Active vacuolar H⁺ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P₂ loss under PIKfyve or Vps34 deficiency

Abstract

The two evolutionarily conserved mammalian lipid kinases Vps34 and PIKfyve are involved in an important physiological relationship, whereby the former produces phosphatidylinositol (PtdIns) 3P that is used as a substrate for PtdIns(3,5)P2 synthesis by the latter. Reduced production of PtdIns(3,5)P2 in proliferating mammalian cells is phenotypically manifested by the formation of multiple translucent cytoplasmic vacuoles, readily rescued upon exogenous delivery of PtdIns(3,5)P2 or overproduction of PIKfyve. Although the aberrant vacuolation phenomenon has been frequently used as a sensitive functional measure of localized PtdIns(3,5)P2 reduction, cellular factors governing the appearance of cytoplasmic vacuoles under PtdIns3P-PtdIns(3,5)P2 loss remain elusive. To gain further mechanistic insight about the vacuolation process following PtdIns(3,5)P2 reduction, in this study we sought for cellular mechanisms required for manifestation of the aberrant endomembrane vacuoles triggered by PIKfyve or Vps34 dysfunction. The latter was achieved by various means such as pharmacological inhibition, gene disruption, or dominant-interference in several proliferating mammalian cell types. We report here that inhibition of V-ATPase with bafilomycin A1 as well as inactivation of the GTP-GDP cycle of Rab5a GTPase phenotypically rescued or completely precluded the cytoplasmic vacuolization despite the continued presence of inactivated PIKfyve or Vps34. Bafilomycin A1 also restored the aberrant EEA1-positive endosomes, enlarged upon short PIKfyve inhibition with YM201636. Together, our work identifies for the first time that factors such as active V-ATPase or functional Rab5a cycle are acting coincidentally with the PtdIns(3,5)P2 reduction in triggering formation of aberrant cytoplasmic vacuoles under PIKfyve or Vps34 dysfunction.

Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma

“…Such an observation is consistent with the recent report that endolysosome acidification is critical for vacuole formation downstream of PI(3,5)P 2 depletion. 51 Although our new observations suggest that the CLCN7/OSTM1 transporter is required for endolysosomal trafficking defects that contribute to apilimod cytotoxicity, further studies will be required to define specific mechanisms that couple CLCN7/OSTM1 function to PI(3,5)P 2 availability.…”

Indolyl-Pyridinyl-Propenone-Induced Methuosis through the Inhibition of PIKFYVE

“… 10 Accordingly, the inhibition of select lipid kinases or phosphatases by chemical or genetic perturbation can induce cellular vacuolization. 12 , 13 …”

New pieces in the complex puzzle of aberrant vacuolation. Focus on “Active vacuolar H+ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P2 loss under PIKfyve or Vps34 deficiency”

“…In this issue of American Journal of Physiology-Cell Physiology, Compton et al (1) provide new insights into the mechanisms involved in accumulation of large cytosolic vacuoles that affect cellular viability. The authors inactivate Vps34 or PIKfyve using pharmacological or genetic approaches.…”

“…These results suggested that the H ϩ V-ATPase display pH-independent activities through a mechanism that remains to be identified. Alternatively, bafilomycin A1 may also impact on the aberrant vacuoles formation via inhibition of heterotypic and/or homotypic endosome fusion (2) (1,9). In these conditions, the fusion of early endosomal vesicles generates an abnormal accumulation of large intracellular vacuoles.…”

“…In these conditions, the fusion of early endosomal vesicles generates an abnormal accumulation of large intracellular vacuoles. Compton et al (1) established that Rab5 activity is absolutely required for this phenomenon, while bafilomycin A1 inhibits the process. Modulation of Rab5 activity, inhibition of Vps34 or discovery of the molecular mechanism by which bafilomycin A1 inhibits vacuolation might offer new strategies to induce cancer cell death through aberrant vacuolation.…”