Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R-SNAREs (soluble NSF (N-ethylmaleimide-sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R-SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.     

Elucidation of the protein composition of mouse seminal vesicle fluid

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology.     

Bicaudal‐D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule‐based motors is essential for cell organisation and function. The Bicaudal‐D (BicD) protein participates in the transport of a subset of cargoes by the minus‐end‐directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co‐precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin‐mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high‐frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin‐associated trafficking processes and show a novel requirement for microtubule‐based motor transport in the synaptic vesicle cycle.     

Preparation, stability, and in vitro performance of vesicles made with diblock copolymers

Vesicles made completely from diblock copolymers-polymersomes-can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, approximately 100 nm vesicles of PEO-PEE (polyethyleneoxide-polyethylethylene) or PEO-PBD (polyethyleneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.     

The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis

One characteristic linking members of the synaptotagmin family to endocytosis is their ability to bind the heterotetrameric AP2 complex via their C2B domain. By using CD4/synaptotagmin 1 chimeras, we found that the internalization signal of synaptotagmin 1 lies at the extreme COOH-terminus of the protein and can function in the absence of the C2B domain that contains the AP2 binding site. However, although not essential for internalization, the C2B domain of synaptotagmin 1 appeared to control the recognition of the internalization motif. By mutagenesis, two sites have been identified that modify regulation by the C2B domain in the neuroendocrine PC12 cell line. Mutation of a dilysine motif in the beta sandwich core of the domain eliminates endocytosis. This site is known to be a site of protein-protein interaction. Mutations in the calcium binding region, or in its close proximity, also affect internalization in PC12 cells. In fibroblasts, the C2B domain inhibits the COOH-terminal internalization signal, resulting in an absence of internalization in those cells. Thus, internalization of synaptotagmin 1 is controlled by the presence of a latent internalization signal in the COOH-terminal region and a regulatory region in the C2B domain. We propose that internalization of synaptotagmin 1 is regulated in this way to allow it to couple the processes of endocytosis and calcium-mediated exocytosis in cells of the neuroendocrine lineage.     

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.     

Possible roles of beta-catenin in evagination of the optic primordium in rat embryos

The roles of beta-catenin in evagination of the optic primordium in rat embryos were studied using immunostaining. High levels of beta-catenin appeared transiently in the evaginating optic primordium. Evagination of the optic primordium was suppressed in embryos treated with LiCl. In deficient optic vesicles of these embryos, accumulation of beta-catenin was decreased. Deficient optic vesicles also showed suppression of cyclin D1 accumulation and bromodeoxyuridine incorporation, no break in the deposition of laminin and type IV collagen at the basement membrane (BM) and prevention of the change in distribution of microtubules and microfilaments. These results suggest that beta-catenin regulates cell proliferation, breakdown of BM and changes in cell shape in the evaginating optic primordium to cause optic vesicle formation.     

SNARE membrane trafficking dynamics in vivo

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.     

频繁使用染发剂对小鼠染色体畸变率影响的研究

研究了多次接触７ 种染发剂对不同组小鼠骨髓和生殖细胞染色体畸变率的影响。结果发现, ７ 种染发剂均导致出现较高的染色体畸变率,３ 种能引起小鼠骨髓细胞染色体畸变率显著上升, 其中以粉末状染发剂的影响最为严重。４ 种染发剂对进行生殖细胞染色体畸变实验的小鼠均产生显著影响, 尤以氧化型染发剂最为明显。