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TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Shambharkar PB, Bittinger M, Latario B, Xiong Z, Bandyopadhyay S, Davis V, Lin V, Yang Y, Valdez R, Labow MA - PLoS ONE (2015)

Bottom Line: TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux.Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis.Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, 100 Technology Square, Cambridge, Massachusetts, United States of America.

ABSTRACT
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0127480.g002: 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.

Mentions: Cellular localization of a TMEM203-GFP fusion protein was examined by imaging with ER, mitochondria and plasma membrane (PM) markers. Based on the linescan co-lozalization tool TMEM203-GFP was predominately co-localized with the ER marker protein in HeLa cells (Fig 2A). Considering the localization of TMEM203 and TMEM203’s effect on cytoplasmic calcium levels, we asked if TMEM203 was associated with calcium modulatory proteins complexed within the ER [38–40]. Endogenous IP3R, SERCA2 and STIM1 proteins were co-precipitated with TMEM203-FLAG expressed in transfected HEK293 cells, whereas an ER resident membrane protein unrelated to the SOCE complex, INSIG-1 was not (Fig 2B). TMEM-203-FLAG protein was also detected after immunoprecipitation with antibody to endogenous STIM1 (S3 Fig). Thus ER expressed TMEM203 was associated with proteins critical for regulation of calcium influx and efflux into the ER as well STIM1, the calcium sensor for SOCE.


TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Shambharkar PB, Bittinger M, Latario B, Xiong Z, Bandyopadhyay S, Davis V, Lin V, Yang Y, Valdez R, Labow MA - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440627&req=5

pone.0127480.g002: 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.
Mentions: Cellular localization of a TMEM203-GFP fusion protein was examined by imaging with ER, mitochondria and plasma membrane (PM) markers. Based on the linescan co-lozalization tool TMEM203-GFP was predominately co-localized with the ER marker protein in HeLa cells (Fig 2A). Considering the localization of TMEM203 and TMEM203’s effect on cytoplasmic calcium levels, we asked if TMEM203 was associated with calcium modulatory proteins complexed within the ER [38–40]. Endogenous IP3R, SERCA2 and STIM1 proteins were co-precipitated with TMEM203-FLAG expressed in transfected HEK293 cells, whereas an ER resident membrane protein unrelated to the SOCE complex, INSIG-1 was not (Fig 2B). TMEM-203-FLAG protein was also detected after immunoprecipitation with antibody to endogenous STIM1 (S3 Fig). Thus ER expressed TMEM203 was associated with proteins critical for regulation of calcium influx and efflux into the ER as well STIM1, the calcium sensor for SOCE.

Bottom Line: TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux.Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis.Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, 100 Technology Square, Cambridge, Massachusetts, United States of America.

ABSTRACT
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

No MeSH data available.


Related in: MedlinePlus