Abstract：
Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome \"fusion-fission\" cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.
摘要：
溶酶体膜含有协调溶酶体功能和动力学的多种磷酸肌醇(PtdIns)脂质。溶酶体上的PtdIns库受到多种PtdIns激酶和磷酸酶的作用的严格调节；然而，PtdIns在溶酶体功能和动力学中的具体作用目前尚不清楚,需要进一步研究.以前表明，PIKfyve，从PtdIns（3）P合成PtdIns（3,5）P2的脂质激酶，控制溶酶体“聚变裂变”循环动力学，自噬体周转，和内吞货物运输。此外，INPP4B,水解PtdIns(3,4)P2形成PtdIns(3)P的PtdIns4-磷酸酶，正在成为一种在溶酶体生物发生和其他溶酶体功能中起作用的癌症相关蛋白。这里,我们研究了在Inpp4b缺陷小鼠胚胎成纤维细胞中破坏PIKfyve功能的后果.通过共聚焦荧光成像，我们观察到大量扩大的溶酶体的形成，伴随着内吞贩运的加剧减少，破坏溶酶体融合-裂变动力学，和抑制自噬。最后,3H-肌醇标记的PtdIns和PtdIns免疫荧光染色的HPLC闪烁定量，我们观察到,由于磷脂酰肌醇3-激酶催化亚基VPS34酶活性的过度激活,溶酶体PtdIns(3)P水平在Inpp4b缺陷型细胞中显著升高.总之,我们的研究确定了一个新的信号轴，维持正常的溶酶体稳态和动力学，其中包括Inpp4b的催化功能，PIKfyve,VPS34
