Mast cell granule motility and exocytosis is driven by dynamic microtubule formation and kinesin-1 motor function
Fig 5
Knock-down of the kinesin-1 heavy chain, Kif5b, inhibits granule translocation to the plasma membrane and exocytosis.
A) Comparison of Kif5b levels in different RBL-2H3 knock-down strains. Kif5b knock-down strains were generated with three different specific shRNAs and a non-specific scrambled shRNA as a control. Immunoblot analysis shows reduction in Kif5b in all three strains treated with specific shRNAs, with strain 479 showing the greatest reduction. Tubulin levels remained similar. B) Exocytosis assay of RBL-2H3 strains that were unstimulated (black bars), or antigen-stimulated for 30 min (hatched bars). The Kif5b knock-down strain showed a significant reduction in exocytosis compared to the scrambled control and wild-type strains. Exocytosis was assayed as the percent β-hexosaminidase released of total, normalized to wild-type stimulated samples. Statistical analysis was by two-tailed unpaired Student’s t-test (NS, not significant; ***, p = 0.0043; **, p = 0.029; n = 4). C and D) Immunofluorescence microscopy of RBL-2H3 strains showing the intracellular distribution of CD63+ granules. Control cells (C) or Kif5b knock-down cells (D) were left unstimulated or antigen-stimulated for 30 min, fixed and stained for nuclei (blue), microtubules (green), or CD63+ granules (red). Scale bar, 10 μm. Profile plots (right panels) show the levels of CD63+ granules distributed in the indicated cross-sections. Kif5b knock-down did not affect microtubule projections after stimulation, however CD63+ granules accumulated in the perinuclear region (D’, red), while in control cells CD63+ granules were distributed across the entire cross-section (C’, red).