Department of Cell and Developmental Biology (CDB)

The Role of Rab GTPases in Regulating Plasma Membrane Receptor Recycling, Cell motility, and Cytokinesis

Eukaryotic cells compartmentalize biological functions in a series of membrane-bound organelles. The unique composition of each compartment is maintained despite the continuous movement of proteins and lipids within the cell. To achieve that proteins are specifically targeted to various subcellular compartments. Furthermore, regulated protein targeting also plays a key role in plasma membrane receptor recycling, cell motility and cytokinesis (cell division).

Cells achieve protein targeting through the use of transport vesicles equipped with complex arrays of proteins that regulate vesicle formation, transport, and fusion. Small Rab GTPases are the key proteins involved in membrane traffic. Rabs function as "address" tags of transport vesicles by recruiting various effector proteins to the membranes (Figure 1). Critical questions in understanding the roles of Rab proteins are the identity and specificity of these effectors. Since most Rab GTPases have multiple effector proteins, the mechanisms and regulation of their binding are essential for understanding of Rab functions in membrane traffic.

The identification of Rab binding proteins and unders​tanding their function has been main focus of our laboratory in a last few years. The work in my laboratory led to identification of the novel family of Rab binding proteins, known as FIPs. Furthermore, we have shown that different Rab-FIP complexes play a key role in regulating many different cellular functions, including plasma membrane receptor recycling, cell motility, and cytokinesis . Three main projects are being currently investigated in the laboratory: (1) structure/function analysis of Rab-FIP protein complexes; (2) the role of Rab-FIP complexes in regulating recycling and targeting of plasma membrane receptors; and (3) the role of Rab-FIP complexes in epithelial-to-mesenchymal transition. To address these questions we use a variety of methods, including structural studies using circular dichroism, x-ray crystallography and NMR, immunoprecipitations, immunofluorescence and time-lapse microscopy, mutant analysis, permeabilized cell assays, proteomics, affinity chromatography, and yeast-two hybrid screens.

(1) Structure/function analysis of Rab-FIP protein complexes

Understanding the structure of proteins is the key step in determining their function in the cell. Thus, determining the properties of Rab and FIP interactions has been one of the major focuses in the lab. The current work concentrates on determining the structure of Rab-FIP complex (collaboration with Dr. Bill Weis, Stanford University) as well as kinetic properties of Rab-FIP complex formation.
Structural information from above studies is then used to analyze the role of Rab and FIP interactions in vivo. Several microscopy assays are used for that purpose. That includes fluorescent energy transfer (FRET) as well as time-lapse microscopy analysis (for cool movie depicting the fusion of transport vesicle/tubule containing GFP-labeled FIP see Figure 2).

(2) The role of Rab-FIP complexes in regulating recycling and targeting of plasma membrane receptors

Endocytosis and recycling of receptors back to plasma membrane plays a key role in regulating the response of cells to various extracellular ligands. Our laboratory have shown that Rab11/RCP (RCP belongs to FIP protein family) protein complex regulates the transport of receptors from endosomes back to plasma membrane. Thus, the current interest in the laboratory is to understand the molecular machinery regulating Rab11/RCp complex formation and function.

(3) The role of Rab-FIP complexes in epithelial-to-mesenchymal transition.

Epithelial cancers are the most common cancers and include colorectal, prostate, lung, and breast cancers. Epithelial tumor development is usually accompanied by the loss of polarity and acquisition of fibroblast-like motile phenotype, the process known as epithelial-to-mesenchymal transition (Figure 3). Thus, understanding the mechanism that controls the epithelial-to-mesenchymal transition will be the major step in our ability to detect and treat epithelial cancers. Several proteins, including ARF6 GTPase and Exocyst have recently emerged as an important regulator of the epithelial-to-mesenchymal transition. ARF6 regulates actin cytoskeletal rearrangements, stability of cell-cell adhesion, and polarized membrane insertion at the leading edge of lamellipodia/filopodia. Exocyst, on the other hand, regulates the delivery and targeting of plasma membrane needed for the extension of lamellipodia/filopodia. Recently we have determined that Rab11 associates with both ARF6 and exocyst through the help of FIP proteins. Thus, on of laboratories interests is to understand the role of this binding in regulating epithelial-to-mesenchymal transition.​

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.