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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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A SPCA1 point mutant impairs TGN Ca2+ uptake and secretory cargo sorting. (A–D) HeLa cells expressing SPCA1-wt and mutants were treated with control and SPCA1 siRNAs. Media and lysates from these cells were Western blotted with Cathepsin D (A) and Cab45 (C) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (B) and Cab45 (D) normalized to internal Cathepsin D and Cab45 values, respectively. (E) HeLa cells expressing either siRNA-resistant SPCA1-wt-HA or -mut4-HA were transfected with SPCA1 siRNA and subsequently with Go-D1-cpv. Ca2+ entry into the TGN was analyzed in wt (n = 8) or mut4 (n = 15)-expressing cells. 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.
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fig10: A SPCA1 point mutant impairs TGN Ca2+ uptake and secretory cargo sorting. (A–D) HeLa cells expressing SPCA1-wt and mutants were treated with control and SPCA1 siRNAs. Media and lysates from these cells were Western blotted with Cathepsin D (A) and Cab45 (C) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (B) and Cab45 (D) normalized to internal Cathepsin D and Cab45 values, respectively. (E) HeLa cells expressing either siRNA-resistant SPCA1-wt-HA or -mut4-HA were transfected with SPCA1 siRNA and subsequently with Go-D1-cpv. Ca2+ entry into the TGN was analyzed in wt (n = 8) or mut4 (n = 15)-expressing cells. 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.

Mentions: To confirm the ss-HRP secretion phenotype with additional sorting markers, control HeLa cells and HeLa cells expressing siRNA-resistant SPCA1-wt and -mut4 were transfected with control or SPCA1 siRNA, respectively. Cell pellets and supernatants were analyzed for the presence of Cab45 and Cathepsin D by Western blotting. The data clearly showed that SPCA1-wt rescued Cab45 and Cathepsin D sorting, whereas the -mut4 did not (Fig. 10, A–D). Finally, we measured the TGN Ca2+ uptake in HeLa cells stably expressing SPCA1-wt and -mut4 treated with SPCA1 siRNA and transfected with Go-D1-cpv. SPCA1-wt rescued the siRNA-induced defect of Ca2+ uptake, whereas SPCA1-mut4 did not (Fig. 10 E).


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)

A SPCA1 point mutant impairs TGN Ca2+ uptake and secretory cargo sorting. (A–D) HeLa cells expressing SPCA1-wt and mutants were treated with control and SPCA1 siRNAs. Media and lysates from these cells were Western blotted with Cathepsin D (A) and Cab45 (C) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (B) and Cab45 (D) normalized to internal Cathepsin D and Cab45 values, respectively. (E) HeLa cells expressing either siRNA-resistant SPCA1-wt-HA or -mut4-HA were transfected with SPCA1 siRNA and subsequently with Go-D1-cpv. Ca2+ entry into the TGN was analyzed in wt (n = 8) or mut4 (n = 15)-expressing cells. 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4151145&req=5

fig10: A SPCA1 point mutant impairs TGN Ca2+ uptake and secretory cargo sorting. (A–D) HeLa cells expressing SPCA1-wt and mutants were treated with control and SPCA1 siRNAs. Media and lysates from these cells were Western blotted with Cathepsin D (A) and Cab45 (C) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (B) and Cab45 (D) normalized to internal Cathepsin D and Cab45 values, respectively. (E) HeLa cells expressing either siRNA-resistant SPCA1-wt-HA or -mut4-HA were transfected with SPCA1 siRNA and subsequently with Go-D1-cpv. Ca2+ entry into the TGN was analyzed in wt (n = 8) or mut4 (n = 15)-expressing cells. 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.
Mentions: To confirm the ss-HRP secretion phenotype with additional sorting markers, control HeLa cells and HeLa cells expressing siRNA-resistant SPCA1-wt and -mut4 were transfected with control or SPCA1 siRNA, respectively. Cell pellets and supernatants were analyzed for the presence of Cab45 and Cathepsin D by Western blotting. The data clearly showed that SPCA1-wt rescued Cab45 and Cathepsin D sorting, whereas the -mut4 did not (Fig. 10, A–D). Finally, we measured the TGN Ca2+ uptake in HeLa cells stably expressing SPCA1-wt and -mut4 treated with SPCA1 siRNA and transfected with Go-D1-cpv. SPCA1-wt rescued the siRNA-induced defect of Ca2+ uptake, whereas SPCA1-mut4 did not (Fig. 10 E).

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