Mitochondrial Ca2+flickers on endoplasmic reticulum (ER)-mitochondrial contact sites to suppress store-operated Ca2+entry

Mitochondrial Ca2+ flickers on endoplasmic reticulum (ER)-mitochondrial contact sites to suppress store-operated Ca2+ entry | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Mitochondrial Ca 2+ flickers on endoplasmic reticulum (ER)-mitochondrial contact sites to suppress store-operated Ca 2+ entry View ORCID Profile Yu-Chiao Lin , View ORCID Profile Feng-Chiao Tsai doi: https://doi.org/10.1101/2025.01.17.633482 Yu-Chiao Lin 1 Institute of Pharmacology, College of Medicine, National Taiwan University Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yu-Chiao Lin Feng-Chiao Tsai 1 Institute of Pharmacology, College of Medicine, National Taiwan University 2 Department of Internal Medicine, National Taiwan University Hospital Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Feng-Chiao Tsai For correspondence: tsaifc{at}ntu.edu.tw Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Successful Ca 2+ signaling requires appropriate Ca 2+ storage and buffering by endoplasmic reticulum (ER) and mitochondria. Recent research has elucidated how Ca 2+ storage in ER is controlled by STIM1-mediated store-operated Ca 2+ entry (SOCE). However, how cells employ mitochondrial Ca 2+ buffering to maintain Ca 2+ homeostasis has remained elusive. Here, with the use of mitochondria-tethered Ca 2+ sensor, we noticed local Ca 2+ flickering within individual mitochondria. Those Ca 2+ flickers were generated on ER-mitochondrial contact sites (EMC), as indicated by their downregulation under EMC breakdown and upregulation under EMC induction. Surprisingly, EMC breakdown increased SOCE while EMC induction reduced SOCE. Further investigations revealed that EMC effect on SOCE was not through biological functions of mitochondria or through STIM1 regulators, but via IP 3 R-VDAC1-driven mitochondrial Ca 2+ flickers on EMC. Those flickers depleted peri-EMC Ca 2+ inside ER, resulting in STIM1 sequestration around EMC to cause SOCE reduction. Moreover, EMC breakdown also increased availability of STIM1 to bind with microtubule plus ends, preventing STIM1 over-activation and SOCE upregulation. Overall, ER, mitochondria and microtubules constitute a self-sufficient system to control Ca 2+ homeostasis, driven by mitochondrial Ca 2+ flickers to reduce SOCE and to prevent intracellular Ca 2+ overload. Introduction Ca 2+ signaling is fundamental to cellular life, orchestrating physiological processes from gene expression to cell death 1 – 5 . Precise spatial and temporal control of intracellular Ca 2+ homeostasis is maintained through sophisticated interplay between Ca 2+ channels and their regulatory proteins, which move Ca 2+ through plasma membrane and Ca 2+ -storing organelles, most importantly endoplasmic reticulum (ER) and mitochondria. ER serves as the primary intracellular Ca 2+ reservoir, with its Ca 2+ level dynamically regulated by coordinating inward Ca 2+ pumps, mainly sarcoplasmic / endoplasmic reticulum Ca 2+ -ATPase (SERCA), with outward Ca 2+ channels, notably inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 6 – 8 . Moreover, when ER Ca 2+ storage is depleted, the cell activates store-operated calcium entry (SOCE). This process is initiated by the ER Ca 2+ sensor STIM1, which oligomerizes and translocates to ER-plasma membrane (PM) junctions upon ER Ca 2+ depletion 9 , 10 . STIM1 then activates ORAI1 channels at these ER-PM junctions, facilitating Ca 2+ influx to replenish depleted store 11 , 12 . Thus, precise regulation of STIM1 dynamics is essential for appropriate SOCE activation and intracellular Ca 2+ homeostasis. While ER Ca 2+ depletion serves as the master factor of STIM1 activation, multiple factors have been reported contributing to STIM1 regulation, including post-translational modifications, regulatory proteins, and cytoskeletal elements 13 – 15 . Unfortunately, how those factors coordinate with each other for optimal STIM1 activation has not been clarified. Furthermore, in contrast to our comprehensive understanding of ER-mediated Ca 2+ storage as shown above, how mitochondria regulate Ca 2+ homeostasis has remained mystic. In addition to the big mystery that proteins responsible for mitochondrial Ca 2+ efflux, termed as mitochondrial permeability transition (MPT), have yet to be identified, much about mitochondrial Ca 2+ influx has also remained unknown. Recent evidence indicated that mitochondrial Ca 2+ influx occurred through voltage-dependent anion channels (VDACs) on the outer membrane and mitochondrial calcium uniporter (MCU) complex on the inner membrane 16 – 18 , while components and structures of MCU have not been fully elucidated. Recent reports also suggested the presence of mitochondrial Ca 2+ influx on ER-mitochondria contact sites (EMCs), which represent specialized domains where these organelles are tethered in close proximity (10-30 nm) 19 – 21 through protein complexes including mitofusin 2 (MFN2) 22 , 23 , PDZD8 24 and FUNDC1 22 , 25 – 27 . Thus, EMCs were speculated to create privileged space for Ca 2+ transfer, with IP 3 Rs and VDACs serving as key channels for Ca 2+ movement from ER to mitochondria 28 – 32 . However, how EMCs- mediated Ca 2+ transfer occurred remained unclear. Its implication on intracellular Ca 2+ homeostasis also required further exploration. Here, we report the unexpected discovery of spontaneous, rapid Ca 2+ flickers within individual mitochondria. These localized Ca 2+ signals were eliminated by knockdown of EMCs proteins MFN2 and PDZD8 and created by induction of EMCs formation, confirming that EMCs create privileged sites for that dynamic Ca 2+ transfer between ER and mitochondria. We further uncovered an unexpected function of EMCs in suppressing SOCE through STIM1 sequestration, where EMCs depleted local ER Ca 2+ to keep STIM1 accumulation on EMCs, which was away from ORAI1. Interestingly, this regulatory mechanism interacted with microtubule-mediated STIM1 mobilization, revealing a complex interplay between ER, mitochondrial and microtubules in SOCE regulation. Altogether, our work identifies EMCs as critical regulatory hubs for cellular Ca 2+ signaling and highlights an unrecognized cooperation between organelle contacts, cytoskeletal elements, and Ca 2+ homeostasis. Result Using protein-based fluorescent mitochondrial Ca 2+ indicators in HeLa cells and HepG2 cells, we noticed spontaneous Ca 2+ flickers within individual mitochondria ( Fig. 1A , S1A, Video S1, S2 ). Mitochondrial tracing revealed that these Ca 2+ flickers occurred in most mitochondria ( Fig. 1B ) with the frequency of ∼ 1.5 min -1 per mitochondrion (mean ± s.d. = 1.5 ± 0.5 min -1 per mitochondrion in HepG2 cells) ( Fig. 1C ). Normalization of Ca 2+ signals revealed that peaks of Ca 2+ flickers ranged ∼ 0.9 to 1.3-Fold of average signals within individual mitochondria (mean ± s.d. = 1.1 ± 0.1 Fold in HepG2 cells) ( Fig. 1C ). Literature revealed mitochondrial basal Ca 2+ levels to be 100-200 nM, so amplitudes of mitochondrial Ca 2+ flickers probably fell on the range of 90-260 nM. In addition, individual Ca 2+ flickers lasted 6 to 33 seconds (mean ± s.d. = 19.4 ± 6.9 sec in HepG2 cells) ( Fig. 1C ). Regarding that most Ca 2+ channels followed rapid dynamics of opening in nano- to milli-second, long durations of mitochondrial Ca 2+ flickers reflected that each flicker originated from opening of multiple Ca 2+ channels in semi-synchronous manners. Finally, induction of mitochondrial superoxide production using mitoPQ 33 did not increase mitochondrial Ca 2+ flickers ( Fig. S1B, C ), indicating that mitochondrial Ca 2+ flickers were not directly generated by the recently reported mitochondrial flashes (mitoflashes) 34 . Download figure Open in new tab Figure 1. ER-mitochondrial contacts regulate spontaneous mitochondrial Ca 2+ flickers. (A) Representative time-lapse images showing spontaneous mitochondrial Ca 2+ flickers in a HeLa cell. Scale bar, 10 µm(left), and 1 µm (right). (B) Heatmap visualization of mitochondrial Ca 2+ dynamics from (A). Each row represents single mitochondria tracked using the MATLAB Mitometer algorithm. Left panel: mitochondrial Ca 2+ levels indicated by color intensity. Right panel: Frame-to-frame changes in mitochondrial Ca 2+ levels (ΔF = Ft- Ft-1). (C) Characterization of mitochondrial Ca 2+ dynamics. Analysis of individual mitochondrial Ca 2+ flickers showing the flicker frequency (events per mitochondrion; top), peak amplitude (per flicker, middle), and flicker duration (seconds; bottom), with median values indicated by red vertical lines. (D) Analysis of Ca 2+ flicker patterns and periodicity. Left panels: Alignment of mitochondrial Ca 2+ traces centered at peak amplitude (top, mean ± SEM) and their corresponding autocorrelation analysis (bottom), demonstrating temporal periodicity of Ca 2+ events. Right panel: Frequency distribution histogram of Ca 2+ flickers per 49 frames (blue bars) fitted with a Poisson distribution (red dotted line). The close agreement between mean (12.95) and variance (12.42) confirms the stochastic nature of these events. (E) Representative heatmaps of time-lapse mitochondrial Ca 2+ levels in shLuc control cells (shCTL), MFN2-knockdown cells (shMFN2), and PDZD8-knockdown cells (shPDZD8). (F) Percentage of mitochondria exhibiting Ca 2+ flickers (gray bars) or without Ca 2+ flickers (black bars) in shCTL, shMFN2 and shPDZD8 cells. (G) Quantification of the frequency of mitochondrial Ca 2+ flickers per mitochondrion in shCTL, shMFN2, and shPDZD8 cells. (H) Quantification of the peak amplitude of mitochondrial Ca 2+ flickers in shCTL, shMFN2, and shPDZD8 cells. (I) Quantification of the duration of each mitochondrial Ca 2+ flicker in shCTL, shMFN2, and shPDZD8 cells. (J) Schematic illustration of rapamycin-induced contacts between ER and mitochondria facilitating Ca 2+ flow from ER to mitochondria. (K) Representative time-lapse images showing inducible ER-mitochondrial contacts after rapamycin treatment in cells co-expressing FRB-ER-YFP and OMM-FKBP-Turquoise. Scale bar, 10 µm. (L) Representative images of a HeLa cell co-expressing FRB-ER-YFP, OMM- FKBP-Turquoise, and mito-LAR-GECO1.2 before and after 100nM rapamycin treatment. Mitochondrial Ca 2+ level was monitored by mito-LAR-GECO1.2. Scale bar, 10 µm(left), and 1 µm (right). (M) Quantitative analysis of mitochondrial Ca 2+ level at ER-mitochondrial contact sites (EMCs, Red) versus non-EMC mitochondria (non-EMCs, black) following rapamycin treatment. shCTL data in (F-I) corresponded to the measurements shown in (C). Data are presented as SEM in (G-I,M). *P < 0.05; **P < 0.01; ***P < 0.001. Periodicity analysis did not reveal significant temporal oscillation in these Ca 2+ flickers ( Fig. 1D ). Interestingly, the mean value of flicker frequency equaled its variance value, indicating that occurrence of mitochondrial Ca 2+ flickers followed Poisson distribution ( Fig. 1D ), i.e. a stochastic process with preset probability. Together with that ER served as the major intracellular Ca 2+ store and formed extensive contacts with mitochondria, we assumed that mitochondrial Ca 2+ flickers were generated by stochastic contact between ER and mitochondria. Thus, we investigated whether breakdown of these ER-mitochondrial contact sites (EMCs) altered these flickers. Indeed, disrupting EMCs proteins MFN2 and PDZD8 significantly reduced the percentage of mitochondria exhibiting Ca 2+ flickers ( Fig. 1E, F ), along with decreased flickering frequency and amplitudes ( Fig. 1G, H & S1D ), while flickering durations remained largely unchanged ( Fig. 1J & S1E ). In addition to above temporal analysis of mitochondrial Ca 2+ flickers, we also compared Ca 2+ signals between individual mitochondria, noticing that they could be divided into two groups—those with low (“Basal”) Ca 2+ and those with high (“Flickers”) Ca 2+ levels ( Fig. S1F ). Knocking down MFN2 or PDZD8 did not alter basal mitochondrial Ca 2+ but reduced signal intensities of mitochondrial Ca 2+ flickers ( Fig. S1G ), compatible with results from mitochondrial tracing analyses ( Fig. 1E-I ). We finally tested whether direct physical induction of EMCs facilitated mitochondrial Ca 2+ flickers by employing the rapamycin-inducible tethering system to our mitochondrial Ca 2+ detecting platform ( Fig. 1J, K , Video S3) . Indeed, induction of EMCs by rapamycin rapidly increased mitochondrial Ca 2+ at contact sites ( Fig. 1L, M ), confirming that EMCs served as privileged sites for the creation of mitochondrial Ca 2+ flickers. Regarding the pivotal roles of ER and mitochondria on intracellular Ca 2+ storage, our next question was whether these EMCs-mediated mitochondrial Ca 2+ flickers regulated Ca 2+ homeostasis. Specifically, we eliminated EMCs by knocking down their molecules MFN2, PDZD8 or FUNDC1, followed by examining whether EMCs elimination altered SOCE, a major mechanism for cellular Ca 2+ replenishment when intra-ER Ca 2+ was depleted. Before SOCE assays, cells with EMCs elimination were treated with colchicine to avoid potential SOCE interference by ER-microtubule interaction 35 , 36 . As EMCs-mediated mitochondrial Ca 2+ flickers might reduce intra- ER Ca 2+ resulting in SOCE activation, we expected that elimination of EMCs would reduce SOCE. Surprisingly, we saw the opposite: Although EMCs elimination increased intra-ER Ca 2+ levels as expected ( Fig. 2A , 2B; Fig. S2A, S2B ), SOCE was also increased ( Fig. 2A and 2C ). Indeed, knockdown of either IP 3 R or VDAC1 also increased SOCE ( Fig. S2D, 2E ), supporting that EMCs elimination increased SOCE by blocking ER-to-mitochondria Ca 2+ flow to generate mitochondrial Ca 2+ flickers. We further noticed that STIM1 oligomerization with puncta formation, which was an indicator of SOCE activation, was strengthened by EMCs elimination ( Fig. 2D , 2E; S2F, S2G ). EMCs elimination also enhanced STIM1-ORAI1 colocalization ( Fig. S1H, S1I ), another indicator of SOCE activation. Moreover, knockdown of STIM1 and ORAI1 abolished effects of EMCs elimination on intra-ER Ca 2+ ( Fig. S2C ) and SOCE ( Fig. 2F ), confirming the specific effect of EMCs on SOCE regulation. All above results indicated that EMCs-mediated mitochondrial Ca 2+ flickers suppressed SOCE, which was further verified by two rescue experiments. First, we restored protein levels of MFN2 or PDZD8 in cells with MFN2 or PDZD8 knockdown, showing that EMCs restoration reversed both STIM1 activation ( Fig. 2G , 2H ) and SOCE activity ( Fig. 2I ) caused by EMCs elimination. Second, we induced EMCs formation in cells with EMCs elimination using the rapamycin-inducible system described above ( Fig. 1J , 1K, Video S3 ). Excitingly, EMCs induction not only reduced SOCE as expected ( Fig. 2J ; S2J ) but also abolished effects of EMCs elimination on SOCE ( Fig. 2K ; S2K ). Altogether, our knockdown and rescue experiments confirmed the necessity and sufficiency of EMCs-mediated mitochondrial Ca 2+ flickers for the suppression of SOCE. Download figure Open in new tab Figure 2. Knocking down ER-mitochondrial tethering proteins upregulated store-operated Ca 2+ entry. (A) Fura-2 assay showed the relative Ca 2+ level after ER-Ca 2+ was depleted by thapsigargin (TG), followed by the addition of CaCl 2 . Cells were pretreated with 10µM colchicine for 3 hours to depolymerize microtubules. (B) Quantification of the peak value of Ca2+ leakage from ER in (A). The value indicated higher Ca 2+ storage within ER in cells knocked down MFN2, PDZD8 and FUNDC1. (C) Store-operated Ca 2+ entry (SOCE) quantification from (A), demonstrating increased SOCE in MFN2-, PDZD8-, and FUNDC1-knockdown cells (n=150 cells per condition; *** p<0.001). (D) Representative images of mCherry-STIM1 puncta formation following TG treatment in colchicine-pretreated cells. Scale bar, 10µm. (E) Quantification of STIM1 puncta intensity showing enhanced puncta formation in MFN2- and PDZD8-depleted cells. (F) Representative images showing the rescue of enlarged STIM1 puncta by MFN2 or PDZD8 re-expression in respective knockdown cells. Scale bar, 10µm. (G) Quantification of STIM1 puncta intensity fold change in cells knocked down of MFN2 and its rescue, and knocked down of PDZD8 and its rescue. (n=941,264,228,175 and 76 cells, ***p<0.001). (H) Quantification of SOCE activity, which showed the rescue of upregulated SOCE by re-expressing MFN2 and PDZD8 in respective knockdown cells. (n = 1097, 552, 216 and 1006, 780, 84 cells; ****p < 0.0001) (I) Knocking down of STIM1 and ORAI1 in MFN2- and PDZD8-depleted cells eliminated the upregulated SOCE. (n=150 cells per condition; ***p<0.001) (J) Inducing ER-mitochondrial contacts suppress SOCE. Left panel: FRET/CFP ratio analysis demonstrates rapid and stable formation of EMCs upon rapamycin addition (red), while DMSO control shows no changes (blue). Right panel: Cytosolic-Ca 2+ level detected by R-GECO1 showing significantly reduced SOCE in cells with artificially induced EMCs. (K) Formation of artificial ER-mitochondrial contacts rescue the enhanced SOCE phenotype in MFN2- and PDZD8-knockdown cells, as demonstrated by comparing rapamycin versus DMSO treatment. (n =47, 39, 34, 127, 67 and 50 cells, respectively, **p<0.01, ***p<0.001) (L) Schematic model illustrating potential mechanisms that may counteract the effects of MFN2 or PDZD8 knockdown on SOCE regulation. (M) Blocking electron transport chain rotenone/antimycin A did not suppress the enhanced SOCE in MFN2- and PDZD8-knockdown cells. (n =200 cells, respectively, ***p<0.001) (N) SARAF knockdown did not suppress the enhanced SOCE in MFN2- and PDZD8-knockdown cells. (n = 200 cells in each group, ***<0.001) (O) mPTP inhibition completely abolishes the enhanced SOCE in MFN2- and PDZD8-knockdown cells. (n =200 cells in each group) We further investigated the mechanism how EMCs-mediated mitochondrial Ca 2+ flickers reduced SOCE. First, mRNA and protein levels of STIM1 and ORAI1 remained unchanged in MFN2- or PDZD8-knockdown cells ( Fig. S3A-S3D ), suggesting that EMCs elimination did not increase SOCE via upregulation of SOCE proteins. Second, effects of EMCs elimination by protein knockdown could be fully rescued by EMCs induction via rapamycin-inducible system ( Fig. 2J , 2K ), indicating that SOCE was probably directly altered by approximation between ER and mitochondria rather than specific protein interaction on EMCs. Based on above we exhausted remaining possibilities how EMCs suppressed SOCE and generated three hypotheses ( Fig. 2L ): (a) via alteration of mitochondrial functions by mitochondrial Ca 2+ flickers, (b) via regulation of STIM1 activities by STIM1 regulators, and (c) via ER-to-mitochondria Ca 2+ flow changing STIM1 dynamics. We then examined if EMCs-mediated SOCE suppression could be abolished by perturbation of any above processes. For alteration of mitochondrial functions ( Fig. 2La ), we utilized the inhibitor of electron transport chain rotenone / antimycin A ( Fig. 2M , S3E ), inhibitor of ATP synthase oligomycin A ( Fig. S3F ) and inhibitor of mitochondrial membrane potential FCCP ( Fig. S3G ), showing that disruption of mitochondrial functions did not abolish effects of EMCs elimination on SOCE. For