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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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Effects of deletions of acp1+ and/or acp2+ on the accumulation, disappearance, and movements of actin, fimbrin, and Aip1p in endocytic patches. (A) Localization of capping protein subunits in the absence of the other subunit. Negative-contrast fluorescence micrographs of representative cells expressing Acp1p-mEGFP in wild-type and acp2Δ cells (left column) or Acp2p-mEGFP in wild-type and acp1Δ cells (right column). Scale bar: 5 μm. (B–E) Time zero corresponds to the peak of actin. Color code: black, mEGFP-actin; red, mEGFP-Aip1p. 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); +, acp1∆ cells; x, acp2∆ cells; and ◻, double mutant acp1∆/acp2∆ cells. Data sets were aligned according to the two-color data in Figures S1 and S2. (B) Actin net assembly rates in patches of wild-type, acp1∆, acp2∆, and acp1∆/acp2∆ cells. (C–E) Comparisons of the numbers of molecules over time in endocytic patches of wild-type cells with (C) acp1∆ cells, (D) acp2∆ cells, and (E) double mutant acp1∆/acp2∆ cells.
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Figure 3: Effects of deletions of acp1+ and/or acp2+ on the accumulation, disappearance, and movements of actin, fimbrin, and Aip1p in endocytic patches. (A) Localization of capping protein subunits in the absence of the other subunit. Negative-contrast fluorescence micrographs of representative cells expressing Acp1p-mEGFP in wild-type and acp2Δ cells (left column) or Acp2p-mEGFP in wild-type and acp1Δ cells (right column). Scale bar: 5 μm. (B–E) Time zero corresponds to the peak of actin. Color code: black, mEGFP-actin; red, mEGFP-Aip1p. 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); +, acp1∆ cells; x, acp2∆ cells; and ◻, double mutant acp1∆/acp2∆ cells. Data sets were aligned according to the two-color data in Figures S1 and S2. (B) Actin net assembly rates in patches of wild-type, acp1∆, acp2∆, and acp1∆/acp2∆ cells. (C–E) Comparisons of the numbers of molecules over time in endocytic patches of wild-type cells with (C) acp1∆ cells, (D) acp2∆ cells, and (E) double mutant acp1∆/acp2∆ cells.

Mentions: Deletion of the genes for either capping protein subunit eliminated the accumulation of the other capping protein subunit in patches (Figure 3A), so the barbed ends of actin filaments were not capped independently by either subunit of heterodimeric capping protein. These results are consistent with the idea that both Acp1p and Acp2p are necessary to form an active heterodimeric protein (Yamashita et al., 2003) that caps barbed ends of actin filaments in vitro (Isenberg et al., 1980; Wear et al., 2003; Yamashita et al., 2003; Kim et al., 2010) and in vivo (Hug et al., 1995; Kovar et al., 2005).


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)

Effects of deletions of acp1+ and/or acp2+ on the accumulation, disappearance, and movements of actin, fimbrin, and Aip1p in endocytic patches. (A) Localization of capping protein subunits in the absence of the other subunit. Negative-contrast fluorescence micrographs of representative cells expressing Acp1p-mEGFP in wild-type and acp2Δ cells (left column) or Acp2p-mEGFP in wild-type and acp1Δ cells (right column). Scale bar: 5 μm. (B–E) Time zero corresponds to the peak of actin. Color code: black, mEGFP-actin; red, mEGFP-Aip1p. 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); +, acp1∆ cells; x, acp2∆ cells; and ◻, double mutant acp1∆/acp2∆ cells. Data sets were aligned according to the two-color data in Figures S1 and S2. (B) Actin net assembly rates in patches of wild-type, acp1∆, acp2∆, and acp1∆/acp2∆ cells. (C–E) Comparisons of the numbers of molecules over time in endocytic patches of wild-type cells with (C) acp1∆ cells, (D) acp2∆ cells, and (E) double mutant acp1∆/acp2∆ cells.
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Figure 3: Effects of deletions of acp1+ and/or acp2+ on the accumulation, disappearance, and movements of actin, fimbrin, and Aip1p in endocytic patches. (A) Localization of capping protein subunits in the absence of the other subunit. Negative-contrast fluorescence micrographs of representative cells expressing Acp1p-mEGFP in wild-type and acp2Δ cells (left column) or Acp2p-mEGFP in wild-type and acp1Δ cells (right column). Scale bar: 5 μm. (B–E) Time zero corresponds to the peak of actin. Color code: black, mEGFP-actin; red, mEGFP-Aip1p. 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); +, acp1∆ cells; x, acp2∆ cells; and ◻, double mutant acp1∆/acp2∆ cells. Data sets were aligned according to the two-color data in Figures S1 and S2. (B) Actin net assembly rates in patches of wild-type, acp1∆, acp2∆, and acp1∆/acp2∆ cells. (C–E) Comparisons of the numbers of molecules over time in endocytic patches of wild-type cells with (C) acp1∆ cells, (D) acp2∆ cells, and (E) double mutant acp1∆/acp2∆ cells.
Mentions: Deletion of the genes for either capping protein subunit eliminated the accumulation of the other capping protein subunit in patches (Figure 3A), so the barbed ends of actin filaments were not capped independently by either subunit of heterodimeric capping protein. These results are consistent with the idea that both Acp1p and Acp2p are necessary to form an active heterodimeric protein (Yamashita et al., 2003) that caps barbed ends of actin filaments in vitro (Isenberg et al., 1980; Wear et al., 2003; Yamashita et al., 2003; Kim et al., 2010) and in vivo (Hug et al., 1995; Kovar et al., 2005).

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