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Phosphoregulation of STIM1 Leads to Exclusion of the Endoplasmic Reticulum from the Mitotic Spindle
Current Biology 22, 1487–1493, August 21, 2012 ª2012 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2012.05.057
Report Phosphoregulation of STIM1 Leads to Exclusion of the Endoplasmic Reticulum from the Mitotic Spindle Jeremy T. Smyth,1 Amber M. Beg,1 Shilan Wu,2 James W. Putney, Jr.,2,* and Nasser M. Rusan1,* 1Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA 2Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
Summary The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown [1]. Stromal interaction molecule 1 (STIM1) is an ER Ca2+ sensor that activates store-operated Ca2+ entry (SOCE) [2, 3] and also functions in ER morphogenesis through its interaction with the microtubule +TIP protein end binding 1 (EB1) [4]. We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE [5]. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM110A) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM110A, demonstrating that aberrant association of STIM110A with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired +TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca2+ signaling during mitosis require multimodal STIM1 regulation by phosphorylation. Results and Discussion Phosphorylation Dissociates STIM1 from EB1 during Mitosis During interphase, the endoplasmic reticulum (ER) is organized in interconnected tubules and sheets and associates extensively with microtubules (Figures 1A and 1A’) [1, 6], whereas the ER during mitosis consists predominantly of concentric sheets [7] and is largely excluded from the mitotic spindle in HeLa cells (Figures 1B and 1B’) [7, 8]. Mechanisms that underlie this remodeling during mitosis are unknown; however, exclusion from the spindle suggests that mechanisms that associate the ER with microtubules are negatively regulated. One mechanism of ER-microtubule association involves the ER Ca2+ sensor stromal interaction molecule 1 (STIM1). During interphase, STIM1 mediates ER tubule
*Correspondence: [email protected] (J.W.P.), [email protected] (N.M.R.)
extension through interaction with the microtubule +TIP protein end binding 1 (EB1), which decorates microtubules in short linear structures known as comets [9]. This interphase distribution can be seen using an enhanced yellow fluorescent protein-tagged STIM1 (STIM1WT) and EB1 staining (Figures 1C and 1C’). In mitosis, however, STIM1WT is largely absent from the EB1-dense mitotic spindle (Figure 1D), similar to the ER. We therefore hypothesized that ER exclusion from the mitotic spindle depends on inhibition of the interaction of STIM1 with EB1. In support of this, higher magnification of mitotic cells revealed no evidence of STIM1WT-EB1 colocalization (Figure 1D’), including at the spindle poles where a small accumulation of STIM1WT resides (Figure 1D’, arrow). Examination of subsequent stages of mitosis in fixed (see Figure S1A available online) and live cells (Figure 2F; Movie S1) revealed that dissociation of STIM1WT from EB1 persisted through cytokinesis. One mechanism for dissociation of +TIP proteins from EB1 is phosphorylation near the EB1 interaction site [10, 11]. We therefore hypothesized that the recently described phosphorylation of STIM1 during mitosis [5] inhibits the interaction of STIM1 with EB1. Phosphorylation of STIM1 during mitosis occurs at serine/threonine-proline sites that are recognized by the MPM-2 antibody [12, 13], and several are located near the EB1 interaction site (Figure 2A; Figure S1D). To inhibit mitosis-specific STIM1 phosphorylation, we mutated to alanine all ten of the serines or threonines within candidate MPM-2 recognition sites (STIM110A). STIM110A immunoprecipitated from mitotic HeLa extracts failed to exhibit the molecular weight shift seen with STIM1WT and was not recognized by MPM-2 (Figure 2A), consistent with loss of mitosis-specific phosphorylation. We next analyzed the effect of STIM1 phosphorylation on EB1 interaction using a GST-tagged EB1 (GSTEB1) pull-down assay [14]. Phosphorylated STIM1WT from mitotic cells interacted very weakly with GST-EB1 compared to STIM1WT from asynchronous cells, and this interaction was fully restored with nonphosphorylatable STIM110A from mitotic cells (Figure 2B). Thus, phosphorylation negatively regulates the STIM1-EB1 interaction. Accordingly, and in striking contrast to STIM1WT, STIM110A localized extensively to the spindle, both in interpolar and astral regions (Figure 2D). Higher magnification revealed STIM110A comets associated with EB1 comets (Figure 2D’), suggesting that spindle localization of STIM110A is due to EB1 association. Further, STIM110A showed +TIP tracking behavior during metaphase (Figures 2G and 2G’; Movie S2), again suggesting association with and transport by EB1. To determine whether the altered mitotic localization of STIM110A is specifically due to EB1 association, we inhibited EB1 association by mutating isoleucine 644 and proline 645 of the T-R-I-P EB1 interaction site of STIM110A to asparagines (STIM110A_TRNN) [11]. As expected, STIM110A_TRNN from mitotic cell extracts failed to interact with GST-EB1 (Figure 2B), and STIM110A_TRNN also failed to colocalize with EB1 in the mitotic spindle (Figure 2E). Quantitation revealed that greater than 60% of metaphase cells exhibited STIM110A localization in the spindle compared to less than 20% for STIM1WT or STIM110A_TRNN (Figure 2H), showing consistent mislocalization of STIM110A and rescue by STIM110A_TRNN. Examination of subsequent mitotic stages in
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Figure 1. The ER and STIM1 Are Excluded from the Mitotic Spindle (A and B) Interphase (A) or mitotic (B) HeLa cells expressing the ER marker GFP-Sec61b and mCherry-a-tubulin were imaged live by confocal microscopy. Note the similar distribution of ER and a-tubulin in the interphase cell, but the near-complete segregation of ER from spindle microtubules in the mitotic cell. Higher magnifications of the regions within the white boxes are shown in (A’) and (B’). Arrows in (A’) show ER tubules that are aligned along microtubules. (C) An interphase HeLa cell expressing STIM1WT was fixed and stained with anti-EB1 antibody and DAPI. Shown is a maximum projection of deconvolved confocal image stacks covering the entire volume of the cell. Higher magnification of the region within the white box is shown in (C’), and arrows denote STIM1 comets that are associated with EB1 comets. (D) A mitotic HeLa cell expressing STIM1WT was fixed and stained with anti-EB1 antibody and DAPI. Shown is a maximum projection of deconvolved confocal image stacks covering approximately 2.5 mm through one hemisphere of the cell. Note the absence of STIM1WT in the midzone region of the mitotic cell where EB1 is enriched (arrows). Higher magnification of the region within the white box is shown in (D’), and the arrow points to a small accumulation of STIM1WT at the spindle pole. All scale bars are in mm.
fixed (Figure S1A) and live cells (Figure 2F; Movie S1) revealed STIM110A association with the central spindle through abscission. STIM110A also localized to the mitotic spindle in metaphase HEK293 cells (Figure S1B), suggesting similar regulation of STIM1 during mitosis in several cell types. These data collectively indicate that phosphorylation is necessary to dissociate STIM1 from EB1 during mitosis. The ten putative phosphorylation sites mutated in STIM110A span nearly 200 amino acids, but only those closest to the EB1 interaction domain may actually regulate EB1 association. To begin to address this, we mutated to alanine serine 668, the site closest to the EB1 domain that is clearly phosphorylated during mitosis [5], and serine 628, the site closest to the EB1 domain with unknown phosphorylation status (STIM12A)
(Figure S1D). STIM12A partially localized to the mitotic spindle (Figure S1C), whereas mutation of either site alone did not cause spindle localization (data not shown). Therefore, S628 and S668 are likely both phosphorylated during mitosis and coordinately regulate EB1 interaction. However, STIM12A localization to the spindle was not as extensive as STIM110A (Figure S1C), suggesting that additional sites are likely phosphorylated and contribute to regulation of the EB1 interaction. ER Exclusion from the Mitotic Spindle Requires STIM1 Phosphorylation We next asked whether phosphorylation-dependent dissociation of STIM1 from EB1 is an important determinant of spindle exclusion of the ER. Consistent with previous studies [7, 8], the
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Figure 2. Phosphorylation Dissociates STIM1 from EB1 during Mitosis (A) STIM1WT or STIM110A were immunoprecipitated from lysates of asynchronous or mitotic-arrested HeLa cells and analyzed by western blot with anti-GFP antibody (upper blot) and the phosphospecific MPM-2 antibody (lower blot). Note the loss of molecular weight shift of mitotic STIM110A and lack of recognition of mitotic STIM110A by the MPM-2 antibody, both indicative of loss of mitosis-specific phosphorylation of STIM110A. (B–E) GST-EB1 or GST alone immobilized on beads was combined with lysates of asynchronous or mitotic-arrested HeLa cells expressing STIM1WT, STIM110A, or STIM110A-TRNN (labeled 10A_T in the figure) (B). Association of the expressed STIM1 proteins with the GST constructs was then analyzed by western blot with anti-GFP antibody (upper blot). This blot was reprobed with an anti-GST antibody to ensure equal amounts of GST-EB1 or GST beads in each sample (data not shown). The lower blot shows the total amount of each expressed STIM1 protein in the HeLa lysates based on anti-GFP antibody recognition (Input). HeLa cells expressing STIM1WT (C), STIM110A (D), or STIM110A_TRNN (E) were fixed and stained with anti-EB1 antibody and DAPI. Shown are maximum projections of deconvolved confocal image stacks covering approximately 2.5 mm through one hemisphere of the cell. Arrows point to the midzone regions in the STIM1 images; note the significant accumulation of STIM110A in a spindle-like organization in the midzone, whereas the midzone is nearly completely devoid of STIM1WT and STIM110A_TRNN. Magnifications of single focal planes of the regions within the white boxes in (D) are shown in (D’). Arrowheads denote STIM110A comets that are associated with EB1 comets. (F) HeLa cells expressing mCherry-histone 2B (H2B) in combination with STIM1WT (upper row) or STIM110A (lower row) were imaged live through the course of a single mitotic division, beginning in metaphase. Shown are single frames at metaphase, anaphase, and telophase from the full time lapse videos, which are shown in Movie S1. (G) A live metaphase HeLa cell was imaged every 2 s to demonstrate +TIP tracking behavior of STIM110A in the spindle. Stills of the single STIM110A comet in the yellow box are shown in (G’), and the position of the leading tip of the comet is denoted with arrows. The full time-lapse video is shown in Movie S2. (H) The percent of metaphase HeLa cells that exhibited STIM1WT or the indicated mutants localized to the spindle was calculated by blind microscopic observation of fixed cells similar to those shown in (C)–(E). Data are presented as mean 6 SEM from two independent experiments, with greater than 60 cells analyzed per condition; *p < 0.01, one-way ANOVA followed by Tukey-Kramer. All scale bars are in mm.
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Figure 3. Nonphosphorylatable STIM1 Drives ER Tubule Ingression into the Mitotic Spindle (A) HeLa cells expressing STIM1WT (upper row), STIM110A (middle row), or STIM110A_TRNN (lower row) were fixed and stained with antibodies to calreticulin and a-tubulin and DAPI. Shown are single focal planes from deconvolved confocal stacks. Arrows in the calreticulin images point to the location of the spindle based on a-tubulin staining; note the significant calreticulin staining in the spindle region of the STIM110A cell, whereas calreticulin staining is nearly completely absent in this region in the STIM1WT and STIM110A_TRNN cells. (B) A HeLa cell expressing STIM1WT and ER-targeted dsRed (ER-dsRed) was imaged live through the course of a single mitosis beginning in metaphase. Shown are single frames at metaphase, anaphase, and telophase from the full time lapse video, which is shown in Movie S3. The arrow in the telophase ER-dsRed image points to the midbody region inside the ingressing cytokinetic furrow. (C) Same as (B), but for a cell expressing STIM110A. Note the significant accumulation of ER-dsRed tubules in the midbody region (arrow) compared to (B). The full time-lapse video is shown in Movie S4. (D) The percent of metaphase HeLa cells expressing the indicated STIM1 constructs that exhibited ER tubules within the spindle was calculated by blind microscopic observation of fixed cells similar to those shown in (A). Data are presented as mean 6 SEM from two independent experiments, with greater than 50 cells analyzed per condition; *p < 0.01, one-way ANOVA followed by Tukey-Kramer. All scale bars are in mm.
ER was largely excluded from the spindle from metaphase through telophase in wild-type (WT) HeLa cells based on localization of the ER marker GFP-Sec61b (Figure S2A; also Figure 1B). Small clusters of ER often emanated from the spindle
poles, but this represents only a very small fraction of total ER [7]. STIM1WT expression did not alter mitotic ER organization based on localization of the ER marker calreticulin in fixed cells (Figure 3A) and ER-targeted dsRed in live cells (Figure 3B;
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Movie S3). Further, siRNA depletion of STIM1 had no discernable effect on mitotic ER localization (Figures S2B and S2B’). Thus, ER localization during mitosis, including the small clusters at spindle poles, is independent of STIM1, suggesting that phosphorylation of STIM1 in WT mitotic cells completely suppresses its function. In contrast, STIM110A-expressing cells exhibited extensive ingression of ER tubules into the metaphase spindle (Figures 3A and 3C; Movie S4). Spindle-localized ER tubules were seen in 64% of STIM110Aexpressing metaphase cells, compared to 18% for STIM1WT (Figure 3D). In live imaging of STIM110A-expressing cells, numerous ER tubules extended through the contractile ring during abscission, in contrast to the limited localization of ER inside the contractile ring with STIM1WT (Figures 3B and 3C, arrows). Thus, phosphorylation of STIM1 is necessary for exclusion of the ER from the mitotic spindle through cytokinesis. Furthermore, STIM110A_TRNN rescued spindle exclusion compared to STIM110A (Figures 3A and 3D), demonstrating that altered ER distribution caused by STIM110A is due to association with EB1. Significantly, STIM110A provides the first opportunity to examine whether ER mislocalization during mitosis affects cell division. One possibility is that substantial augmentation of spindle-localized ER tubules affects spindle organization or function. However, STIM110A did not alter spindle morphology (Figure 3A) or DNA congression to the metaphase plate (Figure 2F; Movie S1). Further, despite accumulation of ER tubules through the contractile ring in STIM110A-expressing cells, cytokinesis was not visibly impaired (Movies S3 and S4). Alterations to the timing of mitosis, if observed, would also suggest mitotic defects. However, there was no difference in the time of progression from metaphase through telophase in cells expressing STIM110A compared to STIM1WT (Figures 2F and 3B and 3C). To analyze the timing of mitosis more robustly, we calculated mitotic indices of asynchronous cells. Mitotic index reflects the average time spent in mitosis of a cell population. Consistent with the live imaging results, the mitotic index of STIM1WT-expressing HeLa cells was 5.74% 6 0.61% compared to 4.11% 6 0.49% for STIM110A, not a statistically significant difference (p > 0.05, Student’s t test). There was also no significant difference in the mitotic index of BJ cells, a nontransformed, primary human fibroblast cell line (0.91% 6 0.40% for STIM1WT versus 0.72% 6 0.14% for STIM110A; p > 0.05, Student’s t test). Thus, ER mislocalization driven by STIM110A did not cause obvious mitotic defects in the cell types tested. However, changes in ER localization may be detrimental in other physiological contexts or cell types. Notably, cell division in two-dimensional cell culture may not recapitulate the process as it occurs in three-dimensional tissues, particularly with respect to the structure and partitioning of cellular components. Further, cellular defects, such as chromosome instability or altered organelle inheritance, may accumulate over multiple divisions due to ER disorganization, with dire consequences that may not have been apparent here. These possibilities can be addressed by generating STIM110A-expressing transgenic animals. Phosphorylation Is the Primary Mechanism that Dissociates STIM1 from EB1 To determine whether phosphorylation is sufficient to dissociate STIM1 from EB1, we created a putative phosphomimetic STIM1 by mutating all ten MPM-2-directed serines and threonines to glutamates (STIM110E) [15]. We hypothesized that STIM110E would not exhibit +TIP tracking during interphase,
indicative of loss of EB1 interaction. Consistent with this, the robust +TIP tracking seen with STIM1WT was nearly completely absent with STIM110E; only a limited number of short-lived STIM110E comets were visible (Figure 4A; Movie S5). Loss of +TIP tracking of STIM110E, however, was not as complete as with STIM1TRNN, wherein the EB1 interaction site is mutated (Figure 4A; Movie S5). Therefore, factors in addition to phosphorylation, such as alterations to microtubule dynamics or cellular geometry, may also contribute to STIM1 dissociation from EB1 during mitosis to a limited degree. Alternatively, glutamates may not fully mimic phosphorylation [16], and this may also account for the remaining +TIP tracking of STIM110E. Nonetheless, the significant abrogation of +TIP tracking with STIM110E, in combination with the strong association of STIM110A with EB1 during mitosis, demonstrates that phosphorylation is the primary mechanism that dissociates STIM1 from EB1 during mitosis. Phosphorylation Differentially Regulates EB1 Interaction and SOCE Activation The effects of mitosis-specific phosphorylation on STIM1 function are not limited to EB1 dissociation, as phosphorylation has also been suggested, based on a STIM1 truncation mutant, to suppress SOCE activation by STIM1 during mitosis; the functional significance of this remains unknown [5, 17]. In agreement with this, we found that our nonphosphorylatable STIM110A fully supported SOCE in mitotic cells, whereas STIM1WT did not (Figure S3). This is likely not due to restoration of the EB1 interaction by STIM110A, because STIM1 activation of SOCE is independent of its EB1 interaction [9]. It is possible, therefore, that phosphorylation differentially regulates these two independent STIM1 functions. In support of this, we show that STIM110E is a poor phosphomimetic for inhibition of SOCE activation, because STIM110E activates SOCE responses in interphase cells that are not significantly different from those activated by STIM1WT (Figures 4B and 4C). Therefore, glutamates more effectively recapitulate the effects of phosphorylation on the STIM1-EB1 interaction than on SOCE activation, suggesting that the mechanisms by which phosphorylation regulates these two functions, such as conformational changes or altered oligomerization, likely differ. Future work will focus on delineating which phosphorylation sites regulate the STIM1-EB1 interaction and which regulate SOCE activation. Conclusions The association of the ER with microtubules is remodeled during mitosis, although the extent of ER exclusion from the mitotic spindle varies by cell type [7, 18–23]. Mechanisms that underlie this remodeling are unknown and accordingly, the functional significance is also unclear. Our finding that phosphorylation of STIM1 is an important determinant of ER reorganization during mitosis is therefore a significant advance, because it provides the first mechanistic insight into mitotic ER structural remodeling. Further, STIM110A, which results in ectopic localization of ER tubules in the mitotic spindle, offers a molecular tool with which to probe the functional consequences of ER mislocalization during mitosis. Future studies will use STIM110A to address the importance of proper mitotic ER partitioning and function for normal organismal development and physiology. It will also be important to understand how other associations of the ER with microtubules are modified during mitosis, because STIM1
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Figure 4. STIM1 Phosphomimetic Mutant Differentially Affects +TIP Tracking and SOCE
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(A) Interphase HeLa cells expressing STIM1WT, STIM110E, or STIM1TRNN were imaged every second for 30 s to capture +TIP movements of the expressed constructs. The upper row shows the first frame from each time series, and the lower row shows a maximum projection of all 30 frames from the full time lapse. Insets in the maximum projection images show higher magnifications of the regions inside the yellow boxes, and arrows denote two STIM110E tracks indicative of limited +TIP tracking. Note that the STIM110E images contain three separate cells. Full time-lapse videos are shown in Movie S5. (B) SOCE was measured by depleting ER Ca2+ stores with thapsigargin (2 mM) for 15 min in nominal extracellular Ca2+, followed by sequential additions of 0.3 mM and 1.0 mM extracellular Ca2+. Shown are data from interphase WT HeLa cells or cells coexpressing STIM1WT or STIM110E with Orai1. Each trace is the averaged response of all cells measured for each experiment. Note that the differences between the WT and coexpression traces represent the SOCE responses that are induced by the coexpressed proteins (see Supplemental Experimental Procedures for further explanation). (C) For experiments described in (B), the peak SOCE response of each cell to addition of 0.3 mM and 1.0 Ca2+ was calculated, and data are presented as mean 6 SEM. Data are from two independent experiments, with at least 25 cells measured per experiment; differences were not statistically significant (p > 0.05, Student’s t test). All scale bars are in mm.
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phosphorylation is likely only one of many mechanisms that regulate mitotic ER morphology. STIM1 now joins a growing list of +TIP proteins whose interactions with EB1 are regulated by phosphorylation [11, 24], suggesting a paradigm of functional remodeling of microtubule tip-associated complexes during specific cellular processes. Interestingly, +TIP tracking by the EB1-interactors CLASP2 and SLAIN2 is also inhibited during mitosis by multisite phosphorylation [24, 25]. Thus, remodeling of the EB1-associated complex of +TIPS may be necessary during mitosis, and the functional significance of this remodeling requires significant future investigation. Supplemental Information Supplemental Information includes three figures, Supplemental Experimental Procedures, and five movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2012.05.057. Acknowledgments This research was supported by the Intramural Research Program of the NIH, National Heart, Lung and Blood Institute, and National Institute of Environmental Health Sciences. We acknowledge Christian Combs in the NHLBI Light Microscopy Core Facility, Jeff Tucker and Agnes Janoshazi in the NIEHS Confocal Core Facility, and Leigh Samsel and Pradeep Dagur in the NHLBI Flow Cytometry Core Facility for assistance with experiments. We also thank Brian Galletta, Dorothy Lerit, Stephen Shears, and Carmen Williams for helpful comments on the manuscript.
Received: February 17, 2012 Revised: May 3, 2012 Accepted: May 31, 2012 Published online: June 28, 2012 References 1. Friedman, J.R., and Voeltz, G.K. (2011). The ER in 3D: a multifunctional dynamic membrane network. Trends Cell Biol. 21, 709–717. 2. Liou, J., Kim, M.L., Heo, W.D., Jones, J.T., Myers, J.W., Ferrell, J.E., Jr., Meyer, T., and Meyer, T. (2005). STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241. 3. Roos, J., DiGregorio, P.J., Yeromin, A.V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J.A., Wagner, S.L., Cahalan, M.D., et al. (2005). STIM1, an essential and conserved component of store-operated Ca2+ channel function. J. Cell Biol. 169, 435–445. 4. Smyth, J.T., Hwang, S.-Y., Tomita, T., DeHaven, W.I., Mercer, J.C., and Putney, J.W. (2010). Activation and regulation of store-operated calcium entry. J. Cell. Mol. Med. 14, 2337–2349. 5. Smyth, J.T., Petranka, J.G., Boyles, R.R., DeHaven, W.I., Fukushima, M., Johnson, K.L., Williams, J.G., and Putney, J.W., Jr. (2009). Phosphorylation of STIM1 underlies suppression of store-operated calcium entry during mitosis. Nat. Cell Biol. 11, 1465–1472. 6. Park, S.H., and Blackstone, C. (2010). Further assembly required: construction and dynamics of the endoplasmic reticulum network. EMBO Rep. 11, 515–521. 7. Lu, L., Ladinsky, M.S., and Kirchhausen, T. (2009). Cisternal organization of the endoplasmic reticulum during mitosis. Mol. Biol. Cell 20, 3471–3480. 8. Jesch, S.A., Mehta, A.J., Velliste, M., Murphy, R.F., and Linstedt, A.D. (2001). Mitotic Golgi is in a dynamic equilibrium between clustered and free vesicles independent of the ER. Traffic 2, 873–884.
