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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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Expression of CFL-1-S3E that does not bind to SPCA1 and actin causes missorting of secretory cargo. (A) HeLa cells were transfected with control or ADF and CFL-1 siRNA. In parallel, ADF and CFL-1 knockdown HeLa cells were transfected with siRNA-resistant rat HA-tagged CFL-1 (rCFL-1) wt or rCFL-1-S3E plasmids, and analyzed by SDS-PAGE and Western blotting with CFL-1 and ADF antibodies. (B) Cells described in A were transfected with ss-HRP-Flag. Medium from these cells was analyzed for HRP secretion. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (C) HeLa cells transfected with ADF/CFL-1 siRNA, ss-HRP-Flag, and HA-rCFL-1 wt or rCFL-1-S3E were incubated at 20°C for 2 h in the presence of cycloheximide to arrest HRP in the TGN. Cells were then shifted to 37°C, and the localization of HRP was analyzed with an HRP antibody in confocal microscopy. Bars, 5 µm. (D) 100 cells expressing ss-HRP-Flag and HA-rCFL-1 wt or rHA-CFL-1-S3E were counted in three different experiments at 20°C and 37°C. HeLa cells were transfected with control and ADF/CFL-1 siRNA and incubated for 40 h. Then ADF/CFL-1 knockdown cells were either transfected with a control plasmid or with rCFL-1 wt or rCFL-1-S3E. (E and F) Media and lysates from these cells were Western blotted with Cathepsin D (E) and Cab45 (F) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (E) and Cab45 (F) normalized to internal Cathepsin D and Cab45 values, respectively.
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fig7: Expression of CFL-1-S3E that does not bind to SPCA1 and actin causes missorting of secretory cargo. (A) HeLa cells were transfected with control or ADF and CFL-1 siRNA. In parallel, ADF and CFL-1 knockdown HeLa cells were transfected with siRNA-resistant rat HA-tagged CFL-1 (rCFL-1) wt or rCFL-1-S3E plasmids, and analyzed by SDS-PAGE and Western blotting with CFL-1 and ADF antibodies. (B) Cells described in A were transfected with ss-HRP-Flag. Medium from these cells was analyzed for HRP secretion. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (C) HeLa cells transfected with ADF/CFL-1 siRNA, ss-HRP-Flag, and HA-rCFL-1 wt or rCFL-1-S3E were incubated at 20°C for 2 h in the presence of cycloheximide to arrest HRP in the TGN. Cells were then shifted to 37°C, and the localization of HRP was analyzed with an HRP antibody in confocal microscopy. Bars, 5 µm. (D) 100 cells expressing ss-HRP-Flag and HA-rCFL-1 wt or rHA-CFL-1-S3E were counted in three different experiments at 20°C and 37°C. HeLa cells were transfected with control and ADF/CFL-1 siRNA and incubated for 40 h. Then ADF/CFL-1 knockdown cells were either transfected with a control plasmid or with rCFL-1 wt or rCFL-1-S3E. (E and F) Media and lysates from these cells were Western blotted with Cathepsin D (E) and Cab45 (F) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (E) and Cab45 (F) normalized to internal Cathepsin D and Cab45 values, respectively.

