MECHANISMS OF GOLGI VESICULATION DURING MITOSIS

我们重建了高尔基体膜的破碎和分散 通过将透化的正常大鼠肾（NRK）细胞与 有丝分裂胞浆和ATP再生系统。 我们发现 有丝分裂胞质溶胶耗尽p34 cdc 2激酶（有丝分裂所必需的激酶）， 进入有丝分裂）引起高尔基体分裂。 耗尽或 胞浆丝裂原活化蛋白激酶激酶的失活 （MEK 1），然而，抑制高尔基体片段化。 将MEK 1添加回MEK 1 耗尽的有丝分裂胞质液恢复高尔基体碎片化。 我们发现 下游（和相关的）胞质MAPK激酶，ERK 1和ERK 2不是 这一过程所需的。 有趣的是，一种MAPK激酶， 与ERK 2具有广泛交叉反应性的抗体与 高尔基体膜。 我们这项提案的总体目标是 了解MEK 1被激活的机制，并测试我们的 假设高尔基体MAPK激酶是直接下游靶点， 激活MEK 1。 我们的研究应该能找出 称为MEK 1活化所必需的MEK 1-激酶， 参与分散破碎的高尔基体膜，最后， 高尔基体膜相关蛋白，包括MEK 1底物 需要高尔基体分裂。 这些研究应该提供一个根据- 高尔基体膜经历广泛的 有丝分裂开始时的分裂。

We have reconstituted the fragmentation and dispersal of Golgi membranes by incubation of permeabilized Normal Rat Kidney (NRK) cells with mitotic cytosol and an ATP regenerating system at 32 C. We find that mitotic cytosol depleted of p34cdc2 kinase (the kinase necessary for entry into mitosis) causes Golgi fragmentation. Depletion or inactivation of the cytosolic Mitogen activated protein kinase kinase (MEK1), however, inhibits Golgi fragmentation. Adding back MEK1 to MEK1 depleted mitotic cytosol restores Golgi fragmentation. We find that the downstream (and the relevant) cytosolic MAPkinase, ERK1 and ERK2 are not required for this process. Interestingly, a MAPkinase, recognized by an antibody with broad cross-reactivity to ERK2 is tightly associated with Golgi membranes. Our overall goals for this proposal are to understand the mechanism by which MEK1 is activated and test our hypothesis that Golgi MAPkinase is the immediate downstream target of activated MEK1. Our studies should reveal the identity of a regulator termed MEK1-kinase necessary for MEK1 activation, components that are involved in dispersal of the fragmented Golgi membranes and finally the Golgi membrane associated proteins, including the MEK1 substrate required Golgi fragmentation. These studies should provide an under- standing of the mechanism by which Golgi membranes undergo extensive fragmentation at the onset of mitosis.