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http://dx.doi.org/10.1016/j.cub.2012.05.057
Report Phosphoregulation of STIM1 Leads to Exclusion of the Endoplasmic Reticulum from the Mitotic Spindle Jeremy T. Smyth,1 Amber M. Beg,1 Shilan Wu,2 James W. Putney, Jr.,2,* and Nasser M. Rusan1,* 1Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA 2Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
Summary The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown [1]. Stromal interaction molecule 1 (STIM1) is an ER Ca2+ sensor that activates store-operated Ca2+ entry (SOCE) [2, 3] and also functions in ER morphogenesis through its interaction with the microtubule +TIP protein end binding 1 (EB1) [4]. We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE [5]. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM110A) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM110A, demonstrating that aberrant association of STIM110A with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired +TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca2+ signaling during mitosis require multimodal STIM1 regulation by phosphorylation. Results and Discussion Phosphorylation Dissociates STIM1 from EB1 during Mitosis During interphase, the endoplasmic reticulum (ER) is organized in interconnected tubules and sheets and associates extensively with microtubules (Figures 1A and 1A’) [1, 6], whereas the ER during mitosis consists predominantly of concentric sheets [7] and is largely excluded from the mitotic spindle in HeLa cells (Figures 1B and 1B’) [7, 8]. Mechanisms that underlie this remodeling during mitosis are unknown; however, exclusion from the spindle suggests that mechanisms that associate the ER with microtubules are negatively regulated. One mechanism of ER-microtubule association involves the ER Ca2+ sensor stromal interaction molecule 1 (STIM1). During interphase, STIM1 mediates ER tubule
*Correspondence: [email protected] (J.W.P.), [email protected] (N.M.R.)
extension through interaction with the microtubule +TIP protein end binding 1 (EB1), which decorates microtubules in short linear structures known as comets [9]. This interphase distribution can be seen using an enhanced yellow fluorescent protein-tagged STIM1 (STIM1WT) and EB1 staining (Figures 1C and 1C’). In mitosis, however, STIM1WT is largely absent from the EB1-dense mitotic spindle (Figure 1D), similar to the ER. We therefore hypothesized that ER exclusion from the mitotic spindle depends on inhibition of the interaction of STIM1 with EB1. In support of this, higher magnification of mitotic cells revealed no evidence of STIM1WT-EB1 colocalization (Figure 1D’), including at the spindle poles where a small accumulation of STIM1WT resides (Figure 1D’, arrow). Examination of subsequent stages of mitosis in fixed (see Figure S1A available online) and live cells (Figure 2F; Movie S1) revealed that dissociation of STIM1WT from EB1 persisted through cytokinesis. One mechanism for dissociation of +TIP proteins from EB1 is phosphorylation near the EB1 interaction site [10, 11]. We therefore hypothesized that the recently described phosphorylation of STIM1 during mitosis [5] inhibits the interaction of STIM1 with EB1. Phosphorylation of STIM1 during mitosis occurs at serine/threonine-proline sites that are recognized by the MPM-2 antibody [12, 13], and several are located near the EB1 interaction site (Figure 2A; Figure S1D). To inhibit mitosis-specific STIM1 phosphorylation, we mutated to alanine all ten of the serines or threonines within candidate MPM-2 recognition sites (STIM110A). STIM110A immunoprecipitated from mitotic HeLa extracts failed to exhibit the molecular weight shift seen with STIM1WT and was not recognized by MPM-2 (Figure 2A), consistent with loss of mitosis-specific phosphorylation. We next analyzed the effect of STIM1 phosphorylation on EB1 interaction using a GST-tagged EB1 (GSTEB1) pull-down assay [14]. Phosphorylated STIM1WT from mitotic cells interacted very weakly with GST-EB1 compared to STIM1WT from asynchronous cells, and this interaction was fully restored with nonphosphorylatable STIM110A from mitotic cells (Figure 2B). Thus, phosphorylation negatively regulates the STIM1-EB1 interaction. Accordingly, and in striking contrast to STIM1WT, STIM110A localized extensively to the spindle, both in interpolar and astral regions (Figure 2D). Higher magnification revealed STIM110A comets associated with EB1 comets (Figure 2D’), suggesting that spindle localization of STIM110A is due to EB1 association. Further, STIM110A showed +TIP tracking behavior during metaphase (Figures 2G and 2G’; Movie S2), again suggesting association with and transport by EB1. To determine whether the altered mitotic localization of STIM110A is specifically due to EB1 association, we inhibited EB1 association by mutating isoleucine 644 and proline 645 of the T-R-I-P EB1 interaction site of STIM110A to asparagines (STIM110A_TRNN) [11]. As expected, STIM110A_TRNN from mitotic cell extracts failed to interact with GST-EB1 (Figure 2B), and STIM110A_TRNN also failed to colocalize with EB1 in the mitotic spindle (Figure 2E). Quantitation revealed that greater than 60% of metaphase cells exhibited STIM110A localization in the spindle compared to less than 20% for STIM1WT or STIM110A_TRNN (Figure 2H), showing consistent mislocalization of STIM110A and rescue by STIM110A_TRNN. Examination of subsequent mitotic stages in
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Figure 1. The ER and STIM1 Are Excluded from the Mitotic Spindle (A and B) Interphase (A) or mitotic (B) HeLa cells expressing the ER marker GFP-Sec61b and mCherry-a-tubulin were imaged live by confocal microscopy. Note the similar distribution of ER and a-tubulin in the interphase cell, but the near-complete segregation of ER from spindle microtubules in the mitotic cell. Higher magnifications of the regions within the white boxes are shown in (A’) and (B’). Arrows in (A’) show ER tubules that are aligned along microtubules. (C) An interphase HeLa cell expressing STIM1WT was fixed and stained with anti-EB1 antibody and DAPI. Shown is a maximum projection of deconvolved confocal image stacks covering the entire volume of the cell. Higher magnification of the region within the white box is shown in (C’), and arrows denote STIM1 comets that are associated with EB1 comets. (D) A mitotic HeLa cell expressing STIM1WT was fixed and stained with anti-EB1 antibody and DAPI. Shown is a maximum projection of deconvolved confocal image stacks covering approximately 2.5 mm through one hemisphere of the cell. Note the absence of STIM1WT in the midzone region of the mitotic cell where EB1 is enriched (arrows). Higher magnification of the region within the white box is shown in (D’), and the arrow points to a small accumulation of STIM1WT at the spindle pole. All scale bars are in mm.
