Rab11 Regulates the Mast Cell Exocytic Response.
Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell-mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high-affinity receptor, FcϵRI, on the cell surface. Here we use the endosomal v-SNARE VAMP8, and the lysosomal hydrolase β-hexosaminidase (β-Hex), each C-terminally fused to super-ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin-tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with total internal reflection (TIRF) microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated β-Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F-actin with jasplakinolide inhibits antigen-stimulated exocytosis but is not additive with S25N Rab11-mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853-1301, USA.
Joshua D
JD
Wilson
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853-1301, USA.
Sarah A
SA
Shelby
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853-1301, USA.
David
D
Holowka
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853-1301, USA.
Barbara
B
Baird
D000818:Animals; D015550:Cell Degranulation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005089:Exocytosis / Q000276:immunology / Q000502:physiology*; D005470:Fluorometry; D006801:Humans; D008407:Mast Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D051381:Rats; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
F-actin; Rab11; TIRF; VAMP8; degranulation; exocytosis; recycling endosome