Role of phosphoinositides in endosomal trafficking

磷脂酰肌醇(PI)是与膜重塑有关的脂质第二信使。PtdIns3P和PtdIns(3，5)P2存在于细胞膜上，在膜转运中起重要作用。此外，越来越多的人类遗传性疾病(包括肌病和神经病)与调节这些内体PI周转的酶的突变有关。PtdIns5P的特征还很少，但它与信号转导和囊泡运输有关。最近发现了两种催化PtdIns(4，5)P2生成PtdIns5P的PtdIns(4，5)P2 4-Ptase(PtdIns(4，5)P2 4-Ptase type I和PtdIns 4-Ptase II)。我们发现，无论是PtdIns(4，5)P2 4-Ptase I或II的过表达，都可以诱导晚期内体/溶酶体在微管组织中心(MTOC)上的随时间变化的逆行聚集，但对早期内体或高尔基体没有影响。溶酶体的逆行运输被诺可达唑或Dynamitin的表达所抑制，表明微管和动力蛋白马达是聚集所必需的。溶酶体聚集导致EGFR降解延迟，自噬小体成熟缺陷，并分泌溶酶体水解酶，提示PtdIns5P在膜运输中起作用。此外，同时耗尽两种内源性PtdIns(4，5)P2 4-Ptase I和II会导致溶酶体分散到细胞外围。 PI的功能研究中的一个问题是，PI-激酶或磷酸酶长时间的过度表达可能会影响多个信号和运输事件。为了克服这一问题，我们对先前描述的雷帕霉素/FRB/FKBP系统进行了修改，以实现在加入雷帕霉素后，PtdIns(4，5)P2 4-Ptase I的催化结构域可诱导地重新聚集到晚期内体/溶酶体。该系统将允许实时监测PtdIns5P水平变化对内体分布的影响。

Phosphoinositides (PIs) are lipid second messengers implicated in membrane remodeling. PtdIns3P and PtdIns(3,5)P2 are present on endosomal compartments where they play an important role in membrane trafficking. In addition, an increasing number of human genetic diseases (including myopathy and neuropathies) are associated to mutations in enzymes regulating the turnover of these endosomal PIs. PtdIns5P is far less characterized but it has been implicated in signal transduction and vesicular transport. Recently, two PtdIns(4,5)P2 4-Ptases (PtdIns(4,5)P2 4-Ptase type I and II) that catalyze the hydrolysis of PtdIns(4,5)P2 to PtdIns5P have been identified. We found that over-expression of either PtdIns(4,5)P2 4-Ptase I or II, induced a time-dependent retrograde aggregation of late endosomes/lysosomes over the microtubule-organizing center (MTOC) but had no effect on early endosomes or Golgi. Retrograde trafficking of lysosomes was inhibited by nocodazol or by expression of dynamitin, indicating that microtubules and dynein motors are required for the aggregation. Lysosomal clustering led to delayed degradation of EGFR, defective autophagosome maturation, and secretion of lysosomal hydrolases, thus suggesting a role of PtdIns5P in membrane trafficking. Moreover, simultaneous depletion of both, endogenous PtdIns(4,5)P2 4-Ptase I and II induced dispersion of lysosomes to the cell periphery. One of the problems in studying the function of PIs is that prolonged over-expression of PI-kinases or phosphatases may affect multiple signaling and trafficking events. To overcome this problem we modified the previously described rapamycin/FRB/FKBP system to attain inducible recruitment of the catalytic domain of PtdIns(4,5)P2 4-Ptase I to late endosomes/lysosomes upon addition of rapamycin. This system will allow monitoring the effect of changes in the levels of PtdIns5P in the distribution of endosomes in real time.