Exocytosis Of Secretory Organelles Research Articles

Signalling mechanisms for Toll‐like receptor‐activated neutrophil exocytosis: key roles for interleukin‐1‐receptor‐associated kinase‐4 and phosphatidylinositol 3‐kinase but not Toll/IL‐1 receptor (TIR) domain‐containing adaptor inducing IFN‐β (TRIF)

Lipopolysaccharide (LPS) stimulates exocytosis in neutrophils. The signalling molecules involved in the regulation of this mechanism are currently unknown. Using neutrophils from interleukin-1-receptor-associated kinase (IRAK)-4- and Toll/IL-1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-deficient mice, we dissected the signalling pathways that control exocytosis. We analysed exocytosis of peroxidase-negative and azurophilic granules by following the mobilization of the beta2-integrin subunit CD11b and myeloperoxidase (MPO)-containing granules, respectively. IRAK-4-null neutrophils showed marked defects in both peroxidase-negative and azurophilic granule exocytosis in response to LPS. In contrast, the exocytic response to LPS of TRIF-deficient neutrophils was not different from that of wild-type cells. No differences were observed in the exocytosis of secretory organelles between IRAK-4-null and wild-type neutrophils when they were stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA). Electron microscopy analysis showed that no morphological abnormalities were present in the granules of IRAK-4-deficient neutrophils, suggesting that the lack of exocytic response to LPS is not attributable to developmental abnormalities. Using pharmacological inhibitors, we found that p38 mitogen-activated protein kinase (p38MAPK) is essential for the exocytosis of all neutrophil secretory organelles in response to LPS. Interestingly, we found that phosphatidylinositol 3-kinase (PI3K) is essential for azurophilic granule exocytosis but not for the mobilization of other neutrophil granules in response to LPS. Azurophilic granule exocytosis in response to Listeria monocytogenes was dependent on PI3K but not IRAK-4 activity, suggesting that alternative signalling pathways are activated in IRAK-4-deficient neutrophils exposed to whole bacteria. Our results identified IRAK-4, p38MAPK and PI3K as important regulatory components with different roles in the signalling pathways that control Toll-like receptor ligand-triggered neutrophil exocytosis.

On the role of intravesicular calcium in the motion and exocytosis of secretory organelles

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient towards the cytosol (5.5 vs. 7.2) promoted by the V-ATPase activity. This gradient of pH is mainly responsible for the accumulation of amines. The secretory vesicles contain large amounts of total Ca2+, but the free intragranular [Ca2+], the mechanisms for Ca2+ uptake and release from the granules and their physiological relevance regarding exocytosis are still matters of debate.We have recently shown that disruption of the pH gradient of secretory vesicles slowed down exocytosis. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that the V-ATPase inhibitor bafilomycin A1 directly released Ca2+ from freshly isolated vesicles. Accordingly, vesicle alkalinization released Ca2+ from the granules to the cytosol, measured with fura-2 in intact chromaffin cells. Using TIRFM in cells over-expressing the EGFP-labeled synaptobrevin (VAMP2-EGFP) protein, we have then shown that the Ca2+ released from the vesicles to the cytosol in the presence of bafilomycin, dramatically increased the granule motion of chromaffin- or PC12-derived granules, and triggered exocytosis (measured by amperometry).We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of the local cytosolic Ca2+ around the vesicles and in two of the major functions of secretory cells, vesicle motion and exocytosis.1

Molecular determinants of exocytosis.

Membrane fusion processes occur throughout the cell. Among those, exocytosis of secretory organelles is probably one of the fastest fusion events animal cells can achieve. Structural and functional studies have provided a conceptual framework and a starting point for our mechanistic understanding of how SNARE proteins may contribute to the final step in exocytosis.