分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: The integrins, a family of transmembrane proteins, function in cell-to-cell and cell-to-extracellular matrix (ECM) adhesive interactions, and influence cell signaling of cell growth and differentiation. Expression of integrin 6 in three bladder cancer cell lines, HCV29, KK47 and YST1 were quantitatively analyzed by LC-MS using stable isotope labeling by amino acids in cell culture (SILAC), a simple and powerful proteomic strategy. The results showed that the non-invasive bladder cancer cell line KK47 expressed the highest level of integrin alpha 6. The expression of integrin alpha 6 in invasive bladder cancer cell line YTS1 was also higher than in normal bladder epithelial cell line HCV29. Furthermore, these results were confirmed by Western blotting, qPCR, immunohistochemistry and flow cytometry. Clinical data of mRNA 1TGA6 expression pattern from open-access database (www.oncomine.org) showed the same result during bladder cancer progression. All these indicated that integrin alpha 6 is associated with the invasion progress of the bladder cancer. The preliminary data in this study may sparkle the fundamental role of integrin 6 in the research of bladder cancer.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: Talin, as the activator of integrin and the adaptor between the cytoskeleton and integrin, plays a key role in a series of processes such as cell adhesion and migration. The activation of integrin involves F3 subdomain of Talin-FERM domain binding the cytoplasmic tail of integrin beta-subunit. Talin has two states: auto-inhibited and activated. We previously reported the auto-inhibition complex structure of Talin F2F3/R9, in which the integrin binding site F3 interacts with R9(1654 similar to 1822 a.a.) of Talin-ROD, such that integrin cannot be activated. However, besides F3 and R9, it remains unclear what structural or functional roles the other domains of the 270 ku Talin play in the regulation of its activation. Here we solved the crystal structures of Talin R9-R10 (1654 similar to 1973 a.a.) and R10-R11 (1815 similar to 2140 a.a.), respectively. R9, R10 and R11 are all 5-helix bundles. R9 and R10 is joined together by a long alpha-helix instead of a flexible loop, and the two bundles are located at the opposite sides of the long helix with an angle of about 150 degrees. The linker between R10 and R11 is stabilized by neighboring hydrogen bonds, forming an angle of about 120 between the two bundles. These angles observed in our crystal structures are consistent with the previously reported SAXS and EM results. After superimposition of R9-10, R10-11 with previously reported structures of R7-8 and R11-12, a model of R7-12 was acquired, which adopts an elongated linear conformation, except that R8 protrudes from the ROD. According to this model, R10-12 does not intrude the interaction between F3 and R9, whereas R8 not only masks the F3 binding site of R9, but also might electrostatically hinders F2F3 approaching via its unique positively charged surface. This hypothesis was further verified by the results of size exclusion chromatography. Our work provides a new structural basis for studying the mechanism of Talin auto-inhibition.
分类: 生物学 >> 生物物理学 >> 生物力学与生物流变学 提交时间: 2016-05-12
摘要: Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Sensory dendrites innervate peripheral tissues through cell-cell interactions that are poorly understood. The proprioceptive neuron PVD in C. elegans extends regular terminal dendritic branches between muscle and hypodermis. We found that the PVD branch pattern was instructed by adhesion molecule SAX-7/L1CAM, which formed regularly spaced stripes on the hypodermal cell. The regularity of the SAX-7 pattern originated from the repeated and regularly spaced dense body of the sarcomeres in the muscle. The extracellular proteoglycan UNC-52/Perlecan linked the dense body to the hemidesmosome on the hypodermal cells, which in turn instructed the SAX-7 stripes and PVD dendrites. Both UNC-52 and hemidesmosome components exhibited highly regular stripes that interdigitated with the SAX-7 stripe and PVD dendrites, reflecting the striking precision of subcellular patterning between muscle, hypodermis, and dendrites. Hence, the muscular contractile apparatus provides the instructive cues to pattern proprioceptive dendrites.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-11
摘要: Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.