Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and

Localized PtdIns 3,5-P-2 synthesis to regulate early endosome dynamics and fusion. Am J Physiol Cell Physiol 291: C393-C404, 2006. First published March 8, 2006; doi:10.1152/ajpcell.00019.2006. - Perturbations in the intracellular PtdIns 3,5-P-2 pool or the downstream transmission of PtdIns 3,5-P-2 signals often result in a gradual development of gross morphological changes in the pleiomorphic multivesicular endosomes, culminating with the appearance of cytoplasmic vacuoles. To identify the onset of PtdIns 3,5-P-2 functional requirements along the endocytic system, in this study we characterized the morphological changes associated with early expression of the dominant-negative kinase-deficient form (K1831E) of the PtdIns 3,5-P-2-producing kinase PIKfyve, before the formation of cytoplasmic vacuoles in transfected COS cells. Enlarged PIKfyve(K1831E)-positive vesicles co-localizing with dilated EEA1- and Rab5a(WT)-positive perinuclear endosomes were observed (WT, wild type). This was dependent on the presence of active forms of Rab5 and the generation of PtdIns 3-P-enriched platforms on early endosomess. Because PIKfyve(WT) did not substantially colocalize with EEA1- or Rab5-positive endosomes in COS cells, the dynamic PIKfyve-catalyzed PtdIns 3-to-PtdIns 3,5-P-2 switch was suggested to drive away PIKfyveWT from early endosomes toward later compartments. Late endosomes/ lysosomes marked by LAMP1 or Rab7 were dislocated from their typical perinuclear position upon PIKfyve(K1831E) early expression. Cytosols derived from cells stably expressing PIKfyveK1831E stimulated endosome fusion in vitro, whereas PIKfyve(WT)-enriched cytosols had the opposite effect, consistent with PtdIns 3,5-P-2 production negatively regulating the endosome fusion. Together, our data indicate that PtdIns 3,5-P-2 defines specific endosome platforms at the onset of the degradation pathway to regulate the complex process of membrane remodeling and dynamics.