Aquaporin 1 (AQP1) regulates plasma membrane bleb formation by facilitating bleb retraction phase in cancer cells

Cancer cells migrate by using different membrane protrusions including plasma membrane blebs. Blebs are formed when the plasma membrane delaminates from the underlying cortex due to actomyosin contractility-induced increase in cytoplasmic pressure, and this causes influx of water and cytosolic fluid through aquaporins into the detached membrane resulting in its enlargement. In this study, we used a combination of molecular biology and microscopy techniques to examine the involvement of aquaporin 1 (AQP1) in bleb formation, and report for the first time that, siRNA-mediated knockdown of AQP1 inhibited bleb formation in both HT1080 and ACHN cell lines embedded in 3D matrigel matrix. In HT1080 cells, blebs resurfaced 48 h post-siRNA transfection, and this paralleled AQP1 protein expression, as shown by Western blotting. By using confocal and phase-contrast time-lapse microscopy to track bleb dynamics, we demonstrate that the re-emergent blebs had longer lifespan due to longer time of expansion, stabilization and retraction when compared with wildtype blebs. Furthermore, overexpression of GFPtagged AQP1 significantly increased bleb size, and also reduced bleb lifespan by conferring on the cells a shorter and faster bleb retraction time. To unravel the mechanism underpinning AQP1-facilitated bleb retraction, our data showed a requirement for the sodium-hydrogen exchanger (Na + /H + ) activity, as inhibition of the pump's activity attenuated bleb retraction. Similarly, and importantly, we demonstrate that GFP-AQP1 overexpression sufficiently induced blebbing phenotype in non-blebbing MDA-MB-231 cell line. Thus, AQP1 is a potential therapeutic target in combating cancer cell migration.