Cellular Function Of The ADP-ribosylation Factor 6 (Arf6

Arf 6 GTdR调节质膜（PM）和内体区室之间的膜运输，并影响皮质肌动蛋白细胞骨架的动力学。在许多细胞中，该途径是PM蛋白遵循的途径，PM蛋白缺乏网格蛋白定位序列，因此不依赖于网格蛋白被内吞入细胞。在内化后，该膜的一部分在Arf 6活化后回缩至PM。通过Arf 6调节的途径的膜的运动是所需的动态，皮质肌动蛋白重排细胞扩散，突起的形成，和Rac介导的皱褶过程中观察到的。为了了解Arf 6如何执行这些双重功能并识别潜在的Arf 6介质，我们一直在使用模型转染细胞系统，该系统使我们能够跟踪活细胞和固定细胞中的膜动力学和细胞形状变化。我们发现Arf 6通过调节磷脂酰肌醇4-磷酸5-激酶（PIP 5-kinase）的运输和活性发挥作用，PIP 5-kinase是一种产生磷脂酰肌醇4，5-二磷酸（PIP 2）的酶。我们将PIP 2定位于具有丛曲蛋白同源结构域（PH）嵌合体的细胞中，该嵌合体特异性识别膜结合的PIP 2和绿色荧光蛋白（GFP）。发现PIP 2与PM相关，并且也在Arf 6相关的内体膜上。通过表达Arf 6的GTP交换因子EFA 6实现Arf 6的活化，导致突起的形成，膜被摄取到富含PIP 2的大胞饮体中，并且这种内化的膜再循环回到PM。这些“活化的”细胞继续突出并循环膜，因为内源性Arf GTP酶活化蛋白（GAP）仍然可以使Arf 6-GTP活化，从而允许Arf 6的持续活化和失活。相比之下，组成型活性Arf 6，Q67 L的表达导致富含PIP 2的膜内化到细胞中，这些膜的融合产生更大的空泡，并阻止该膜再循环回到PM。人PIP 5-激酶α的过表达模拟了用Arf 6 Q67 L观察到的效果，表明由Arf 6调节的PIP 5-激酶和PIP 2周转对于通过Arf 6内体系统的运输是关键的。

The Arf6 GTPase regulates membrane traffic between the plasma membrane (PM) and an endosomal compartment and influences the dynamics of the cortical actin cytoskeleton. In many cells this pathway is the route followed by PM proteins that lack clathrin localization sequences and hence are endocytosed into cells independently of clathrin. After internalization, a portion of this membrane recycles back to the PM upon Arf6 activation. The movement of membrane through this Arf6-regulated pathway is required for the dynamic, cortical actin rearrangements observed during cell spreading, protrusion formation, and Rac-mediated ruffling. To understand how Arf6 carrries out these dual functions and to identify potential Arf6 mediators, we have been using a model transfected-cell system that allows us to follow membrane dynamics and cell shape alterations in live and fixed cells. We find that Arf6 functions by regulating the trafficking and activity of phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). We localized PIP2 in cells with a chimera of a plextrin homology domain (PH) that specifically recognizes membrane-bound PIP2 and green fluorescent protein (GFP). PIP2 was found associated with the PM and also on the Arf6-associated endosomal membranes. Activation of Arf6 achieved by expression of a GTP exchange factor for Arf6, EFA6, resulted in the formation of protrusions, the uptake of membrane into PIP2-enriched macropinosomes and the recycling of this internalized membrane back to the PM. These "activated" cells continue to protrude and cycle membrane since endogenous Arf GTPase activating proteins (GAPs) can still inactivate Arf6-GTP allowing continual activation and inactivation of Arf6. By contrast, expression of constitutively active Arf6, Q67L, resulted in the internalization of PIP2-enriched membrane into the cell, the fusion of these membranes generating larger vacuoles and a block in recycling of that membrane back to the PM. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6Q67L demonstrating that PIP 5-kinase and PIP2 turnover regulated by Arf6 are critical for trafficking through the Arf6 endosomal system.