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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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Establishment of an in vitro reconstitution assay. (A) Establishment of the F-actin bead assay with controls: Ni-NTA beads, His-Sumo, His-Sumo-CFL-1, or mutant His-Sumo-CFL-1-S3E protein were attached to Ni-NTA beads (also depicted in the scheme). Immunofluorescence staining was performed using a primary anti-His antibody and a secondary Alexa Fluor 488 antibody to visualize the recombinant proteins on beads. (B) Immobilized proteins on beads were incubated with polymerized Atto-488 actin (also depicted in the scheme), and the actin signal was monitored by epifluorescence microscopy. In case F-actin was recruited, a bright fluorescent signal would have been detectable on the surface of the bead. Only immobilized CFL-1 recruited F-actin to beads. (C) His-Sumo-SPCA1-L1 or His-Sumo-SPCA1-L2-C1 were attached to beads and subsequently incubated with either recombinant CFL-1 or CFL-1-S3E without the His-Sumo tag, as depicted in the scheme. Staining with His or CFL-1, Alexa Fluor 488– or Alexa Fluor 594–specific antibodies, and subsequent confocal imaging revealed that CFL-1 binds SPCA1-L2-C1 exclusively, whereas CFL-1-S3E failed to bind. Bars, 10 µm.
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fig2: Establishment of an in vitro reconstitution assay. (A) Establishment of the F-actin bead assay with controls: Ni-NTA beads, His-Sumo, His-Sumo-CFL-1, or mutant His-Sumo-CFL-1-S3E protein were attached to Ni-NTA beads (also depicted in the scheme). Immunofluorescence staining was performed using a primary anti-His antibody and a secondary Alexa Fluor 488 antibody to visualize the recombinant proteins on beads. (B) Immobilized proteins on beads were incubated with polymerized Atto-488 actin (also depicted in the scheme), and the actin signal was monitored by epifluorescence microscopy. In case F-actin was recruited, a bright fluorescent signal would have been detectable on the surface of the bead. Only immobilized CFL-1 recruited F-actin to beads. (C) His-Sumo-SPCA1-L1 or His-Sumo-SPCA1-L2-C1 were attached to beads and subsequently incubated with either recombinant CFL-1 or CFL-1-S3E without the His-Sumo tag, as depicted in the scheme. Staining with His or CFL-1, Alexa Fluor 488– or Alexa Fluor 594–specific antibodies, and subsequent confocal imaging revealed that CFL-1 binds SPCA1-L2-C1 exclusively, whereas CFL-1-S3E failed to bind. Bars, 10 µm.

Mentions: To elucidate how F-actin binds to the SPCA1–CFL-1 complex, we established a reconstitution assay to visualize the process. To set up the assay, we attached His-Sumo, His-Sumo-CFL-1-S3E, and His-Sumo-CFL-1 to Ni-NTA beads. As a negative control, we incubated Ni-NTA beads with BSA. To visualize the proteins, beads were incubated with an anti-His antibody and an Alexa Fluor 488–labeled secondary antibody (Fig. 2 A), and analyzed by fluorescence microscopy. The His-tagged proteins were visible on the beads in equal amounts, whereas the BSA-incubated Ni-NTA beads did not show a fluorescent signal (Fig. 2 A). To test if actin can be recruited to CFL-1 under these conditions, we incubated the beads with fluorescently labeled F-actin and monitored them by fluorescence microscopy. F-actin strongly associated to His-Sumo-CFL-1 beads but not to beads alone, His-Sumo, or His-Sumo-CFL-1-S3E (Fig. 2 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)

Establishment of an in vitro reconstitution assay. (A) Establishment of the F-actin bead assay with controls: Ni-NTA beads, His-Sumo, His-Sumo-CFL-1, or mutant His-Sumo-CFL-1-S3E protein were attached to Ni-NTA beads (also depicted in the scheme). Immunofluorescence staining was performed using a primary anti-His antibody and a secondary Alexa Fluor 488 antibody to visualize the recombinant proteins on beads. (B) Immobilized proteins on beads were incubated with polymerized Atto-488 actin (also depicted in the scheme), and the actin signal was monitored by epifluorescence microscopy. In case F-actin was recruited, a bright fluorescent signal would have been detectable on the surface of the bead. Only immobilized CFL-1 recruited F-actin to beads. (C) His-Sumo-SPCA1-L1 or His-Sumo-SPCA1-L2-C1 were attached to beads and subsequently incubated with either recombinant CFL-1 or CFL-1-S3E without the His-Sumo tag, as depicted in the scheme. Staining with His or CFL-1, Alexa Fluor 488– or Alexa Fluor 594–specific antibodies, and subsequent confocal imaging revealed that CFL-1 binds SPCA1-L2-C1 exclusively, whereas CFL-1-S3E failed to bind. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Establishment of an in vitro reconstitution assay. (A) Establishment of the F-actin bead assay with controls: Ni-NTA beads, His-Sumo, His-Sumo-CFL-1, or mutant His-Sumo-CFL-1-S3E protein were attached to Ni-NTA beads (also depicted in the scheme). Immunofluorescence staining was performed using a primary anti-His antibody and a secondary Alexa Fluor 488 antibody to visualize the recombinant proteins on beads. (B) Immobilized proteins on beads were incubated with polymerized Atto-488 actin (also depicted in the scheme), and the actin signal was monitored by epifluorescence microscopy. In case F-actin was recruited, a bright fluorescent signal would have been detectable on the surface of the bead. Only immobilized CFL-1 recruited F-actin to beads. (C) His-Sumo-SPCA1-L1 or His-Sumo-SPCA1-L2-C1 were attached to beads and subsequently incubated with either recombinant CFL-1 or CFL-1-S3E without the His-Sumo tag, as depicted in the scheme. Staining with His or CFL-1, Alexa Fluor 488– or Alexa Fluor 594–specific antibodies, and subsequent confocal imaging revealed that CFL-1 binds SPCA1-L2-C1 exclusively, whereas CFL-1-S3E failed to bind. Bars, 10 µm.
Mentions: To elucidate how F-actin binds to the SPCA1–CFL-1 complex, we established a reconstitution assay to visualize the process. To set up the assay, we attached His-Sumo, His-Sumo-CFL-1-S3E, and His-Sumo-CFL-1 to Ni-NTA beads. As a negative control, we incubated Ni-NTA beads with BSA. To visualize the proteins, beads were incubated with an anti-His antibody and an Alexa Fluor 488–labeled secondary antibody (Fig. 2 A), and analyzed by fluorescence microscopy. The His-tagged proteins were visible on the beads in equal amounts, whereas the BSA-incubated Ni-NTA beads did not show a fluorescent signal (Fig. 2 A). To test if actin can be recruited to CFL-1 under these conditions, we incubated the beads with fluorescently labeled F-actin and monitored them by fluorescence microscopy. F-actin strongly associated to His-Sumo-CFL-1 beads but not to beads alone, His-Sumo, or His-Sumo-CFL-1-S3E (Fig. 2 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