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stratum: Biological Overview | References

BIOLOGICAL OVERVIEW

The basement membrane (BM), a sheet of extracellular matrix lining the basal side of epithelia, is essential for epithelial cell function and integrity, yet the mechanisms that control the basal restriction of BM proteins are poorly understood. In epithelial cells, a specialized pathway is dedicated to restrict the deposition of BM proteins basally. This study reports the identification of a factor in this pathway, a homolog of the mammalian guanine nucleotide exchange factor (GEF) Mss4, which is named Stratum (CG7787). The loss of Stratum leads to the missecretion of BM proteins at the apical side of the cells, forming aberrant layers in close contact with the plasma membrane. This study found that Rab8 GTPase acts downstream of Stratum in this process. Altogether, these results uncover the importance of this GEF/Rab complex in specifically coordinating the basal restriction of BM proteins, a critical process for the establishment and maintenance of epithelial cell polarity (Devergne, 2017).

One of the common characteristics of epithelial tissues is the presence of a specialized sheet of extracellular matrix (ECM) at their basal side, called the basement membrane. BMs are cell-adherent extracellular scaffolds composed of proteins such as type IV Collagen (Coll IV), laminins, and heparan sulfate proteoglycans such as Perlecan (Pcan). BMs interact with the basal side of epithelial cells via cellular receptors such as Integrin and Dystroglycan. In addition to providing tissue support, BMs are essential for embryonic and organ morphogenesis and adult functions. The BM has been shown to act as a signaling platform for the regulation of epithelial polarity. The BM can direct the orientation of the apico-basal axis of epithelial cells, resulting in the formation of a basal domain on the side contacting the BM and an apical domain on the opposite side. The loss of integrity and misregulation of the BM have been associated with tumor metastasis. Despite the significance of the BM in both normal and abnormal epithelial cells, the molecular mechanisms ensuring accurate basal secretion of BM proteins remain largely elusive (Devergne, 2017).

Epithelial cells exhibit a pronounced apico-basal polarity. Polarized intracellular trafficking is a critical process required to establish and maintain epithelial cell polarity by delivering newly synthesized and recycled proteins to their correct destinations. In polarized epithelial cells, a pathway is specifically dedicated to the basal restriction of BM components. It is composed of the guanine nucleotide exchange factor (GEF) Crag (Calmodulin-binding protein related to a Rab3 guanosine diphosphate [GDP]/guanosine triphosphate [GTP] exchange protein) and its guanosine triphosphatase (GTPase) Rab10, as well as the phosphoinositide phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and the protease-like protein Scarface (Denef, 2008, Devergne, 2014, Lerner, 2013, Sorrosal, 2010; Devergne, 2017 and references therein).

To study the mechanisms leading to the basal restriction of BM proteins in polarized epithelial cells, the highly polarized follicular epithelium (FE) of the Drosophila melanogaster ovary was used as a model system. The FE consists of a monolayer epithelium composed of highly polarized cells, called follicle cells (FCs), which surrounds the germline cells. As is typical of epithelial cells, FCs contain different membrane domains: an apical domain facing the germline, a basolateral domain, and junctional domains. Components of the BM, such as Pcan and Coll IV, are actively secreted basally by FCs during egg chamber maturation, thus establishing the FE as an excellent model for the basal restriction of BM proteins in epithelial cells (Devergne, 2017).

Using this model system, a GEF/RabGTPase complex has been identified, composed of the GEF Stratum (Strat) and the Rab8GTPase, which controls the basal restriction of BM proteins in polarized epithelial cells. The loss of one of these partners leads to the apical mislocalization of BM components. Although Rab8GTPase has a diffuse cytoplasmic localization in the FE, Strat is basally enriched; this suggests that Strat restricts Rab8GTPase activation basally, leading to basal secretion of BM proteins. In addition, this study shows that other factors involved in polarized BM deposition, including PI(4,5)P2 and Crag, control intracellular levels of Strat (Devergne, 2017).

The GEF Crag and its RabGTPase partner Rab10 play critical roles in directing the basal secretion of BM proteins in polarized epithelial cells (Denef, 2008, Lerner, 2013). To identify factors that control the polarized intracellular trafficking and secretion of BM proteins, a Drosophila protein interaction map (DPiM) was used to find Rab10 interacting partners. One strong interactor was a putative GEF encoded by the gene CG7787. Because GEFs are critical for the control of intracellular trafficking, its role in BM secretion was investigated (Devergne, 2017).

