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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

Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203  mice.Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203  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  testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl2.
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pone.0127480.g007: Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 mice.Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 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 testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl2.

Mentions: Single cell suspensions of testes from WT and Tmem203 mice were prepared and ratiometric calcium measurements in equivalent gated populations consisting chiefly of round spermatids [36] were performed using flow cytometry. Calcium flux was measured in response to release of intracellular calcium stores before (Fig 7A and 7B) and after addition of extracellular calcium (Fig 7C and 7D). The Tmem203 cells exhibited modestly lower basal cytoplasmic calcium levels at rest. The rises in intracellular calcium immediately after store depletion were modestly lower than WT mice. Tmem203 cells exhibited a reduction of cytosolic calcium after store depletion by TG in contrast to WT cells which showed a continuous rise in cytoplasmic calcium. Tmem203 cells showed a more pronounced reduction of cytosolic calcium after the peak levels induced by ionomycin (Fig 7B). After addition of calcium to the media after store depletion by either TG or Ionomycin, Tmem203 cells exhibited significantly reduced cytoplasmic calcium accumulation compared to WT cells (Fig 7C and 7D). These data are consistent with both reduced calcium uptake and increased cellular extrusion predicted by the expression profiling data which showed reduced expression of calcium import channels and increased expression of the predominant cellular calcium pump. These data suggest that the defective spermiogenesis in Tmem203 deficient mice is due to alterations in calcium homeostasis.


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)

Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203  mice.Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203  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  testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440627&req=5

pone.0127480.g007: Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 mice.Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 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 testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl2.
Mentions: Single cell suspensions of testes from WT and Tmem203 mice were prepared and ratiometric calcium measurements in equivalent gated populations consisting chiefly of round spermatids [36] were performed using flow cytometry. Calcium flux was measured in response to release of intracellular calcium stores before (Fig 7A and 7B) and after addition of extracellular calcium (Fig 7C and 7D). The Tmem203 cells exhibited modestly lower basal cytoplasmic calcium levels at rest. The rises in intracellular calcium immediately after store depletion were modestly lower than WT mice. Tmem203 cells exhibited a reduction of cytosolic calcium after store depletion by TG in contrast to WT cells which showed a continuous rise in cytoplasmic calcium. Tmem203 cells showed a more pronounced reduction of cytosolic calcium after the peak levels induced by ionomycin (Fig 7B). After addition of calcium to the media after store depletion by either TG or Ionomycin, Tmem203 cells exhibited significantly reduced cytoplasmic calcium accumulation compared to WT cells (Fig 7C and 7D). These data are consistent with both reduced calcium uptake and increased cellular extrusion predicted by the expression profiling data which showed reduced expression of calcium import channels and increased expression of the predominant cellular calcium pump. These data suggest that the defective spermiogenesis in Tmem203 deficient mice is due to alterations in calcium homeostasis.

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