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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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F-actin is recruited to SPCA1-L2-C1 via CFL-1 in vitro. (A) His-Sumo tag, His-Sumo-SPCA1-L1, and His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads were incubated with recombinant CFL-1 or CFL-1-S3E in the presence of growing actin filaments labeled with Atto-488. Actin recruitment was assessed by fluorescence microscopy. (B) For the quantification the mean intensity of a circular region of interest (ROI), 12 pixels in diameter adjacent to the rim of the bead and 1 in the background were measured. The ratio of these two intensities is plotted for each experimental condition. A ratio of 1 indicates no increase of fluorescent signal over the background. Error bars represent SEM. n = 20. (C) His-Sumo-SPCA1-L2-C1 on beads was either incubated with CFL-1, actin, and KMEI buffer or with CFL-1 preincubated with untagged SPCA1-L2-C1, actin, and KMEI buffer. (D) The quantification was performed by measuring the absolute fluorescence intensities of a circular region of ROI, 12 pixels in diameter adjacent to the rim of the bead. Error bars represent SEM. n = 20. (E) To visualize the binding of His-Sumo-SPCA1-L2, CFL-1, and actin to the beads (scheme, left), full-length SPCA1-L2 and CFL-1 were labeled with fluorophores. Alexa-568-SPCA1-L2 was immobilized on beads as described earlier and incubated with Alexa-647-CFL-1 and Atto-488 actin induced to polymerize by adding KMEI buffer. As negative controls, Ni-NTA beads were incubated with labeled CFL-1 and actin. Bars, 10 µm.
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fig3: F-actin is recruited to SPCA1-L2-C1 via CFL-1 in vitro. (A) His-Sumo tag, His-Sumo-SPCA1-L1, and His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads were incubated with recombinant CFL-1 or CFL-1-S3E in the presence of growing actin filaments labeled with Atto-488. Actin recruitment was assessed by fluorescence microscopy. (B) For the quantification the mean intensity of a circular region of interest (ROI), 12 pixels in diameter adjacent to the rim of the bead and 1 in the background were measured. The ratio of these two intensities is plotted for each experimental condition. A ratio of 1 indicates no increase of fluorescent signal over the background. Error bars represent SEM. n = 20. (C) His-Sumo-SPCA1-L2-C1 on beads was either incubated with CFL-1, actin, and KMEI buffer or with CFL-1 preincubated with untagged SPCA1-L2-C1, actin, and KMEI buffer. (D) The quantification was performed by measuring the absolute fluorescence intensities of a circular region of ROI, 12 pixels in diameter adjacent to the rim of the bead. Error bars represent SEM. n = 20. (E) To visualize the binding of His-Sumo-SPCA1-L2, CFL-1, and actin to the beads (scheme, left), full-length SPCA1-L2 and CFL-1 were labeled with fluorophores. Alexa-568-SPCA1-L2 was immobilized on beads as described earlier and incubated with Alexa-647-CFL-1 and Atto-488 actin induced to polymerize by adding KMEI buffer. As negative controls, Ni-NTA beads were incubated with labeled CFL-1 and actin. Bars, 10 µm.

Mentions: To test the actin recruitment to the SPCA1 domains in the presence or absence of CFL-1 as well as CFL-1-S3E, the proteins were attached to the beads and incubated with fluorescently labeled actin as described in the Materials and methods. Actin did not associate to His-Sumo, or to His Sumo-SPCA1-L1 in either the absence or the presence of CFL-1 or CFL-1-S3E (Fig. 3 A). In contrast, although His-Sumo-SPCA1-L2-C1 did not interact with actin in the absence of CFL-1 or in the presence of CFL-1-S3E, the actin filaments were strongly recruited after recombinant CFL-1 was added (Fig. 3 A). To quantify our results, we examined 10 beads in three independent experiments and quantified the amount of labeled actin recruited to the beads by calculating the fluorescence ratio of the area close to the bead surface to the intensity of the background (Fig. 3 B).


