Cytoskeletal Remodeling and Exocytosis During Mast Cell Activation is Controlled by the RhoA GEF, GEF-H1

• Author / Creator

  Guo, Yitian

• Mast cells are tissue-resident immune cells that play important roles in health and diseases. Mast cells release their granule contents under antigen-stimulation via the FcεRI signaling pathway. The process of regulated exocytosis in mast cells is known as degranulation which participates in allergic and inflammatory disorders, such as asthma. Therefore, it is important to elucidate the detailed mechanisms of mast cell exocytosis. Previously, we and others have shown that Rho proteins (Rac1, Cdc42, RhoA), members of small monomeric G proteins and molecular switches, regulate mast cell degranulation. Rho proteins are master regulators of the cytoskeleton including actin and microtubules, suggesting that cytoskeleton remodeling is involved in controlling granule transport in mast cells. Rho proteins are activated by RhoGEFs.
  Here, we investigated the roles of cytoskeleton remodeling and RhoGEFs in regulating mast cell exocytosis. In Chapter 3, we primarily used live-cell imaging to analyze cytoskeleton remodeling and granule transport in real-time during antigen-stimulation of RBL-2H3 cells. Granule transport to the cell periphery was found to be coordinated with de novo microtubule formation rather than F-actin. Kinesore, a drug that activates the microtubule motor kinesin-1 in the absence of cargo, inhibited microtubule-granule association and significantly reduced exocytosis, but had no effect on cell morphology. Immunofluorescence microscopy showed granules accumulated in the perinuclear region after kinesore treatment. The depolymerization of microtubules with nocodazole or colchicine also resulted in a significant defect in exocytosis and prevention of granule movement; however, cell morphology was also significantly affected. Furthermore, enriched granule fractions showed kinesin-1 levels increased in antigen-stimulated cells, whereas they were reduced by kinesore pre-treatment. Results of granule co-fractionation assays suggested that cargo adaptors recruitment to granules was independent of the kinesin-1 motor association. Altogether, results in Chapter 3 showed that mast cell granules associate with microtubules and are driven by kinesin-1 to facilitate exocytosis.
  In Chapter 4, RhoGEFs required for mast cell exocytosis were investigated using RBL-2H3 cells as model mast cells. RT-PCR was used to profile the expression levels of RhoGEFs. High levels and selective mast cell expression suggested that Vav1, P-Rex1, α-PIX, β-PIX and GEF-H1 may act as candidates. Silencing of Vav1, P-Rex1, α-PIX, β-PIX expression by RNA interference (RNAi) did not alter granule movement or exocytosis in antigen-stimulated cells. The activation of Rac1 was downregulated, but not completely, in Vav1, P-Rex1, and α-PIX depleted cells. These results ruled out independent functional roles for these RhoGEFs in mast cell exocytosis. Importantly, silencing of GEF-H1 significantly disrupted cell spreading, granule movement and exocytosis. Re-introduction of an RNAi-resistant mutant of GEF-H1 restored cell morphology and granule localization in GEF-H1-depleted cells when stimulated. Moreover, RhoA, but not Rac1, was found to be a downstream target by GEF-H1. Morphologically, the knockdown of GEF-H1 suppressed stress fiber formation, a function of RhoA, without altering cell ruffling or lamellipodia formation. Re-introduction of Rho-G14V, a constitutively active mutant of RhoA, restored normal cell morphology in antigen-stimulated GEF-H1-depleted cells. In addition, focal adhesion (FA) formation was found to participate in granule exocytosis. Inhibition of FA formation by PF-573228 significantly reduced antigen-stimulated exocytosis. GEF-H1 depletion led to the reduced formation of FAs in antigen-stimulated cells, which correlated with defective exocytosis. Furthermore, GEF-H1 underwent activation via the FcεRI signaling pathway, but was found to be independent of microtubule dynamics. Activation of GEF-H1 was dependent on the Syk kinase regardless of other kinases including Src, Fyn, Lck, MEK1/2, PI3K and FAK. The Syk inhibitor, GS-9973, suppressed cell spreading, granule movement and exocytosis in antigen-stimulated RBL-2H3 cells. Assays of co-localization and co-fractionation of enriched granules did not identify interactions between GEF-H1 and Exo70, an important component of the exocytosis machinery. Taken together, the GEF-H1-RhoA signaling axis transduces antigen stimulation signals from FcεRI to the exocytosis machinery in mast cells, which we show involves the formation of FAs.

• Subjects / Keywords

  • mast cell
  • RhoGEF
  • GEF-H1
  • ARHGEF2
  • exocytosis
  • cytoskeletal remodeling

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-kxv8-q360

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.