In APCs, antigen processing compartments often have the phenotypic characteristics of multivesicular bodies (MVB). In studies examining MHC-II trafficking to antigen processing compartments we found that activated (but not resting) B cells secrete significant amounts of their total pool of pMHC-II on MVB-derived vesicles termed exosomes. Surprisingly, it was predominantly Ii-free surface pMHC-II that internalized and trafficked to these MVB, highlighting a previously unrecognized transport pathway followed by pMHC-II. We also found that interaction of antigen-loaded B cells with antigen-specific T cells stimulates exosome release from B cells, and these exosomes in turn can stimulate primed (but not nave) T cells to proliferate. Our results support a model that T cell stimulated exosome release from activated B cells serves to augment T cell responses. The movement of proteins and lipids from one intracellular compartment to another is carried out by a well-orchestrated process of transport vesicle formation, vesicle docking with a target compartment, and finally vesicle-target membrane fusion. The proteins that catalyze membrane fusion are termed SNAREs. We have been investigating the role of distinct SNARE isoforms in regulating secretory granule exocytosis from immune cells using a variety of SNARE knock-out mice and have found that VAMP-8, but not VAMP-2 or VAMP-3, regulates mast cell secretory granule exocytosis. Surprisingly, VAMP-8-deletion only affects serotonin exocytosis but not histamine or TNF-alpha exocytosis, showing for the first time that secretory granule heterogeneity exists in mast cells. We are currently using these mice to examine the role of specific SNARE proteins in regulated exocytosis from other immune cells. SNAP-23 is a protein that regulates the ability of transport vesicles to dock and fuse with the plasma membrane. Although SNAP-23 is thought to have a ubiquitous function in all cells, there is no in vivo demonstration that SNAP-23 is a functionally important molecule. We have created a SNAP-23 knock-out mouse and determined that SNAP-23 null embryos die by E3.5, thereby precluding us from identifying a biological function of SNAP-23. We have therefore made a conditional knock-out of SNAP-23 using Cre/lox technology and found the deletion of SNAP-23 in any given cell type leads to the death of those cells. By transiently deleting SNAP-23 in fibroblasts we have found that SNAP-23 deletion leads to rapid cell death, demonstrating an essential role for SNAP-23 in regulating cell survival.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011035-06
Application #
8763316
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2013
Total Cost
$279,623
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Wu, Zhengli; Chen, Xiaochun; Liu, Fang et al. (2014) Calpain-1 contributes to IgE-mediated mast cell activation. J Immunol 192:5130-9
Suh, Young Ho; Terashima, Akira; Petralia, Ronald S et al. (2010) A neuronal role for SNAP-23 in postsynaptic glutamate receptor trafficking. Nat Neurosci 13:338-43
Berger, Adam C; Roche, Paul A (2009) MHC class II transport at a glance. J Cell Sci 122:1-4
Puri, Niti; Roche, Paul A (2008) Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms. Proc Natl Acad Sci U S A 105:2580-5
Suh, Young Ho; Pelkey, Kenneth A; Lavezzari, Gabriela et al. (2008) Corequirement of PICK1 binding and PKC phosphorylation for stable surface expression of the metabotropic glutamate receptor mGluR7. Neuron 58:736-48
Lee, Jeong Ho; Lee, Jinu; Choi, Kyu Yeong et al. (2008) Calmodulin dynamically regulates the trafficking of the metabotropic glutamate receptor mGluR5. Proc Natl Acad Sci U S A 105:12575-80
Walseng, Even; Bakke, Oddmund; Roche, Paul A (2008) Major histocompatibility complex class II-peptide complexes internalize using a clathrin- and dynamin-independent endocytosis pathway. J Biol Chem 283:14717-27