Biomedical subjects

Thorsten Lang

Publications and source records attributed to Thorsten Lang.

11 recordsLinked to original sources

PubMed2006

Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity.

Membrane fusion in the secretory pathway is mediated by soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Different fusion steps are thought to be effected by independent sets of SNAREs, but it is unclear whether specificity is determined by an intrinsic specificity of SNARE pairing or by upstream factors. Using a newly developed microscopy-based assay, we have investigated the SNARE specificity of homotypic early endosomal fusion. We show that early endosomes contain multiple sets of SNAREs, including, in addition to the putative early endosomal SNAREs, those involved in exocytosis and in fusion of late endosomes. We demonstrate that fusion is largely mediated by a complex formed by syntaxin 13, syntaxin 6, vti1a, and VAMP4, whereas the exocytic and late endosomal SNAREs play little or no role in the reaction. In contrast, proteoliposomes reconstituted with early endosomal SNAREs promiscuously fuse with liposomes containing exocytotic or late endosomal SNAREs. We conclude that the specificity of SNARE pairing does not suffice to determine the specificity of organelle fusion.

Animals↗

PubMed2006

The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane.

In the plasma membrane, syntaxin 1 and syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct syntaxin 1 clustering and that additional cytoplasmic protein-protein interactions, primarily involving the SNARE motif, are required. Apparently no specific cofactors are needed because i), clusters form equally well in nonneuronal cells, and ii), as revealed by nanoscale subdiffraction resolution provided by STED microscopy, the number of clusters directly depends on the syntaxin 1 concentration. For syntaxin 4 clustering the N-terminal domain and the linker region are also dispensable. Moreover, clustering is specific because in both cluster types syntaxins mutually exclude one another at endogenous levels. We suggest that the SNARE motifs of syntaxin 1 and 4 mediate specific syntaxin clustering by homooligomerization, thereby spatially separating sites for different biological activities. Thus, syntaxin clustering represents a mechanism of membrane patterning that is based on protein-protein interactions.

Amino Acid Motifs↗

PubMed2006

Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes.

Munc18-1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18-1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18-1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18-1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18-1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25-deficient mice are used. We conclude that Munc18-1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.

Animals↗

PubMed2005

Alternative splicing of SNAP-25 regulates secretion through nonconservative substitutions in the SNARE domain.

The essential membrane fusion apparatus in mammalian cells, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consists of four alpha-helices formed by three proteins: SNAP-25, syntaxin 1, and synaptobrevin 2. SNAP-25 contributes two helices to the complex and is targeted to the plasma membrane by palmitoylation of four cysteines in the linker region. It is alternatively spliced into two forms, SNAP-25a and SNAP-25b, differing by nine amino acids substitutions. When expressed in chromaffin cells from SNAP-25 null mice, the isoforms support different levels of secretion. Here, we investigated the basis of that different secretory phenotype. We found that two nonconservative substitutions in the N-terminal SNARE domain and not the different localization of one palmitoylated cysteine cause the functional difference between the isoforms. Biochemical and molecular dynamic simulation experiments revealed that the two substitutions do not regulate secretion by affecting the property of SNARE complex itself, but rather make the SNAP-25b-containing SNARE complex more available for the interaction with accessory factor(s).

Alternative Splicing↗

PubMed2005

Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells.

During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] level in bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2 affect secretion. Membrane levels of PI(4,5)P2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2 signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase I, or infusion of PI(4,5)P2 through the patch pipette, increased the PI(4,5)P2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2 from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5)P2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2 levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2 level secretion scales with plasma membrane PI(4,5)P2 content and that PI(4,5)P2 has an early effect on secretion by regulating the number of vesicles ready for release.

Animals↗

PubMed2005

A dual function for Munc-18 in exocytosis of PC12 cells.

Munc-18 interacts with the SNARE protein syntaxin and is supposed to influence transmitter release by controlling the formation of exocytosis-relevant SNARE complexes. Here, we used combined biochemical and physiological analyses to study the role of the Munc-18/syntaxin interaction in large dense core vesicle (LDCV) exocytosis of neuroendocrine PC12 cells. We compared two Munc-18 mutants carrying mutations in the syntaxin-binding region and show that Munc-18's membrane association depends on direct binding to syntaxin. The data suggest that perturbation of syntaxin binding inhibits neurotransmitter release upstream of the individual fusion event implying an essential role of the Munc-18/syntaxin complex leading to exocytosis. Furthermore, we show that a Munc-18 mutant lacking any syntaxin binding has a stimulatory effect on secretion, and provide evidence that the Munc-18/Mint1 interaction may constitute a second pathway for Munc-18 to regulate exocytosis. We propose that Munc-18 represents a dynamic link between syntaxin-related and Mint1-related mechanisms, both involved in the control of LDCV exocytosis in neuroendocrine cells.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis.

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.

Amino Acid Motifs↗

PubMed2003

Membrane fusion.

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Fusion reactions share common features, but are catalyzed by diverse proteins. These proteins mediate the initial recognition of the membranes that are destined for fusion and pull the membranes close together to destabilize the lipid/water interface and to initiate mixing of the lipids. A single fusion protein may do everything or assemblies of protein complexes may be required for intracellular fusion reactions to guarantee rigorous regulation in space and time. Cellular fusion machines are adapted to fit the needs of different reactions but operate by similar principles in order to achieve merging of the bilayers.

Amino Acid Motifs↗

PubMed2002

Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells.

During exocytosis, secretory granules fuse with the plasma membrane and discharge their content into the extracellular space. The exocytosed membrane is then reinternalized in a coordinated fashion. A role of clathrin-coated vesicles in this process is well established, whereas the involvement of a direct retrieval mechanism (often called kiss and run) is still debated. Here we report that a significant population of docked secretory granules in the neuroendocrine cell line PC12 fuses with the plasma membrane, takes up fluid-phase markers, and is retrieved at the same position. Fusion allows for complete discharge of small molecules, whereas GFP-labeled neuropeptide Y (molecular mass approximately equal 35 kDa) is only partially released. Retrieved granules were preferentially associated with dynamin. Furthermore, recapture is inhibited by guanosine 5'-[gamma-thio]triphosphate and peptides known to block dynamin function. We conclude that secretory granules can be recaptured immediately after formation of an exocytotic opening by an endocytic reaction that is spatially and temporally coupled to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion, but is not a reversal of the fusion reaction.

Animals↗

PubMed2002

SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs.

During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane-bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events.

Animals↗

PubMed2002

Fusion of vacuoles--where are the membranes, and where are the holes?

In the February 8th issue of Cell, Wang et al. report the surprising finding that vacuolar fusion occurs at the periphery of the contact area of the vacuoles and not by the expansion of a central fusion pore. During fusion, a disk of boundary membrane is excised and left behind within the fused vacuoles.

Eukaryotic Cells↗