Department of Cell and Developmental Biology (CDB)

ONGOING AND FUTURE RESEARCH

Research in my laboratory has three major research programs, all designed to study different aspects of cell polarity during division, epithelial tissue formation and cancer metastasis.

Project #1. The mechanisms regulating apical lumen formation during epithelial tissue polarization and morphogenesis

Epithelium is a tissue composed of polarized cells, which line the body’s organs and perform specialized functions. Since epithelial cells act as barriers and selective transporters, the significance of epithelial polarization is clear. Failure of epithelial cells to appropriately polarize leads to a variety of diseases, including but not limited to, polycystic kidney disease, microvillus inclusion disease, cystic fibrosis, and certain metastatic cancers. As the result, the polarization of epithelial cells is a highly regulated event that is conserved across various organisms. The plasma membrane (PM) of epithelial cells is divided into the apical and basolateral domains, which are distinct in both lipid and protein composition (Figure 1). Specialized protein complexes, the tight junctions and the adherens junction, maintain the integrity of these two discrete apical and basolateral domains. Tight junction formation is crucial for initiating the polarity program of the cell, and is regulated by the cell’s polarity complexes (Crumbs, PAR, and Scribble complexes), as it marks the separation of the apical and lateral domains.

Apical lumen formation using 3D tissue culture system. Tissue morphogenesis requires the coordination of, and therefore communication between, entire groups of epithelial cells within three-dimensional (3D) space. The final result of these processes is the formation of epithelial tubes or end buds, in which all epithelial cells are properly oriented such that their apical domain faces the central apical lumen (Figure 1). Since much of the work on epithelial polarity has been conducted using two-dimensional epithelial cell models, the process of apical lumen formation in 3D-environment remains poorly understood and is the main focus of this project. One of the prevailing models of de novo lumen formation is known as hollowing model. It was proposed that specialized apical recycling endosomes are formed and exocytosed to the site of the forming lumen. The fusion of these endosomes with the PM initiates the formation of the apical central lumen (Figure 1). While the requirement of apical recycling endosomes for apical lumen formation is now well established, how these specialized endosomes are formed, transported and targeted to a single apical lumen formation site remains largely unclear. What is known, is that the apical membrane initiation site (AMIS) is marked by the recruitment of several tight junction proteins and the Exocyst protein complex, before the delivery and fusion of apical endosomes containing various polarity proteins, such as Rab11, FIP5, Crumbs3, TUBA and gp135 (Figure 1). Thus, it was suggested that tight junction proteins or the Exocyst complex within AMIS mediate the targeting of apical endosomes to the site of forming apical lumen. However, the identity and regulation of proteins mediating endocytic targeting to the AMIS remains to be determined, and is one of the goals of this research project.

The use of zebrafish intestine development as a model to study apical lumen formation. The evidence for the hollowing model of lumen formation in epithelial cells is largely derived from studies conducted in 3D tissue culture systems. Thus, it remains unclear whether the same molecular machinery is used during epithelial tissue morphogenesis in vivo. Only recently some evidence for the hollowing-dependent lumen formation come into light, through studies in gut and endothelial blood vessel development in zebrafish embryos. We are currently collaborating with Dr. Bruce Appel’s lab to use zebrafish intestine tube development as a model system for testing the roles of endocytic transport during apical lumen formation.

Project #2. The mechanisms regulating endocytic organelles and cytoskeleton during cell division

The last step of cell division is a physical separation of two daughter cells via process known as cytokinesis. After replication of the genetic material, mother cell divides by the formation of the cleavage furrow that constricts cytoplasm leaving two daughter cells connected by a thin intracellular bridge (ICB). The resolution of this bridge (abscission) results in separation of two daughter cells. Although mechanisms that govern abscission are not fully understood, recent evidence suggest that endosomes, actin cytokeleton and ESCRT protein complex play critical role in this process. The focus of this project is to dissect the roles of endosomes and ESCRT complex during cytokinesis and investigate the cross-talk between the ESCRT, cytoskeleton and endocytic membrane transport.

Recycling endosomes (RE) have emerged as important players in mediating abscission. Originally it was proposed that REs initiate abscission by fusing with each other and the plasma membrane, thus building a separating membrane in a manner similar to a formation of phragmoplast in plant cells. However, recent data from our laboratory have shown that fusion of REs instead mediates a formation of a “secondary ingression” (Figure 2). Rab11, a small monomeric GTPase, plays a major role in regulating RE delivery to the ICB via binding to the FIP3 effector protein. The FIP3/Rab11 complex accumulates at the ICB during mitosis and is known to be required for late stages of cell division. Our recent data show that Rab11/FIP3-endosomes deliver known actin regulating protein p50RhoGAP to the furrow during late telophase. Thus, we hypothesize that FIP3-dependent delivery of p50RhoGAP is required for actin depolymerization during late telophase in mammalian cells. Investigating the role and regulation of Rab11/FIP3 endosomes and actin cytoskeleton during abscission is main focus of this project.

Project #3. The mechanisms regulating MMP targeting to forming invadopodia during breast cancer cell invasion

Although breast cancer is one of the leading causes of death for women, the mechanisms regulating the development and metastasis of breast carcinomas are not well defined. Multiple studies of breast carcinomas suggest that the progression of tumors is dependent to a large extent on the intrinsic properties of cancer cells, such as their ability to migrate and invade. Indeed, increased cell migration is a key event in malignant tumors and its prevention may slow the malignant evolution.

