Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking.

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells.

The path of pre-ribosomes through the nuclear pore complex revealed by electron tomography.

Determining the path of single ribonucleoprotein (RNP) particles through the 100 nm-wide nuclear pore complex (NPC) by fluorescence microscopy remains challenging due to resolution limitation and RNP labeling constraints. By using high-pressure freezing and electron tomography, here we captured snapshots of the translocation of native RNP particles through NPCs in yeast and analyzed their trajectory at nanometer-scale resolution. Morphological and functional analyses indicate that these particles mostly correspond to pre-ribosomes.

Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells.

Annexin A2 (AnxA2) is a calcium and lipid binding protein involved in neuroendocrine secretion. We have previously demonstrated that AnxA2 participates in the formation and/or stabilization of lipid microdomains required for structural and spatial organization of the exocytotic machinery in chromaffin cells. However, the regulation of AnxA2 is not fully understood.

Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis.

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells.