An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry

Abstract

The STIM1–ORAI1 pathway of store-operated Ca2+ entry is an essential component of cellular Ca2+ signalling1. STIM1 senses depletion of intracellular Ca2+ stores in response to physiological stimuli, and relocalizes within the endoplasmic reticulum to plasma-membrane-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca2+ channels. Here we use a genome-wide RNA interference screen in HeLa cells to identify filamentous septin proteins as crucial regulators of store-operated Ca2+ entry. Septin filaments and phosphatidylinositol-4,5-bisphosphate (also known as PtdIns(4,5)P2) rearrange locally at endoplasmic reticulum–plasma membrane junctions before and during formation of STIM1–ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PtdIns(4,5)P2 reorganization and efficient STIM1–ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud and the primary cilium, and to serve as membrane diffusion barriers and/or signalling hubs in cellular processes such as vesicle trafficking, cell polarity and cytokinesis2,3,4. Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1–ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes.

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Figure 1: Mammalian septin proteins are essential regulators of NFAT activation and store-operated Ca2+ influx.
Figure 2: Septin depletion impairs STIM1-ORAI1 colocalization at ER–plasma membrane junctions.
Figure 3: Septin 4 relocalizes at the plasma membrane after ER Ca2+ store depletion.
Figure 4: Rearrangement of septin 4 at the plasma membrane is required for ORAI1 cluster formation.
Figure 5: Septins organize PIP2 domains surrounding STIM1–ORAI1 clusters at ER–plasma membrane junctions.

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Acknowledgements

We thank C. Shamu, S. Rudnicki, S. Johnston and D. Wrobel for screening support; A. Carpenter and M. Bray for CellProfiler optimization; P. Meraner and Y. Zhou for cell lines and constructs; S. Schmid, J. Fitzpatrick and the Waitt Advanced Biophotonics Center at the Salk Institute for access to TIRF microscopes; C. Junker for the RFP–ER construct; S. Field for the PLCδ-PH–eGFP plasmid; and S. Schmid for manuscript review. The work was supported by National Institutes of Health (NIH) R01 grants AI040127 and AI084167(to A.R. and P.G.H.), NIH RC4 grant AI092763 (to A.R. and S.S.); a Fellowship from the Canadian Institutes for Health Research and a Special Fellowship from The Leukemia & Lymphoma Society (to S.S.); postdoctoral fellowship QU298/1-1 from the Deutsche Forschungsgemeinschaft (to A.Q.); NIH grant K08 HL107451 (to M.J.); a postdoctoral scholarship from the Knut & Alice Wallenberg Foundation (to R.N.); NIH R01 grant R01GM73165 (to S. Schmid, which supports M.M.).

Author information

The genome-wide screen was designed, optimized and performed by S.S. with assistance from B.B.; S.S. validated septins as regulators of NFAT, store-operated Ca2+ entry, STIM1–ORAI1 colocalization and ORAI1 cluster formation, with assistance from G.M.F. for confocal imaging, CellProfiler analyses and qRT–PCR. A.Q. performed single-cell Ca2+ imaging, electrophysiology, TIRFM, STIM1–ORAI1 colocalization, line-scan analysis and FCF experiments, with assistance from M.M. for TIRF microscopy. M.J. and R.N. performed bioinformatic analyses. P.G.H. and A.R. provided overall direction and supervised project planning and execution. S.S., A.Q., P.G.H. and A.R. wrote the manuscript with input from other authors.

Correspondence to Anjana Rao or Patrick G. Hogan.

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Competing interests

A.R. and P.G.H. are founders of Calcimedica, Inc (La Jolla, California). The remaining authors state that they have no competing interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10, Supplementary Methods, Supplementary Tables 1-2, a Supplementary Discussion, Supplementary References and the legend for the Supplementary Data (see separate file). (PDF 3259 kb)

Supplementary Data

This file contains the data for the candidate regulators of NFAT nuclear import (see Supplementary Information file for legend). (XLS 213 kb)

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Sharma, S., Quintana, A., Findlay, G. et al. An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry. Nature 499, 238–242 (2013). https://doi.org/10.1038/nature12229

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