Fig 1.
TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.
(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30).
Fig 2.
TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores.
(A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl2 was added to record SOCE.
Fig 3.
Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells.
(A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) & n = 54 (TMEM203 siRNA)].
Table 1.
Tmem203 null male mice are sterile.
Fig 4.
Tmem203 null mice completely lack mature spermatozoa in epididymis.
(A) Computer assisted sperm analyzer based analysis of epididymis preparations from wild type and Tmem203 null mice showed complete absence (*) of mature spermatozoa in Tmem203 null mice. Data is representative of two independent experiments. (B) Representative photomicrographs illustrating hematoxylin and eosin (H&E)-stained sections of proximal epididymis (caput; upper left and right panels) and distal epididymis (cauda; lower left and right panels) from a 48-week-old wild type mouse (upper and lower left panels) and from a 48-week-old Tmem203 null mice (upper and lower right panels). Note the complete absence of mature spermatozoa in the epididymis of the Tmem203 null mice compared to the wild type mice in which numerous mature spermatozoa are observed; tubular lumina of the epididymis from Tmem203 null mice contains eosinophilic proteinaceous material mixed with cellular debris. Scale bars = 50 μm.
Fig 5.
Tmem203 null mice exhibit a disruption of spermiogenesis.
(A-B) Propidium iodide based DNA flow cytometry analysis of testicular cell suspensions from wild-type (red tracer) and Tmem203 null mice (green tracer) at 35 day (A) or 30 week (B) (n = 2 or 3 for each genotype). Arrows highlight the differences between the wild-type and Tmem203 null samples. Abbreviations: haploid-condensed (1n-C)-elongated spermatids; haploid (1n) round spermatids; diploid (2n)—Sertoli cells, spermatogonia; S-ph, spermatogonia synthesizing DNA and the tetraploid (4n)—pachytene spermatocytes and G2 spermatogonia (C) Representative photomicrographs illustrating hematoxylin and eosin (H&E) stained sections of Stage VII seminiferous tubules from a 48-week-old wild type mouse (left panels) and from a 48-week-old cMAC knockout mouse (right panels). Compared to the seminiferous tubule from the wild type mouse (left panels) the predominant morphological changes observed in the seminiferous tubule of the cMAC knockout mouse (right panels) are characterized by an overall subtle, relative reduction in numbers of late stage post-meiotic spermatids (steps 9–16), degenerative intracytoplasmic vacuolar changes most prominent in step 16 spermatids and complete lack of spermiation (disengagement of step 16 spermatozoa from the Sertoli cell and release into the tubular lumen). Lower panels illustrate higher magnification of areas enclosed by square boxes in the upper left and right panels. Scale bars = 50 μm (upper panels) and 25 μm (lower panels). (D-E) Representative transmission electron micrographs of Stage VII seminiferous tubules from a 32-week-old wild type mouse (left panels) and from a 32-week-old Tmem203 null mouse (right panels). Labeled are step 7 spermatocytes (7), residual bodies (rb), endoplasmic reticulum (er) and degenerate, misshapen spermatid heads (asterisks) mitochondrial sheath (ms) surrounding the outer dense fibers, axoneme and axoneme complex of microtubules. Phagocytosis by Sertoli cells of degenerate spermatids is illustrated in both the top and bottom panel on the right for the Tmem203 null mouse. Residual bodies (rb) contain dense aggregations of RNA, lipid, clear vesicles, multivesicular bodies and other organelles. Scale bars = 5.0 μm (for D),2.0 μm (for E).
Fig 6.
Gene expression profiling in Tmem203 null mouse testes indicates aberrant expression of key calcium channels and pumps.
(A) A spotfire based visualization of differential gene expression displaying fold changes in gene expression versus FDR corrected P value obtained from a microarray based RNA expression analysis from a set of five Tmem203 null and wild type mice. (B) Ingenuity based pathways enrichment of differentially expressed genes in Tmem203 null mouse testes. For the analysis the pathways showing a significant change with a p value of >2 were considered. (C) Quantitative real time PCR analysis of RNA obtained from WT and Tmem203 null mice testes for genes differentially expressed in the calcium signaling pathway selected from (B). Expression level of mentioned genes in Tmem203 null mice testes relative to WT testes expression level after normalization to Gapdh. Data represents 4 replicates (+/- Std Dev) and validated in 2 or more RNA preparations from Tmem203 null and WT testes. *—Transcript not detected in Tmem203 null mice testes.
Fig 7.
Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice.
Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl2.