Cellular vacuolization caused by overexpression of the PIKfyve-binding deficient Vac14L156R is rescued by starvation and inhibition of vacuolar-ATPase

Abstract

Phosphoinositides (PI) and converting enzymes are crucial determinants of organelle identity and morphology. One important endolysosomal specific PI is PI(3,5)P, generated by the PIKfyve kinase, which orchestrates in combination with Vac14 and Fig4. Dysfunction of this complex leads to large intracellular vacuoles in various cell types and is linked to neurological diseases. Here, we characterize the vacuolization phenotype caused by overexpression of the PIKfyve binding deficient mutant Vac14 in podocytes, which represent specialized cells of the kidney. Vacuolization of podocytes, which was associated with strong maturation defects in the endolysosomal system, could be completely rescued by starvation or treatment of cells with the v-ATPase inhibitor Bafilomycin A1. Moreover, we elucidated a strong and reversible de-vacuolization effect of the cholesterol export inhibitor U18666A, which was accompanied by increased basification of the lysosomal pH values. Taken together, our data give new hints to potential therapeutic targets in the treatment of disease linked to intracellular vacuolization.

The Fusarium mycotoxin, 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) induces vacuolization in HepG2 cells

“…This may change the membrane properties, and several studies have revealed that this may result in transient vacuolization (Young et al 2019;Young et al 2016), as here seen for AOD. Transient vacuolization might be due increased formation or reduced processing of vacuoles involved in endocytosis, autophagy or by swelling of lysosomes (Choy et al 2018;Schulze et al 2017; J o u r n a l P r e -p r o o f Shubin et al 2016). The proteomic analysis (Table 1) showing that AOD affect the level of several proteins involved in the phagosome pathways; support the hypothesis that AOD may affect various parts of the vesicular transport systems.…”

“…Interestingly, inhibition of the v-ATPase with concanamycin A, or incubation with chloroquine, which make the lysosomes basic, completely blocked the AOD-induced vacuolization indicating that the vacuolization is dependent of acidic lysosomes. Similarly, bafilomycin A1 has been found to inhibit vacuolization induced by a number of well-known lysosomal trapping compounds (Marceau et al 2012;Schulze et al 2017;Shubin et al 2016). Furthermore, these type of compounds will also reduce lysosomal degradation capacity (Seglen and Gordon 1980) as here seen following AOD exposure.…”

Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast

“…, 2006 ), suggesting that the a2 isoform could be susceptible to PI(4)P levels in maturing phagosomes. V-ATPase activity has also been linked to vacuolization of podocytes arising from altered PI(3,5)P 2 metabolism ( Schulze et al. , 2017 ), but the a-subunit isoform present in the vacuoles is unknown.…”

Interaction of the late endo-lysosomal lipid PI(3,5)P2 with the Vph1 isoform of yeast V-ATPase increases its activity and cellular stress tolerance

“…We have limited understanding of how these neurodegenerative diseases arise from PI(3,5)P2 deficiency. Although the diseases are neurodegenerative in nature, extensive vacuolation is found in all studied cell types with a loss of PI(3,5)P2 (24,45). In yeast and some mammalian cell lines, inhibition of V-ATPase reverses the large vacuolation phenotype of PI(3,5)P2 deficiency (43,44).…”

“…The mechanism behind the enlargement is incompletely understood (42,43). Cells chronically or acutely deprived of V-ATPase activity do not undergo such vacuolar enlargement (43)(44)(45). We tested whether V-ATPase activation by PI(3,5)P2 was required to maintain normal vacuolar morphology.…”