CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic beta cells.

CAPS1 and CAPS2 regulate dense-core vesicle release of transmitters and hormones in neuroendocrine cells, but their precise roles in the secretory process remain enigmatic. Here we show that CAPS2(-/-) and CAPS1(+/-);CAPS2(-/-) mice, despite having increased insulin sensitivity, are glucose intolerant and that this effect is attributable to a marked reduction of glucose-induced insulin secretion. This correlates with diminished Ca(2+)-dependent exocytosis, a reduction in the size of the morphologically docked pool, a decrease in the readily releasable pool of secretory vesicles, slowed granule priming, and suppression of second-phase (but not first-phase) insulin secretion. In beta cells of CAPS1(+/-);CAPS2(-/-) mice, the lowered insulin content and granule numbers were associated with an increase in lysosome numbers and lysosomal enzyme activity. We conclude that although CAPS proteins are not required for Ca(2+)-dependent exocytosis to proceed, they exert a modulatory effect on insulin granule priming, exocytosis, and stability.

Recent progress in freeze-fracturing of high-pressure frozen samples.

Pancreatic tissue, bacteria and lipid vesicles were high-pressure frozen and freeze-fractured. In addition to the normal holder, a new type of high-pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low-mass copper platelets, as used for propane-jet freezing. Both the standard and the new suspension holder allowed us to make cryo-fractures without visible ice crystal damage. High-pressure frozen rat pancreas tissue samples were cryo-fractured and cryo-sectioned with a new type diamond knife in the microtome of a freeze-etching device. The bulk fracture faces and blockfaces were investigated in the frozen-hydrated state by use of a cryo-stage in an in-lens SEM. Additional structures can be made visible by controlled sublimation of ice ('etching'), leading to a better understanding of the three-dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life-like state, preventing the artefacts associated with conventional fixation techniques.

Structure and composition of the fusion pore.

Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, alpha-fodrin and the calcium channels alpha1c and beta3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.