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Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast.

Berro J, Pollard TD - Mol. Biol. Cell (2014)

Bottom Line: Capping protein and Aip1p help maintain the high density of actin filaments in meshwork by keeping actin filaments close enough for cross-linking.Our experiments also reveal new cellular functions for Acp1p and Acp2p independent of their capping activity.We identified two independent pathways that control polarization of endocytic sites, one depending on acp2(+) and aip1(+) during interphase and the other independent of acp1(+), acp2(+), and aip1(+) during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8103 Nanobiology Institute, Yale University, New Haven, CT 06520-8103 Institut Camille Jordan, UMR CNRS 5208, Université de Lyon, 69622 Villeurbanne-Cedex, France Centre de Génétique et de Physiologie Moléculaire et Cellulaire, UMR CNRS 5534, Université de Lyon, 69622 Villeurbanne-Cedex, France.

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Synergy between capping protein and Aip1p in actin patches revealed by deletions of each protein. Symbols and lines: dark lines are average values; light lines are ± 1 SD of the mean values; plain lines, wild-type cells (same as Figure 1A); (●) aip1∆ cells. Color code: black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; red, mEGFP-Aip1p; green, fimbrin Fim1p-mEGFP; and teal, sum of Acp2p and Aip1p. Time zero is the peak of actin. Data sets were aligned in time using Fim1p-mCherry according to the two-color data in Figures S1 and S2. (A) Numbers of tagged molecules over time in endocytic patches of wild-type and aip1∆ strains. (B) Normalized intensities over time of actin (first row) and capping protein subunit Acp1p (second row) relative to fimbrin in (first column) wild-type cells and (second column) aip1Δ deletion mutant (see Figures S2 and S5 for delay measurements and statistical analysis). (C) Fimbrin occupancy of filaments in wild-type cells over time (ratio of mEGFP-Act1p/5% to Acp1p-mEGFP or Acp2p-mEGFP molecules measured in A). The teal dashed line is the ratio between the number of actin subunits and the sum of Acp2p and Aip1p molecules in wild-type cells. (D) Net actin assembly rates in patches of (plain line) wild-type and (●) aip1∆ strains.
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Figure 2: Synergy between capping protein and Aip1p in actin patches revealed by deletions of each protein. Symbols and lines: dark lines are average values; light lines are ± 1 SD of the mean values; plain lines, wild-type cells (same as Figure 1A); (●) aip1∆ cells. Color code: black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; red, mEGFP-Aip1p; green, fimbrin Fim1p-mEGFP; and teal, sum of Acp2p and Aip1p. Time zero is the peak of actin. Data sets were aligned in time using Fim1p-mCherry according to the two-color data in Figures S1 and S2. (A) Numbers of tagged molecules over time in endocytic patches of wild-type and aip1∆ strains. (B) Normalized intensities over time of actin (first row) and capping protein subunit Acp1p (second row) relative to fimbrin in (first column) wild-type cells and (second column) aip1Δ deletion mutant (see Figures S2 and S5 for delay measurements and statistical analysis). (C) Fimbrin occupancy of filaments in wild-type cells over time (ratio of mEGFP-Act1p/5% to Acp1p-mEGFP or Acp2p-mEGFP molecules measured in A). The teal dashed line is the ratio between the number of actin subunits and the sum of Acp2p and Aip1p molecules in wild-type cells. (D) Net actin assembly rates in patches of (plain line) wild-type and (●) aip1∆ strains.

Mentions: If Aip1p caps the barbed ends of actin filaments as proposed by Okada et al. (2002), capping protein Acp1p/Acp2p might compensate for the loss of Aip1p. Indeed, in the aip1∆ deletion strain, endocytic patches accumulated ∼50% more capping protein (from 152 ± 17 SD to 223 ± 36 SD Acp1p-mEGFP molecules) and 35% more actin (from 6271 ± 1049 SD to 8438 ± 1261 SD molecules) (Figure 2A and Table 1). Strikingly, the peaks of both Acp1p and Acp2p shifted ∼4 s to the actin disassembly phase (Figures 2, A and B, S1, and S2), whereas both capping protein subunits peaked at the same time as actin in wild-type cells (Figures 1A, 2B, S1, and S2). The number of molecules of capping protein in aip1Δ cells mirrored the sum of the number of molecules of capping and Aip1p in wild type but on a slightly different timescale (Figure 2A, teal dashed line). These data are consistent with the hypothesis that capping protein replaces Aip1p during disassembly of actin in aip1Δ mutants. This hypothesis is further supported by equal net rates of actin assembly and disassembly in patches of wild-type and aip1Δ cells (Figure 2D), because these rates suggest that the number of free barbed ends is the same in both strains.


Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast.