fixed (Figure S1A) and live cells (Figure 2F; Movie S1) revealed STIM110A association with the central spindle through abscission. STIM110A also localized to the mitotic spindle in metaphase HEK293 cells (Figure S1B), suggesting similar regulation of STIM1 during mitosis in several cell types. These data collectively indicate that phosphorylation is necessary to dissociate STIM1 from EB1 during mitosis. The ten putative phosphorylation sites mutated in STIM110A span nearly 200 amino acids, but only those closest to the EB1 interaction domain may actually regulate EB1 association. To begin to address this, we mutated to alanine serine 668, the site closest to the EB1 domain that is clearly phosphorylated during mitosis [5], and serine 628, the site closest to the EB1 domain with unknown phosphorylation status (STIM12A)
(Figure S1D). STIM12A partially localized to the mitotic spindle (Figure S1C), whereas mutation of either site alone did not cause spindle localization (data not shown). Therefore, S628 and S668 are likely both phosphorylated during mitosis and coordinately regulate EB1 interaction. However, STIM12A localization to the spindle was not as extensive as STIM110A (Figure S1C), suggesting that additional sites are likely phosphorylated and contribute to regulation of the EB1 interaction. ER Exclusion from the Mitotic Spindle Requires STIM1 Phosphorylation We next asked whether phosphorylation-dependent dissociation of STIM1 from EB1 is an important determinant of spindle exclusion of the ER. Consistent with previous studies [7, 8], the
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Figure 2. Phosphorylation Dissociates STIM1 from EB1 during Mitosis (A) STIM1WT or STIM110A were immunoprecipitated from lysates of asynchronous or mitotic-arrested HeLa cells and analyzed by western blot with anti-GFP antibody (upper blot) and the phosphospecific MPM-2 antibody (lower blot). Note the loss of molecular weight shift of mitotic STIM110A and lack of recognition of mitotic STIM110A by the MPM-2 antibody, both indicative of loss of mitosis-specific phosphorylation of STIM110A. (B–E) GST-EB1 or GST alone immobilized on beads was combined with lysates of asynchronous or mitotic-arrested HeLa cells expressing STIM1WT, STIM110A, or STIM110A-TRNN (labeled 10A_T in the figure) (B). Association of the expressed STIM1 proteins with the GST constructs was then analyzed by western blot with anti-GFP antibody (upper blot). This blot was reprobed with an anti-GST antibody to ensure equal amounts of GST-EB1 or GST beads in each sample (data not shown). The lower blot shows the total amount of each expressed STIM1 protein in the HeLa lysates based on anti-GFP antibody recognition (Input). HeLa cells expressing STIM1WT (C), STIM110A (D), or STIM110A_TRNN (E) were fixed and stained with anti-EB1 antibody and DAPI. Shown are maximum projections of deconvolved confocal image stacks covering approximately 2.5 mm through one hemisphere of the cell. Arrows point to the midzone regions in the STIM1 images; note the significant accumulation of STIM110A in a spindle-like organization in the midzone, whereas the midzone is nearly completely devoid of STIM1WT and STIM110A_TRNN. Magnifications of single focal planes of the regions within the white boxes in (D) are shown in (D’). Arrowheads denote STIM110A comets that are associated with EB1 comets. (F) HeLa cells expressing mCherry-histone 2B (H2B) in combination with STIM1WT (upper row) or STIM110A (lower row) were imaged live through the course of a single mitotic division, beginning in metaphase. Shown are single frames at metaphase, anaphase, and telophase from the full time lapse videos, which are shown in Movie S1. (G) A live metaphase HeLa cell was imaged every 2 s to demonstrate +TIP tracking behavior of STIM110A in the spindle. Stills of the single STIM110A comet in the yellow box are shown in (G’), and the position of the leading tip of the comet is denoted with arrows. The full time-lapse video is shown in Movie S2. (H) The percent of metaphase HeLa cells that exhibited STIM1WT or the indicated mutants localized to the spindle was calculated by blind microscopic observation of fixed cells similar to those shown in (C)–(E). Data are presented as mean 6 SEM from two independent experiments, with greater than 60 cells analyzed per condition; *p < 0.01, one-way ANOVA followed by Tukey-Kramer. All scale bars are in mm.
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Figure 3. Nonphosphorylatable STIM1 Drives ER Tubule Ingression into the Mitotic Spindle (A) HeLa cells expressing STIM1WT (upper row), STIM110A (middle row), or STIM110A_TRNN (lower row) were fixed and stained with antibodies to calreticulin and a-tubulin and DAPI. Shown are single focal planes from deconvolved confocal stacks. Arrows in the calreticulin images point to the location of the spindle based on a-tubulin staining; note the significant calreticulin staining in the spindle region of the STIM110A cell, whereas calreticulin staining is nearly completely absent in this region in the STIM1WT and STIM110A_TRNN cells. (B) A HeLa cell expressing STIM1WT and ER-targeted dsRed (ER-dsRed) was imaged live through the course of a single mitosis beginning in metaphase. Shown are single frames at metaphase, anaphase, and telophase from the full time lapse video, which is shown in Movie S3. The arrow in the telophase ER-dsRed image points to the midbody region inside the ingressing cytokinetic furrow. (C) Same as (B), but for a cell expressing STIM110A. Note the significant accumulation of ER-dsRed tubules in the midbody region (arrow) compared to (B). The full time-lapse video is shown in Movie S4. (D) The percent of metaphase HeLa cells expressing the indicated STIM1 constructs that exhibited ER tubules within the spindle was calculated by blind microscopic observation of fixed cells similar to those shown in (A). Data are presented as mean 6 SEM from two independent experiments, with greater than 50 cells analyzed per condition; *p < 0.01, one-way ANOVA followed by Tukey-Kramer. All scale bars are in mm.
