Limits...
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.

Show MeSH

Related in: MedlinePlus

The interaction of CFL-1 and actin with SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A, top) Schematic representation of GFP-HA (aa 1–239), SPCA1-L1-HA (aa 124–252), and SPCA1-L2-C1-HA (aa 549–680) fusion constructs cloned into the retroviral expression vector pLPCX. (A, bottom) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were visualized with an HA antibody and analyzed by confocal microscopy. (B) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were lysed, and lysates were incubated with µMACS anti-HA magnetic microbeads. GFP-HA–, SPCA1-L1-HA–, and SPCA1-L2-C1-HA–associated proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting using HA, CFL-1, or β-actin antibodies, respectively. (C) HeLa cells stably expressing GFP-HA, SPCA1-L1-HA, and SPCA1-L2-C1-HA were incubated for 2 h at 20°C and subsequently permeablized with digitonin, washed, and then fixed with formaldehyde before incubation with anti-SPCA1 (green) or anti–CFL-1 (red) antibodies. (D) TGN localization of CFL-1 under different conditions in Triton X-100–permeabilized (images in Fig. S2) versus digitonin-permeabilized cells was determined by counting 100 cells per condition in three independent experiments. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: The interaction of CFL-1 and actin with SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A, top) Schematic representation of GFP-HA (aa 1–239), SPCA1-L1-HA (aa 124–252), and SPCA1-L2-C1-HA (aa 549–680) fusion constructs cloned into the retroviral expression vector pLPCX. (A, bottom) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were visualized with an HA antibody and analyzed by confocal microscopy. (B) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were lysed, and lysates were incubated with µMACS anti-HA magnetic microbeads. GFP-HA–, SPCA1-L1-HA–, and SPCA1-L2-C1-HA–associated proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting using HA, CFL-1, or β-actin antibodies, respectively. (C) HeLa cells stably expressing GFP-HA, SPCA1-L1-HA, and SPCA1-L2-C1-HA were incubated for 2 h at 20°C and subsequently permeablized with digitonin, washed, and then fixed with formaldehyde before incubation with anti-SPCA1 (green) or anti–CFL-1 (red) antibodies. (D) TGN localization of CFL-1 under different conditions in Triton X-100–permeabilized (images in Fig. S2) versus digitonin-permeabilized cells was determined by counting 100 cells per condition in three independent experiments. Bars, 5 µm.

Mentions: We reported previously that actin and CFL-1 interact with SPCA1 at the TGN to facilitate Ca2+-dependent secretory cargo sorting (von Blume et al., 2009, 2011). Therefore, we aimed to investigate whether the interaction between CFL-1 and SPCA1-L2-C1 is crucial for this process. To address this, we generated stable cell lines transduced with either GFP-HA as a control, SPCA1-L1-HA, or SPCA1-L2-C1-HA (Fig. 4 A, scheme). First, we analyzed the localization of the proteins by immunofluorescence microscopy. The cells overexpressing GFP-HA, L1-HA, or L2-C1-HA were fixed and stained with an antibody against HA. All the HA-tagged proteins showed a comparable localization in the cytosol or the nucleus (Fig. 4 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)

The interaction of CFL-1 and actin with SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A, top) Schematic representation of GFP-HA (aa 1–239), SPCA1-L1-HA (aa 124–252), and SPCA1-L2-C1-HA (aa 549–680) fusion constructs cloned into the retroviral expression vector pLPCX. (A, bottom) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were visualized with an HA antibody and analyzed by confocal microscopy. (B) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were lysed, and lysates were incubated with µMACS anti-HA magnetic microbeads. GFP-HA–, SPCA1-L1-HA–, and SPCA1-L2-C1-HA–associated proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting using HA, CFL-1, or β-actin antibodies, respectively. (C) HeLa cells stably expressing GFP-HA, SPCA1-L1-HA, and SPCA1-L2-C1-HA were incubated for 2 h at 20°C and subsequently permeablized with digitonin, washed, and then fixed with formaldehyde before incubation with anti-SPCA1 (green) or anti–CFL-1 (red) antibodies. (D) TGN localization of CFL-1 under different conditions in Triton X-100–permeabilized (images in Fig. S2) versus digitonin-permeabilized cells was determined by counting 100 cells per condition in three independent experiments. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: The interaction of CFL-1 and actin with SPCA1-L2-C1 is crucial for protein sorting at the TGN. (A, top) Schematic representation of GFP-HA (aa 1–239), SPCA1-L1-HA (aa 124–252), and SPCA1-L2-C1-HA (aa 549–680) fusion constructs cloned into the retroviral expression vector pLPCX. (A, bottom) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were visualized with an HA antibody and analyzed by confocal microscopy. (B) HeLa cells stably transfected with GFP-HA, SPCA1-L1-HA, or SPCA1-L2-C1-HA were lysed, and lysates were incubated with µMACS anti-HA magnetic microbeads. GFP-HA–, SPCA1-L1-HA–, and SPCA1-L2-C1-HA–associated proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting using HA, CFL-1, or β-actin antibodies, respectively. (C) HeLa cells stably expressing GFP-HA, SPCA1-L1-HA, and SPCA1-L2-C1-HA were incubated for 2 h at 20°C and subsequently permeablized with digitonin, washed, and then fixed with formaldehyde before incubation with anti-SPCA1 (green) or anti–CFL-1 (red) antibodies. (D) TGN localization of CFL-1 under different conditions in Triton X-100–permeabilized (images in Fig. S2) versus digitonin-permeabilized cells was determined by counting 100 cells per condition in three independent experiments. Bars, 5 µm.
Mentions: We reported previously that actin and CFL-1 interact with SPCA1 at the TGN to facilitate Ca2+-dependent secretory cargo sorting (von Blume et al., 2009, 2011). Therefore, we aimed to investigate whether the interaction between CFL-1 and SPCA1-L2-C1 is crucial for this process. To address this, we generated stable cell lines transduced with either GFP-HA as a control, SPCA1-L1-HA, or SPCA1-L2-C1-HA (Fig. 4 A, scheme). First, we analyzed the localization of the proteins by immunofluorescence microscopy. The cells overexpressing GFP-HA, L1-HA, or L2-C1-HA were fixed and stained with an antibody against HA. All the HA-tagged proteins showed a comparable localization in the cytosol or the nucleus (Fig. 4 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