Ca2 Second Messenger for Control of Flagellar Movement

钙离子作为第二信使在鞭毛节拍控制中的作用将被研究。拟议的实验的主要目的是利用生化程序识别鞭毛轴丝中可能参与控制鞭毛弯曲形状的钙结合蛋白。对从莱茵衣藻分离的轴丝进行生化分离，以确定与鞭毛节拍控制有关的钙调蛋白(CaM)在轴丝下的分布。使用125I-CaM或生物素标记的CaM和亲和素过氧化物酶的凝胶覆盖程序将监测CaM结合蛋白在轴丝中的分布。将从轴丝制备外部二重微管，以确定这些结构是否含有CaM、CaM结合蛋白或其他感兴趣的钙结合蛋白。最后，将制备针对特定CaM结合蛋白的多克隆抗血清，并将其用于免疫标记研究，以检测这种“候选”蛋白在轴丝和外二联体微管中的分布。这些研究应该阐明钙通过识别可能控制鞭毛形状的关键钙结合蛋白来改变鞭毛波形的机制，并总体上拓宽我们对涉及钙作为第二信使的过程的理解。

The role of calcium ions as a second messenger in the control of flagellar beat will be examined. The major objective of the proposed experiments is to identify, using biochemical procedures, calcium- binding proteins in the flagellar axoneme that may be involved in controlling the shape of flagellar bends. Biochemical fractionation of axonemes isolated from Chlamydomonas reinhardtii will be carried out to determine the sub-axonemal distribution of calmodulin (CaM), which has been implicated in the control of flagellar beat. The distribution of CaM-binding proteins in the axoneme will be monitored by gel overlay procedures using 125-I-CaM or biotinylated CaM and avidin-peroxidase. Outer doublet microtubules will be prepared from axonemes to determine whether these structures contain CaM, CaM-binding proteins, or other calcium binding proteins of interest. Finally, polyclonal antisera directed against a specific CaM-binding protein will be prepared and used in immunolabelling studies to examine the distribution of this "candidate" protein wthin the axoneme and the outer doublet microtubules. These studies should clarify the mechanisms by which calcium alters flagellar waveform by identifying key calcium-binding proteins that may control flagellar shape, as well as generally broaden our understanding of processes that involve calcium as a second messenger.