regulation of STIM1 activities ( Fig. 2Lb ), we employed RNA interference targeting putative STIM1 regulators SARAF 15 ( Fig. 2N , S3H ), STIMATE 14 ( Fig. S3I ) and septins 5 (specifically septin-4 37 and septin-7 4 , 38 , 39 ) ( Fig. S3J ), showing that disruption of STIM1 regulators did not abolish effects of EMCs elimination on SOCE. For ER- to-mitochondria Ca 2+ flow ( Fig. 2Lc ), inhibitors of mitochondrial Ca 2+ uniporter (MCU) DS16570511 (DS; Fig. S3K, S3N ), MCUi4 ( Fig. S3L, S3O ) and ruthenium red (RuR; Fig. S3M ) also failed to abolish effects of EMCs elimination on SOCE. However, cyclosporin A (CysA), a putative inhibitor of VDAC1, completely extinguished effects of EMCs elimination on SOCE ( Fig. 2O ). Moreover, knockdown of either IP 3 R (shITPR; Fig. 3A , 3B ) or VDAC1 ( Fig. 3C , 3D ) also obliterated effects of EMCs elimination on SOCE. All above results confirmed that EMCs suppressed SOCE neither by altering mitochondrial functions nor via STIM1 regulators, but directly through ER-to-mitochondria Ca 2+ flow. Download figure Open in new tab Figure 3. ER-Mitochondrial Contacts Regulate SOCE through IP3R- and VDAC1-Dependent Local Ca2+ Depletion and STIM1 Sequestration (A) Fura-2 assay showing SOCE responses in cells simultaneous knockdown of IP3R and either MFN2 or PDZD8. (B) Quantification of SOCE from (A) showing IP3R is essential for the phenotype of enhanced SOCE in the MFN2- and PDZD8-knockdown cells. (n = 200 cells in each group, ***p <0.001) (C) Relative Ca 2+ level in cells double knockdown of VDAC1 and either MFN2, PDZD8. (D) Quantification of SOCE from (C) showing VDAC1 is required for shMFN2- and shPDZD8-upregulated SOCE. (E) Schematic model depicting how Ca 2+ flow through IP3Rs and VDAC1 generates low [Ca2+] within ER at ER-mitochondrial contact sites, resulting in local STIM1 sequestration and prevention of excessive STIM1 activation. (F) Representative images showing local ER-Ca 2+ level at rapamycin-induced ER-mitochondrial contact sites. HeLa cells co-express FRB-ER-YFP, OMM- FKBP-Turquoise and ER-LAR-GECO1, with ER-LAR-GECO1 indicating [Ca 2+ ] within ER. Scale bar, 10µm (left panel), 2µm (right panel). (G) Kymograph showing the concurrent decrease of ER-[Ca 2+ ] at EMCs (lower panel) and stable ER-[Ca 2+ ] at non-EMCs ER (upper panel) after rapamycin addition. (H) Quantification of relative ER-[Ca 2+ ] demonstrates rapid and significant Ca 2+ decrease specifically at ER-mitochondrial contact sites (red) compared to non- contact ER regions (black) upon contact formation. (I) Representative images showing STIM1 accumulation at rapamycin-induced contact regions (highlighted by red dots circle). (J) Kymographs showing concurrent increases in STIM1 signals at EMCs (upper panel) after rapamycin addition, compare with that at non-EMC ER (lower panel). (K) Quantification of STIM1 levels show specific accumulation at ER- mitochondrial contact sites (red) compared to non-contact ER regions (black) from (J). The fact that ER-to-mitochondria Ca 2+ flow at EMCs generates mitochondrial Ca 2+ flickers to significantly increase mitochondrial Ca 2+ levels ( Fig. S1F, S1G ) suggests that intra-ER Ca 2+ around EMCs should be significantly reduced. We thus speculated that reduction of local intra-ER Ca 2+ resulted in STIM1 oligomerization at EMCs ( Fig. 3E ), similarly to what occurred at ER-plasma membrane junction sites during intra-ER Ca 2+ depletion. If so, EMCs would serve as hubs for STIM1 sequestration and SOCE suppression, while EMCs elimination would increase the pool of STIM1 augmenting SOCE activation. To prove this hypothesis, we examined whether intra-ER Ca 2+ was locally decreased at EMCs, and whether STIM1 was locally increased at EMCs. Using above-mentioned rapamycin-inducible system to create and quantify signals on EMCs ( Fig. S4A-C ), we observed rapid decrease in local ER Ca 2+ levels using the ER-specific Ca 2+ indicator ER-LAR-GECO1 ( Fig. 3F - H) , with simultaneous increase of local STIM1 signals ( Fig. 3I-K ). These results provided direct evidence for STIM1 sequestration and SOCE suppression by ER-to- mitochondrial Ca 2+ flow at EMCs. Finally, we explored whether microtubules exerted effects on EMCs-mediated SOCE suppression, as literature unveiled important interactions between microtubules versus mitochondria, ER and STIM1. To begin with, cells with EMCs elimination and SOCE assays were compared in the absence ( Fig. 2A ) vs. presence of colchicine ( Fig. 4A ), which suppressed polymerization of microtubules. Interestingly, while EMCs elimination still increased intra-ER Ca 2+ in the presence of microtubules ( Fig. 4A , 4B ), it failed to further increase SOCE when intra-ER Ca 2+ was completely depleted ( Fig. 4A , 4C ). Together with above results that EMCs elimination increased SOCE by increasing the pool of STIM1, this experiment suggested that microtubules counteracted with STIM1 to abolish EMCs effects on SOCE when Ca 2+ was depleted. Indeed, literature reported that EB1 at the microtubule plus-end bound to STIM1, restricting its translocation to ER-plasma membrane junctions for SOCE activation. We therefore proposed the presence of competition for STIM1 between EMCs and EB1 under Ca 2+ depletion ( Fig. 4D ). As expected, accumulation of STIM1 signals on EB1-labeled microtubule plus-ends was promoted by EMCs elimination ( Fig. 4E-G ), supporting our competition model. Furthermore, the competition between EMCs and microtubule plus-ends for STIM1 could be relieved not only by STIM1 over-expression ( Fig. 4H ), but also by transient elimination of microtubules using rapamycin-inducible spastin-mediated microtubule cleavage 40 , ( Fig. 4I , S4D- F ), resulting in the augmentation of SOCE activation during EMCs elimination. These results confirmed that microtubule plus-ends competed with EMCs for STIM1 regulating SOCE activities during Ca 2+ depletion. Ultimately, while EB1 over- expression reduced SOCE as expected ( Fig. 4J ), the reduction was abolished by EMCs elimination, indicating that EMCs were also required for ER (STIM1)- microtubule (EB1) interaction and SOCE suppression. Overall, our investigation unveiled an integrated, self-sufficient machinery for intracellular Ca 2+ homeostasis, within which ER, mitochondria and microtubules interacted with each other maintaining proper SOCE activities ( Fig. 4K ). Download figure Open in new tab Figure 4. Microtubules could reduce shMFN2- and shPDZD8-effects on SOCE by competing for STIM1. (A) Fura-2 assay showing cytosolic [Ca 2+ ] after sequential treatment with TG and CaCl 2 . (B) Quantification of the ER Ca 2+ release peak from (A), showing enhanced ER Ca 2+ storage in either MFN2- and PDZD8-knockdown cells without colchicine treatment. (C) Quantification of Ca 2+ influx peak from (A), showing SOCE amplitude in MFN2- and PDZD8-knockdown cells without colchicine treatment. (D) Schematic representation of the proposed mechanism whereby EB1-binding hinders local STIM1 activation during Ca 2+ flow from ER to mitochondria. (E) Representative images showing mCherry-STIM1 and EB1 distribution in shCTL, shMFN2, and shDPZD8 cells. Scale bar, 1µm. (F) Quantitative analysis of STIM1 enrichment on EB1-positive structures. Each dot represents a single cell, and lines represent linear regression fits for each condition. (G) Pearson’s correlation coefficient (R) analysis of STIM1 and EB1 colocalization. Bar graph shows mean ± SEM of Pearson’s R values between STIM1 and EB1 signals across multiple cells for each condition. (n= 50 cells) (H) Quantification of SOCE peak values showing STIM1 overexpression enhances the elevated SOCE response in MFN2- and PDZD8-knockdown cells. Two-way ANOVA p <0.001. (I) STIM1 puncta intensity measurements in MFN2- and PDZD8-knockdown cells after microtubule depolymerization by recruiting engineered microtubule-cleaving enzymes, (J) Overexpressing EB1 suppressed SOCE activity. (K) Schematic model illustrating the dual regulation of STIM1 by EMCs and microtubules. The left panel shows STIM1 accumulation at EMC region due to locally reduced ER-Ca 2+ level; middle panel illustrated enhanced SOCE activation when STIM1 is free from both EMC and microtubule binding; right panel demonstrates how STIM1-EB1 interactions along microtubules compete with STIM1 accumulation at EMCs, providing an opposite regulatory mechanism for SOCE activity. Discussion Our research unraveled the spontaneous flickering phenomenon of mitochondrial Ca 2+ on ER-mitochondrial contact sites (EMCs), resulting in the suppression of store- operated Ca 2+ entry (SOCE) and reduction of intracellular Ca 2+ storage. This mitochondrial Ca 2+ flickering implicates that activities of mitochondria can be controlled individually, probably through manipulation of EMCs in temporal and spatial manners. Linkage between these Ca 2+ flickers and SOCE also indicates significant impacts of local organellar Ca 2+ on global Ca 2+ homeostasis. Individual mitochondrial activities have been noticed in literature but have not been clarified. While early studies showed that mitochondrial Ca 2+ responded to cytosolic Ca 2+ change 41 – 44 , Patcher et al. reported miniature Ca 2+ signals in single mitochondria triggered by ryanodine receptors 45 , indicating critical roles of ER to initiate Ca 2+ activities in individual mitochondria. Wang et al. later reported superoxide flashes in single mitochondria 46 , but their relationship with mitochondria Ca 2+ was not elucidated. Recent studies further unveiled the potential existence of ER-to-mitochondrial Ca 2+ flow on EMCs 47 , followed by our present work, which comprehensively