Mentions: To further substantiate the significance of the formation of the complex for sorting and TGN Ca2+ entry, we aimed at elucidating the consequence of impaired CFL-1 and actin binding to SPCA1. As described previously, SPCA1 binds to active CFL-1, and this binding recruits CFL-1 to the TGN (von Blume et al., 2011). We have also reported previously that inactivation of CFL-1 by overexpression of LIMK, which phosphorylates CFL-1, or knockdown of slingshot, which dephosphorylates CFL-1, interferes with protein secretion (von Blume et al., 2009). To test if an inactive CFL-1 phosphomimetic S3E mutant that does not bind to SPCA1 interferes with Ca2+-dependent protein sorting, HeLa cells were transfected with control or ADF/CFL-1 siRNAs as well as a siRNA-resistant HA-tagged rat CFL-1 (rCFL-1) or rCFL-1-S3E, respectively. To test the knockdown and the expression of CFL-1 and the siRNA-resistant mutants, lysates were analyzed by Western blotting with CFL-1 and ADF antibodies, respectively. ADF/CFL-1 was successfully depleted by siRNAs, and overexpression of HA-rCFL-1 wild type (wt) and S3E in ADF/CFL-1–depleted cells occurred at similar levels (Fig. 7 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)

Expression of CFL-1-S3E that does not bind to SPCA1 and actin causes missorting of secretory cargo. (A) HeLa cells were transfected with control or ADF and CFL-1 siRNA. In parallel, ADF and CFL-1 knockdown HeLa cells were transfected with siRNA-resistant rat HA-tagged CFL-1 (rCFL-1) wt or rCFL-1-S3E plasmids, and analyzed by SDS-PAGE and Western blotting with CFL-1 and ADF antibodies. (B) Cells described in A were transfected with ss-HRP-Flag. Medium from these cells was analyzed for HRP secretion. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (C) HeLa cells transfected with ADF/CFL-1 siRNA, ss-HRP-Flag, and HA-rCFL-1 wt or rCFL-1-S3E were incubated at 20°C for 2 h in the presence of cycloheximide to arrest HRP in the TGN. Cells were then shifted to 37°C, and the localization of HRP was analyzed with an HRP antibody in confocal microscopy. Bars, 5 µm. (D) 100 cells expressing ss-HRP-Flag and HA-rCFL-1 wt or rHA-CFL-1-S3E were counted in three different experiments at 20°C and 37°C. HeLa cells were transfected with control and ADF/CFL-1 siRNA and incubated for 40 h. Then ADF/CFL-1 knockdown cells were either transfected with a control plasmid or with rCFL-1 wt or rCFL-1-S3E. (E and F) Media and lysates from these cells were Western blotted with Cathepsin D (E) and Cab45 (F) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (E) and Cab45 (F) normalized to internal Cathepsin D and Cab45 values, respectively.
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fig7: Expression of CFL-1-S3E that does not bind to SPCA1 and actin causes missorting of secretory cargo. (A) HeLa cells were transfected with control or ADF and CFL-1 siRNA. In parallel, ADF and CFL-1 knockdown HeLa cells were transfected with siRNA-resistant rat HA-tagged CFL-1 (rCFL-1) wt or rCFL-1-S3E plasmids, and analyzed by SDS-PAGE and Western blotting with CFL-1 and ADF antibodies. (B) Cells described in A were transfected with ss-HRP-Flag. Medium from these cells was analyzed for HRP secretion. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (C) HeLa cells transfected with ADF/CFL-1 siRNA, ss-HRP-Flag, and HA-rCFL-1 wt or rCFL-1-S3E were incubated at 20°C for 2 h in the presence of cycloheximide to arrest HRP in the TGN. Cells were then shifted to 37°C, and the localization of HRP was analyzed with an HRP antibody in confocal microscopy. Bars, 5 µm. (D) 100 cells expressing ss-HRP-Flag and HA-rCFL-1 wt or rHA-CFL-1-S3E were counted in three different experiments at 20°C and 37°C. HeLa cells were transfected with control and ADF/CFL-1 siRNA and incubated for 40 h. Then ADF/CFL-1 knockdown cells were either transfected with a control plasmid or with rCFL-1 wt or rCFL-1-S3E. (E and F) Media and lysates from these cells were Western blotted with Cathepsin D (E) and Cab45 (F) antibodies. Western blots from three independent experiments were quantified by densitometry. Bar graphs represent the densitometry values of external Cathepsin D (E) and Cab45 (F) normalized to internal Cathepsin D and Cab45 values, respectively.
Mentions: To further substantiate the significance of the formation of the complex for sorting and TGN Ca2+ entry, we aimed at elucidating the consequence of impaired CFL-1 and actin binding to SPCA1. As described previously, SPCA1 binds to active CFL-1, and this binding recruits CFL-1 to the TGN (von Blume et al., 2011). We have also reported previously that inactivation of CFL-1 by overexpression of LIMK, which phosphorylates CFL-1, or knockdown of slingshot, which dephosphorylates CFL-1, interferes with protein secretion (von Blume et al., 2009). To test if an inactive CFL-1 phosphomimetic S3E mutant that does not bind to SPCA1 interferes with Ca2+-dependent protein sorting, HeLa cells were transfected with control or ADF/CFL-1 siRNAs as well as a siRNA-resistant HA-tagged rat CFL-1 (rCFL-1) or rCFL-1-S3E, respectively. To test the knockdown and the expression of CFL-1 and the siRNA-resistant mutants, lysates were analyzed by Western blotting with CFL-1 and ADF antibodies, respectively. ADF/CFL-1 was successfully depleted by siRNAs, and overexpression of HA-rCFL-1 wild type (wt) and S3E in ADF/CFL-1–depleted cells occurred at similar levels (Fig. 7 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