Biomedical subjects

Martin Wienisch

Publications and source records attributed to Martin Wienisch.

4 recordsLinked to original sources

PubMed2006

Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical.

Upon exocytosis, synaptic vesicle proteins are released into the plasma membrane and have to be retrieved by compensatory endocytosis. When green fluorescent protein-labeled versions of the vesicle proteins synaptobrevin-2 and synaptotagmin-1 are overexpressed in rat hippocampal neurons, up to 30% are found on axonal membranes under resting conditions. To test whether and to what extent these plasma membrane-stranded proteins participate in exo-endocytic cycling, a new proteolytic approach was used to visualize the fate of newly exocytosed proteins separately from that of the plasma membrane-stranded ones. We found that both pools were mixed and that endocytosed vesicles were largely composed of previously stranded molecules. The degree of nonidentity of vesicular proteins exo- and endocytosed depended on stimulus duration. By using an antibody to the external domain of synaptotagmin-1, we estimated that under physiological conditions a few percent of vesicular proteins were located near the active zone, from where they were preferentially recycled upon stimulation.

Animals↗

PubMed2006

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling.

Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling.

Adaptor Protein Complex 2↗

PubMed2005

Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy.

The clathrin triskelion is composed of three light chain (LC) and three heavy chain (HC) subunits. Cellular control of clathrin function is thought to be aimed at the LC subunit, mainly on the basis of structural information. To test this hypothesis in vivo, we used evanescent-wave photobleaching recovery to study clathrin exchange from single pits using LC (LCa and LCb) and HC enhanced green fluorescent protein fusion constructs. The recovery signal was corrected for cytosolic diffusional background, yielding the pure exchange reaction times. For LCa, we measured an unbinding time constant tau(LEa) = 18.9 +/- 1.0 seconds at room temperature, faster than previously published; for LCb, we found tau(LCb) = 10.6 +/- 1.9 seconds and for HC tau(HC) = 15.9 +/- 1.0 seconds. Sucrose treatment, ATP or Ca(2+) depletion blocked exchange of LCa completely, but only partially of HC, lowering its time constant to tau = 10.0 +/- 0.9 seconds, identical to the one for LCb exchange. The latter was also not blocked by Ca(2+) depletion or sucrose. We conclude that HCs bound both to LCa and to LCb contribute side by side to pit formation in vivo, but the affinity of LCa-free HC in pits is reduced, and the Ca(2+)- and ATP-mediated control of clathrin function is lost.

Adenosine Triphosphate↗

PubMed2004

Monitoring clathrin-mediated endocytosis during synaptic activity.

To visualize clathrin redistribution during endocytosis in hippocampal boutons, we used a fusion protein of clathrin light chain with enhanced green fluorescent protein. Both high potassium and electric field stimulation lead after a stimulus-dependent delay to a transient increase of fluorescence in synapses, but a slight and transient decrease in adjacent axonal segments. We conclude that the rise and fall of the signal in boutons, with decay kinetics remarkably similar to previous estimates of the endocytic time course, reflects coat assembly and disassembly. Thus, we could selectively measure clathrin-mediated endocytosis and separate its kinetics from other modes of membrane retrieval in CNS synapses. A long-lasting delay preceding the fluorescent transients shows that endocytosis during the first few seconds of continuing stimulation cannot be mediated by newly formed clathrin-coated pits. Therefore, a fast mode of endocytosis is either clathrin-independent or involves preassembled (easily retrievable) clathrin lattices at sites of endocytosis.

Action Potentials↗