Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells.

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

Remodelling of Ca2+ handling organelles in adult rat ventricular myocytes during long term culture.

It is well known that for cardiomyocytes, isolation and culturing induce largely unknown remodelling processes. We analysed changes in the structure of cell compartments with optical techniques such as confocal microscopy and fluorescence redistribution after photobleaching employing adenoviral-mediated transduction of targeted fluorescent proteins and small molecule dyes. We identified characteristic remodelling processes: the T-tubular membrane system was gradually lost by a process referred to as "sequential pinching off", in an outward direction. Mitochondria fell in one of three classes, very small (0.9 microm length), medium long (1.8 microm) or extended shape (3.6 microm) organelles. Over the culturing time mitochondria gradually fused. Bleaching of individual mitochondria revealed association between apparently separated mitochondria by "tunnelling" via sub-resolution organelle-tubes. This tunnelling process was increasing over the culturing time. A gradual loss of the cross-striation arrangement in the endoplasmic/sarcoplasmic reticulum was visualised. Analysis of large populations of Ca(2+) sparks by video-rate confocal 2D-scanning revealed significant albeit small changes of these elementary SR-Ca(2+) release events in adult cardiomyocytes that could be related to changes in SR-Ca(2+) content rather than resting Ca(2+) concentration. In conclusion, primary isolated cardiomyocytes from adult hearts undergo a well-defined, but reproducible subcellular remodelling during optimised long term culture.

Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.