小反刍兽疫病毒H和F蛋白在细胞融合中的作用

【目的】研究小反刍兽疫病毒囊膜糖蛋白(血凝素蛋白和融合蛋白)在病毒囊膜和宿主细胞膜融合过程中所发挥的作用。【方法】制备构建成功的小反刍兽疫病毒囊膜糖蛋白和病毒受体SLAM、Nectin4的真核表达质粒pCMV-HA-H、pCAGGS-Flag-F、pCMV-Myc-SLAM和pCMV-Myc-Nectin 4,将其组合转染至CHO-K1细胞,通过显微观察和间接免疫荧光技术分析小反刍兽疫病毒H和F蛋白在病毒融合过程中的功能。【结果】除空白对照组和重组质粒单独转染组细胞中没有发现合胞体外,其余组细胞中均出现了合胞体,而且F和H蛋白共转染组合胞体的数目明显较多;并在共表达H、F蛋白的细胞中观察到了蛋白分布极化的帽子现象。【结论】PPRV F蛋白是病毒囊膜和细胞膜融合的必需蛋白,但需要与PPRV H共同作用才能使病毒成功入侵靶细胞。

Liquid-liquid phase separation in biology: mechanisms,physiological functions and human diseases

Cells are compartmentalized by numerous membrane-enclosed organelles and membraneless compartments to ensure that a wide variety of cellular activities occur in a spatially and temporally controlled manner. The molecular mechanisms underlying the dynamics of membrane-bound organelles, such as their fusion and fission, vesicle-mediated trafficking and membrane contactmediated inter-organelle interactions, have been extensively characterized. However, the molecular details of the assembly and functions of membraneless compartments remain elusive. Mounting evidence has emerged recently that a large number of membraneless compartments, collectively called biomacromolecular condensates, are assembled via liquid-liquid phase separation(LLPS). Phase-separated condensates participate in various biological activities, including higher-order chromatin organization,gene expression, triage of misfolded or unwanted proteins for autophagic degradation, assembly of signaling clusters and actin-and microtubule-based cytoskeletal networks, asymmetric segregations of cell fate determinants and formation of pre-and post-synaptic density signaling assemblies. Biomacromolecular condensates can transition into different material states such as gel-like structures and solid aggregates. The material properties of condensates are crucial for fulfilment of their distinct functions, such as biochemical reaction centers, signaling hubs and supporting architectures. Cells have evolved multiple mechanisms to ensure that biomacromolecular condensates are assembled and disassembled in a tightly controlled manner. Aberrant phase separation and transition are causatively associated with a variety of human diseases such as neurodegenerative diseases and cancers. This review summarizes recent major progress in elucidating the roles of LLPS in various biological pathways and diseases.

Vertebrate Dynein-f depends on Wdr78 for axonemal localization and is essential for ciliary beat

Motile cilia and flagella are microtubule-based organelles important for cell locomotion and extracellular liquid flow through beating. Although axonenal dyneins that drive ciliary beat have been extensively studied in unicellular Chlamydomonas, to what extent such knowledge can be applied to vertebrate is poorly known. In Chlamydomonas, Dynein-f controls flagellar waveforms but is dispensable for beating. The flagellar assembly of its heavy chains (HCs) requires its intermediate chain (IC) IC140 but not IC138. Here we show that, unlike its Chlamydomonas counte『part, vertebrate Dynein-f is essential for ciliary beat. We confirmed that Wdr78 is the vertebrate orthologue of IC3 Wdr78 associated with Dynein-f subunits such as Dnah2 (a HC) and Wdr63 (IC140 orthologue). It was expressed as a motile cilium-specific protein in mammalian cells. Depletion of Wdr78 or Dnah2 by RNAi paralyzed mouse ependymal cilia. Zebrafish Wdr78 morphants displayed ciliopathy-related phenotypes, such as curved bodies, hydrocephalus, abnormal otolith, randomized left-right asymmetry, and pronephric cysts, accompanied with paralyzed pronephric cilia. Furthermore, all the HCs and ICs of Dynein-f failed to localize in the Wdr78-depleted mouse ependymal cilia. Therefore, both the functions and subunit dependency of Dynein-fare altered in evolution, probably to comply with ciliary roles in higher organisms.

Microtubule-bundling protein Spef1 enables mammalian ciliary central apparatus formation

Cilia are cellular protrusions containing nine microtubule(MT)doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions.Cilia require the central pair(CP)apparatus,consisting of two CP MTs covered with projections of CP proteins,for planar strokes.How the CP MTs of such‘9+2’cilia are constructed,however,remains unknown.Here we identify Spef1,an evolutionarily conserved microtubule-bundling protein,as a core CP MT regulator in mammalian cilia.Spef1 was selectively expressed in mammalian cells with 9+2 cilia and specifically localized along the CP.Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6,resulting in rotational beat of the ependymal cilia.Spef1,which binds to MTs through its N-terminal calponin.homologous domain,formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs.Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus.We propose that Spefl bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.

O-GIcNAc transferase regulates centriole behavior and intraflagellar transport to promote ciliogenesis

Dear Editor,O-GIcNAcylation is a nutrient sensor that is particularly sensitive to environmental glucose(Hardiville and Hart,2014).Glucose can be converted to UDP-GIcNAc through the hexosamine biosynthetic pathway,providing a substrate for O-GIcNAcylation.Two enzymes participate in this reversible modification,O-GIcNAc transferase(OGT),which adds a single GIcNAc residue to the serine/threonine sites of proteins,and O-GIcNAcase(OGA),which removes the residue(Yang and Qian,2017).