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Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

Kienzle C, Basnet N, Crevenna AH, Beck G, Habermann B, Mizuno N, von Blume J - J. Cell Biol. (2014)

Bottom Line: How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known.A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner.Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

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Expression of CFL-1-S3E inhibits Ca2+ entry into the TGN. (A) HeLa cells were transfected with ADF/CFL-1 siRNA and transfected with Go-D1-cpv and a control plasmid or HA-rCFL-S3E, then processed for immunofluorescence microscopy with anti-HA and anti-TGN46 antibodies. Bars, 5 µm. (B) HeLa cells were transfected with ADF/CFL-1 siRNA and, subsequently, with Go-D1-cpv and a control plasmid or HA-rCFL-S3E. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23) or rCFL-1-S3E (n = 14). Fluorescent signals reflecting TGN [Ca2+] were presented as ΔR/R0, where R0 is the value obtained before addition of 2.2 mM Ca2+ to the cell’s bathing solution. Data are expressed as the mean ± SEM (error bars). Mean maximum values measured after readdition of Ca2+ were statistically different between control plasmid/HA-rCFL-1-S3E–transfected cells.
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fig8: Expression of CFL-1-S3E inhibits Ca2+ entry into the TGN. (A) HeLa cells were transfected with ADF/CFL-1 siRNA and transfected with Go-D1-cpv and a control plasmid or HA-rCFL-S3E, then processed for immunofluorescence microscopy with anti-HA and anti-TGN46 antibodies. Bars, 5 µm. (B) HeLa cells were transfected with ADF/CFL-1 siRNA and, subsequently, with Go-D1-cpv and a control plasmid or HA-rCFL-S3E. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23) or rCFL-1-S3E (n = 14). Fluorescent signals reflecting TGN [Ca2+] were presented as ΔR/R0, where R0 is the value obtained before addition of 2.2 mM Ca2+ to the cell’s bathing solution. Data are expressed as the mean ± SEM (error bars). Mean maximum values measured after readdition of Ca2+ were statistically different between control plasmid/HA-rCFL-1-S3E–transfected cells.

Mentions: The next question that we aimed to address was whether the expression of the actin and SPCA1 binding–deficient mutant has an effect on TGN Ca2+ uptake. We have shown previously that the knockdown of ADF/CFL-1 significantly impairs TGN Ca2+ uptake. This phenotype could be rescued by the expression of siRNA-resistant rCFL-1 wt (von Blume et al., 2011). To test the effect of CFL-S3E on TGN Ca2+ uptake, HeLa cells were treated with ADF/CFL-1 siRNAs and transfected with either Go-D1-cpv and a control plasmid or HA-rCFL-1-S3E, and processed for immunofluorescence microscopy (Fig. 8 A). The TGN Ca2+ uptake was significantly reduced in ADF/CFL-1–depleted cells expressing HA-rCFL-1-S3E compared with control cells (Fig. 8 B). These data further demonstrate that the binding between SPCA1, actin, and CFL-1 is crucial for Ca2+ pumping at the TGN.


Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

Kienzle C, Basnet N, Crevenna AH, Beck G, Habermann B, Mizuno N, von Blume J - J. Cell Biol. (2014)

Expression of CFL-1-S3E inhibits Ca2+ entry into the TGN. (A) HeLa cells were transfected with ADF/CFL-1 siRNA and transfected with Go-D1-cpv and a control plasmid or HA-rCFL-S3E, then processed for immunofluorescence microscopy with anti-HA and anti-TGN46 antibodies. Bars, 5 µm. (B) HeLa cells were transfected with ADF/CFL-1 siRNA and, subsequently, with Go-D1-cpv and a control plasmid or HA-rCFL-S3E. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23) or rCFL-1-S3E (n = 14). Fluorescent signals reflecting TGN [Ca2+] were presented as ΔR/R0, where R0 is the value obtained before addition of 2.2 mM Ca2+ to the cell’s bathing solution. Data are expressed as the mean ± SEM (error bars). Mean maximum values measured after readdition of Ca2+ were statistically different between control plasmid/HA-rCFL-1-S3E–transfected cells.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig8: Expression of CFL-1-S3E inhibits Ca2+ entry into the TGN. (A) HeLa cells were transfected with ADF/CFL-1 siRNA and transfected with Go-D1-cpv and a control plasmid or HA-rCFL-S3E, then processed for immunofluorescence microscopy with anti-HA and anti-TGN46 antibodies. Bars, 5 µm. (B) HeLa cells were transfected with ADF/CFL-1 siRNA and, subsequently, with Go-D1-cpv and a control plasmid or HA-rCFL-S3E. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23) or rCFL-1-S3E (n = 14). Fluorescent signals reflecting TGN [Ca2+] were presented as ΔR/R0, where R0 is the value obtained before addition of 2.2 mM Ca2+ to the cell’s bathing solution. Data are expressed as the mean ± SEM (error bars). Mean maximum values measured after readdition of Ca2+ were statistically different between control plasmid/HA-rCFL-1-S3E–transfected cells.
Mentions: The next question that we aimed to address was whether the expression of the actin and SPCA1 binding–deficient mutant has an effect on TGN Ca2+ uptake. We have shown previously that the knockdown of ADF/CFL-1 significantly impairs TGN Ca2+ uptake. This phenotype could be rescued by the expression of siRNA-resistant rCFL-1 wt (von Blume et al., 2011). To test the effect of CFL-S3E on TGN Ca2+ uptake, HeLa cells were treated with ADF/CFL-1 siRNAs and transfected with either Go-D1-cpv and a control plasmid or HA-rCFL-1-S3E, and processed for immunofluorescence microscopy (Fig. 8 A). The TGN Ca2+ uptake was significantly reduced in ADF/CFL-1–depleted cells expressing HA-rCFL-1-S3E compared with control cells (Fig. 8 B). These data further demonstrate that the binding between SPCA1, actin, and CFL-1 is crucial for Ca2+ pumping at the TGN.

Bottom Line: How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known.A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner.Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

Show MeSH
Related in: MedlinePlus