InteractiveFly: GeneBrief

Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein: Biological Overview | References

The polarized architecture of epithelia relies on an interplay between the cytoskeleton, the trafficking machinery, and cell-cell and cell-matrix adhesion. Specifically, contact with the basement membrane (BM), an extracellular matrix underlying the basal side of epithelia, is important for cell polarity. However, little is known about how BM proteins themselves achieve a polarized distribution. In a genetic screen in the Drosophila follicular epithelium, mutations were identified in Crag, which encodes a conserved protein with domains implicated in membrane trafficking. Follicle cells mutant for Crag lose epithelial integrity and frequently become invasive. The loss of Crag leads to the anomalous accumulation of BM components on both sides of epithelial cells without directly affecting the distribution of apical or basolateral membrane proteins. This defect is not generally observed in mutants affecting epithelial integrity. It is proposed that Crag plays a unique role in organizing epithelial architecture by regulating the polarized secretion of BM proteins (Denef, 2008)

Epithelia are organized as sheets of tightly adherent cells with distinct apical-basal polarity. Intact tissue architecture is vital for their function, and a loss of epithelial organization is often associated with carcinoma progression and tumor metastasis. The polarized architecture of epithelial cells is evident from the presence of distinct apical and basolateral membrane domains that have different lipid and protein compositions. To establish and maintain these separate membrane domains, newly synthesized and recycled proteins need to be delivered to the correct location, a process that requires the sorting of proteins into different apical and basolateral transport vesicles, followed by their transport to, and fusion with, specific regions of the plasma membrane. Protein localization is subsequently maintained through interactions with the underlying cytoskeleton and other cortical protein complexes and through intercellular junctions that inhibit diffusion between the apical and basolateral membrane domains (Denef, 2008)

In order for apical and basolateral transport vesicles to reach and fuse with the correct membrane region, initial cell surface asymmetries need to be established. This is achieved by a combination of external cues, including sites of contact with neighboring cells and with the extracellular matrix (ECM). Of particular interest in this light is the basement membrane (BM), a specialized sheet of ECM contacting the basal side of epithelial tissues. The BM is composed primarily of the secreted glycoproteins Laminin, Collagen IV, and Nidogen and the heparan sulfate proteoglycan Perlecan. Interactions between the BM and epithelial cells are mediated by a variety of cell surface receptors including integrins and Dystroglycan. Evidence from genetic analyses in model organisms and experiments in cultured cells has indicated an important role for the BM in generating epithelial cell polarity (Li, 2003; O'Brien, 2002). Therefore, the accumulation of BM on the basal side of the epithelium only is crucial to convey the positional information necessary to maintain a polarized architecture (Denef, 2008)

Even though substantial progress has been made in understanding the mechanisms that regulate the polarized trafficking of basolateral membrane proteins (Mostov, 2003; Rodriguez-Boulan, 2005), very little is known about how basally secreted proteins, such as BM components, are sorted and delivered specifically to the basal side of epithelial cells. Different pathways for the polarized trafficking of basolateral membrane and secreted proteins have been proposed, yet no components specifically regulating the polarized secretion of BM proteins have been identified (Denef, 2008)

To study the establishment and maintenance of epithelial architecture, the follicular epithelium (FE) in Drosophila, which forms a monolayer of somatic cells surrounding the germline during oogenesis, was used. The follicle cells (FC) that make up the FE display a distinct apical-basal polarity, apparent in the presence of different membrane and cortical domains, apically localized adherens junctions, and the polarized organization of the cytoskeleton. As in other epithelia, the basal side of the FE is in contact with the BM. The apical side, however, contacts the germline rather than a lumen or the external environment. Cues on the basal, apical, and lateral sides of the FC are required to establish and maintain a fully polarized epithelial phenotype (Denef, 2008)

To further elucidate the molecular mechanisms controlling epithelial organization, advantage of the stem-cell-derived nature of the FE and a genetic mosaic screen was performed to identify genes required for this process. Multiple alleles were identified of Crag (Calmodulin-binding protein related to a Rab3 GDP-GTP exchange protein), an evolutionarily conserved gene for which no biological function had previously been described. FC mutant for Crag often lose epithelial characteristics and become motile and invasive. Strikingly, prior to inducing a loss of epithelial integrity, the absence of Crag causes the aberrant accumulation of the BM components Perlecan, Laminin, and Collagen IV on both sides of the epithelium, a defect that was not generally observed in mutants disrupting epithelial structure. It is proposed that Crag plays a unique role in regulating epithelial architecture by specifically controlling the polarized secretion of BM proteins. While it has been suggested that BM components need specific factors to ensure their accurate basal secretion, Crag is the first protein identified to be required for such a process (Denef, 2008)

