Rab11 Regulates the Mast Cell Exocytic Response

Abstract

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 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.

Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling

“…Some outcomes of the rapid depolymerization of cortical actin that occurs within seconds of antigen addition (Wollman and Meyer, 2012; Wilson et al. , 2016; Supplemental Figure S3) may be roughly approximated by latrunculin and cytochalasin treatments.…”

Regulation of Exocytosis and Mitochondrial Relocalization by Alpha-Synuclein in a Mammalian Cell Model

“…Previous studies reported that the disordered C-terminus of a-syn ( Figure 1) mediates interactions with proteins involved in exocytosis, including the v-SNARE VAMP2 26 and Rab family proteins, 27 which also participate in stimulated exocytosis of REs in RBL cells. 23 We found that truncated a-syn 1-102 (C-terminal residues 103-140 deleted) inhibits thapsigargin mediated exocytosis similarly to Wt a-syn ( Figure 2F,G). We also tested Wt a-syn tagged with mRFP on its C-terminus (Wt syn-mRFP), considering that a fluorescent tag the size of mRFP (~27 kDa) may sterically disrupt some cellular interactions of a-syn (~14 kDa) and found it to be similarly inhibitory ( Figure S1B, E).…”

“…The DNP-antigen crosslinks IgE/FceRI on the cell surface to initiate a signaling cascade, activating protein kinase C (PKC) and triggering Ca 2+ release from intracellular stores, followed by store operated Ca 2+ entry from the extracellular medium and consequent exocytosis of secretory lysosomes and REs. 23 Figure 2D).…”

“…We showed previously that stimulated exocytosis of REs is triggered by both antigen and thapsigargin. 23 Total internal reflection microscopy (TIRFM) shows vividly that low expression levels of Wt a-syn inhibit thapsigargin-stimulated exocytosis (Figure 2A,B and supplemental Movies S1A,B). To view this time course by microscopy, cells were co-transfected with DNA for human a-syn or vector control (pcDNA), together with the reporter VAMP8-pHluorin, a v-SNARE protein conjugated to a fluorophore that is quenched when localized to the mildly acidic environment of the RE.…”

“…VAMP8-pHluorin fluorescence increases markedly after stimulated trafficking of REs to, and exocytosis at, the plasma membrane, thereby encountering the neutral pH environment of the extracellular medium. 23 Addition of NH4 + Clat the end of the time course neutralizes the pH in all cellular compartments and provides a means of normalizing the level of the stimulated exocytosis. TIRFM further reveals that, in contrast to lower a-syn expression levels which Figure 2.…”