Figure 1.
Expression profiles of calcium transporters in lactation.
Mouse mammary tissue, starting from day 10 prior to parturition, was evaluated for the expression of (A) SPCA1, and (B) SPCA2 by Western blotting. Loading was normalized relative to tissue DNA concentrations, and expressed relative to starting levels at Day -10. C) RT-PCR of mRNA for isoforms of SPCA, Orai, PMCA and STIM proteins at distinct time points of mammary development, including pre-pregnancy (virgin), parturition (Day 0), lactation (Day 5) and involution (Day 5 after removal of pups). Transcripts are grouped as early, mid and unchanged, according to their time of induction following lactation on Day 0. D) SPCA2 was immunoprecipitated from lactating mouse mammary tissue using polyclonal rabbit anti-hSPCA2 peptide antibody or rabbit IgG as control. Orai1 was detected as a co-immunoprecipitate by immunoblotting (IB).
Figure 2.
Immunofluorescence microscopy of SPCA1, SPCA2, Orai1 and STIM1 in lactating mouse tissue.
Confocal microscopy imaging of sections treated as described under Experimental procedures. A) SPCA1 co-localizes with the Golgi marker, GM130. B) SPCA2 has a diffuse distribution, with little co-localization with Golgi marker, GM130. C) Orai1 (left panel) and STIM1 (right panel) show basolateral and reticular localization, respectively. Scale bar: 100 µm.
Figure 3.
Expression of SPCA2 and Orai1 in mammospheres.
A) Schema for induction of SCp2 cells into mammospheres. B) Cells in monolayer migrate and assemble into mammospheres following induction. C) Semi-quantitative RT-PCR showing that SPCA2, Orai1–3 and STIM1–2 are induced along with known markers β-casein and PMCA2 in the transition of SCp2 cell into differentiated mammospheres. D–F) Confocal sections of mammosphere showing indirect immunofluorescence of the indicated proteins; asterisks mark some areas of co-localization. Orthogonal sections are shown with the merged image. The cartoons indicate approximate location of optical plane through the mammosphere. D) Confocal section of a single layered mammosphere shows vesicular distribution of SPCA2 with some colocalization in the vicinity of E-cadherin as seen in the orthogonal view. E) Differentiated mammosphere showing Orai1 limited to the basal membrane with some SPCA2 punctae just below the plasma membrane, seen in orthogonal view. F) Top view of mammosphere shown in (E). Colocalized SPCA2 (green) and Orai1 (red) appear yellow. G) Secondary anti-mouse or anti-rabbit antibody alone shows no staining of mammosphere.
Figure 4.
Effect of SPCA2 and Orai1 knockdown on mammosphere formation and Ca2+ influx.
A) Western blot of SCp2 lysates derived from cells expressing shSc (scrambled shRNA), shSPCA2 and shOrai1 showing effectiveness of knockdown. B) RT-PCR of SPCA, Orai, SERCA2b and STIM1 isoforms in SCp2 cells following treatment with shRNA as indicated. Note that all three Orai isoforms were depleted in cells treated with shOrai1 virus. C) Comparison of small, aborted spheroids with mammospheres in SCp2 cells. D) Quantification of spheroids and mammosphere types in shRNA treated SCp2 cells ten days after lactogenic induction (* p≤0.05; ** p≤0.005). E) SOCE is drastically reduced in SCp2 monolayer cells treated with shSPCA2 and shOrai1 constructs (results averaged from n = 76 cells for shEV; n = 98 for shSPCA2; n = 123 for shOrai1). Fold change is the normalized change in fluorescence ratio (340/380 nm) of Fura-2. Inset: Fura2 fluorescence ratios showing baseline levels of free Ca2+ are lower in knockdown.
Figure 5.
Depletion of SPCA2 blocks cell surface trafficking of Orai1.
A–B) Immunofluorescence labeling of mSPCA2 shows less vesicular distribution of SPCA2 in cells treated with shOrai1. C-D) Immunofluorescence labeling of mOrai1 in SPCA2 knockdown cells shows retention to the perinuclear region only. SPCA2 and Orai1 signals from conjugated anti-rabbit and anti-mouse (AlexaFluor 288 and 388, respectively) secondary antibody were pseudocolored for ease of comparison.
Figure 6.
Ectopic expression of hSPCA2 constructs restores Ca2+ influx and Orai1 trafficking.
A) Immunofluorescence staining of mOrai1 shows trafficking defect in shSPCA2 cells (top panel). When the C-terminal end of hSPCA2, hSPCA2-C is ectopically expressed mOrai1 staining appears more vesicular (middle panel). Full-length hSPCA2 fully rescues mOrai1 trafficking, showing both plasma membrane and vesicular localization seen (bottom panel). B) SOCE is restored for shSPCA2 treated cells ectopically expressing either full-length hSPCA2 or C-terminal domain, hSPCA2C (results averaged from n = 111 cells for EV/GST; n = 63 for shSPCA2/CMV-EV; n = 67 for shSPCA2/CMV-hSPCA2C and n = 75 for shSPCA2/CMV-shSPCA2). Fold change is the normalized change in fluorescence ratio (340/380 nm) of Fura-2. Error bars reflect standard deviation from the mean for each measurement and time point using a Student’s t test.
Figure 7.
Store Independent Ca2+ Entry requires SPCA2 and Orai1.
A) SICE is virtually abolished in monolayer cells expressing shRNA constructs for SPCA2 and Orai1 constructs (results averaged from n = 91cells for shSc; n = 82 for shSPCA2; n = 92 for shOrai1). Error bars reflect standard deviation from the mean for each measurement at each time point. B) SICE is restored and elevated in shSPCA2 treated cells expressing either hSPCA2 or hSPCA2C (results averaged from n = 94 cells for EV/GST; n = 74 for shSPCA2/CMV-EV; n = 91 for shSPCA2/CMV-hSPCA2C and n = 104 for shSPCA2/CMV-shSPCA2). C) Fluorescence image of Fura-2AM loaded in monolayer cells, and mammospheres. (D) Store independent Ca2+ influx in monolayer cells and differentiated mammospheres. After a brief (∼20 s) incubation in nominally Ca2+ free extracellular medium, readdition of Ca2+ (2 mM) elicits Ca2+ entry that is transient in monolayer cells but elevated and sustained in mammospheres (n = 129 and n = 84 for monolayer and mammosphere cells, also respectively). E) Basal Ca2+ levels are similar in mammospheres, relative to monolayer cells as seen by Fura-2 fluorescence ratio. F) Store-dependent Ca2+ influx in monolayer cells and differentiated mammospheres. Addition of thapsigargin (Tg) empties the internal (ER) stores in nominally Ca2+-free medium. Upon readdition of extracellular Ca2+ (2 mM), Ca2+ influx is slightly higher in mammospheres (n = 89 and n = 67 for monolayer and mammosphere cells, respectively). Fold change is the normalized change in fluorescence ratio (340/380 nm) of Fura-2.