First, this study shows that Crag mutant cells lose the polarized distribution of the BM components Pcan, Lam, and Coll IV. Instead of being present exclusively on the basal side of the epithelium, these BM proteins were found to accumulate both basally and apically in the absence of Crag. This was true for all mutant clones analyzed, regardless of their position within the epithelium and even in the absence of any other discernible phenotypes. This contrasts with apical, junctional, and basolateral membrane proteins, which have altered distributions in Crag mutant cells only when epithelial structure is severely disturbed. The failure to restrict the accumulation of BM components to the basal side of the FE is therefore likely the primary defect in Crag mutant cells. Second, the mistargeting of BM components in Crag mutant cells appears to be a specific consequence of the loss of Crag function and not a general defect observed in the absence of other proteins known to regulate epithelial organization. FC mutant for par-6, crb, or dlg and thus lacking the activity of any one of three major polarity complexes show no alterations in the accumulation of BM proteins. Crag may therefore be the first factor identified to specifically control the accurate deposition of BM on the basal side of epithelial cells (Denef, 2008)

Based on the well-established role of the BM in regulating epithelial organization, it is proposed that the presence of BM components on both sides of the FE is responsible for the loss of epithelial integrity in the absence of Crag. Studies in both model organisms and cultured cells have pointed to a central role for the BM in regulating epithelial polarity and tissue organization (Li, 2003; Miner, 2004; Yurchenco, 2004). In particular, contact with the BM has been shown to direct the orientation of the apical-basal axis of epithelia, with the apical pole forming opposite to the side contacting the BM (O'Brien, 2002). Exposure of the apical surface of cultured epithelial cysts or monolayers to either collagen or laminin triggers a reversal of polarity (see O'Brien, 2001; Yu, 2005). Polarity reversal is completed only after the BM present on the original basal side of the epithelium has been degraded, and it is preceded by a loss of apical membrane identity, a dispersal of basolateral proteins, and the formation of multiple cell layers (Schwimmer, 1995; Wang, 1990; Zuk, 1996). This artificially induced situation shows noticeable similarities with the behavior of Crag mutant FC where BM components are found in contact with both sides of the epithelium. Cells form multiple layers, the cortical localization of apical proteins is lost, and basolateral proteins are found along the entire cell surface. Thus, the presence of a BM on both sides of the FE in the absence of Crag can account for the observed phenotypes. Even though the significance of the BM in organizing epithelial architecture has been well established, the results underscore the importance of maintaining a polarized deposition of BM proteins on the basal side of epithelial cells (Denef, 2008)

Interestingly, epithelial disorganization is not restricted to Crag mutant cells but is also seen in neighboring wild-type cells. This nonautonomy can be explained by the observation that Pcan, Lam, and Coll IV were found on the apical side of wild-type cells neighboring mutant cells, presumably due to their diffusion within the extracellular space (Denef, 2008)

How could the absence of Crag protein lead to the aberrant deposition of BM components on the apical side of epithelial cells? Previous work in tissue culture has suggested that epithelial cells secrete BM proteins predominantly from their basal side and has indicated the existence of an active sorting and targeting process mediating this polarized secretion. Furthermore, it has been suggested that different trafficking routes for basally secreted and basolateral transmembrane proteins exist. It is thus conceivable that Crag plays a role in specifically controlling the polarized secretion of BM proteins (Denef, 2008)

A direct role for Crag in regulating membrane trafficking is suggested by the presence of a conserved DENN domain at its amino terminus. DENN domains are found in a large number of proteins in nearly all eukaryotes. The precise function of the DENN domain remains to be elucidated, but its presence in a number of proteins involved in endocytosis or exocytosis, most notably in a number of Rab interactors, led to the hypothesis that the DENN domain regulates membrane trafficking (Allaire, 2006; Clague, 2005; Falbel, 2003; Levivier, 2001; Miyoshi, 2004). Moreover, the localization of Crag to the cell cortex and to recycling and early endosome membranes is consistent with a role in membrane trafficking (Denef, 2008)