demonstrated the dynamics of EMCs-mediated Ca 2+ flickering in single mitochondria. Since Ca 2+ activities among single mitochondria are not synchronous to each other, they may function differentially from each other to achieve concise temporal and spatial control of cellular tasks. Our experiments revealed that EMCs elimination increased intra-ER Ca 2+ storage ( Fig. S2A, B ) by enhancing SOCE. Previous studies have attributed the elevated ER Ca 2+ to reduced Ca 2+ transfer from ER to mitochondria 20 , 48 – 50 , suggesting that disrupted ER-mitochondrial Ca 2+ communication leads to Ca 2+ accumulation in the ER. However, we showed that elevated ER Ca 2+ level primarily depended on enhanced SOCE activity, as evidenced by its abolition upon STIM1 and ORAI knockdown ( Fig. S2C ). Notably, inhibiting mitochondrial Ca 2+ uptake through MCU failed to extinguish the elevated ER Ca 2+ levels in EMCs-deficient cells, further supporting that reduced ER to mitochondrial Ca 2+ transfer was not the primary cause of ER Ca 2+ increase under EMCs elimination ( Fig. S3K-M ). Moreover, our finding that EMCs suppressed SOCE indicates the importance of EMCs-mediated local Ca 2+ transfer on the regulation of global intracellular Ca 2+ storage. Importantly, we identified the mechanism how EMCs suppress SOCE. Depletion of intra-ER Ca 2+ has been known to alter STIM1 conformation, resulting in STIM1 oligomerization to open ORAI1 on the plasma membrane, triggering Ca 2+ inflow from extracellular space 10 , 31 , 51 , 52 . On top of that, we demonstrated that EMCs-mediated mitochondrial Ca 2+ flickers depleted ER Ca 2+ locally, leading to STIM1 accumulation (probably by oligomerization) at EMCs. By sequestering STIM1 at EMCs, cells can reduce the pool of STIM1 for SOCE activation. This finding highlights the dual roles of ER on Ca 2+ homeostasis, (1) by regulating STIM1 activities based on bulk ER Ca 2+ levels, and (2) by regulating STIM1 availabilities via EMCs-mediated local Ca 2+ dynamics. Our results further demonstrate dual functions of EMCs on Ca 2+ signaling, by enhancing mitochondrial Ca 2+ flickers locally while reducing Ca 2+ storage globally at the same time. Though more research is required, recent studies have demonstrated the importance of ER-to-mitochondrial Ca 2+ flow on mitochondrial functions 19 , 53 – 55 . Thus, Ca 2+ flickers from single mitochondria are expected to achieve temporal and spatial control of mitochondrial functions. On top of that, EMCs-mediated SOCE reduction will protect mitochondria from Ca 2+ overload by spatial restriction of STIM1 to reduce SOCE activation. This coordination between mitochondrial Ca 2+ flickers and global SOCE through EMCs represents an elegant mechanism that links structural organization to cellular functions. Finally, our work disclosed interactions between EMCs and microtubules regarding Ca 2+ regulation. Previous studies reported that microtubules interacted with STIM1 through EB1 binding, restricting STIM1 translocation to ER-plasma membrane junctions 35 . Results from our experiments also supported that microtubules competed with EMCs for STIM1 binding and SOCE suppression when Ca 2+ was depleted. Moreover, the suppressive effect of EB1 over-expression on SOCE was abolished by EMCs elimination, indicating complex interactions between ER, mitochondria and microtubules for Ca 2+ regulation. More studies will be required to untangle those interactions. In conclusion, we observed spontaneous mitochondrial Ca 2+ flickering, which was generated by ER-to-mitochondria Ca 2+ flow at EMCs. This Ca 2+ flow further depleted local ER Ca 2+ to restrict STIM1 at EMCs, resulting in suppression of SOCE and decrease of intracellular Ca 2+ storage. In addition, microtubules interacted with EMCs to prevent Ca 2+ overload under EMCs elimination. Hence, ER, mitochondria and microtubules constitute a self-sufficient system regulating Ca 2+ homeostasis through SOCE. RESOURCE AVAILABILITY Lead contact Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Feng-Chiao Tsai ( fengchiaotsai{at}ntu.edu.tw ). MATERIALS AVAILABILITY All unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Data and code availability Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request. AUTHOR CONTRIBUTIONS Yu-Chiao Lin. designed and performed experiments, analyzed data, and wrote the original draft. Feng-Chiao Tsai, analyzed data and wrote the original draft. DECLARATION OF INTERESTS The authors declare no competing interests. Supplemental information Video S1. Spontaneous mitochondrial Ca 2+ flickers, related to Figure 1A . Video S2. Mitochondrial [Ca 2+ ] along with mitochondrial-targeted GFP, related to Figure S1A. Video S3. Rapamycin induced ER-mitochondrial contacts, related to Figure 1K . Download figure Open in new tab Figure S1. Spontaneous Ca 2+ flickers within individual mitochondria that regulated by EMCs, related to figure 1. (A) Representative images of mitochondrial-[Ca 2+ ] along with mitochondrial- targeted GFP. (B) Heatmap visualization of mitochondrial Ca 2+ dynamics in cells pretreated with mitoPQ for 1 hours to induce superoxide accumulation. (C) Characterization of mitochondrial Ca 2+ dynamics from (B). Analysis of individual mitochondrial Ca 2+ flickers showing the flicker frequency (events per mitochondrion; top), peak amplitude (per flicker, middle), and flicker duration (seconds; bottom), with median values indicated by red vertical lines. (D) Alignment of mitochondrial Ca 2+ flickers showing reduced amplitude in shMFN2 and shPDZD8 cells. (E) Heatmap visualization of aligned Ca 2+ flickers showing unchanged duration of Ca 2+ flickers. (F) Histogram of mitochondrial [Ca 2+ ] demonstrates right shift of flickers signal in shMFN2 and shPDZD8 cells. (G) Quantification of the mitochondrial [Ca 2+ ] showing reduced mitochondrial Ca 2+ flickers in shMFN2 and shPDZD8 cells. Download figure Open in new tab Figure S2. Store-operated Ca 2+ entry reduced in EMCs deficient cells, related to figure 2. (A) Measurement of ER-[Ca 2+ ] using GCaMPer, indicating upregulation of ER- [Ca2+] in MFN2-knocked down cells. (B) Measurement of ER-[Ca 2+ ] using T1ER showing increased ER-[Ca 2+ ] in shMFN2 and shPDZD8 cells. (C) Knocking down STIM1 and ORAI1 (shS1 + shO1) eliminated the upregulating ER-[Ca 2+ ] in shMFN2 cells. (D) Fura-2 assay showing the Ca 2+ entry after ER Ca 2+ leakage. (E) Knocking down IP3R (shITPR) and VDAC1 upregulated SOCE. (F) Live-cell imaging of TG-induced STIM1 activation. Scale bar, 10µm(left), 1µm (right). (G) Quantification of time-lapse STIM1 puncta signal after TG treatmen. (H) Representative images showing colocalization of STIM1 and ORAI1. (I) Knocking down MFN2 and PDZD8 increased the colocalization of STIM1 and ORAI1. (J) Inducing FRB-YFP and FKBP-Turquoise oligomerization (left) did not significantly affect SOCE (right), as the control experiment for Figure 2J . (K) SOCE assay in cells with induced oligomerization between FKBP-Turquoise and FRB-ER-YFP (left), OMM-FKBP-Turquoise, and FRB-ER-YFP (right), related to Figure 2K . Download figure Open in new tab Figure S3. Analysis of STIM1/ORAI1 expression and SOCE regulation by various inhibitors, related to Figure 2. (A-B) qPCR analysis showing relative expression levels of STIM1 (A) and ORAI1 (B) in control and knockdown cells. (C-D) Western blot analysis showing protein levels of STIM1, ORAI1 in control and EMCs deficient cells. (E-G) SOCE activity in cells pretreated with vehicle control (DMSO, E), oligomycin A (olgA, F) and FCCP (G), related to Figure 2La. (H-J) SOCE activity in cells knocked down with siCTL (H), siSTIMATE (I) and siSep4 & 7 (J), related to Figure 2Lb. (K-M) SOCE peak in cells pretreated with DS16570511 (DS, K), MCUi4 (L) and ruthenium red (RuR, M), related to Figure 2Lc . (N-O) ER-[Ca 2+ ] upregulated in EMCs deficient cells pretreated with MCU inhibitors DS16570511 (DS, N) and MCUi4 (O). Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001). Download figure Open in new tab Figure S4. Dynamic analysis of induced ER-mitochondrial contacts and microtubule manipulation, related to Figure 3-4. (A-C) Kymograph analysis showing the spatiotemporal dynamics of FRB-ER- YFP (A), OMM-FKBP-Turquoise (B) and FRET signal (C). (D) Time-lapse imaging showing microtubule disruption after rapamycin-induced contact between EMTB-CFP-FRB and dSpastin3Q-FKBP-YFP. (E) Representative images showing microtubule patterns in DMSO- and rapamycin-treated cells. 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Share Mitochondrial Ca 2+ flickers on endoplasmic reticulum (ER)-mitochondrial contact sites to suppress store-operated Ca 2+ entry Yu-Chiao Lin , Feng-Chiao Tsai bioRxiv 2025.01.17.633482; doi: https://doi.org/10.1101/2025.01.17.633482 Share This Article: Copy Citation Tools Mitochondrial Ca 2+ flickers on endoplasmic reticulum (ER)-mitochondrial contact sites to suppress store-operated Ca 2+ entry Yu-Chiao Lin , Feng-Chiao Tsai bioRxiv 2025.01.17.633482; doi: https://doi.org/10.1101/2025.01.17.633482 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7626) Biochemistry (17654) Bioengineering (13878) Bioinformatics (41895) Biophysics (21430) Cancer Biology (18569) Cell Biology (25471) Clinical Trials (138) Developmental Biology (13366) Ecology (19876) Epidemiology (2067) Evolutionary Biology (24294) Genetics (15593) Genomics (22480) Immunology (17719) Microbiology (40331) Molecular Biology (17155) Neuroscience (88500) Paleontology (666) Pathology (2829) Pharmacology and Toxicology (4818) Physiology (7635) Plant Biology (15116) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9817) Zoology (2269)