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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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The interaction of CFL-1 and actin to SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A) HeLa cells expressing ss-HRP were transduced with a pLPCX plasmid (control), SPCA1-L1-HA, or SPCA1-L2-C1-HA. Cells were subsequently lysed and proteins were separated by SDS-PAGE. The presence of SPCA1-L1-HA and SPCA1-L2-C1-HA was determined by Western blotting with an anti-HA antibody. (B) Cell culture supernatants of cells described in A were analyzed for HRP activity by chemiluminescence. Error bars show mean ± SD of external HRP activity normalized to internal HRP activity of three independent experiments. Datasets were statistically significant when P < 0.01 (**). (C) HeLa cells stably expressing SPCA1-L1-HA or SPCA1-L2-C1-HA were incubated at 20°C for 2 h in the presence of cycloheximide to accumulate HRP in the TGN. Cells were subsequently shifted to 37°C, and the localization of HRP was analyzed by fluorescence microscopy with an anti-HRP antibody. To quantify the results, 100 cells expressing SPCA1-L1-HA or SPCA-L2-C1-HA in three different experiments at 20°C and 37°C were counted. Bars, 5 µm. (D and E) Media and lysates from the same cells were Western blotted with specific antibodies against Cathepsin D (D) or Cab45 (E). Western blots from three independent experiments were quantified by densitometry using the ImageJ software. Bar graphs represent the densitometry values of external Cathepsin D (D) and Cab45 (E) normalized to internal Cathepsin D and Cab45 values, respectively.
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fig5: The interaction of CFL-1 and actin to SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A) HeLa cells expressing ss-HRP were transduced with a pLPCX plasmid (control), SPCA1-L1-HA, or SPCA1-L2-C1-HA. Cells were subsequently lysed and proteins were separated by SDS-PAGE. The presence of SPCA1-L1-HA and SPCA1-L2-C1-HA was determined by Western blotting with an anti-HA antibody. (B) Cell culture supernatants of cells described in A were analyzed for HRP activity by chemiluminescence. Error bars show mean ± SD of external HRP activity normalized to internal HRP activity of three independent experiments. Datasets were statistically significant when P < 0.01 (**). (C) HeLa cells stably expressing SPCA1-L1-HA or SPCA1-L2-C1-HA were incubated at 20°C for 2 h in the presence of cycloheximide to accumulate HRP in the TGN. Cells were subsequently shifted to 37°C, and the localization of HRP was analyzed by fluorescence microscopy with an anti-HRP antibody. To quantify the results, 100 cells expressing SPCA1-L1-HA or SPCA-L2-C1-HA in three different experiments at 20°C and 37°C were counted. Bars, 5 µm. (D and E) Media and lysates from the same cells were Western blotted with specific antibodies against Cathepsin D (D) or Cab45 (E). Western blots from three independent experiments were quantified by densitometry using the ImageJ software. Bar graphs represent the densitometry values of external Cathepsin D (D) and Cab45 (E) normalized to internal Cathepsin D and Cab45 values, respectively.

Mentions: Next, we wanted to know the functional consequence of SPCA1-L2-C1 overexpression for TGN protein sorting in these cells. In recent years we have established robust assays to determine CFL-1– and SPCA1-dependent TGN sorting (von Blume et al., 2009, 2011, 2012). First, HeLa cells stably expressing signal sequence HRP (ss-HRP) were transduced with a control plasmid, SPCA1-L1-HA, or SPCA1-L2-C1-HA. To test the expression of the HA-tagged proteins, lysates were analyzed by Western blotting with an HA antibody. The cells expressed SPCA1-L1-HA and SPCA1-L2-C1-HA at similar levels (Fig. 5 A). The HRP assay was performed by incubating the cells with serum-free medium, and the secretion of HRP was measured by chemiluminescence as described previously (Bard et al., 2006).


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)

The interaction of CFL-1 and actin to SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A) HeLa cells expressing ss-HRP were transduced with a pLPCX plasmid (control), SPCA1-L1-HA, or SPCA1-L2-C1-HA. Cells were subsequently lysed and proteins were separated by SDS-PAGE. The presence of SPCA1-L1-HA and SPCA1-L2-C1-HA was determined by Western blotting with an anti-HA antibody. (B) Cell culture supernatants of cells described in A were analyzed for HRP activity by chemiluminescence. Error bars show mean ± SD of external HRP activity normalized to internal HRP activity of three independent experiments. Datasets were statistically significant when P < 0.01 (**). (C) HeLa cells stably expressing SPCA1-L1-HA or SPCA1-L2-C1-HA were incubated at 20°C for 2 h in the presence of cycloheximide to accumulate HRP in the TGN. Cells were subsequently shifted to 37°C, and the localization of HRP was analyzed by fluorescence microscopy with an anti-HRP antibody. To quantify the results, 100 cells expressing SPCA1-L1-HA or SPCA-L2-C1-HA in three different experiments at 20°C and 37°C were counted. Bars, 5 µm. (D and E) Media and lysates from the same cells were Western blotted with specific antibodies against Cathepsin D (D) or Cab45 (E). Western blots from three independent experiments were quantified by densitometry using the ImageJ software. Bar graphs represent the densitometry values of external Cathepsin D (D) and Cab45 (E) normalized to internal Cathepsin D and Cab45 values, respectively.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4151145&req=5

fig5: The interaction of CFL-1 and actin to SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A) HeLa cells expressing ss-HRP were transduced with a pLPCX plasmid (control), SPCA1-L1-HA, or SPCA1-L2-C1-HA. Cells were subsequently lysed and proteins were separated by SDS-PAGE. The presence of SPCA1-L1-HA and SPCA1-L2-C1-HA was determined by Western blotting with an anti-HA antibody. (B) Cell culture supernatants of cells described in A were analyzed for HRP activity by chemiluminescence. Error bars show mean ± SD of external HRP activity normalized to internal HRP activity of three independent experiments. Datasets were statistically significant when P < 0.01 (**). (C) HeLa cells stably expressing SPCA1-L1-HA or SPCA1-L2-C1-HA were incubated at 20°C for 2 h in the presence of cycloheximide to accumulate HRP in the TGN. Cells were subsequently shifted to 37°C, and the localization of HRP was analyzed by fluorescence microscopy with an anti-HRP antibody. To quantify the results, 100 cells expressing SPCA1-L1-HA or SPCA-L2-C1-HA in three different experiments at 20°C and 37°C were counted. Bars, 5 µm. (D and E) Media and lysates from the same cells were Western blotted with specific antibodies against Cathepsin D (D) or Cab45 (E). Western blots from three independent experiments were quantified by densitometry using the ImageJ software. Bar graphs represent the densitometry values of external Cathepsin D (D) and Cab45 (E) normalized to internal Cathepsin D and Cab45 values, respectively.
Mentions: Next, we wanted to know the functional consequence of SPCA1-L2-C1 overexpression for TGN protein sorting in these cells. In recent years we have established robust assays to determine CFL-1– and SPCA1-dependent TGN sorting (von Blume et al., 2009, 2011, 2012). First, HeLa cells stably expressing signal sequence HRP (ss-HRP) were transduced with a control plasmid, SPCA1-L1-HA, or SPCA1-L2-C1-HA. To test the expression of the HA-tagged proteins, lysates were analyzed by Western blotting with an HA antibody. The cells expressed SPCA1-L1-HA and SPCA1-L2-C1-HA at similar levels (Fig. 5 A). The HRP assay was performed by incubating the cells with serum-free medium, and the secretion of HRP was measured by chemiluminescence as described previously (Bard et al., 2006).

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