Role of phosphoinositides in endosomal trafficking

磷酸肌苷（PIs）是参与膜重构的脂质第二信使。PtdIns3P和PtdIns(3,5)P2存在于内体腔室中，在膜运输中起重要作用。此外，越来越多的人类遗传疾病（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。ptdin5p的特征要少得多，但它与信号转导和囊泡运输有关。最近，已经鉴定出两个PtdIns(4,5)P2 4- ptase (PtdIns(4,5)P2 4- ptase type I和II)，它们催化PtdIns(4,5)P2水解为PtdIns5P。我们发现PtdIns(4,5)P2 4- ptase I或II的过度表达诱导了后期内体/溶酶体在微管组织中心（MTOC）上的时间依赖性逆行聚集，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动力蛋白的表达所抑制，这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，自噬体成熟缺陷，溶酶体水解酶分泌不足，从而提示PtdIns5P在膜运输中的作用。此外，同时消耗两种内源性PtdIns(4,5)P2 4- ptase I和II诱导溶酶体向细胞周围分散。

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.