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Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies
Shivangi M Inamdar1, Shu-Chan Hsu2, Charles Yeaman1
1Molecular and Cellular Biology Program, University of IowaIowa City, IA, USA; Department of Anatomy and Cell Biology, University of IowaIowa City, IA, USA.
Frontiers in Cell and Developmental Biology
|July 5, 2016
Summary
Investigating the exocyst complex in epithelial cells revealed distinct Sec6 protein configurations. These changes in exocyst structure correlate with its function in exocytosis and localization during cell polarization.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The exocyst complex is crucial for spatial regulation of exocytosis, mediating vesicle tethering to the plasma membrane.
- Mechanisms governing exocyst assembly, disassembly, localization, and function remain incompletely understood.
- Understanding exocyst dynamics is key to deciphering regulated secretion processes.
Purpose of the Study:
- To investigate configurational changes of the exocyst complex, specifically Sec6, during transitions in exocytosis.
- To map epitopes on Sec6 and correlate their accessibility with exocyst function and localization in epithelial cells.
- To explore the role of Ral GTPases in regulating exocyst localization and configuration.
Main Methods:
- Utilized a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe Sec6 epitopes in MDCK cells.
- Employed gel filtration and co-immunoprecipitation to study Sec6-Sec8 interactions and complex formation.
- Performed RNA interference (RNAi) for Sec6 and RalA, alongside epitope mapping using fusion proteins.
Main Results:
- Over 20 mAbs recognized distinct Sec6 epitopes, labeling different subcellular structures (cytosolic, nuclear, apical junctional complex, desmosomes, ER, intermediate filaments).
- Epitope accessibility varied between non-polarized and polarized cells, indicating redistribution or concealment during polarization.
- RalA RNAi reduced exposure of Sec6 C-terminal epitopes at the plasma membrane, suggesting Ral GTPase involvement.
- Differential co-immunoprecipitation revealed distinct interaction patterns of Sec6 with other exocyst subunits (Sec5, -10, -15, Exo70, -84, Sec3, Sec8) depending on the antibody used (N-terminal vs. C-terminal).
Conclusions:
- Sec6 exhibits distinct configurations linked to its subcellular localization and function in exocytosis.
- Epitope accessibility changes during epithelial polarization suggest dynamic regulation of the exocyst complex.
- The exocyst complex undergoes molecular rearrangements or associates with accessory proteins, modulating Sec6 conformation during activation and localization.
