MOTILE MODELS OF TELEOST RETINAL CONES

这项研究计划的总体目标是使用裂解细胞模型 以探讨硬骨视网膜锥体的作用机制和调控。 肌动蛋白依赖的细胞收缩和微管依赖的细胞 伸长率。我们已经开发了获得ATP依赖的程序 硬骨骨锥模型中收缩和伸长的重新激活 用1%Brij-58裂解。通过改变cAMP和钙水平我们可以产生 要么重新激活伸长，要么收缩足够广泛 长度变化(20-40u)以允许详细的定量。我们将分析 再活化条件和不同探针定量的影响 再激活运动的速率和程度及薄层电子显微镜观察 模特们。我们特别建议1)研究肌球蛋白、肌球蛋白 轻链激酶和钙调蛋白在调节锥体收缩中的作用 探讨基于动力蛋白的微管间滑动的可能性 为锥体伸长提供动力。建议的研究包括 核苷酸、离子、pH、钒酸根和氢氧化钠的影响分析 重新激活的伸长和收缩，免疫荧光定位和 用血小板肌球蛋白抗体进行铁蛋白抗体定位研究， 钙调蛋白、纤毛动力蛋白和卵动力蛋白的影响 外源性钙调蛋白、平滑肌肌球蛋白和肌球蛋白轻链激酶 再激活收缩和微管的形态分析 伸长锥形肌样体内的极性和小管间联系。

The general objective of this research program is to use lysed-cell models of teleost retinal cones to investigate the mechanisms and regulation of actomyosin-dependent cell contraction and microtubule-dependent cell elongation. We have developed procedures for obtaining ATP-dependent re-activation of both contraction and elongation in models of teleost cones lysed with 1% Brij-58. By altering cAMP and calcium levels we can produce either reactivated elongation or contraction with sufficiently extensive length changes (20-40u) to permit detailed quantitation. We will analyze the effects of reactivation conditions and various probes by quantifying rates and extents of reactivated movement and by thin section TEM of models. We propose specifically 1) to examine the roles of myosin, myosin light chain kinase and calmodulin in regulating cone contraction and 2) to investigate the possibility that dynein-based inter-microtubule sliding provides motive force for cone elongation. Proposed studies include analyses of the effects of nucleotides, ions, pH, vanadate and EHNA on reactivated elongation and contraction, immunofluorescence localization and ferritin-antibody localization studies with antibodies to platelet myosin, calmodulin, and ciliary and egg dynein, investigations of the effects of exogenous calmodulin, smooth muscle myosin and myosin light chain kinase on reactivated contraction, and morphological analyses of microtubule polarities and intertubule associations in elongating cone myoids.