Apical Pcan in Crag mutant cells originates from newly synthesized protein and not from transcytosed basal protein because Crag pcan double mutant clones that maintain basal Pcan do not show apical Pcan. Assuming that in wild-type FC newly synthesized BM components are secreted exclusively on the basal side, Crag could act by ensuring one or more steps during their polarized secretion. Crag may facilitate the sorting of newly synthesized BM components. In the absence of Crag, inaccurate sorting would lead to the packaging of BM proteins in vesicles destined for both apical and basolateral secretion. The colocalization of Crag protein with the recycling endosome (RE) marker Rab11 may point to a role for Crag in protein sorting, as the RE has been shown to be a hub for the sorting of both newly synthesized and endocytosed apical and basolateral proteins. Alternatively, Crag may act at a later step and direct the polarized transport, targeting, or fusion of vesicles containing BM components specifically with the basal membrane. Interestingly, since Crag is absent from the basal cortex in FC, Crag would play a repulsive role, inhibiting the targeting or fusion of BM protein-containing vesicles with apical and lateral membranes. In the absence of Crag, transport vesicles would be allowed to fuse with basolateral as well as apical membranes, leading to the observed apolar distribution of Lam, Coll IV and Pcan (Denef, 2008)

Even though evidence from previous work points toward the active sorting and polarized secretion of BM proteins, the possibility remains that BM components are secreted both apically and basally in wild-type FC but stabilized only on the basal side, or removed preferentially apically, for instance through degradation by localized matrix metalloproteases. While these scenarios require additional assumptions, Crag could mediate either of these processes and regulate the basal accumulation of BM proteins more indirectly, yet specifically (Denef, 2008)

In summary, a crucial role has been uncovered for the conserved protein Crag in the regulation of epithelial architecture of the Drosophila FE. Crag appears to specifically affect the polarized trafficking of BM components. Even though the existence of different trafficking routes for basally secreted and basolateral transmembrane proteins has been suggested previously, Crag is the first specific component required to ensure the polarized accumulation of BM on the basal side of epithelial cells. It therefore serves to define this process and provides a clear entry point into its molecular analysis. It will be interesting to elucidate the molecular machinery through which Crag regulates the polarized deposition of BM components (Denef, 2008)

Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate

The basement membrane (BM), a specialized sheet of the extracellular matrix contacting the basal side of epithelial tissues, plays an important role in the control of the polarized structure of epithelial cells. However, little is known about how BM proteins themselves achieve a polarized distribution. This study identifies phosphatidylinositol 4,5-bisphosphate (PIP2) as a critical regulator of the polarized secretion of BM proteins. A decrease of PIP2 levels, in particular through mutations in Phosphatidylinositol synthase (Pis) and other members of the phosphoinositide pathway, leads to the aberrant accumulation of BM components at the apical side of the cell without primarily affecting the distribution of apical and basolateral polarity proteins. In addition, PIP2 controls the apical and lateral localization of Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a factor specifically required to prevent aberrant apical secretion of BM. It is proposed that PIP2, through the control of Crag's subcellular localization, restricts the secretion of BM proteins to the basal side (Devergne, 2014).

Retinal targets for calmodulin include proteins implicated in synaptic transmission

Ca2+ influxes regulate multiple events in photoreceptor cells including phototransduction and synaptic transmission. An important Ca2+ sensor in Drosophila vision appears to be calmodulin since a reduction in levels of retinal calmodulin causes defects in adaptation and termination of the photoresponse. These functions of calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the TRP and TRPL ion channels, NinaC and INAD. To identify additional calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, a screen was conducted for retinal calmodulin-binding proteins. Eight additional calmodulin-binding proteins were found that are expressed in the Drosophila retina. These included six targets that are related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein, UNC13, and a protein, CRAG, related to Rab3 GTPase exchange proteins. Two other calmodulin-binding proteins included Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission (Xu, 1998).

