Lysosome enlargement in PIKfyve inhibited cells proceeds through homotypic lysosome fusion rather than growth of individual lysosomes

Abstract

Phosphoinositides are important lipids that modulate various functions including signal transduction, membrane trafficking, and organelle identity. Phosphatidylinositol‐3,5‐bisphosphate [PI(3,5)P2] is a phosphoinositide found on lysosomes where it controls a wide array of functions such as stress‐induced signalling, autophagic flux, ion‐channel activity, trafficking and endolysosome morphology. Lysosomes are degradative organelles where macromolecules, foreign pathogens, and intracellular organelles arrive through endocytosis, phagocytosis and autophagy for degradation, respectively. PI(3,5)P2 is synthesized from phosphatidylinositol‐3‐phosphate [PI(3)P] via the lipid kinase PIKfyve. One of the most dramatic phenotypes in cells deficient for PI(3,5)P2 synthesis is the enlargement of lysosomes. However, the dynamics and mechanism of lysosome enlargement due to PIKfyve inhibition remains mostly unexplored. We aimed to distinguish whether lysosome swelling proceeds through growth of individual lysosomes or if multiple lysosomes fuse together culminating in enlarged lysosomes in cells treated with apilimod, a specific inhibitor of PIKfyve. Using a combination of spinning disc microscopy and volumetric/particle counting analysis, we show that lysosome enlargement in PIKfyve‐inhibited cells results from homotypic lysosome fusion rather than by growth of individual lysosomes. This is supported by a decrease in lysosome number and increase in volume of individual lysosomes, while total lysosome volume remained constant. These effects are reversed upon washing of apilimod. Consistent with this, disruption of microtubules and microtubule‐associated motor complexes, dynein and kinesin‐1, reduced the rate of lysosome enlargement in PIKfyve‐inhibited cells, while accelerating shrinkage of lysosomes during apilimod removal. We propose a model whereby lysosomes undergo constant kiss‐and‐run (fusion and fission) events. In the absence of PIKfyve, lysosomes “kiss” but fail to “run”, leading to a shift towards homotypic fusion, which increases the size of individual lysosomes and a reduction in total lysosome number.Support or Funding InformationThis work was made possible by NSERC and Canada Research Chair Program.