How Filopodia Respond to Calcium in the Absence of a Calcium-binding Structural Protein: They Use Rapid Transit

Abstract

During directional locomotion, cells must reorient themselves in response to attractive or repulsive cues. Filopodia are narrow actin-based protrusions whose prevalence at the leading edge of a migrating cell is related to the persistence of locomotion. Although there is a marked absence of calcium-binding components in their structure, they responded to store-operated calcium entry (SOCE). Here, we used a two-phase protocol to determine how they responded. In the first phase, extracellular calcium was removed and ER calcium lowered by blocking reuptake through the calcium pump. This was known to activate stromal interacting molecule (STIM) and cause its microtubule-mediated translocation to the cell surface. In the second phase, extracellular calcium and calcium influx into the ER were restored. ER depletion caused filopodia to increase, followed by a spontaneous decrease that was blocked by inhibiting endocytosis. The intracellular calcium concentration increased during depletion, while the size of the exchangeable compartment of vesicles, measured by fluid-phase marker uptake, shrank. When SOCE mediators and the aquaporin, AQP4, were localized, STIM and transient receptor potential canonical (TPRC) channels occupied vesicular profiles side-by-side in linear arrays. STIM1 was translocated, as expected. TRPC1 was initially in a rapidly recycling pool (RRP) where it partially colocalized with Vamp2. Calcium restoration caused TRPC1 exocytosis, while STIM1 reverted toward its original pattern associated with the ER. The exchangeable compartment was restored and this enabled filopodia extension, which was blocked by inhibitors of TRPC1/4/5 and endocytosis. That vesicle recycling was essential for extension during calcium readdition was indicated by reversal of the effect of endocytosis inhibitors in the depletion and readdition phases. The results suggest that SOCE regulates the size of the RRP in epithelial cells, and vesicle recycling is the immediate mechanism affecting filopodia extension. The conclusions are discussed in light of factors regulating protrusion formation, namely surface tension and vesicle trafficking.