To test the involvement of CG7787 in BM polarity, the expression of CG7787 in FCs was knocked down by RNAi. The distribution of BM proteins was monitored using the GFP-protein trap lines Pcan-GFP and Coll IV-GFP that reflect the endogenous localization of these proteins (Denef, 2008, Devergne, 2014). CG7787-depleted epithelial cells present an accumulation of Pcan and Coll IV on both their basal and their apical surfaces, indicating that CG7787 is required for polarized BM deposition. Because BM proteins accumulate in an apical sheet in CG7787-depleted cells, the gene was named stratum (strat) (Devergne, 2017).

The gene strat encodes a GEF, based on predicted conserved protein domains, that belongs to the MSS4 family of proteins. In particular, mammalian Mss4 (mammalian suppressor of yeast Sec4), also called RABIF (Rab interacting factor), has been shown to interact with Rabs belonging to the same subfamily, including Rab1, Rab3, Rab8, and Rab10, all of which are involved in secretion. To assess whether Strat is the Drosophila homolog of mammalian Mss4/RabIF, human Mss4/RabIF (hMss4)was expressed in strat-knocked down FCs. hMss4 expression partially rescues the defects associated with the loss of Strat, indicating that Strat is the functional homolog of human Mss4/RabIF (Devergne, 2017).

To confirm the phenotype observed in strat RNAi-expressing FCs, four strat mutant lines were generated by ethyl methanesulfonate (EMS) mutagenesis. Homozygous mutant FC clones were generated using the flippase/flippase recognition target (Flp/FRT) system. In strat mutant FCs, Pcan (Trol) accumulates apically. Expression of a full-length strat transgene rescued this mislocalization phenotype, indicating that strat mutant alleles were generated. Overall, these data confirm Stratum as an essential factor for the basal restriction of BM proteins in epithelial cells. Moreover, quantification of the BM mislocalization phenotype observed in strat mutant clones indicates that the apical accumulation of BM components progressively increases during egg chamber maturation (Devergne, 2017).

However, Strat does not globally control the apico-basal polarity of FCs. The polarized distribution of other classes of proteins that undergo polarized intracellular trafficking localize normally, indicating that Strat is a member of a pathway specifically dedicated to the polarized sorting of BM proteins in epithelial cells (Devergne, 2017).

A better understanding of this biological pathway requires a careful analysis of its different members. To do so, it as decided to better characterize the apical mislocalization of BM components observed in strat mutant FCs. Super-resolution three-dimensional structured illumination microscopy (3D-SIM) was used and the plasma membrane marker mCD8-RFP (red fluorescent protein) was expressed exclusively in the FE to observe the distribution of Coll IV-GFP in relation to mCD8-RFP. 3D-SIM has a resolution of 120 nm in xy axes, allowing the distribution of BM proteins to be uprecisely determined with respect to the FE plasma membrane. The spatial distribution and levels of these components were quantified by measuring fluorescence intensity with an optical section through FCs. The basal and apical membranes can be visualized by the two most extreme red mCD8-RFP peaks. As expected in wild-type (WT) FCs, only one peak of Coll IV (Coll IV-GFP, green) was observed on the basal side, and it co-localizes tightly with the basal plasma membrane of the cells; in addition, no apical Coll IV peak was observed (Devergne, 2017).

In contrast, in strat-knocked down FCs, an additional Coll IV peak was observed at the apical membrane that is also tightly associated with the plasma membrane. Moreover, the Coll IV peak is apical to the apical plasma membrane peak, indicating that Coll IV is also found outside of the apical plasma membrane. Thus, the loss of Strat leads to the apical secretion of BM proteins. These data, confirmed by 3D reconstruction, suggest that the putative GEF Strat is not required for secretion per se but rather for the directionality of secretion. The same observation was made in Crag-knocked down FCs, suggesting that Crag and Strat are both involved in directionality of secretion (Devergne, 2017).

3D-SIM imaging also revealed that the apical deposition of Coll IV is different from its basal deposition. The pixel distribution shows that the 'sheet' of Coll IV is thicker apically (between 300 and 600 nM) than basally (less than 100 nM, below the resolution of SIM). The apical FC membrane contains microvilli and is therefore topologically thicker than the basal membrane. Moreover, it is unknown whether the mechanisms needed to establish a properly assembled BM are present apically. Altogether, these data suggest that the loss of Strat leads to the apical secretion of Coll IV, which associates with the apical plasma membrane with an aberrant organization (Devergne, 2017).

Next, attempts were made to identify the RabGTPase or RabGTPases that function with Stratum in polarized BM deposition. Because Rab10 is involved in polarized BM secretion in epithelial cells, Rab10 was tested as a potential Strat interactor (Devergne, 2017).

