1Medical Biochemistry and Molecular Biology, Saarland University, 2Experimental and Clinical Pharmacology and Toxicology, Saarland University
Lang, S., Schäuble, N., Cavalié, A., Zimmermann, R. Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms. J. Vis. Exp. (53), e2730, doi:10.3791/2730 (2011).
In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.
1. Preparation of stock solutions
2. Gene silencing in HeLa cells
In order to study the contribution of a certain protein to ER Ca2+ efflux, the respective gene has to be efficiently silenced with two different siRNAs (Fig. 1). In addition, the effect of silencing has to be overcome by expression of the respective wild type gene. Typically we use siRNAs that are directed against the coding and non-coding (UTR) region, respectively, of the gene of interest. Employing UTR-directed siRNA provides a convenient way for complementation.
3. Complementation of HeLa cells
In order to rescue the phenotype of SEC61A1 silencing, the SEC61A1 cDNA was inserted into the multiple cloning site (MCS) of a pCDNA3-internal ribosomal entry site (IRES)-GFP-vector that contained the cytomegalovirus (CMV) promoter, the MCS, the IRES, plus the green fluoresecent protein (GFP) coding sequence.
4. Live cell calcium imaging
5. Representative Results:
So far, we addressed the question of whether silencing of the SEC61A1 gene affects calcium (Ca2+) leakage from the ER (Fig. 1). The SEC61A1 gene was silenced by two different siRNAs in HeLa cells for 96 hours. While silencing hardly affected cell growth and viability, protein transport into the ER of semi-permeabilized cells was almost completely inhibited. Furthermore, the SEC61A1 silenced cells were severely affected with respect to Ca2+ leakage from the ER. The effect of gene silencing was reversed by expression of the SEC61A1 gene. Thus, Sec61 complexes that are present in the ER membrane of all nucleated cells form Ca2+ leak channels that can be expected to play a crucial role in Ca2+ homeostasis. However, the presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of Ca2+ from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM) binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. At the cellular level, two different CaM antagonists stimulated Ca2+ release from the ER in the presence but not in the absence of Sec61 channels (Figs 4 and 5). The latter was not observed when Sec61 channels were present that contained mutations in the IQ motif of Sec61α. Thus, Ca2+-CaM is involved in limiting Ca2+ leakage from the ER (Fig. 6).
Figure 1. Flow chart. See text for details.
Figure 2. Data analysis for silencing efficiency. Silencing was evaluated by Western-blot analysis using antibodies that were directed against Sec61α and β-actin (loading control). The primary antibodies were visualized by using ECL Plex secondary antibodies and fluorescence imaging.
Figure 3. Data analysis for transfection efficiency. Images were recorded on a fluorescence microscope equipped with cooled CCD camera. Transformation efficiency can be determined by dividing the number of cells displaying GFP fluorescence by cells counted in the brightfield mode.
Figure 4. Screen shots from live cell calcium imaging of HeLa cells in the presence of control- or SEC61A1-siRNA and the presence or absence of the CaM-antagonist ophiobolin A. HeLa cells were treated with siRNA directed against SEC61A1 (B) or a negative control siRNA (A) for 96 h as indicated. These cells were loaded with the calcium indicator FURA-2 AM and incubated in a Ca2+ free buffer containing 0.5 mM EGTA, then buffer or ophiobolin A (Ophio A) in buffer was added and the incubation resumed. After 10 min, Ca2+ release was initiated by applying thapsigargin (TG) in the absence of external Ca2+ and the incubation was continued. Screen shots from the continuous calcium imaging were taken at the indicated times.
Figure 5. Data analysis for live cell calcium imaging experiments. (A) Kinetic and quantitative analysis of a series of experiments as depicted in Fig. 4A. Cytoslic [Ca2+] was estimated from ratio measurements by an established calibration method2. The effect of thapsigargin is shown as bar diagram. (B-D) Cells were treated with control siRNA or the indicated SEC61A1-siRNA for 48 h and then transformed with either control vector (C), or SEC61A1 expression plasmid (C), or mutant SEC61A1 expression plasmid (D) as indicated. After 48 h, calcium imaging experiments were carried out as in Figs 4 and 5A. Statistical analysis of the changes in the cytosolic Ca2+ concentration after the addition of thapsigargin in experiments such as presented in A are shown. P values < 0.001 were defined as significant by unpaired t test and are indicated by three asterisks (***), n.s., not significant. The numbers of cells that were analyzed are indicated. Average values are given, error bars represent standard errors of the means (s.e.m.). We note that these examples were adapted from ref. 13.
Figure 6. These data indicate that the release of nascent chains from the Sec61 complex indeed leads to calcium release from the ER and the formation of a calcium nanodomain around the cytosolic mouth of the Sec61 complex. This calcium is bound by calmodulin and calcium-calmodulin closes the Sec61 complex.”
In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling. Here, we have described an experimental system that allows us to directly address the role of one potential Ca2+ leak channel and to characterize its putative regulatory mechanisms13. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.
We note that only viable cells should be analyzed and that overall viability should be above 80%. Therefore, cell viability is routinely evaluated employing Nuclear-ID blue/green cell viability reagent according to the manufacturer’s protocol. Furthermore, statistical analysis of the changes in the cytosolic Ca2+ concentration is mandatory. Therefore, the experiments have to be carried out for four different batches of cells and two coverslips with at least 20 cells have to be analyzed for each condition in a single experiment. We note that the various experiments have to be carried out at the same time after seeding on the cover slips.
No conflicts of interest declared.
S.L. was supported by a fellowship from the DFG (Graduate Research School 845). This work was supported by grants from the DFG (SFB 530/C1 & FOR 967) and by HOMFOR.
|Penicillin/ Streptomycin||PAA Laboratories||P11-010|
|Fugene HD||Roche Group||04709713|
|Nuclear-ID Blue/Green cell viability reagent||Enzo Life Sciences||ENZ-53004|
|Ophiobolin A||Enzo Life Sciences||ALX-270-109|
|Countess® Automated Cell Counter||Invitrogen|
|Typhoon-Trio imaging system||GE Healthcare|
|TE2000-S microscopewith DS-5Mc camera||Nikon Instruments|
|iMIC microscope with polychrome V||Till Photonics|