Rab27b regulates mast cell granule dynamics and secretion

Abstract

The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome-related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b. Here we used genetically modified mice to study the involvement of the Rab27 proteins in mast cells, which play key roles in allergic responses. Both Rab27a and Rab27b isoforms are expressed in bone marrow-derived mast cells (BMMC) and localize to secretory granules. Nevertheless, secretory defects as measured by beta-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo were found only in Rab27b and double Rab27 knockout (KO) mice. Immunofluorescence studies suggest that a subset of Rab27b and double Rab27-deficient BMMCs exhibit mild clustering of granules. Quantitative analysis of live-cell time-lapse imaging revealed that BMMCs derived from double Rab27 KO mice showed almost 10-fold increase in granules exhibiting fast movement (>1.5 microm/s), which could be disrupted by nocodazole. These results suggest that Rab27 proteins, particularly Rab27b, play a crucial role in mast cell degranulation and that their action regulates the transition from microtubule to actin-based motility.

Figure 1. Rab27 proteins in BMMCs

A) Bone marrow-derived mast cells derived from C57BL/6J wild-type, Rab27a KO (Rab27a^ash/ash), Rab27b KO (Rab27b^−/−) and double KO (Rab27a^ash/ashRab27b^−/−) mice were subjected to immunoblot analysis as described under Materials and Methods. B) Bone marrow-derived mast cells derived from C57BL/6J wild-type mice were subjected to immunofluorescence using anti-Rab27a, anti-Rab27b and anti-serotonin antibodies. Representative single midsection images by confocal microscopy are shown. Inserted images are enlarged. Bar, 10 μm.

Figure 2. >Passive cutaneous anaphylaxis assays in Rab27-deficient mice

Rab27a^ash/ash(ash/ash) or heterozygote littermate (ash/+) mice (A), Rab27b^−/− (−/−) or heterozygote littermate (−/+) mice (B), and Rab27a^ash/ashRab27b^−/− (−/−) or heterozygote littermate (−/+) mice (C) were subjected to PCA assays as described under Materials and Methods. Data presented are the mean with standard error of the mean of at least nine independent experiments. *p < 0.05 (unpaired Student's t-test).

Figure 3. Antigen-induced β-hexosaminidase release in BMMCs derived from Rab27-deficient mice

Bone marrow-derived mast cells derived from Rab27a^ash/ash (ash/ash) or heterozygote littermate (ash/+) mice (A), Rab27b^−/− (−/−) or heterozygote littermate (−/+) mice (B) and Rab27a^ash/ashRab27b^−/− (−/−) or heterozygote littermate (−/+) mice (C) were subjected to β-hexosaminidase release assay as described under Materials and Methods. After sensitization with anti-dinitrophenyl IgE, the cells were stimulated with various concentration of DNP–BSA for 20 min. Each point was determined in triplicate and presented as mean with standard error of the mean. The experiments shown are representative of at least three independent experiments. *p < 0.05 (Mann–Whitney test).

Figure 4. Immunofluorescence analysis of mast cell secretory granules in BMMCs derived from Rab27-deficient mice

Bone marrow-derived mast cells derived from Rab27a^ash/+Rab27b^−/+ (Double het), Rab27a^ash/ash (Rab27a KO), Rab27b^−/− (Rab27b KO) and Rab27a^ash/ashRab27b^−/− (Double KO) were subjected to immunofluorescence as described under Materials and Methods, and stained with anti-histamine (green) and anti-alpha-tubulin (red) antibodies. Representative projected images using confocal microscopy as described under Materials and Methods are shown. Bar, 5 μm.

Figure 5. Electron microscopy analysis of BMMCs

A) Ultrathin sections of epon-embedded BMMCs from the indicated strains. Bar, 2 μm. B) Ultrathin cryosections of BMMCs from EGFP-Rab27a mice were double immunogold labeled for Rab27b (PAG15) and Rab27a (PAG10). Rab27a was detected with anti-GFP. Labeling for both Rab27b (arrowheads) and Rab27a (arrows) could be detected on the same granules Bar, 500 nm. Inset: example of a type II granule containing an electron-dense core and small internal vesicles. Bar, 250 nm. PM, plasma membrane.

Figure 6. Secretory granule dynamics in BMMCs

Bone marrow-derived mast cells derived from Rab27a^ash/+Rab27b^−/+ (Double het) (A), or Rab27a^ash/ashRab27b^−/− (Double KO) mice in the absence (B) or presence of nocodazole (C) were labeled with LysoTracker Red DND-99 and subjected to TIRF microscopy as described under Materials and Methods. Arrowheads point to granules that remain still and arrows to granules that exhibit fast movement (<1.5 μm/s, see also Table 1). Selected frames from time-lapse recordings are presented. Bar, 2.5 μm.