Berro J, Pollard TD - Mol. Biol. Cell (2014)

Synergy between capping protein and Aip1p in actin patches revealed by deletions of each protein. Symbols and lines: dark lines are average values; light lines are ± 1 SD of the mean values; plain lines, wild-type cells (same as Figure 1A); (●) aip1∆ cells. Color code: black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; red, mEGFP-Aip1p; green, fimbrin Fim1p-mEGFP; and teal, sum of Acp2p and Aip1p. Time zero is the peak of actin. Data sets were aligned in time using Fim1p-mCherry according to the two-color data in Figures S1 and S2. (A) Numbers of tagged molecules over time in endocytic patches of wild-type and aip1∆ strains. (B) Normalized intensities over time of actin (first row) and capping protein subunit Acp1p (second row) relative to fimbrin in (first column) wild-type cells and (second column) aip1Δ deletion mutant (see Figures S2 and S5 for delay measurements and statistical analysis). (C) Fimbrin occupancy of filaments in wild-type cells over time (ratio of mEGFP-Act1p/5% to Acp1p-mEGFP or Acp2p-mEGFP molecules measured in A). The teal dashed line is the ratio between the number of actin subunits and the sum of Acp2p and Aip1p molecules in wild-type cells. (D) Net actin assembly rates in patches of (plain line) wild-type and (●) aip1∆ strains.
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Related In: Results  -  Collection

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Figure 2: Synergy between capping protein and Aip1p in actin patches revealed by deletions of each protein. Symbols and lines: dark lines are average values; light lines are ± 1 SD of the mean values; plain lines, wild-type cells (same as Figure 1A); (●) aip1∆ cells. Color code: black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; red, mEGFP-Aip1p; green, fimbrin Fim1p-mEGFP; and teal, sum of Acp2p and Aip1p. Time zero is the peak of actin. Data sets were aligned in time using Fim1p-mCherry according to the two-color data in Figures S1 and S2. (A) Numbers of tagged molecules over time in endocytic patches of wild-type and aip1∆ strains. (B) Normalized intensities over time of actin (first row) and capping protein subunit Acp1p (second row) relative to fimbrin in (first column) wild-type cells and (second column) aip1Δ deletion mutant (see Figures S2 and S5 for delay measurements and statistical analysis). (C) Fimbrin occupancy of filaments in wild-type cells over time (ratio of mEGFP-Act1p/5% to Acp1p-mEGFP or Acp2p-mEGFP molecules measured in A). The teal dashed line is the ratio between the number of actin subunits and the sum of Acp2p and Aip1p molecules in wild-type cells. (D) Net actin assembly rates in patches of (plain line) wild-type and (●) aip1∆ strains.
Mentions: If Aip1p caps the barbed ends of actin filaments as proposed by Okada et al. (2002), capping protein Acp1p/Acp2p might compensate for the loss of Aip1p. Indeed, in the aip1∆ deletion strain, endocytic patches accumulated ∼50% more capping protein (from 152 ± 17 SD to 223 ± 36 SD Acp1p-mEGFP molecules) and 35% more actin (from 6271 ± 1049 SD to 8438 ± 1261 SD molecules) (Figure 2A and Table 1). Strikingly, the peaks of both Acp1p and Acp2p shifted ∼4 s to the actin disassembly phase (Figures 2, A and B, S1, and S2), whereas both capping protein subunits peaked at the same time as actin in wild-type cells (Figures 1A, 2B, S1, and S2). The number of molecules of capping protein in aip1Δ cells mirrored the sum of the number of molecules of capping and Aip1p in wild type but on a slightly different timescale (Figure 2A, teal dashed line). These data are consistent with the hypothesis that capping protein replaces Aip1p during disassembly of actin in aip1Δ mutants. This hypothesis is further supported by equal net rates of actin assembly and disassembly in patches of wild-type and aip1Δ cells (Figure 2D), because these rates suggest that the number of free barbed ends is the same in both strains.

Bottom Line: Capping protein and Aip1p help maintain the high density of actin filaments in meshwork by keeping actin filaments close enough for cross-linking.Our experiments also reveal new cellular functions for Acp1p and Acp2p independent of their capping activity.We identified two independent pathways that control polarization of endocytic sites, one depending on acp2(+) and aip1(+) during interphase and the other independent of acp1(+), acp2(+), and aip1(+) during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8103 Nanobiology Institute, Yale University, New Haven, CT 06520-8103 Institut Camille Jordan, UMR CNRS 5208, Université de Lyon, 69622 Villeurbanne-Cedex, France Centre de Génétique et de Physiologie Moléculaire et Cellulaire, UMR CNRS 5534, Université de Lyon, 69622 Villeurbanne-Cedex, France.

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Related in: MedlinePlus