ARTICLE https://doi.org/10.1038/s41467-021-24739-9 OPEN KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals ✉ ✉ Nagendra Babu Thillaiappan 1,2 , Holly A. Smith1, Peace Atakpa-Adaji1 & Colin W. Taylor 1 2+ 2+ Regulation of IP3 receptors (IP3Rs) by IP3 and Ca allows regenerative Ca signals, the 2+ smallest being Ca puffs, which arise from coordinated openings of a few clustered IP3Rs. 2+ Cells express thousands of mostly mobile IP3Rs, yet Ca puffs occur at a few immobile IP3R 1234567890():,; clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas- induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma mem- brane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ con- centration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional 2+ 2+ IP3R clusters and results in more Ca puffs and larger global Ca signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites 2+ 2+ where store-operated Ca entry occurs, licenses IP3Rs to evoke Ca puffs and global 2+ cytosolic Ca signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry. 1 Department of Pharmacology, Tennis Court Road, Cambridge, UK. 2 Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar ✉ University, Doha, Qatar. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:4514 | https://doi.org/10.1038/s41467-021-24739-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24739-9 2+ ytosolic Ca signals regulate diverse activities in all from EGFP-IP3R1 HeLa cells in the same ratio as their overall 1 fi eukaryotic cells . Most of these signals are due to the expression, con rming that EGFP-IP3R1 subunits assemble with C 2+ 20 opening of Ca -permeable channels, which then allow the other IP3R subtypes . Thus, EGFP-IP3R1 puncta probably 2+ fl Ca to ow into the cytosol across either the plasma membrane report the presence of all three IP3R subtypes in EGFP-IP3R1 (PM) or the membranes of intracellular organelles, primarily the HeLa cells. endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate recep- We first considered whether Ca2+ leaking across the PM 2+ 2+ tors (IP3Rs), the most widely expressed of these Ca channels, might provide a local increase in cytosolic free Ca concentra- 2+ 2+ reside in the membranes of the ER from which they release Ca tion ([Ca ]c) that then selectively stimulates IP3Rs immediately 2,3 2+ 2+ when they bind IP3 . This Ca flux delivers Ca to the cytosol beneath the PM. Previous work with SH-SY5Y neuroblastoma and to the cytosolic surface of other organelles, notably cells9 and human embryonic kidney (HEK) cells11 established 4 5 2+ 2+ 2+ mitochondria and lysosomes . By depleting the ER of Ca ,IP3- that Ca puffs do not require extracellular Ca . We confirmed evoked Ca2+ release also stimulates store-operated Ca2+ entry these findings in HeLa cells by demonstrating that the frequency (SOCE) across the PM. Stromal interaction molecule 1 (STIM1), and amplitude of Ca2+ puffs evoked by photolysis of caged 2+ 2+ which straddles the ER membrane, detects the loss of ER Ca IP3 (ci-IP3) were unaffected by removing extracellular Ca through its luminal Ca2+-binding sites causing it to unfurl (Fig. 1a, b). We conclude that licensing cannot be due to 2+ cytosolic domains. These domains reach across narrow mem- sensitization of IP3Rs near the PM by Ca leaking across brane contact sites (MCS) between the ER and PM to contact the PM. 2+ 2+ 20 Orai Ca channels, causing them to open and allow Ca to The immobility of licensed IP3Rs alongside the PM , the fl 6,7 23 ow into the cell through the SOCE pathway .IP3Rs are presence of a cortical actin cytoskeleton in most animal cells , 2+ 24–26 fi therefore Ca -signalling hubs: in all animal cells, they link the evidence that IP3Rs interact with actin , and identi cation of 27 extracellular stimuli that evoke the formation of IP3 to delivery of actin-binding proteins as partners of IP3Rs in HeLa cells led us 2+ Ca from the ER to the cytosol or other organelles, and to consider whether actin might anchor licensed IP3Rs. Total to the regulation of Ca2+ entry across the PM through internal reflection fluorescence microscopy (TIRFM) revealed SOCE. that 27 ± 7% of EGFP-IP3R1 clusters colocalize with actin fi fi IP3 is not alone suf cient to stimulate the opening of an IP3R. laments (Fig. 1c, Supplementary Movie 1); this fraction is 8 Instead, IP3 binding to all four subunits of a tetrameric IP3R similar to the immobile fraction of peripheral IP3Rs (~25%) primes it to respond to Ca2+, which then evokes channel reported previously20. Live-cell imaging confirmed that immobile 3 opening . In the presence of IP3,IP3Rs can thereby evoke regen- IP3R puncta selectively associate with actin (Fig. 1d, Supplemen- erative