ER was largely excluded from the spindle from metaphase through telophase in wild-type (WT) HeLa cells based on localization of the ER marker GFP-Sec61b (Figure S2A; also Figure 1B). Small clusters of ER often emanated from the spindle
poles, but this represents only a very small fraction of total ER [7]. STIM1WT expression did not alter mitotic ER organization based on localization of the ER marker calreticulin in fixed cells (Figure 3A) and ER-targeted dsRed in live cells (Figure 3B;
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Movie S3). Further, siRNA depletion of STIM1 had no discernable effect on mitotic ER localization (Figures S2B and S2B’). Thus, ER localization during mitosis, including the small clusters at spindle poles, is independent of STIM1, suggesting that phosphorylation of STIM1 in WT mitotic cells completely suppresses its function. In contrast, STIM110A-expressing cells exhibited extensive ingression of ER tubules into the metaphase spindle (Figures 3A and 3C; Movie S4). Spindle-localized ER tubules were seen in 64% of STIM110Aexpressing metaphase cells, compared to 18% for STIM1WT (Figure 3D). In live imaging of STIM110A-expressing cells, numerous ER tubules extended through the contractile ring during abscission, in contrast to the limited localization of ER inside the contractile ring with STIM1WT (Figures 3B and 3C, arrows). Thus, phosphorylation of STIM1 is necessary for exclusion of the ER from the mitotic spindle through cytokinesis. Furthermore, STIM110A_TRNN rescued spindle exclusion compared to STIM110A (Figures 3A and 3D), demonstrating that altered ER distribution caused by STIM110A is due to association with EB1. Significantly, STIM110A provides the first opportunity to examine whether ER mislocalization during mitosis affects cell division. One possibility is that substantial augmentation of spindle-localized ER tubules affects spindle organization or function. However, STIM110A did not alter spindle morphology (Figure 3A) or DNA congression to the metaphase plate (Figure 2F; Movie S1). Further, despite accumulation of ER tubules through the contractile ring in STIM110A-expressing cells, cytokinesis was not visibly impaired (Movies S3 and S4). Alterations to the timing of mitosis, if observed, would also suggest mitotic defects. However, there was no difference in the time of progression from metaphase through telophase in cells expressing STIM110A compared to STIM1WT (Figures 2F and 3B and 3C). To analyze the timing of mitosis more robustly, we calculated mitotic indices of asynchronous cells. Mitotic index reflects the average time spent in mitosis of a cell population. Consistent with the live imaging results, the mitotic index of STIM1WT-expressing HeLa cells was 5.74% 6 0.61% compared to 4.11% 6 0.49% for STIM110A, not a statistically significant difference (p > 0.05, Student’s t test). There was also no significant difference in the mitotic index of BJ cells, a nontransformed, primary human fibroblast cell line (0.91% 6 0.40% for STIM1WT versus 0.72% 6 0.14% for STIM110A; p > 0.05, Student’s t test). Thus, ER mislocalization driven by STIM110A did not cause obvious mitotic defects in the cell types tested. However, changes in ER localization may be detrimental in other physiological contexts or cell types. Notably, cell division in two-dimensional cell culture may not recapitulate the process as it occurs in three-dimensional tissues, particularly with respect to the structure and partitioning of cellular components. Further, cellular defects, such as chromosome instability or altered organelle inheritance, may accumulate over multiple divisions due to ER disorganization, with dire consequences that may not have been apparent here. These possibilities can be addressed by generating STIM110A-expressing transgenic animals. Phosphorylation Is the Primary Mechanism that Dissociates STIM1 from EB1 To determine whether phosphorylation is sufficient to dissociate STIM1 from EB1, we created a putative phosphomimetic STIM1 by mutating all ten MPM-2-directed serines and threonines to glutamates (STIM110E) [15]. We hypothesized that STIM110E would not exhibit +TIP tracking during interphase,
indicative of loss of EB1 interaction. Consistent with this, the robust +TIP tracking seen with STIM1WT was nearly completely absent with STIM110E; only a limited number of short-lived STIM110E comets were visible (Figure 4A; Movie S5). Loss of +TIP tracking of STIM110E, however, was not as complete as with STIM1TRNN, wherein the EB1 interaction site is mutated (Figure 4A; Movie S5). Therefore, factors in addition to phosphorylation, such as alterations to microtubule dynamics or cellular geometry, may also contribute to STIM1 dissociation from EB1 during mitosis to a limited degree. Alternatively, glutamates may not fully mimic phosphorylation [16], and this may also account for the remaining +TIP tracking of STIM110E. Nonetheless, the significant abrogation of +TIP tracking with STIM110E, in combination with the strong association of STIM110A with EB1 during mitosis, demonstrates that phosphorylation is the primary mechanism that dissociates STIM1 from EB1 during mitosis. Phosphorylation Differentially Regulates EB1 Interaction and SOCE Activation The effects of mitosis-specific phosphorylation on STIM1 function are not limited to EB1 dissociation, as phosphorylation has also been suggested, based on a STIM1 truncation mutant, to suppress SOCE activation by STIM1 during mitosis; the functional significance of this remains unknown [5, 17]. In agreement with this, we found that our nonphosphorylatable STIM110A fully supported SOCE in mitotic cells, whereas STIM1WT did not (Figure S3). This is likely not due to restoration of the EB1 interaction by STIM110A, because STIM1 activation of SOCE is independent of its EB1 interaction [9]. It is possible, therefore, that phosphorylation differentially regulates these two independent STIM1 functions. In support of this, we show that STIM110E is a poor phosphomimetic for inhibition of SOCE activation, because STIM110E activates SOCE responses in interphase cells that are not significantly different from those activated by STIM1WT (Figures 4B and 4C). Therefore, glutamates more effectively recapitulate