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. 2008 Feb 12;18(3):177-82.
doi: 10.1016/j.cub.2007.12.050. Epub 2008 Jan 31.

STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER

Ilya Grigoriev  1 , Susana Montenegro Gouveia, Babet van der Vaart, Jeroen Demmers, Jeremy T Smyth, Srinivas Honnappa, Daniël Splinter, Michel O Steinmetz, James W Putney Jr, Casper C Hoogenraad, Anna Akhmanova
Affiliations

STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER

Ilya Grigoriev et al. Curr Biol. .

Abstract

Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.

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Figures

Figure 1
Figure 1. STIM1 interacts with EB1
A,B,F. GST pull down assays with the indicated GST fusions; extracts of HEK293 cells overexpressing GFP-STIM1, GFP-STIM1-C3 mutant or GFP alone were used in A and F, and the purified full length EB1 protein in B. Coomassie-stained gels are shown for GST fusions; other proteins were detected by Western blotting with antibodies against GFP (A, F) or EB1 (B). C, D. HeLa cells were transfected with GFP-STIM1 or GFP-STIM1-C3 mutant, fixed and stained for the endogenous EB1. The insets show enlargements of the boxed areas. In the overlay GFP-STIM1 is shown in green and EB1 in red. Bars, 10 μm. E. Immunoprecipitation from extracts of HeLa cells with the rabbit polyclonal antibody against EB1 or a control rabbit serum. The lane marked “extr.” shows 5% of the input. Dynactin subunit p150Glued, a known EB1 partner, was used as a positive control, and GM130, a protein associated with the cytoplasmic side of the Golgi, as a negative control. G. Mapping of the minimal MT plus end binding domain of STIM1 by colocalization with EB1 in fixed HeLa cells. A scheme of STIM1 protein structure and the deletion mutants is shown; S, signal peptide; EF, EF hand; SAM, sterile α motif domain; TM, transmembrane domain; ERM, ezrin-radixin-moesin domain; CC, coiled coil; S/P, serine-proline rich domain; KK, lysine-rich domain.
Figure 2
Figure 2. GFP-STIM1 colocalizes with ER and MT plus ends in live cells
A. Simultaneous imaging of GFP-STIM1 (green in overlay) and DsRed2-ER (red in overlay) in a transiently transfected MRC5-SV cell. B,C. Simultaneous imaging of GFP-STIM1 (green in overlay) and EB3-mRFP (red in overlay) in a transiently transfected MRC5-SV cell; a single frame is shown in B; successive frames from Video 4 are shown in C (time is indicated above the panels). GFP-STIM1 comets are indicated by green arrows and EB3-mRFP comets are highlighted by red arrows. D. Simultaneous imaging of GFP-STIM1 (green in overlay) and mCherry-α-tubulin (red in overlay) in a transiently transfected MRC5-SV cell. Successive frames are shown; time is indicated above the panels. Tips of extending/retracting ER tubules and MTs are indicated by green and red arrows, respectively. Bars, 3 μm.
Figure 3
Figure 3. Analysis of GFP-STIM1 dynamics in control cells and after Ca2+ store depletion
A. FRAP analysis of GFP-STIM1 behavior. Each panel, with the exception of the panel marked “FRAP” (which shows a single frame), represents superimposition of 5 successive frames with a 1s interval. Note that recovery of diffuse ER signal in the bleached area precedes the appearance of GFP-STIM1 comets (indicated by red arrows). B. The average GFP-STIM1 intensity ratio of two regions inside and outside of the photobleached area in HeLa cells (measured as described in [14]). Left: control cells, n = 20; cells after addition of 2 μM thapsigargin (TG), n = 13 cells; middle: control cells, n = 7; 2 μm thapsigargin n = 12 cells; right: GFP-STIM, n = 20 cells; EB3-mRFP, n = 7 cells. C. Representative frames of simultaneous two-color video of an MRC5-SV cell expressing GFP-STIM1 and EB3-RFP before and 120s after the addition of 2 μM thapsigargin in normal culture medium. Kymographs illustrating the changes of fluorescent intensity over time in the indicated boxed areas are shown on the right. In kymographs motile comets appear as slopes and immobile structures as vertical lines. Bars, 5 μm.
Figure 4
Figure 4. EB1 and STIM1 are required for TAC-mediated ER extension
A. Western blot analysis of extracts of HeLa cells 3 days after transfection with the indicated siRNAs. B. HeLa cells were transfected with the siRNA EB1#1; two days later the cells were transfected with GFP-STIM1, cultured for one more day, fixed and stained for EB1. Bar, 5 μm. C. HeLa cells transfected with GFP-STIM1 were treated with taxol, fixed and stained for EB1. D, E. HeLa cells were transfected with the indicated siRNAs; one day later cells were transfected with plasmid DNA, cultured for two more days and used for dual color imaging. The following combinations of fluorescent markers were used: mCherry-α-tubulin (stably expressed in HeLa cells) together with transiently expressed YFP-ER; transiently expressed EB3-mRFP and YFP-ER, transiently expressed EB3-mRFP and GFP-STIM1; mCherry-α-tubulin (stably expressed in HeLa cells) together with transiently expressed GFP-STIM1. D. Number of TACs, determined as the events of colocalization of ER tubule protrusion (detected with YFP-ER or GFP-STIM1) with EB3 comets or with growing MT plus ends. E. Number of sliding events, determined as the events of ER protrusion (detected with YFP-ER or GFP-STIM1), which did not colocalize with EB3 comets or with growing MT plus ends. Number of analysed cells: ER-MT: control, n = 20; EB1 #1, n = 20, EB1 #2, n = 15. ER-EB3: control, n = 20; STIM1 #1, n = 20, STIM1 #2, n = 20. STIM1-EB3: n = 20. STIM1-MT: control, n=10; EB1#1, n = 15, EB1 #2, n = 15. Values obtained in EB1 or STIM1 siRNA-treated cells that were significantly different from the corresponding values in cells treated with the control siRNAs are indicated by asterisks (p<0.001, ***; p<0.01, **; p<0.05, *; p>0.05, n.s., Kolmogorov-Smirnov test).
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