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)

F-actin is recruited to SPCA1-L2-C1 via CFL-1 in vitro. (A) His-Sumo tag, His-Sumo-SPCA1-L1, and His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads were incubated with recombinant CFL-1 or CFL-1-S3E in the presence of growing actin filaments labeled with Atto-488. Actin recruitment was assessed by fluorescence microscopy. (B) For the quantification the mean intensity of a circular region of interest (ROI), 12 pixels in diameter adjacent to the rim of the bead and 1 in the background were measured. The ratio of these two intensities is plotted for each experimental condition. A ratio of 1 indicates no increase of fluorescent signal over the background. Error bars represent SEM. n = 20. (C) His-Sumo-SPCA1-L2-C1 on beads was either incubated with CFL-1, actin, and KMEI buffer or with CFL-1 preincubated with untagged SPCA1-L2-C1, actin, and KMEI buffer. (D) The quantification was performed by measuring the absolute fluorescence intensities of a circular region of ROI, 12 pixels in diameter adjacent to the rim of the bead. Error bars represent SEM. n = 20. (E) To visualize the binding of His-Sumo-SPCA1-L2, CFL-1, and actin to the beads (scheme, left), full-length SPCA1-L2 and CFL-1 were labeled with fluorophores. Alexa-568-SPCA1-L2 was immobilized on beads as described earlier and incubated with Alexa-647-CFL-1 and Atto-488 actin induced to polymerize by adding KMEI buffer. As negative controls, Ni-NTA beads were incubated with labeled CFL-1 and actin. Bars, 10 µm.
© Copyright Policy - openaccess
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fig3: F-actin is recruited to SPCA1-L2-C1 via CFL-1 in vitro. (A) His-Sumo tag, His-Sumo-SPCA1-L1, and His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads were incubated with recombinant CFL-1 or CFL-1-S3E in the presence of growing actin filaments labeled with Atto-488. Actin recruitment was assessed by fluorescence microscopy. (B) For the quantification the mean intensity of a circular region of interest (ROI), 12 pixels in diameter adjacent to the rim of the bead and 1 in the background were measured. The ratio of these two intensities is plotted for each experimental condition. A ratio of 1 indicates no increase of fluorescent signal over the background. Error bars represent SEM. n = 20. (C) His-Sumo-SPCA1-L2-C1 on beads was either incubated with CFL-1, actin, and KMEI buffer or with CFL-1 preincubated with untagged SPCA1-L2-C1, actin, and KMEI buffer. (D) The quantification was performed by measuring the absolute fluorescence intensities of a circular region of ROI, 12 pixels in diameter adjacent to the rim of the bead. Error bars represent SEM. n = 20. (E) To visualize the binding of His-Sumo-SPCA1-L2, CFL-1, and actin to the beads (scheme, left), full-length SPCA1-L2 and CFL-1 were labeled with fluorophores. Alexa-568-SPCA1-L2 was immobilized on beads as described earlier and incubated with Alexa-647-CFL-1 and Atto-488 actin induced to polymerize by adding KMEI buffer. As negative controls, Ni-NTA beads were incubated with labeled CFL-1 and actin. Bars, 10 µm.
Mentions: To test the actin recruitment to the SPCA1 domains in the presence or absence of CFL-1 as well as CFL-1-S3E, the proteins were attached to the beads and incubated with fluorescently labeled actin as described in the Materials and methods. Actin did not associate to His-Sumo, or to His Sumo-SPCA1-L1 in either the absence or the presence of CFL-1 or CFL-1-S3E (Fig. 3 A). In contrast, although His-Sumo-SPCA1-L2-C1 did not interact with actin in the absence of CFL-1 or in the presence of CFL-1-S3E, the actin filaments were strongly recruited after recombinant CFL-1 was added (Fig. 3 A). To quantify our results, we examined 10 beads in three independent experiments and quantified the amount of labeled actin recruited to the beads by calculating the fluorescence ratio of the area close to the bead surface to the intensity of the background (Fig. 3 B).

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