the effects of phosphorylation on the STIM1-EB1 interaction than on SOCE activation, suggesting that the mechanisms by which phosphorylation regulates these two functions, such as conformational changes or altered oligomerization, likely differ. Future work will focus on delineating which phosphorylation sites regulate the STIM1-EB1 interaction and which regulate SOCE activation. Conclusions The association of the ER with microtubules is remodeled during mitosis, although the extent of ER exclusion from the mitotic spindle varies by cell type [7, 18–23]. Mechanisms that underlie this remodeling are unknown and accordingly, the functional significance is also unclear. Our finding that phosphorylation of STIM1 is an important determinant of ER reorganization during mitosis is therefore a significant advance, because it provides the first mechanistic insight into mitotic ER structural remodeling. Further, STIM110A, which results in ectopic localization of ER tubules in the mitotic spindle, offers a molecular tool with which to probe the functional consequences of ER mislocalization during mitosis. Future studies will use STIM110A to address the importance of proper mitotic ER partitioning and function for normal organismal development and physiology. It will also be important to understand how other associations of the ER with microtubules are modified during mitosis, because STIM1
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Figure 4. STIM1 Phosphomimetic Mutant Differentially Affects +TIP Tracking and SOCE
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(A) Interphase HeLa cells expressing STIM1WT, STIM110E, or STIM1TRNN were imaged every second for 30 s to capture +TIP movements of the expressed constructs. The upper row shows the first frame from each time series, and the lower row shows a maximum projection of all 30 frames from the full time lapse. Insets in the maximum projection images show higher magnifications of the regions inside the yellow boxes, and arrows denote two STIM110E tracks indicative of limited +TIP tracking. Note that the STIM110E images contain three separate cells. Full time-lapse videos are shown in Movie S5. (B) SOCE was measured by depleting ER Ca2+ stores with thapsigargin (2 mM) for 15 min in nominal extracellular Ca2+, followed by sequential additions of 0.3 mM and 1.0 mM extracellular Ca2+. Shown are data from interphase WT HeLa cells or cells coexpressing STIM1WT or STIM110E with Orai1. Each trace is the averaged response of all cells measured for each experiment. Note that the differences between the WT and coexpression traces represent the SOCE responses that are induced by the coexpressed proteins (see Supplemental Experimental Procedures for further explanation). (C) For experiments described in (B), the peak SOCE response of each cell to addition of 0.3 mM and 1.0 Ca2+ was calculated, and data are presented as mean 6 SEM. Data are from two independent experiments, with at least 25 cells measured per experiment; differences were not statistically significant (p > 0.05, Student’s t test). All scale bars are in mm.
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phosphorylation is likely only one of many mechanisms that regulate mitotic ER morphology. STIM1 now joins a growing list of +TIP proteins whose interactions with EB1 are regulated by phosphorylation [11, 24], suggesting a paradigm of functional remodeling of microtubule tip-associated complexes during specific cellular processes. Interestingly, +TIP tracking by the EB1-interactors CLASP2 and SLAIN2 is also inhibited during mitosis by multisite phosphorylation [24, 25]. Thus, remodeling of the EB1-associated complex of +TIPS may be necessary during mitosis, and the functional significance of this remodeling requires significant future investigation. Supplemental Information Supplemental Information includes three figures, Supplemental Experimental Procedures, and five movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2012.05.057. Acknowledgments This research was supported by the Intramural Research Program of the NIH, National Heart, Lung and Blood Institute, and National Institute of Environmental Health Sciences. We acknowledge Christian Combs in the NHLBI Light Microscopy Core Facility, Jeff Tucker and Agnes Janoshazi in the NIEHS Confocal Core Facility, and Leigh Samsel and Pradeep Dagur in the NHLBI Flow Cytometry Core Facility for assistance with experiments. We also thank Brian Galletta, Dorothy Lerit, Stephen Shears, and Carmen Williams for helpful comments on the manuscript.
Received: February 17, 2012 Revised: May 3, 2012 Accepted: May 31, 2012 Published online: June 28, 2012 References 1. Friedman, J.R., and Voeltz, G.K. (2011). The ER in 3D: a multifunctional dynamic membrane network. Trends Cell Biol. 21, 709–717. 2. Liou, J., Kim, M.L., Heo, W.D., Jones, J.T., Myers, J.W., Ferrell, J.E., Jr., Meyer, T., and Meyer, T. (2005). STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241. 3. Roos, J., DiGregorio, P.J., Yeromin, A.V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J.A., Wagner, S.L., Cahalan, M.D., et al. (2005). STIM1, an essential and conserved component of store-operated Ca2+ channel function. J. Cell Biol. 169, 435–445. 4. Smyth, J.T., Hwang, S.-Y., Tomita, T., DeHaven, W.I., Mercer, J.C., and Putney, J.W. (2010). Activation and regulation of store-operated calcium entry. J. Cell. Mol. Med. 14, 2337–2349. 5. Smyth, J.T., Petranka, J.G., Boyles, R.R., DeHaven, W.I., Fukushima, M., Johnson, K.L., Williams, J.G., and Putney, J.W., Jr. (2009). Phosphorylation of STIM1 underlies suppression of store-operated calcium entry during mitosis. Nat. Cell Biol. 11, 1465–1472. 6. Park, S.H., and Blackstone, C. (2010). Further assembly required: construction and dynamics of the endoplasmic reticulum network. EMBO Rep. 11, 515–521. 7. Lu, L., Ladinsky, M.S., and Kirchhausen, T. (2009). Cisternal organization of the endoplasmic reticulum during mitosis. Mol. Biol. Cell 20, 3471–3480. 8. Jesch, S.A., Mehta, A.J., Velliste, M., Murphy, R.F., and Linstedt, A.D. (2001). Mitotic Golgi is in a dynamic equilibrium between clustered and free vesicles independent of the ER. Traffic 2, 873–884.
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