Fili N, Calleja V, Woscholski R, Parker P J, Larijani B
Cell Biophysics Laboratory and Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Proc Natl Acad Sci U S A. 2006 Oct 17;103(42):15473-8. doi: 10.1073/pnas.0607040103. Epub 2006 Oct 9.
It is increasingly recognized that the compartmental organization of signaling processes has a profound influence on cellular behavior. However, our inability to influence these compartmental events in a spatially restricted and acute manner limits our understanding of causation. To determine whether local compartmental loss of a phosphoinositide disrupts the normal traffic of specific cargoes through endosomes, we developed the use of a regulated dimerization device, here designed to compartmentally modify the phosphoinositide content of Rab5-positive endosomes. This modification is effected through the specific regulated recruitment of the 3-phosphatase myotubularin to endosomal membranes in intact cells. The selective manipulation of endosomal phosphatidylinositols (PIs) demonstrates that it is the phosphatidylinositol 3-phosphate (PtdIns3P) or its metabolite PtdIns(3,5)P2 within this compartment that determines the normal maturation of the endosomal compartment and the flux of receptors through it. On local loss of PtdIns3P/PtdIns(3,5)P2, the endosomal compartment itself fails to continue its normal maturation process, leading to the microtubule-dependent tubularization of the endosomal network. Furthermore, it is shown that endosomal PtdIns3P/PtdIns(3,5)P2 is necessary for transferrin receptor traffic through this compartment while having an effect on EGF receptor (EGFR) entry into and sorting from this endosome compartment. The ability to acutely and selectively influence compartmental behavior as exemplified here for endomsomes clearly illustrates the power of the approach used to dissect the role of localized signals and events.
人们越来越认识到，信号传导过程的区室化组织对细胞行为有着深远影响。然而，我们无法以空间受限且急性的方式影响这些区室事件，这限制了我们对因果关系的理解。为了确定磷酸肌醇的局部区室缺失是否会破坏特定货物通过内体的正常运输，我们开发了一种受调控的二聚化装置，此处设计用于在区室水平上改变Rab5阳性内体的磷酸肌醇含量。这种改变是通过在完整细胞中将3 - 磷酸酶肌管素特异性地受调控募集到内体膜上来实现的。对内体磷脂酰肌醇（PI）的选择性操纵表明，正是该区室内的磷脂酰肌醇3 - 磷酸（PtdIns3P）或其代谢产物PtdIns(3,5)P2决定了内体区室的正常成熟以及受体通过它的通量。当PtdIns3P/PtdIns(3,5)P2局部缺失时，内体区室本身无法继续其正常成熟过程，导致内体网络的微管依赖性管状化。此外，研究表明内体PtdIns3P/PtdIns(3,5)P2对于转铁蛋白受体通过该区室的运输是必需的，同时对表皮生长因子受体（EGFR）进入和从这个内体区室分选有影响。如这里以内体为例所展示的那样，能够急性且选择性地影响区室行为，清楚地说明了用于剖析局部信号和事件作用的方法的强大之处。