To assess whether Rab10 functions downstream of Stratum, a constitutively active form of Rab10 (Rab10CA), was used. Constitutively active forms of RabGTPases remain bound to GTP and thus do not require a GEF for activation. If Strat functions as a GEF for Rab10 in polarized BM deposition, the expression of Rab10CA (yellow fluorescent protein [YFP]-Rab10CA) may rescue the apical mislocalization of BM proteins observed in strat-deficient cells. The expression of Rab10CA in strat-knocked FCs did not rescue the BM mislocalization phenotype. Although a negative result is difficult to interpret, these data might suggest one of the following: (1) Strat is not a GEF for Rab10 during polarized BM deposition, (2) Strat functions as a GEF for another Rab or other Rabs that also control this process, or (3) the expression of Rab10CA is not strong enough to suppress the strat phenotype. Because the expression of YFP-tagged Rab10CA could not be detected in the FE, the latter hypothesis seems unlikely (Devergne, 2017).

To determine whether Strat interacts with other RabGTPases during BM polarity, Rab8 was examined. In Drosophila, Rab8 and Rab10 are paralogs, sharing an amino acid sequence identity of 67%. In addition, mammalian Mss4/RabIF can act as a GEF for Rab8a. First, the effects of Rab8 on BM proteins were examined. In FCs mutant for Rab8, knocked down for Rab8, or expressing a dominant-negative form of Rab8 (Rab8DN), mislocalization was observed of Pcan or Coll IV, indicating that Rab8 is also involved in polarized BM secretion in epithelial cells. To assess whether Rab8 acts downstream of Strat, a constitutively active (CA) form of Rab8 (YFP-Rab8CA) was expressed in strat knockdown FCs. This resulted in a partial rescue of the phenotype associated with the loss of strat, suggesting that Rab8 acts downstream of Strat in the process of polarized BM deposition. In addition, the expression of wild-type full-length Rab8 did not rescue the phenotype associated with the loss of Strat, suggesting that Strat activates the GTPase activity of Rab8 (Devergne, 2017).

Finally, to determine whether Strat and Rab8 interact physically, co-immunoprecipitation (coIP) was performed of tagged Rab8 (YFPMYC-Rab8) and Stratum (Strat-HA [C-term HA tagged Stratum]), and they were found to interact in ovary extracts. Altogether, these results suggest that Strat acts as a GEF for Rab8 during the basal restriction of BM deposition. This conclusion is supported by the data that Rab8a interacts with Mss4 in mammalian cells and has weak GEF activity for Rab8a in vitro. It was also shown that Strat phenotype is rescued by hMss4, suggesting that Strat and hMss4 share similar activities. Thus, another GEF/Rab complex, Strat/Rab8, was identified in addition to Crag/Rab10, that is involved in BM deposition in epithelial cells (Devergne, 2017).

Three non-exclusive mechanisms have been proposed to explain the basal secretion of BM proteins: (1) BM-containing vesicles are directly targeted to the basal side of polarized cells, (2) BM-containing vesicles are blocked apically, and (3) BM proteins are secreted on both sides of epithelial cells but are degraded or endocytosed apically. The intracellular localization of components involved in this process, such as Stratum and Rab8, may provide insight into how these factors restrict BM proteins basally. First, this study assessed the subcellular localization of Rab8 using endogenously tagged YFP-Myc-tagged-Rab8 (YFPMyc-Rab8). In the FE, YFPMyc-Rab8 is detected diffusely throughout the cytoplasm and is non-polarized during early and mid-stages of oogenesis. YFPMyc-Rab8 accumulates in intracellular puncta, which may represent endosomes and/or vesicles. More specifically, Rab8 partially co-localizes with early and recycling endosome and Golgi markers. This subcellular localization is consistent with the known role of Rab8 in regulating vesicular transport from the Golgi to the plasma membrane. YFPMyc-Rab8 becomes slightly enriched at the basal side of the FCs starting in stages 9 to 10. In contrast to Rab8, Strat (Strat-HA) has a diffuse intracellular localization earlier in oogenesis but quickly assumes a pronounced polarized distribution, accumulating basally in FCs. This observation suggests that Strat restricts the activity of Rab8 basally to allow proper basal deposition of BM proteins. Alternatively, because mammalian Mss4 protein has been shown to have only weak GTPase activity compared to other GEFs, Mss4 may act as a chaperone, allowing interacting Rab proteins to be properly activated where and when they are needed in the cell. Therefore, Strat may play one or both of these roles to restrict Rab8 activity basally and thus direct BM protein-containing vesicles toward the basal side of the cell (Devergne, 2017).

