Epithelia consist from polarized cells that are capable of selectively transporting substances across epithelial monolayer. This selective transport is achieved by the partitioning of the plasma membrane into distinct domains: apical and basolateral, with both of these plasma membrane compartments having distinct lipid and protein compositions. Since the fidelity of trans-epithelial protein transport is crucial to a variety of epithelial functions, epithelial cells have developed complicated mechanisms to ensure correct transport of proteins. Rab11 GTPases are the members of small monomeric GTPase super-family that has been implicated in regulating endocytic membrane transport. Rab GTPase work by recruiting various effector proteins to the distinct cellular compartments. Thus, understanding the role of these effector proteins is a key step in understanding the function of epithelial cell. In the last six years several Rab11-binding proteins have been identified, which include Rab11 family interacting proteins, also known as FIPs. Works from several laboratories, including ours, have shown that Rip11/FIP5 member of FIP family regulate polarized protein transport in epithelial cells. Furthermore, it was shown that FIPs act as scaffolding factors allowing the assembly of specific sorting/transport complexes required for the epithelial protein traffic. Based on recently published results and on preliminary data, we propose the following hypotheses. First, that Rip11/FIP5 mediates protein transport to apical plasma membrane. Second, that Rip11/FIP5 regulates apical protein transport by sequential recruiting of sortin nexin 18 (SNX18) and kinesin II to the endocytic membranes. Thus, the main goal of this proposal is to further characterize Rip11/FIP5-SNX18 and Rip11/FIP5-Kinesin II complex formation and determine their role on epithelial transport. I propose three different aims designed to test these hypotheses. In the aim #1 we will analyze the role of Rip11/FIP5 in regulating apical protein transport. We will use combination of tet-inducible protein knock-down, fluorescence imaging and in vivo transport assays. In the aim #2 and aim #3 we will characterize the binding of Rip11/FIP5 to SNX18 and Kinesin II. We will use the combination of immunoprecipitations, glutathione bead pull-down, yeast two-hybrid and isothermal calorimetry assays. In addition, in aim #2 and aim #3 we will determine the roles of Rip11/FIP5-binding proteins in regulating apical and basolateral membrane traffic. To that end we will use the combination of tet- inducible protein knock-down, fluorescence imaging, site-directed mutagenesis and in vivo transport assays.

Public Health Relevance

The goal of this project is to understand the molecular mechanisms of Rab11-dependent polarized membrane traffic and define the membrane traffic steps that are regulated by each Rab11-Rip11/FIP5 protein complex. Furthermore, this project will also identify the molecular machinery mediating Rab11-dependent endocytic protein sorting. These data will advance our understanding, both conceptually and mechanistically, of the machinery governing protein targeting in epithelial cells. To elucidate the mechanisms of protein targeting is of major importance because the defects in this process cause a variety of pulmonary (cystic fibrosis) and renal (nephrogenic diabetes insipidus) disorders. Thus, new insights into the mechanisms of polarized membrane traffic may lead to the identification of new drug targets as well as new therapeutic approaches aimed at the identification and treatment of membrane traffic disorders.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Mullins, Christopher V
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Colorado Denver
Schools of Medicine
United States
Zip Code
Gibieža, Paulius; Prekeris, Rytis (2018) Rab GTPases and cell division. Small GTPases 9:107-115
Das, Lipsa; Gard, Jaime M C; Prekeris, Rytis et al. (2018) Novel Regulation of Integrin Trafficking by Rab11-FIP5 in Aggressive Prostate Cancer. Mol Cancer Res 16:1319-1331
Jewett, Cayla E; Prekeris, Rytis (2018) Insane in the apical membrane: Trafficking events mediating apicobasal epithelial polarity during tube morphogenesis. Traffic :
Antanavi?i?t?, Ieva; Gibieža, Paulius; Prekeris, Rytis et al. (2018) Midbody: From the Regulator of Cytokinesis to Postmitotic Signaling Organelle. Medicina (Kaunas) 54:
Dionne, Lai Kuan; Peterman, Eric; Schiel, John et al. (2017) FYCO1 regulates accumulation of post-mitotic midbodies by mediating LC3-dependent midbody degradation. J Cell Sci 130:4051-4062
Peterman, E; Prekeris, R (2017) Understanding post-mitotic roles of the midbody during cell differentiation and polarization. Methods Cell Biol 137:173-186
Mandell, Michael A; Jain, Ashish; Kumar, Suresh et al. (2016) TRIM17 contributes to autophagy of midbodies while actively sparing other targets from degradation. J Cell Sci 129:3562-3573
Mangan, Anthony J; Sietsema, Daniel V; Li, Dongying et al. (2016) Cingulin and actin mediate midbody-dependent apical lumen formation during polarization of epithelial cells. Nat Commun 7:12426
Dionne, Lai Kuan; Wang, Xiao-Jing; Prekeris, Rytis (2015) Midbody: from cellular junk to regulator of cell polarity and cell fate. Curr Opin Cell Biol 35:51-8
Prekeris, Rytis (2015) Analyzing the functions of Rab11-effector proteins during cell division. Methods Cell Biol 130:19-34

Showing the most recent 10 out of 36 publications