Current Biology 22, 1487–1493, August 21, 2012 ª2012 Elsevier Ltd All rights reservedhttp://dx.doi.org/10.1016/j.cub.2012.05.057
Phosphoregulation of STIM1 Leads
to Exclusion of the Endoplasmic Reticulum
from the Mitotic Spindle
Jeremy T. Smyth,1Amber M. Beg,1Shilan 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
The endoplasmic reticulum (ER) undergoes significant reor-
ganization between interphase and mitosis, but the under-
lying mechanisms are unknown . 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) . We previ-
ously demonstrated that phosphorylation of STIM1 during
mitosis suppresses SOCE . 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. How-
ever, a nonphosphorylatable STIM1 mutant (STIM110A) colo-
pulling ER tubules into the spindle. This effect was rescued
by mutating the EB1 interaction site of STIM110A, demon-
strating that aberrant association of STIM110Awith EB1 is
responsible for the ER mislocalization. A STIM1 phosphomi-
metic exhibited significantly impaired +TIP tracking in inter-
phase but was ineffective at inhibiting SOCE, suggesting
different mechanisms of regulation of these two STIM1 func-
tions by phosphorylation. Thus, ER spindle exclusion and
ER-dependent Ca2+signaling during mitosis require multi-
modal STIM1 regulation by phosphorylation.
Results and Discussion
Phosphorylation Dissociates STIM1 from EB1 during
During interphase, the endoplasmic reticulum (ER) is orga-
nized 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  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 mecha-
nisms 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
extension through interaction with the microtubule +TIP
protein end binding 1 (EB1), which decorates microtubules in
short linear structures known as comets . 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, STIM1WTis 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 (Fig-
ure 1D’), including at the spindle poles where a small accumu-
lation of STIM1WTresides (Figure 1D’, arrow). Examination of
online) and live cells (Figure 2F; Movie S1) revealed that disso-
ciation of STIM1WTfrom 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 phosphor-
ylation of STIM1 during mitosis  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 ala-
nine all ten of the serines or threonines within candidate
MPM-2 recognition sites (STIM110A). STIM110Aimmunoprecip-
weight shift seen with STIM1WTand was not recognized by
MPM-2 (Figure 2A), consistent with loss of mitosis-specific
phosphorylation. We next analyzed the effect of STIM1 phos-
phorylation on EB1 interaction using a GST-tagged EB1 (GST-
EB1) pull-down assay . Phosphorylated STIM1WTfrom
mitotic cells interacted very weakly with GST-EB1 compared
to STIM1WTfrom asynchronous cells, and this interaction
was fully restored with nonphosphorylatable STIM110Afrom
mitotic cells (Figure 2B). Thus, phosphorylation negatively
regulates the STIM1-EB1 interaction. Accordingly, and in
striking contrast to STIM1WT, STIM110Alocalized extensively
to the spindle, both in interpolar and astral regions (Figure 2D).
Higher magnification revealed STIM110Acomets associated
withEB1 comets(Figure 2D’), suggesting thatspindle localiza-
tion of STIM110Ais 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
ciation, we inhibited EB1 association by mutating isoleucine
644 and proline 645 of the T-R-I-P EB1 interaction site of
STIM110Ato asparagines (STIM110A_TRNN) . As expected,
STIM110A_TRNNfrom mitotic cell extracts failed to interact
with GST-EB1 (Figure 2B), and STIM110A_TRNNalso failed to
colocalize withEB1in the mitoticspindle (Figure 2E).Quantita-
tion revealed that greater than 60% of metaphase cells ex-
hibited STIM110Alocalization in the spindle compared to less
than 20% for STIM1WTor STIM110A_TRNN(Figure 2H), showing
STIM110A_TRNN. Examination of subsequent mitotic stages in
*Correspondence: email@example.com (J.W.P.), firstname.lastname@example.org (N.M.R.)
fixed (Figure S1A) and live cells (Figure 2F; Movie S1) revealed
STIM110Aassociation with the central spindle through ab-
scission. STIM110Aalso localized to the mitotic spindle in
metaphase HEK293 cells (Figure S1B), suggesting similar re-
gulation 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 , and serine 628, the site closest to the EB1
domain with unknown phosphorylation status (STIM12A)
(Figure S1D). STIM12Apartially 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 phos-
phorylated and contribute to regulation of the EB1 interaction.
ER Exclusion from the Mitotic Spindle Requires STIM1
We next asked whether phosphorylation-dependent dissocia-
tion of STIM1 from EB1 is an important determinant of spindle
exclusionoftheER. Consistent withprevious studies[7,8],the
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 STIM1WTwas 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 STIM1WTwas fixed and stained with anti-EB1 antibody and DAPI. Shown is a maximum projection of deconvolved
confocal image stacks covering approximately 2.5 mmthrough one hemisphere of the cell. Note the absenceof STIM1WTinthe 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
STIM1WTat the spindle pole. All scale bars are in mm.
