Ral-regulated interaction between Sec5 and paxillin targets Exocyst to focal complexes during cell migration
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Published on: November 1, 2021
Krystle S Spiczka1, Charles Yeaman
1Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Abstract:
Changes in cellular behavior that cause epithelial cells to lose adhesiveness, acquire a motile invasive phenotype and metastasize to secondary sites are complex and poorly understood. Molecules that normally function to integrate adhesive spatial information with cytoskeleton dynamics and membrane trafficking probably serve important functions in cellular transformation. One such complex is the Exocyst, which is essential for targeted delivery of membrane and secretory proteins to specific plasma membrane sites to maintain epithelial cell polarity. Upon loss of cadherin-mediated adhesion in Dunning R3327-5'A prostate tumor cells, Exocyst localization shifts from lateral membranes to tips of protrusive membrane extensions. Here, it colocalizes and co-purifies with focal complex proteins that regulate membrane trafficking and cytoskeleton dynamics. These sites are the preferred destination of post-Golgi transport vesicles ferrying biosynthetic cargo, such as alpha(5)-integrin, which mediates adhesion of cells to the substratum, a process essential to cell motility. Interference with Exocyst activity impairs integrin delivery to plasma membrane and inhibits tumor cell motility and matrix invasiveness. Localization of Exocyst and, by extension, targeting of Exocyst-dependent cargo, is dependent on Ral GTPases, which control association between Sec5 and paxillin. Overexpression of Ral-uncoupled Sec5 mutants inhibited Exocyst interaction with paxillin in 5'A cells, as did RNAi-mediated reduction of either RalA or RalB. Reduction of neither GTPase significantly altered steady-state levels of assembled Exocyst in these cells, but did change the observed localization of Exocyst proteins.
Insights
The Exocyst complex is crucial for epithelial cell polarity and metastasis. Its relocation and interaction with focal complexes regulate cell motility and invasion by controlling protein delivery to the cell membrane.
Area of Science:
- Cell Biology
- Cancer Biology
- Molecular Biology
Background:
- Epithelial cell transformation, characterized by loss of adhesion and acquisition of invasive phenotypes, is a complex process poorly understood.
- Molecules integrating cell adhesion with cytoskeleton dynamics and membrane trafficking are likely key players in cellular transformation.
- The Exocyst complex is vital for maintaining epithelial cell polarity through targeted delivery of proteins to the plasma membrane.
Purpose of the Study:
- To investigate the role of the Exocyst complex in prostate tumor cell metastasis.
- To elucidate the mechanisms by which Exocyst localization and function are regulated during cellular transformation.
- To determine the impact of Exocyst activity on cell motility and invasiveness.
Main Methods:
- Studied Dunning R3327-5'A prostate tumor cells with altered cadherin-mediated adhesion.
- Analyzed Exocyst localization shifts using microscopy and co-purification assays.
- Investigated the role of Ral GTPases in Exocyst localization and function.
- Assessed the effects of Exocyst interference on cell motility and matrix invasiveness.
Main Results:
- Upon loss of adhesion, Exocyst relocalizes from lateral membranes to protrusive extensions, co-localizing with focal complex proteins.
- Exocyst at these sites mediates the delivery of alpha(5)-integrin, essential for cell-substratum adhesion and motility.
- Interference with Exocyst activity impairs integrin delivery, reducing tumor cell motility and invasiveness.
- Exocyst localization and cargo targeting depend on Ral GTPases, controlling the Sec5-paxillin interaction.
Conclusions:
- The Exocyst complex plays a critical role in regulating prostate tumor cell motility and invasiveness by controlling protein trafficking to the cell surface.
- Ral GTPases are key regulators of Exocyst localization and function, influencing its interaction with focal adhesion machinery.
- Targeting Exocyst function represents a potential therapeutic strategy to inhibit cancer cell metastasis.
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