Helical structures of ESCRT-III are disassembled by VPS4

During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs (endosomal sorting complexes required for transport) interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies; for the budding of some enveloped viruses; and for daughter cell scission in cytokinesis. We found that the ESCRT-III proteins CHMP2A and CHMP3 (charged multivesicular body proteins 2A and 3) could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule, whereas the AAA-type adenosine triphosphatase VPS4 could bind on the inside of the tubule and disassemble the tubes upon adenosine triphosphate hydrolysis. CHMP2A and CHMP3 copolymerized in solution, and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly budding vesicle, catalyzing late steps in budding under the control of VPS4.     

Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery

During cytokinesis, as dividing animal cells pull apart into two daughter cells, the final stage, termed abscission, requires breakage of the midbody, a thin membranous stalk connecting the daughter cells. This membrane fission event topologically resembles the budding of viruses, such as HIV-1, from infected cells. We found that two proteins involved in HIV-1 budding-tumor susceptibility gene 101 (Tsg101), a subunit of the endosomal sorting complex required for transport I (ESCRT-I), and Alix, an ESCRT-associated protein-were recruited to the midbody during cytokinesis by interaction with centrosome protein 55 (Cep55), a centrosome and midbody protein essential for abscission. Tsg101, Alix, and possibly other components of ESCRT-I were required for the completion of cytokinesis. Thus, HIV-1 budding and cytokinesis use a similar subset of cellular components to carry out topologically similar membrane fission events.     

Ceramide triggers budding of exosome vesicles into multivesicular endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.     

Membrane phosphatidylserine regulates surface charge and protein localization

Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx.     

Wnt5a control of cell polarity and directional movement by polarized redistribution of adhesion receptors

Mechanisms by which Wnt pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are incompletely understood. We show that acute responses to Wnt5a involve recruitment of actin, myosin IIB, Frizzled 3, and melanoma cell adhesion molecule into an intracellular structure in a melanoma cell line. In the presence of a chemokine gradient, this Wnt-mediated receptor-actin-myosin polarity (W-RAMP) structure accumulates asymmetrically at the cell periphery, where it triggers membrane contractility and nuclear movement in the direction of membrane retraction. The process requires endosome trafficking, is associated with multivesicular bodies, and is regulated by Wnt5a through the small guanosine triphosphatases Rab4 and RhoB. Thus, cell-autonomous mechanisms allow Wnt5a to control cell orientation, polarity, and directional movement in response to positional cues from chemokine gradients.     

Zebrafish Dpr2 inhibits mesoderm induction by promoting degradation of nodal receptors

Nodal proteins, members of the transforming growth factor-beta (TGFbeta) superfamily, have been identified as key endogenous mesoderm inducers in vertebrates. Precise control of Nodal signaling is essential for normal development of embryos. Here, we report that zebrafish dapper2 (dpr2) is expressed in mesoderm precursors during early embryogenesis and is positively regulated by Nodal signals. In vivo functional studies in zebrafish suggest that Dpr2 suppresses mesoderm induction activities of Nodal signaling. Dpr2 is localized in late endosomes, binds to the TGFbeta receptors ALK5 and ALK4, and accelerates lysosomal degradation of these receptors.     

The cytoskeletal protein vinculin contains transformation-sensitive, covalently bound lipid

Vinculin, which is associated with the cytoskeleton of many cells, has been suggested as a possible linker between microfilament bundles and the plasma membrane. Here it will be shown that fatty acid is covalently attached to vinculin in vivo. Furthermore, in chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus, tsNY68, the acylation of vinculin at the permissive temperature was less than one-third that at the nonpermissive temperature. Thus, the covalent binding of lipid to vinculin is a transformation-sensitive event. The covalent modification of vinculin by lipids could be directly or indirectly involved in its reversible association with membranes. This modification may also provide a mechanism to alter the organization of vinculin within cells and thereby play a regulatory role in anchoring or stabilizing microfilament bundles at plasma membranes.     

Mitotic Golgi partitioning is driven by the membrane-fissioning protein CtBP3/BARS

Organelle inheritance is an essential feature of all eukaryotic cells. As with other organelles, the Golgi complex partitions between daughter cells through the fission of its membranes into numerous tubulovesicular fragments. We found that the protein CtBP3/BARS (BARS) was responsible for driving the fission of Golgi membranes during mitosis in vivo. Moreover, by in vitro analysis, we identified two stages of this Golgi fragmentation process: disassembly of the Golgi stacks into a tubular network, and BARS-dependent fission of these tubules. Finally, this BARS-induced fission of Golgi membranes controlled the G2-to-prophase transition of the cell cycle, and hence cell division.     

Efficient inhibition of the Alzheimer's disease beta-secretase by membrane targeting

beta-Secretase plays a critical role in beta-amyloid formation and thus provides a therapeutic target for Alzheimer's disease. Inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of active enzyme. We have addressed this issue by synthesizing a membrane-anchored version of a beta-secretase transition-state inhibitor by linking it to a sterol moiety. Thus, we targeted the inhibitor to active beta-secretase found in endosomes and also reduced the dimensionality of the inhibitor, increasing its local membrane concentration. This inhibitor reduced enzyme activity much more efficiently than did the free inhibitor in cultured cells and in vivo. In addition to effectively targeting beta-secretase, this strategy could also be used in designing potent drugs against other membrane protein targets.     