At least two of the four novel calmodulin-binding proteins share similarities to components implicated in synaptic transmission. One of these proteins (1441 residues), referred to as CRAG (calmodulin-binding protein related to a Rab3 GDP/GTP exchange protei) due to its similarity to a domain in the recently identified rat Rab3 GDP/GTP exchange protein (rRab3 GEP) and the C. elegans homolog, AEX-3, which has been implicated in synaptic vesicle release. AEX-3 and the rRab3 GEP (1409 and 1602 amino acids, respectively) contain three regions of homology, the first of which (~500 residues) is conserved in CRAG, AEX-3, rRab3 GEP (CAR domain) and the human homolog, MADD (death domain MAP kinase activator). The latter two regions are conserved in AEX-3 and rRab3 GEP (AR1 and AR2), but not CRAG, and are shorter (~100 and 300 residues, respectively) than the CAR homology. The CAR domain in CRAG is ~36 identical over 321 amino acids (residues 95-415) to either rRab3 GEP or AEX-3. In addition, there is weak homology (16%) in the flanking sequences that extended the CAR domain in CRAG to residues 73-490. The C-terminal ~800 residues of CRAG did not share significant primary amino acid sequence homology with the rRab3 GEP, AEX-3, or any other protein in the data banks (Xu, 1998).

Search PubMed for articles about Drosophila Crag

Allaire, P. D., et al. (2006). Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis. J. Neurosci. 26: 13202-13212. PubMed ID: 17182770

Clague, M. J. and Lorenzo, O. (2005). The myotubularin family of lipid phosphatases. Traffic 6: 1063-1069. PubMed ID: 16262718

Denef, N., et al. (2008). Crag regulates epithelial architecture and polarized deposition of basement membrane proteins in Drosophila. Dev. Cell 14: 354-364. PubMed ID: 18331716

Devergne, O., Tsung, K., Barcelo, G. and Schupbach, T. (2014). Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci U S A 111: 7689-7694. PubMed ID: 24828534

Falbel, T. G., et al. (2003). SCD1 is required for cytokinesis and polarized cell expansion in Arabidopsis thaliana. Development 130: 4011-4024. PubMed ID: 12874123

Levivier, E., et al. (2001). uDENN, DENN, and dDENN: indissociable domains in Rab and MAP kinases signaling pathways. Biochem. Biophys. Res. Commun. 287: 688-695. PubMed ID: 11563850

Li, S., et al. (2003). The role of laminin in embryonic cell polarization and tissue organization. Dev. Cell 4: 613-624. PubMed ID: 12737798

Miner, J. H. and Yurchenco, P. D. Laminin functions in tissue morphogenesis. Annu. Rev. Cell Dev. Biol. 20: 255-284. PubMed ID: 15473841

Miyoshi, J. and Takai, Y. (2004). Dual role of DENN/MADD (Rab3GEP) in neurotransmission and neuroprotection. Trends Mol. Med. 10: 476-480. PubMed ID: 15464446

Mostov, K., Su, T. and ter Beest, M. (2003). Polarized epithelial membrane traffic: conservation and plasticity. Nat. Cell Biol. 5: 287-293. PubMed ID: 12669082

O'Brien, L. E., et al. (2001). Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nat. Cell Biol. 3: 831-838. PubMed ID: 11533663

Rodriguez-Boulan, E., Kreitzer, G. and Musch, A. (2005). Organization of vesicular trafficking in epithelia. Nat. Rev. Mol. Cell Biol. 6: 233-247. PubMed ID: 15738988

Schwimmer, R. and Ojakian, G. K. (1995). The alpha 2 beta 1 integrin regulates collagen-mediated MDCK epithelial membrane remodeling and tubule formation. J. Cell Sci. 108: 2487-2498. PubMed ID: 7673363

Wang, A. Z., Ojakian, G. K. and Nelson, W. J. (1990). Steps in the morphogenesis of a polarized epithelium. II. Disassembly and assembly of plasma membrane domains during reversal of epithelial cell polarity in multicellular epithelial (MDCK) cysts. J. Cell Sci. 95: 153-165. PubMed ID: 2351700

Xu, X. Z., et al. (1998). Retinal targets for calmodulin include proteins implicated in synaptic transmission. J. Biol. Chem. 273(47): 31297-307. PubMed ID: 9813038

Yu, W., et al. (2005). Beta1-integrin orients epithelial polarity via Rac1 and laminin. Mol. Biol. Cell 16: 433-445. PubMed ID: 15574881

Yurchenco, P. D., Amenta, P. S. and Patton, B. L. (2004). Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol. 22: 521-538. PubMed ID: 14996432

Zuk, A. and Matlin, K. S. (1996). Apical beta 1 integrin in polarized MDCK cells mediates tubulocyst formation in response to type I collagen overlay. J. Cell Sci. 109: 1875-1889. PubMed ID: 8832410

date revised: 25 November 2014

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