How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

Abstract

AbstractBackgroundFor many cell types, directional locomotion depends on their maintaining filopodia at the leading edge. Filopodia lack any Ca2+-binding structural protein but respond to store-operated Ca2+entry (SOCE).MethodsSOCE was induced by first replacing the medium with Ca2+-free salt solution with cyclopiazonic acid (CPA). This lowers Ca2+in the ER and causes stromal interacting molecule (STIM) to be translocated to the cell surface. After this priming step, CPA was washed out, and Ca2+influx restored by addition of extracellular Ca2+. Intracellular Ca2+levels were measured by calcium orange fluorescence. Regulatory mechanisms were identified by pharmacological treatments. Proteins mediating SOCE were localized by immunofluorescence and analyzed after image processing.ResultsDepletion of the ER Ca2+increased filopodia prevalence briefly, followed by a spontaneous decline that was blocked by inhibitors of endocytosis. Intracellular Ca2+increased continuously for ~ 50 min. STIM and a transient receptor potential canonical (TRPC) protein were found in separate compartments, but an aquaporin unrelated to SOCE was present in both. STIM1- and TRPC1-bearing vesicles were trafficked on microtubules. During depletion, STIM1 migrated to the surface where it coincided with Orai in punctae, as expected. TRPC1 was partially colocalized with Vamp2, a rapidly releasable pool marker, and with phospholipases (PLCs). TRPC1 retreated to internal compartments during ER depletion. Replenishment of extracellular Ca2+altered the STIM1 distribution, which came to resemble that of untreated cells. Vamp2 and TRPC1 underwent exocytosis and became homogeneously distributed on the cell surface. This was accompanied by an increased prevalence of filopodia, which was blocked by inhibitors of TRPC1/4/5 and endocytosis.ConclusionsBecause the media were devoid of ligands that activate receptors during depletion and Ca2+replenishment, we could attribute filopodia extension to SOCE. We propose that the Orai current stimulates exocytosis of TRPC-bearing vesicles, and that Ca2+influx through TRPC inhibits PLC activity. This allows regeneration of the substrate, phosphatidylinositol 4,5 bisphosphate (PIP2), a platform for assembling proteins, e. g. Enabled and IRSp53. TRPC contact with PLC is required but is broken by TRPC dissemination. This explains how STIM1 regulates the cell’s ability to orient itself in response to attractive or repulsive cues.