The polarized localization of Strat differs from Crag, which accumulates at apical and lateral membranes, suggesting that Crag blocks the apical secretion of BM proteins. Both Crag and Stratum GEFs have critical roles to restrict BM proteins basally; however, they are structurally, and perhaps functionally, different. Crag is a 187 kDa multidomain protein composed of three differentially expressed in normal and neoplastic cells (DENN) domains with GEF activity, a Calmodulin binding domain, and a conserved C-terminal domain. In contrast, Stratum is 14 kDa and composed of a single Mss4 domain with weak GEF activity. In view of these structural differences, it is unlikely that Crag and Stratum have the same interactors, regulators, and effectors. In addition, the strikingly different localization of these factors suggests independent roles in this process, because Crag is localized to lateral and apical membranes and Stratum is localized to the basal side of cells. Yet altogether, these proteins allow the specific basal restriction of BM components in epithelial cells (Devergne, 2017).

Recently studies have shown that the proper intracellular distribution of Crag is dependent on the phosphoinositide PI(4,5)P2. A decrease in PI(4,5)P2 levels leads to a loss of Crag apico-basal distribution and the mislocalization of BM proteins (Devergne, 2014). To assess the role of other members of the pathway, such as PI(4,5)P2 and Crag, on Strat localization, the distribution of Strat-HA was determined in Phosphatidylinositol synthase (Pis) and Crag mutant FCs. As was previously observed for Crag, a decrease in PI(4,5)P2 levels in Pis mutant FCs leads to reduced levels of Strat. This phenotype is observed in 49% of mutant clones. The same decrease of Strat can be observed in Crag mutant FCs (in 47% of mutant clones). These data suggest that both PI(4,5)P2 levels and Crag control the levels and distribution of Strat. Because previous work has shown that PI(4,5)P2 controls Crag localization, the decrease of Strat observed in Pis mutant FCs might be due to the loss of Crag. However, the distribution and levels of Crag are not significantly affected in strat mutant FCs. Overall, the loss of Strat observed in the mutant backgrounds highlights the existence of a regulatory mechanism between the two GEF/Rab complexes dedicated to the polarized secretion of BM proteins and should be investigated further (Devergne, 2017).

In conclusion, this study has identified Strat, the homolog of mammalian GEF Mss4/RabIF, and Rab8GTPase as essential regulators in the basal sorting of BM proteins in polarized epithelial cells. This GEF/Rab complex partners to correctly deliver BM protein-containing vesicles basally, an essential process for epithelial cell function. Previous work identified an apical complex involved in this process containing Crag/Rab10 and depending on PI(4,5)P2. This study has found a more basally localized complex, consisting of Strat and Rab8, also required for the exclusive basal localization of BM proteins. These complexes do not function redundantly but both complexes are required independently. A third complex involving Rab10 and Ehbp1 has been described to deliver BM proteins to the basolateral side of the follicle cells in a late differentiation process involved in egg chamber elongation (Isabella, 2016). These findings reveal that the proper positioning of BM proteins is handled by the cell in more complex regulatory pathways than was previously realized (Devergne, 2017).

Search PubMed for articles about Drosophila Stratum

Denef, N., Chen, Y., Weeks, S. D., Barcelo, G. and Schupbach, T. (2008). Crag regulates epithelial architecture and polarized deposition of basement membrane proteins in Drosophila. Dev Cell 14(3): 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(21): 7689-7694. PubMed ID: 24828534

Devergne, O., Sun, G. H. and Schupbach, T. (2017). Stratum, a homolog of the human GEF Mss4, partnered with Rab8, controls the basal restriction of basement membrane proteins in epithelial cells. Cell Rep 18(8): 1831-1839. PubMed ID: 28228250

Isabella, A. J. and Horne-Badovinac, S. (2016). Rab10-mediated secretion synergizes with tissue movement to build a polarized basement membrane architecture for organ morphogenesis. Dev Cell 38(1): 47-60. PubMed ID: 27404358

Lerner, D. W., McCoy, D., Isabella, A. J., Mahowald, A. P., Gerlach, G. F., Chaudhry, T. A. and Horne-Badovinac, S. (2013). A Rab10-dependent mechanism for polarized basement membrane secretion during organ morphogenesis. Dev Cell 24(2): 159-168. PubMed ID: 23369713

Sorrosal, G., Perez, L., Herranz, H. and Milan, M. (2010). Scarface, a secreted serine protease-like protein, regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo. EMBO Rep 11(5): 373-379. PubMed ID: 20379222

date revised: 21 November 2022

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