signals through Ca2+-induced Ca2+ release (CICR). The tary Fig. 1b–e). Cytochalasin D and latrunculin A caused actin smallest of these regenerative events are Ca2+ puffs, which are depolymerization, and as actin filaments retracted many 2+ local cytosolic Ca signals that arise from coordinated openings immobile IP3R puncta retreated with them and remained 2+ fi – of a few clustered IP3Rs as Ca released by an open IP3R sti- associated with residual laments (Supplementary Fig. 1f i, mulates the opening of its neighbours9 (Supplementary Fig. 1a). Supplementary Movies 2, 3)28. We detected no association of 2+ 10,11 fi 29–32 Ca puffs, which can be evoked by all three IP3R subtypes , IP3Rs with intermediate laments (Supplementary Fig. 2, 2+ allow local Ca signalling and they have been thought to be the Supplementary Movie 4). These results suggest that IP3R puncta building blocks for larger Ca2+ signals, although that view has are immobilized alongside the PM by association with recently been challenged12. Cells typically express several thou- actin. 13–15 2+ sand IP3Rs, most of which are mobile ,yetCa puffs, whe- ther evoked by endogenous signalling pathways or by the uniform release of cytosolic IP3 from a caged precursor, occur repeatedly at KRas-induced actin-interacting protein ties IP3Rs to actin. rather few immobile sites within a cell16–21. In seeking to address KRas-induced actin-interacting protein (KRAP), now designated 2+ this long-standing conundrum we recently reported that Ca IP3 receptor-interacting domain-containing protein 2 (ITPRID fi puffs occur at only a few immobile IP3R clusters alongside the PM 2), was originally identi ed as a large actin-binding protein that is and adjacent to the sites where STIM1 accumulates when SOCE is over-expressed in a colon cancer cell line expressing activated 20 33 33,34 activated . We described these responsive IP3R clusters as KRas . KRAP is now known to be widely expressed and to 29,35,36 licensed IP3Rs to suggest an additional level of regulation that associate with all three IP3R subtypes . Results from co- 2+ precedes gating by IP3 and Ca . immunoprecipitation and immunocytochemical analyses suggest Here, we identify this additional form of regulation and that the N-terminal region of KRAP may interact with IP3Rs, demonstrate that IP3Rs are licensed to respond when they are while the C-terminal may interact with actin (Supplementary tethered to actin by KRas-induced actin-interacting protein Fig. 3a); it remains unclear whether these interactions are direct (KRAP). We show that all Ca2+ signals, whether local or global, or via intermediary proteins33,35,37,38. The functions of KRAP are require KRAP-mediated licensing of IP3Rs and that endogenous poorly understood, but it affects both the subcellular distribution 29 2+ 35,39 KRAP limits the capacity of IP3Rs to respond. This additional, of IP3Rs and IP3-evoked Ca release . Proteins related to 2+ 33,40–43 and obligatory, level of IP3R regulation reveals an important role KRAP are also implicated in Ca signalling (Supple- 2+ for the actin cytoskeleton in regulating IP3-evoked Ca signals mentary Fig. 3a). These observations, alongside evidence from the and suggests mechanisms whereby local depletion of the ER HeLa cell interactome suggesting that KRAP and IP3Rs share might control SOCE. partners, including several that interact with actin27 (Supple- mentary Fig. 3b), prompted us to consider whether KRAP might license IP3Rs. Results HeLa cells express KRAP35 (Supplementary Fig. 3c), and Licensed IP3Rs associate with actin. We used HeLa cells with TIRFM and spinning-disc confocal microscopy revealed that fl endogenous type 1 IP3Rs tagged with enhanced green uorescent endogenous KRAP form puncta, some of which colocalize with a protein (EGFP) to address the mechanisms that license immobile subset of EGFP-IP3Rs (Fig. 1e). The colocalization was restricted 2+ 20 IP3Rs adjacent to the PM to evoke Ca puffs .IP3R1 is the to regions close to the PM (Fig. 1f, g, Supplementary Fig. 3d). We major subtype in HeLa cells22, but we showed previously that an used an object-based colocalization method44 to study nearest- antibody to EGFP immunoprecipitated all three IP3R subtypes neighbour distances between IP3R and KRAP puncta. This 2 NATURE COMMUNICATIONS | (2021) 12:4514 | https://doi.org/10.1038/s41467-021-24739-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24739-9 ARTICLE fi con rmed that 28 ± 7% of IP3R puncta colocalized with KRAP, colocalized IP3R-KRAP puncta retreated with residual actin as significantly more than expected from randomly distributed it depolymerized after the addition of latrunculin A (Fig. 1l). puncta (Fig.