The extracellular matrix (ECM) proteins are crucial for regulation of breast cancer cell metastasis. ECM proteins that make up the specialized basement membrane serve as a barrier for the cell motility and invasion. The breach of the biological barriers, such as basement membranes is an important step in cancer cell invasion and metastasis. Basement membrane disruption usually involves a localized degradation of the ECM via the secretion of matrix metalloproteinase’s (MMPs). MMPs can be subdivided into multiple categories dependent on their substrate specificity, such as collagenases, stromelysins, and gelatinases. The main focus of this project is the MMP2 and MMP9 gelatinases. MMP2/9 mainly hydrolyzes the components of basement membrane, such as collagen type IV, thus has been implicated in the regulation of tumor growth and metastasis.

The role of invadopodia formation during cancer cell invasion. Invadopodia (also sometimes referred to as podosome in non-cancer cells) is an actin-rich plasma membrane protrusion located at the ventral side of the cancer cell. Invadopodia is the sites of ECM degradation and has been shown to be induced by Src kinase and mediate cancer cell invasion in vitro and in vivo. It has been demonstrated that MMP2/9 accumulate within the forming invadopodia, where they mediate localized ECM degradation. However, the mechanism mediating this sorting and targeting of MMP2/9 to invadopodia during metastasis remains virtually unknown. Defining these mechanisms and understanding their regulation is the main goal of this proposal.

Schiel, J.A., Simon, G., Castle, D., Christine C.W., D., and Prekeris, R. (2012) FIP3-endosome mediated actin depolymerization and formation of the secondary ingression mediates ESCRT-III recruitment to the abscission site during cytokinesis. Nature Cell Biology. In Press.

Prekeris, R. (2012) The art of “Cut and Run”: the role of Rab14 GTPase in regulating N-cadherin shedding and cell motility. Developmental Cell. 22(9):909-910.

Prekeris, R. (2012) Making the final cut: the role of endosomes during mitotic cell division. Chapter in Book “Membrane Trafficking”.

Collins, L.L., Simon, G.S., Matheson, J., Wu, C., Miller, M.C., Otani, T., Yu, X., Prekeris, R., and G.W. Gould (2012) Rab11-FIP3 is a cell cycle-regulated phosphoprotein. BMC Cell Biology. 13(1):4.

Arras, L., Yang, I., Lackford, B., Riches, D., Prekeris, R., Freedman, J.H., Schwartz, D.A., and Alper, S. (2012) Spatiotemporal inhibition of innate immunity signaling by the TBC1D23 Rab-GAP. The Journal of Immunology. 188(6):2905-2913.

Willenborg, C., Jing, J., Wu, C., Matern, H., Burden, J., and Prekeris, R. (2011) FIP5/Rip11 and SNX18 Interaction Regulates Epithelial Lumen Formation. The Journal of Cell Biology. 195(1):71-86.

Schiel, J., Park, K., Morphew, M.K., Reid, E., Hoenger, A., and Prekeris, R. (2011) Coordinated Endocytic Membrane Fusion and Buckling-Induced Microtubule Severing Mediate Cell Abscission. Journal of Cell Science. 124:1411-1424.

Willenborg, C., and Prekeris, R. (2011) Apical protein transport and lumen morphogenesis in polarized epithelial cells. Bioscience Reports. 31(4):245-256.

Schiel, J. and Prekeris, R. (2010) Making the final cut: Mechanisms mediating the abscission step of cytokinesis. The Scientific World Journal. 10:1424-1434.

Hsu, VW and Prekeris, R. (2010) Mechanistic understanding of transport through the recycling endosome. Current Opinion in Cell Biology. 22(4):528-534.

Jing, J., Junutula. J.R., Wu, C., Burden, J., Peden, A.A., and R. Prekeris (2010) FIP1/RCP binding to Golgin-97 regulates retrograde transport from recycling endosomes to Trans-Golgi Network. Molecular Biology of the Cell. 21(17):3041-3053.

Jing, J., Wilson, G., Tarbutton, E., and R. Prekeris (2009) Rab11-FIP3 is a Rab11 and Arf6 binding proteins that regulates breast cancer motility by modulating actin cytoskeleton. European Journal of Cell Biology. 88(6):325-341.

Jing, J. and Prekeris, R. (2009) Polarized endocytic transport: the roles of Rab11 and Rab11-FIPs in regulating cell polarity. Histology and Histopathology: Cellular and Molecular Biology. 24(9):1171-1180.

Simon, G.C., E. Schonteich, C.C. Wu, D. Ekiert, A. Piekny, X. Yu, G.W. Gould, M. Glotzer and R. Prekeris (2008) Sequential Cyk4/MgcRacGAP binding to ECT2 and Rab11-FIP3 regulates cleavage furrow ingression and abscission during cytokinesis. EMBO J. 27:1791-1803.

Schonteich, E., G.M, Wilson, J. Burden, C.R. Hopkins, K. Anderson, J.R. Goldenring and R. Prekeris (2008) Rip11/FIP5 and Kinesin II complex regulates endocytic protein recycling. Journal of Cell Science. 121:3824-3833.

Schonteich, E., M. Pilli, G.C. Simon, H.T. Matern, J.R. Junutula, D. Sentz, R.K. Holmes and R. Prekeris (2007) Molecular characterization of Rab11-FIP3 binding to Arf GTPases. European Journal of Cell Biology. 86:417-431.

Yu, X., Prekeris, R., and G. W. Gould (2007) Role of endosomal Rab GTPases in cytokinesis. European Journal for Cell Biology. 86:25-35.

Eathiraj, S., Mishra, A., Prekeris, R., and D.G. Lambright (2006) Structural basis for Rab11-mediated recruitment of FIP3 to recycling endosomes during cytokinesis. Journal of Molecular Biology. 364(2):121-135. ​​