Abstract
ORAI1 Ca2+ channels in the plasma membrane (PM) are gated by STIM1 at endoplasmic reticulum (ER)-PM junctions to effect store-dependent Ca2+ entry into cells, but little is known about how local STIM-ORAI signalling at junctions is coordinated with overall cellular architecture. Filamentous septins can specify cytoskeletal rearrangements and have been found recently to modulate STIM-ORAI signalling. Here we show by super-resolution imaging of ORAI1, STIM1, and septin 4 in living cells that septins facilitate Ca2+ signalling indirectly. Septin 4 does not colocalize preferentially with ORAI1 in resting or stimulated cells, assemble stably at ER-PM junctions, or specify a boundary that directs or confines ORAI1 to junctions. Rather, ORAI1 is recruited to junctions solely through interaction with STIM proteins, while septins regulate the number of ER-PM junctions and enhance STIM1-ORAI1 interactions within junctions. Thus septins communicate with STIM1 and ORAI1 through protein or lipid intermediaries, and are favorably positioned to coordinate Ca2+ signalling with rearrangements in cellular architecture.
Highlights
STIM1 and ORAI1 underlie store-dependent Ca2+ influx, a linchpin of local and global cellular signalling and cellular Ca2+ balance[1,2,3,4]
Initial evidence suggested that the modulation occurred only at endoplasmic reticulum (ER)-plasma membrane (PM) junctions— that septins had no effect when the cytoplasmic domain of STIM1, untethered to ER, activated ORAI1 outside of junctions10— raising intriguing questions about whether and how septins 4 and 5 could strengthen STIM-ORAI signalling without targeting STIM1 and ORAI1 themselves
There was no enrichment of ORAI1 above random in the subset of pixels where STIM1 was localized
Summary
STIM1 and ORAI1 underlie store-dependent Ca2+ influx, a linchpin of local and global cellular signalling and cellular Ca2+ balance[1,2,3,4]. ORAI1 calcium signalling, like other cellular calcium signalling pathways, is intensively modulated by mechanisms that target STIM1 and ORAI1 directly[8,9]. It has been less clear how STIM-ORAI signalling is coordinated with other cellular processes. Our results rule out this simple possibility, but delineate several features of PM organization downstream of septins 4/5 that reinforce physiological STIM-ORAI Ca2+ signalling. These include the local density of ER-PM junctions, the internal specialization of junctions for STIM-ORAI signalling, and possibly the local modulation of ORAI1 concentration. The links that we demonstrate from septins to cellular Ca2+ signalling could help to explain how cells coordinate their local Ca2+ responses during physiological signalling with changes in cytoskeletal and PM organization
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