Abstract
ABSTRACTThe vacuolar ATPase (V-ATPase) proton pump sustains cellular pH homeostasis, and its inhibition triggers numerous stress responses. However, the cellular mechanisms involved remain largely elusive in cancer cells. We studied V-ATPase in the prostate cancer (PCa) cell line PC-3, which has characteristics of highly metastatic PCa. V-ATPase inhibitors impaired endo-lysosomal pH, vesicle trafficking, migration, and invasion. V-ATPase accrual in the Golgi and recycling endosomes suggests that traffic of internalized membrane vesicles back to the plasma membrane was particularly impaired. Directed movement provoked co-localization of V-ATPase containing vesicles with F-actin near the leading edge of migrating cells. V-ATPase inhibition prompted prominent F-actin cytoskeleton reorganization. Filopodial projections were reduced, which related to reduced migration velocity. F-actin formed novel cytoplasmic rings. F-actin rings increased with extended exposure to sublethal concentrations of V-ATPase inhibitors, from 24 to 48 h, as the amount of alkalinized endo-lysosomal vesicles increased. Studies with chloroquine indicated that F-actin rings formation was pH-dependent. We hypothesize that these novel F-actin rings assemble to overcome widespread traffic defects caused by V-ATPase inhibition, similar to F-actin rings on the surface of exocytic organelles.
Highlights
Membrane compartmentalization allows eukaryotic cells to organize functions by grouping them in membrane-bound vesicles
Inhibition of pH regulation has been proposed as a therapeutic strategy in cancer cells (Meehan et al, 2017) because V-ATPase is involved in metastasis and is exploited by tumors to survive, proliferate, and resist therapy (Stransky et al, 2016)
To determine the via the membrane-embedded domain (Vo) subunit-a isoform preferentially expressed in PC-3 cells, we used Quantitative real-time PCR (qRT-PCR) to measure relative expression of these four Voa isoforms
Summary
Membrane compartmentalization allows eukaryotic cells to organize functions by grouping them in membrane-bound vesicles. Compartmentalization is maintained through vesicle transport (Cho and Stahelin, 2005; Miaczynska et al, 2004; Pfeffer, 2003) which relies upon differential pH gradients (Casey et al, 2010; Paroutis et al, 2004). Both processes require vacuolar type H+-ATPase [vacuolar ATPase (V-ATPase)] proton pumps (Paroutis et al, 2004; Sobota et al, 2009).
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