家蝇蛋白酶解物体内抗氧化性评价

研究家蝇蛋白酶解物的体内抗氧化作用,可为家蝇幼虫资源的开发利用提供基础。试验将昆明种小鼠随机分为空白对照组(生理盐水)及家蝇蛋白酶解物低剂量组(200μg/g)、中剂量组(400μg/g)、高剂量组(800μg/g),每组10只,检测各组小鼠血清及肝、脑组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)及过氧化氢酶(CAT)的活性。结果显示,家蝇蛋白酶解物可以增强小鼠血清、肝、脑组织中SOD,GSH-PX,CAT的活性,且明显高于空白对照组。说明家蝇蛋白酶解物具有显著的体内抗氧化活性。     

Organization of intracellular reactions with rationally designed RNA assemblies

The rules of nucleic acid base-pairing have been used to construct nanoscale architectures and organize biomolecules, but little has been done to apply this technology in vivo. We designed and assembled multidimensional RNA structures and used them as scaffolds for the spatial organization of bacterial metabolism. Engineered RNA modules were assembled into discrete, one-dimensional, and two-dimensional scaffolds with distinct protein-docking sites and used to control the spatial organization of a hydrogen-producing pathway. We increased hydrogen output as a function of scaffold architecture. Rationally designed RNA assemblies can thus be used to construct functional architectures in vivo.     

Structure and function of an essential component of the outer membrane protein assembly machine

Integral beta-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli, which has one or more polypeptide transport-associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous beta-barrel protein precursors. Analysis of POTRA domain deletions shows which are essential and provides a view of the spatial organization of this assembly machine.     

BAR domains as sensors of membrane curvature: the amphiphysin BAR structure

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.     

Vascular injury and lysis of basement membrane in vitro by neutral protease of human leukocytes

Frozen and thawed granules of human, peripheral-blood leukocytes rapidly produce hemorrhage when injected into animal tissues. The effect is blocked by inhibitors of proteolysis. The granule extract can digest vascular basement membrane in vitro at neutral pH. In addition, basement membranes of blood vessels damaged in vivo by the leukocyte fraction are found to be attenuated when examined by electron microscopy. The proteases of human leukocyte granules differ in several important respects from known lysosomal cathepsins and trypsin-like esterases. Polymorphonuclear neutrophils are a major source of the neutral proteases present in circulating white cells, and release these enzymes during phagocytosis of immune complexes.     

墨西哥湾扇贝初级卵母细胞发育及卵黄发生超微结构的初步研究

采用电子显微镜术,研究了墨西哥湾扇贝Argopecten irradians concentricus的初级卵母细胞发育和卵黄发生。结果表明,墨西哥湾扇贝初级卵母细胞的发育可分为卵黄合成前期、卵黄合成期和成熟期;5种细胞器,即线粒体、高尔基液泡、糙面内质网、多泡小体和微吞饮泡参与了初级卵母细胞卵黄颗粒的形成,其中,线粒体和高尔基液泡是形成卵黄颗粒的主要胞器。卵黄物质的来源有内源性和外源性两种方式,外源性卵黄物质主要来自消化腺中贮存的营养。另外,研究发现核膜泡可能参与了多泡小体的形成。     

Positioning of longitudinal nerves in C. elegans by nidogen

Basement membranes can help determine pathways of migrating axons. Although members of the nidogen (entactin) protein family are structural components of basement membranes, we find that nidogen is not required for basement membrane assembly in the nematode Caenorhabditis elegans. Nidogen is localized to body wall basement membranes and is required to direct longitudinal nerves dorsoventrally and to direct axons at the midlines. By examining migration of a single axon in vivo, we show that nidogen is required for the axon to switch from circumferential to longitudinal migration. Specialized basement membranes may thus regulate nerve position.     

Molecular analysis of protein assembly in muscle development

The challenge presented by myofibril assembly in striated muscle is to understand the molecular mechanisms by which its protein components are arranged at each level of organization. Recent advances in the genetics and cell biology of muscle development have shown that in vivo assembly of the myofilaments requires a complex array of structural and associated proteins and that organization of whole sarcomeres occurs initially at the cell membrane. These studies have been complemented by in vitro analyses of the renaturation, polymerization, and three-dimensional structure of the purified proteins.     

Role of Rab9 GTPase in facilitating receptor recruitment by TIP47

Mannose 6-phosphate receptors (MPRs) deliver lysosomal hydrolases from the Golgi to endosomes and then return to the Golgi complex. TIP47 recognizes the cytoplasmic domains of MPRs and is required for endosome-to-Golgi transport. Here we show that TIP47 also bound directly to the Rab9 guanosine triphosphatase (GTPase) in its active, GTP-bound conformation. Moreover, Rab9 increased the affinity of TIP47 for its cargo. A functional Rab9 binding site was required for TIP47 stimulation of MPR transport in vivo. Thus, a cytosolic cargo selection device may be selectively recruited onto a specific organelle, and vesicle budding might be coupled to the presence of an active Rab GTPase.