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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.

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

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Overexpression of SPC1-L2-C1 inhibits TGN Ca2+ influx. (A) HeLa cells were transfected with the TGN-specific Ca2+ FRET sensor Go-D1-cpv and a control plasmid, SPCA1-HA, or SPCA1-L2-C1-HA. Cells were fixed, stained with anti-HA and anti-TGN46 antibodies, and analyzed by fluorescence microscopy. Bars, 5 µm. (B) HeLa cells were transfected with Go-D1-cpv and a control plasmid, or SPCA1 siRNA and a control plasmid, and either SPCA1-L1-HA or SPCA1-L2-C1-HA, respectively. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23), SPCA1 siRNA (n = 9), SPCA1-L1-HA, or SPCA1-L2-C1-HA (n = 11). 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. Mean maximum values measured after readdition of Ca2+ were statistically different between control/SPCA1-L1-HA and SPCA-L2-C1-HA–transfected cells.
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fig6: Overexpression of SPC1-L2-C1 inhibits TGN Ca2+ influx. (A) HeLa cells were transfected with the TGN-specific Ca2+ FRET sensor Go-D1-cpv and a control plasmid, SPCA1-HA, or SPCA1-L2-C1-HA. Cells were fixed, stained with anti-HA and anti-TGN46 antibodies, and analyzed by fluorescence microscopy. Bars, 5 µm. (B) HeLa cells were transfected with Go-D1-cpv and a control plasmid, or SPCA1 siRNA and a control plasmid, and either SPCA1-L1-HA or SPCA1-L2-C1-HA, respectively. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23), SPCA1 siRNA (n = 9), SPCA1-L1-HA, or SPCA1-L2-C1-HA (n = 11). 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. Mean maximum values measured after readdition of Ca2+ were statistically different between control/SPCA1-L1-HA and SPCA-L2-C1-HA–transfected cells.

Mentions: The results of the sorting assays strongly suggested that the overexpression of SPCA1-L2-C1 interferes with SPCA1 function by capturing the necessary interaction partners required for activation and, thus, sorting. To test this hypothesis, we performed Ca2+ measurements with a TGN-targeted Ca2+ FRET sensor (Lissandron et al., 2010). TGN Ca2+ uptake solely relies on SPCA1, as demonstrated before (Lissandron et al., 2010). HeLa cells were transfected with a control plasmid, SPCA1 siRNA, SPCA1-L1-HA, or SPCA1-L2-C1-HA in combination with the Go-D1-cpv sensor to measure the TGN Ca2+ uptake of the TGN, as described previously (von Blume et al., 2011). The coexpression of Go-D1-cpv and SPCA1-L1-HA or SPCA1-L2-C1-HA was confirmed by immunofluorescence microscopy (Fig. 6 A).


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)

Overexpression of SPC1-L2-C1 inhibits TGN Ca2+ influx. (A) HeLa cells were transfected with the TGN-specific Ca2+ FRET sensor Go-D1-cpv and a control plasmid, SPCA1-HA, or SPCA1-L2-C1-HA. Cells were fixed, stained with anti-HA and anti-TGN46 antibodies, and analyzed by fluorescence microscopy. Bars, 5 µm. (B) HeLa cells were transfected with Go-D1-cpv and a control plasmid, or SPCA1 siRNA and a control plasmid, and either SPCA1-L1-HA or SPCA1-L2-C1-HA, respectively. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23), SPCA1 siRNA (n = 9), SPCA1-L1-HA, or SPCA1-L2-C1-HA (n = 11). 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. Mean maximum values measured after readdition of Ca2+ were statistically different between control/SPCA1-L1-HA and SPCA-L2-C1-HA–transfected cells.
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fig6: Overexpression of SPC1-L2-C1 inhibits TGN Ca2+ influx. (A) HeLa cells were transfected with the TGN-specific Ca2+ FRET sensor Go-D1-cpv and a control plasmid, SPCA1-HA, or SPCA1-L2-C1-HA. Cells were fixed, stained with anti-HA and anti-TGN46 antibodies, and analyzed by fluorescence microscopy. Bars, 5 µm. (B) HeLa cells were transfected with Go-D1-cpv and a control plasmid, or SPCA1 siRNA and a control plasmid, and either SPCA1-L1-HA or SPCA1-L2-C1-HA, respectively. Ca2+ entry into the TGN was measured in Ca2+-depleted cells transfected with control plasmid (n = 23), SPCA1 siRNA (n = 9), SPCA1-L1-HA, or SPCA1-L2-C1-HA (n = 11). 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. Mean maximum values measured after readdition of Ca2+ were statistically different between control/SPCA1-L1-HA and SPCA-L2-C1-HA–transfected cells.
Mentions: The results of the sorting assays strongly suggested that the overexpression of SPCA1-L2-C1 interferes with SPCA1 function by capturing the necessary interaction partners required for activation and, thus, sorting. To test this hypothesis, we performed Ca2+ measurements with a TGN-targeted Ca2+ FRET sensor (Lissandron et al., 2010). TGN Ca2+ uptake solely relies on SPCA1, as demonstrated before (Lissandron et al., 2010). HeLa cells were transfected with a control plasmid, SPCA1 siRNA, SPCA1-L1-HA, or SPCA1-L2-C1-HA in combination with the Go-D1-cpv sensor to measure the TGN Ca2+ uptake of the TGN, as described previously (von Blume et al., 2011). The coexpression of Go-D1-cpv and SPCA1-L1-HA or SPCA1-L2-C1-HA was confirmed by immunofluorescence microscopy (Fig. 6 A).

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