Flagellar Glycopro#ein Dynamics and Whole Cell Locomotion

一类350 kDa糖蛋白在鞭毛衣单胞菌膜平面内的活跃运动负责该生物体的整个细胞滑动运动。质膜蛋白动力学也可能涉及沿固体底物或细胞外基质的许多其他形式的全细胞运动。假设鞭毛膜与固体底物接触启动跨膜信号通路，激活鞭毛膜内马达，负责结合鞭毛膜糖蛋白并向其施加力。某些鞭毛膜糖蛋白与抗碳水化合物单克隆抗体交联是一种人工手段，可以启动信号通路（包括钙内流和蛋白质磷酸化的改变），并导致抗体识别的鞭毛膜蛋白重新分配。目前的研究旨在阐明蛋白质磷酸化在信号通路中的作用，并鉴定和表征负责糖蛋白运动的生物运动，从而确定整个细胞的滑行运动。研究将解决鞭毛膜蛋白和蛋白激酶与350 Kda鞭毛膜糖蛋白的调节关联，以及膜基质部分中磷酸化蛋白和其他蛋白与轴突和脑微管的关联。本文将建立一个利用鞭毛膜囊泡和纯化微管研究鞭毛膜动力学的体外系统。生物运动活动将寻求在膜-基质室使用体外微管滑动试验。膜基质部分中的3S钙特异性atp酶将被纯化并作为候选马达进行表征。将获得针对各种鞭毛膜基质磷酸化蛋白和候选运动蛋白的单克隆抗体，用于研究这些蛋白在鞭毛信号传导和糖蛋白运动和全细胞运动的力产生中的作用。单细胞绿藻衣藻是一种被广泛用于研究各种生物学问题的模式生物。藻类有鞭毛，这使它能够在水介质中自由游动，其功能类似于其他生物中鞭毛推动的运动。因此，鞭毛衣单胞菌已成为研究鞭毛运动机制的高效模型。除了自由游动的运动方式外，衣藻还表现出在固体基质上的滑行运动，这也涉及到鞭毛，或者更具体地说，涉及到鞭毛周围的质膜。这种滑动运动的机制可以追溯到鞭毛膜平面内某些膜糖蛋白的主动运动。很可能这个系统将作为一个很好的通用模型，用于研究膜蛋白在膜平面内的“蓄意”运动。

Active movement of a class of 350 kDa glycoproteins within the plane of the Chlamydomonas flagellar membrane is responsible for whole cell gliding motility in this organism. Plasma membrane protein dynamics may also be involved in many other forms of whole cell locomotion along solid substrates or extracellular matrix. Contact of the flagellar membrane with a solid substrate is hypothesized to initiate a transmembrane signaling pathway that activates an intraflagellar motor responsible for binding to and applying force to the flagellar membrane glycoproteins. Crosslinking of certain populations of flagellar membrane glycoproteins with anti-carbohydrate monoclonal antibodies is an artificial means to initiate the signaling pathway (involving calcium influx and changes in protein phosphorylation) and result in redistribution of the flagellar membrane proteins recognized by the antibodies. The present study is directed towards an elucidation of the role of protein phosphorylation in the signaling pathway and the identification and characterization of the biological motor responsible for glycoproteins movements and hence whole cell gliding motility. Studies will address the regulated association of flagellar membrane proteins and protein kinases with the 350 Kda flagellar membrane glycoproteins, and the association of phosphoproteins and other proteins in the membrane-matrix fraction with axonemal and brain microtubules. An in vitro system for studying flagellar membrane dynamics using inside-out flagellar membrane vesicles and purified microtubules will be developed. Biological motor activity will be sought within the membrane-matrix compartment using in vitro microtubule gliding assays. The 3S calcium-specific ATPase in the membrane -matrix fraction will be purified and characterized as a candidate motor. Monoclonal antibodies to various flagellar membrane-matrix phosphoproteins and to candidate motor proteins will be obtained for use as probes for studying the role of these proteins in flagellar signaling and force production for glycoprotein movement and whole cell locomotion. %%% The unicellular green alga, Chlamydomonas, is a widely used model organism for studying a wide variety of biological questions. The alga has flagellae, which enable it to swim freely in aqueous media, in a manner which is functionally similar to other flagellar-propelled motilities in other organisms. The Chlamydomonas flagellum has therefore been a highly productive model for studying the mechanism of flagellar motility in general. In addition to the free-swimming mode of locomotion, Chlamydomonas also exhibits a gliding motility on solid substrata, which also involves the flagellum, or, more specifically, the plasma membrane surrounding the flagellum. The mechanism of this gliding motility has been traced to the active movement of certain membrane glycoproteins within the plane of the flagellar membrane. It is likely that this system will serve as a good general model for studying "deliberate" movements of membrane proteins within the plane of the membrane.