CIN85 and CD2AP orthologue: Biological Overview | References
Gene name - CIN85 and CD2AP orthologue
Cytological map position- 100A6-100A6
Function - signaling
Symbol - cindr
FlyBase ID: FBgn0027598
Genetic map position - 3R:26,634,349..26,652,542 [+]
Classification - Src homology 3 domains
Cellular location - cytoplasmic
|Recent literature||Yasin, H. W., van Rensburg, S. H., Feiler, C. E. and Johnson, R. I. (2016). The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity. Dev Biol [Epub ahead of print]. PubMed ID: 26772997
Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. This study shows that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. This study has confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer.
|Ketosugbo, K. F., Bushnell, H. L. and Johnson, R. I. (2017). A screen for E3 ubiquitination ligases that genetically interact with the adaptor protein Cindr during Drosophila eye patterning. PLoS One 12(11): e0187571. PubMed ID: 29117266
Ubiquitination is a crucial post-translational modification that can target proteins for degradation. The E3 ubiquitin ligases are responsible for recognizing substrate proteins for ubiquitination, hence providing specificity to the process of protein degradation. This study describes a genetic modifier screen that identified E3 ligases that modified the rough-eye phenotype generated by expression of cindr RNAi transgenes during Drosophila eye development. In total, 36 E3 ligases, as well as 4 Cullins, were identified that modified the mild cindrRNA mis-patterning phenotype. This indicates possible roles for these E3s/Cullins in processes that require Cindr function, including cytoskeletal regulation, cell adhesion, cell signaling and cell survival. Three E3 ligases identified in this screen had previously been linked to regulating JNK signaling.
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, Rab4, PAK2, 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).
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).
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 cortactins 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 cortactins 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).
Search PubMed for articles about Drosophila Cindr
Bogler, O., et al (2000). SETA: a novel SH3 domain-containing adapter molecule associated with malignancy in astrocytes. Neuro. Oncol. 2: 6-15. PubMed ID: 11302255
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
Gout, I., et al. (2000). Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein. EMBO J. 19: 4015-4025. PubMed ID: 10921882
Haglund, K., et al. (2010). Cindr interacts with anillin to control cytokinesis in Drosophila melanogaster. Curr. Biol. 20(10): 944-50. PubMed ID: 20451383
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date revised: 30 January 2011
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