2+] or indirectly by manipulating buffer pH to suppress PMCA activity; however, the possibility cannot be excluded that other Ca2+ channels or transporters may participate. Overall, this study confirms a role for Cindr in synaptic vesicle endocytosis and recycling and highlight an unexpected requirement for presynaptic Ca2+ homeostasis and resulting vesicle exocytosis and release (Ojelade, 2019).

Actin cytoskeletal dynamics have been implicated in synaptic vesicle recycling and plasticity, including at the Drosophila NMJ. Cindr is an actin binding protein, and this study found that cindr loss of function stabilizes F-actin in the Drosophila nervous system, consistent with prior studies in both flies and human cells. Changes in actin could potentially influence many functions and phenotypes addressed in this study. Pharmacological manipulations that disrupt actin have been reported to attenuate ghost bouton formation and impair synaptic vesicle endocytosis. Based on biochemical studies, actin may also bind and regulate human PMCA to modulate Ca2+ homeostasis. In mammals, Synapsins have been shown to tether synaptic vesicles to the actin cytoskeleton, thereby regulating the vesicle reserve pool and activity-dependent, presynaptic plasticity. In Drosophila, mutations in synapsin attenuate the formation of NMJ ghost boutons following stimulation. While loss of cindr leads to an increase in Synapsin, this is unlikely to explain the accompanying ghost bouton formation, because Synapsin overexpression does not cause this phenotype. Although elevations were identified in Synapsin and other presynaptic proteins in Cd2ap null mice, it will be important in future work to directly confirm a conserved role for Cd2ap in the function of mammalian central synapses. In the rat peripheral nervous system, Cd2ap was implicated to participate in signal transduction during axonal outgrowth accompanying collateral sprouting, an important contributor to neuronal plasticity (Ojelade, 2019).

dhat reduced CD2AP expression is protective for AD; however, this interpretation contradicts experimental findings that cindr/CD2AP (1) is required for neuronal homeostasis and synaptic function and (2) knockdown enhances Tau-induced neurodegeneration. Moreover, the true causal variant or variants at the CD2AP locus remain unknown. A sequencing study identified several AD cases harboring a rare, CD2AP coding variant (K633R) predicted to be damaging. Although this finding requires replication, it underscores the importance of fine mapping to definitively identify the responsible allelic variants and their mechanisms. Furthermore, because available brain RNA sequencing data are from bulk tissue, it was not possible to differentiate the impact of CD2AP risk alleles on gene expression in neurons versus other cell types (e.g., astrocytes, endothelial cells, or microglia). CD2AP is broadly expressed, and although the studies define important neuronal requirements, prior work highlights complementary functions in other cell types and tissues. Finally, it is possible that susceptibility genes and the implicated biological pathways have dynamic and pleiotropic relationships with AD phenotypes, even flipping from protection to risk during distinct temporal or cell-type-specific disease phases. For example, while reduced neuronal [Ca2+] may attenuate synaptic strength and thereby increase vulnerability to early AD synaptic dysfunction, the identical perturbation may protect against late excitotoxic mechanisms. Thus, it is possible, if not likely, that CD2AP impinges on multiple targets and pathways to affect AD pathogenesis. In conclusion, although substantial challenges remain, functional dissection of susceptibility genes like CD2AP holds enormous promise for refinement of AD mechanistic models and development of improved therapeutics (Ojelade, 2019).

Search PubMed for articles about Drosophila Cindr

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Cochran, J. N., Rush, T., Buckingham, S. C. and Roberson, E. D. (2015). The Alzheimer's disease risk factor CD2AP maintains blood-brain barrier integrity. Hum Mol Genet 24(23): 6667-6674. PubMed ID: 26358779

Dustin, M.L., et al. (1998). A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94:667-677. PubMed ID: 9741631

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Haglund, K., et al. (2010). Cindr interacts with anillin to control cytokinesis in Drosophila melanogaster. Curr. Biol. 20(10): 944-50. PubMed ID: 20451383

Harrison, B. J., Venkat, G., Lamb, J. L., Hutson, T. H., Drury, C., Rau, K. K., Bunge, M. B., Mendell, L. M., Gage, F. H., Johnson, R. D., Hill, C. E., Rouchka, E. C., Moon, L. D. and Petruska, J. C. (2016). The adaptor protein CD2AP is a coordinator of Neurotrophin signaling-mediated axon arbor plasticity. J Neurosci 36(15): 4259-4275. PubMed ID: 27076424

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Kurakin, A. V., Wu, S. and Bredesen, D. E. (2003). Atypical recognition consensus of CIN85/SETA/Ruk SH3 domains revealed by target-assisted iterative screening. J. Biol. Chem. 278: 34102-34109. PubMed ID: 12829691

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Mattila P.K., Pykäläinen A., Saarikangas J., Paavilainen V.O., Vihinen H., Jokitalo E., Lappalainen P. 2007. Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism. J. Cell Biol. 176: 953-964. PubMed ID: 17371834

Monzo, P., et al. (2005). Clues to CD2-associated protein involvement in cytokinesis, Mol. Biol. Cell 16: 2891-2902. PubMed ID: 15800069

Ojelade, S. A., Lee, T. V., Giagtzoglou, N., Yu, L., Ugur, B., Li, Y., Duraine, L., Zuo, Z., Petyuk, V., De Jager, P. L., Bennett, D. A., Arenkiel, B. R., Bellen, H. J. and Shulman, J. M. (2019). cindr, the Drosophila homolog of the CD2AP Alzheimer's disease risk gene, is required for synaptic transmission and proteostasis. Cell Rep 28(7): 1799-1813. PubMed ID: 31412248

Peters, I., et al. (2006). IGF-binding protein-3 modulates TGF-beta/BMP-signaling in glomerular podocytes. J. Am. Soc. Nephrol. 17: 1644-1656. PubMed ID: 16672319

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Schiffer, M., et al. (2004). A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta-induced apoptosis. J. Biol. Chem. 279: 37004-37012. PubMed ID: 15213232

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Take, H., et al. (2000). Cloning and characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl. Biochem. Biophys. Res. Commun. 268: 321-328. PubMed ID: 10679202

Ubelmann, F., Burrinha, T., Salavessa, L., Gomes, R., Ferreira, C., Moreno, N. and Guimas Almeida, C. (2017). Bin1 and CD2AP polarise the endocytic generation of beta-amyloid. EMBO Rep 18(1): 102-122. PubMed ID: 27895104

Woroniecki, R. P., et al. (2006). Glomerular expression of transforming growth factor-beta (TGF-beta) isoforms in mice lacking CD2-associated protein. Pediatr. Nephrol. 21: 333-338. PubMed ID: 16388393

date revised: 3 January 2020

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