Examining the Role of STRIPAK in the Tight Junctions of Epithelial Cells Open Access

Yang, Chloe (Spring 2022)

Permanent URL: https://etd.library.emory.edu/concern/etds/kw52j935w?locale=en


Background: Striatin family proteins, striatin, SG2NA, and zinedin, serve as scaffolds for striatin interacting phosphatase and kinase (STRIPAK) complexes, which play a role in several cellular processes, including signaling, cell cycle regulation, vesicular trafficking, cell polarity, vascular development, and cardiac functioning. In addition to core components, including striatin, protein phosphatase 2A, Mob3, CCM3, a GCKIII kinase, and STRIP1 or 2, different STRIPAK complexes can also contain additional components. Tight junctions (TJs) maintain cell polarity and protect organisms from their external environment. Striatin was recently shown to be present in tight junctions and to be important for their formation or stability, but what other components of STRIPAK that might be in tight junctions is not known. Moreover, how striatin is targeted to tight junctions is unknown.  To define the STRIPAK complex present in TJs and to begin to study how STRIPAK is localized to junctions and what its role is in TJ formation, remodeling, and stability, we studied the localization of striatin and other STRIPAK components in human epithelial cells at steady-state and during remodeling.

Methods: The localizations of different STRIPAK components and TJ proteins were visualized using immunofluorescence (IF) in human colorectal cancer cells to characterize the STRIPAK complex present at tight junctions before and during TJ remodeling. In addition, stable cell lines expressing PP2A- or CCM3striatin mutants were developed to help probe the relevant protein-protein interactions that are important for TJ formation and stability. 

Results: We demonstrated that STRIPAK core proteins striatin, CCM3, GCKIII kinases Mst3 and Mst4, Mob3, and STRIP1 localize to tight junctions in epithelial cells. We also showed that of two mutually exclusive additional STRIPAK components, SLMAP and CTTNBP2/NL, only SLMAP is present in tight junctions. STRIPAK components CCM3, striatin, and STRIP1 were found to co-localize with junctional proteins during TJ remodeling, but SLMAP did not. PP2A- striatin mutant-expressing cells demonstrated an internalization of TJ scaffolding protein ZO-1.  

Conclusions: Striatin-scaffolded core STRIPAK complexes containing SLMAP likely play a role in vesicular trafficking of TJ proteins to and/or from the membrane.  This may be regulated in a PP2A-dependent manner. These findings provide insight into the regulation of TJ formation and CCM disease.  

Table of Contents

Introduction - 1

Tight Junctions - 1

Tight Junction Assembly - 2

PP2A - 4

STRIPAK Complex - 5

Striatin - 6

GCKIII and CCM3 - 7

Mob3 - 8

STRIP1/2 and associated proteins - 9

Cerebral Cavernous Malformation - 10

Preliminary data and hypothesis - 12

Materials and Methods - 13

Results - 17

Discussion - 29

References - 33

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