Current Biology Vol 22 No 16
Figure 2. Phosphorylation Dissociates STIM1 from EB1 during Mitosis
(A) STIM1WTorSTIM110Awere immunoprecipitated from lysates of asynchronous ormitotic-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 STIM110Aand lack of recog-
nition of mitotic STIM110Aby 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 STIM110Ain a spindle-like organization in the midzone, whereas the midzone
is nearly completely devoid of STIM1WTand STIM110A_TRNN. Magnifications of single focal planes of the regions within the white boxes in (D) are shown
in (D’). Arrowheads denote STIM110Acomets 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 STIM110Ain the spindle. Stills of the single STIM110Acomet 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 STIM1WTor 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.
STIM Phosphorylation Controls ER Spindle Exclusion
ER was largely excluded from the spindle from metaphase
through telophase in wild-type (WT) HeLa cells based on local-
ization of the ER marker GFP-Sec61b (Figure S2A; also Fig-
ure 1B). Small clusters of ER often emanated from the spindle
poles, but this represents only a very small fraction of total ER
. STIM1WTexpression did not alter mitotic ER organization
(Figure 3A) and ER-targeted dsRed in live cells (Figure 3B;
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 STIM110Acell, whereas calreticulin staining is nearly
completely absent in this region in the STIM1WTand STIM110A_TRNNcells.
(B) A HeLa cell expressing STIM1WTand 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.
Current Biology Vol 22 No 16
Movie S3). Further, siRNA depletion of STIM1 had no discern-
able effect on mitotic ER localization (Figures S2B and S2B’).
Thus, ER localization during mitosis, including the small clus-
ters 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 STIM110A-
expressing 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 cytoki-
nesis. Furthermore, STIM110A_TRNNrescued spindle exclusion
compared to STIM110A(Figures 3A and 3D), demonstrating
ciation with EB1.
Significantly, STIM110Aprovides 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, STIM110Adid not alter spindle mor-
phology (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-express-
ing 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 differ-
ence in the time of progression from metaphase through telo-
phase in cells expressing STIM110Acompared 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 reflectstheaverage timespent in mitosisof
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 statis-
tically 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 STIM1WTversus 0.72% 6 0.14% for
STIM110A; p > 0.05, Student’s t test). Thus, ER mislocalization
driven by STIM110Adid 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-dimen-
sional tissues, particularly with respect to the structure and
partitioning of cellular components. Further, cellular defects,
such as chromosome instability or altered organelle inheri-
ganization, with dire consequences that may not have been
apparent here. These possibilities canbe addressed by gener-
ating STIM110A-expressing transgenic animals.
Phosphorylation Is the Primary Mechanism that
Dissociates STIM1 from EB1
To determine whether phosphorylation is sufficient to disso-
ciate STIM1 from EB1, we created a putative phosphomimetic
STIM1 by mutating all ten MPM-2-directed serines and threo-
nines to glutamates (STIM110E) . We hypothesized that
STIM110Ewould not exhibit +TIP tracking during interphase,
indicative of loss of EB1 interaction. Consistent with this, the
absent with STIM110E; only a limited number of short-lived
STIM110Ecomets 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 phos-
phorylation, 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 , 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
STIM110Awith EB1 during mitosis, demonstrates that phos-
phorylation 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 phosphoryla-
tion 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
STIM110Afully supported SOCE in mitotic cells, whereas
of the EB1 interaction by STIM110A, because STIM1 activation
of SOCE is independent of its EB1 interaction . It is possible,
therefore, that phosphorylation differentially regulates these
two independent STIM1 functions. In support of this, we
show that STIM110Eis a poor phosphomimetic for inhibition
of SOCE activation, because STIM110Eactivates SOCE re-
sponses in interphase cells that are not significantly different
from those activated by STIM1WT(Figures 4B and 4C). There-
fore, glutamates more effectively recapitulate the effects of
phosphorylation on the STIM1-EB1 interaction than on SOCE
activation, suggesting that the mechanisms by which phos-
phorylation regulates these two functions, such as conforma-
tional changes or altered oligomerization, likely differ. Future
work will focus on delineating which phosphorylation sites
regulate the STIM1-EB1 interaction and which regulate
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,
spindle, offers a molecular tool with which to probe the func-
tional consequences of ER mislocalization during mitosis.
Future studies will use STIM110Ato address the importance
of proper mitotic ER partitioning and function for normal
organismal development and physiology. It will also be impor-
tant to understand how other associations of the ER with
microtubules are modified during mitosis, because STIM1
STIM Phosphorylation Controls ER Spindle Exclusion
phosphorylation is likely only one of many mechanisms that
regulate mitotic ER morphology.
STIM1 now joins a growing list of +TIP proteins whose inter-
actions with EB1 are regulated by phosphorylation [11, 24],
suggesting a paradigm of functional remodeling of microtu-
bule tip-associated complexes during specific cellular pro-
cesses. Interestingly, +TIP tracking by the EB1-interactors
CLASP2 and SLAIN2 is also inhibited during mitosis by multi-
site 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 includes three figures, Supplemental Experi-
mental Procedures,and five movies and can be found with this article online
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
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(A) Interphase HeLa cells expressing STIM1WT,
STIM110E, or STIM1TRNNwere imaged every
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