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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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Evidence for functional differences between capping protein subunits. (A) Nuclear, cytoplasmic, and total cell concentrations of Acp1p-mEGFP and Acp2p-mEGFP in wild-type cells and single mutants acp1Δ and acp2Δ, and double mutants acp1Δ/aip1Δ. Color code: blue, Acp1p-mEGFP; purple, Acp2p-mEGFP. Empty bars with a filled circle, nuclear concentration; filled bar with an empty circle, cytoplasmic concentration; filled bar, total cell concentration. (B) Western blots with antibodies against GFP for Acp2p-mEGFP in wild-type and acp1Δ cells, and Acp1p-mEGFP in wild-type and acp2Δ cells. Intensities of the bands are proportional to total cell concentrations for comparison with the filled bars in B. Numbers represent the relative intensities of bands in corresponding mutants. (C–E) Time courses with time 0 s at the peak of actin assembly. 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; ●, aip1∆ cells; ◻, double mutant acp1∆/acp2∆ cells; ○, double mutant acp1∆/aip1∆ cells. Color code: black, mEGFP-actin; and green, fimbrin Fim1p-mEGFP. Time zero is the peak of actin. (C) Numbers of mEGFP-actin molecules in all mutants over time. (D) Number of molecules of Fim1p-mEGFP in all mutants over time. (E) Net actin assembly rates in wild-type and acp1Δ/aip1Δ strains over time.
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Figure 4: Evidence for functional differences between capping protein subunits. (A) Nuclear, cytoplasmic, and total cell concentrations of Acp1p-mEGFP and Acp2p-mEGFP in wild-type cells and single mutants acp1Δ and acp2Δ, and double mutants acp1Δ/aip1Δ. Color code: blue, Acp1p-mEGFP; purple, Acp2p-mEGFP. Empty bars with a filled circle, nuclear concentration; filled bar with an empty circle, cytoplasmic concentration; filled bar, total cell concentration. (B) Western blots with antibodies against GFP for Acp2p-mEGFP in wild-type and acp1Δ cells, and Acp1p-mEGFP in wild-type and acp2Δ cells. Intensities of the bands are proportional to total cell concentrations for comparison with the filled bars in B. Numbers represent the relative intensities of bands in corresponding mutants. (C–E) Time courses with time 0 s at the peak of actin assembly. 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; ●, aip1∆ cells; ◻, double mutant acp1∆/acp2∆ cells; ○, double mutant acp1∆/aip1∆ cells. Color code: black, mEGFP-actin; and green, fimbrin Fim1p-mEGFP. Time zero is the peak of actin. (C) Numbers of mEGFP-actin molecules in all mutants over time. (D) Number of molecules of Fim1p-mEGFP in all mutants over time. (E) Net actin assembly rates in wild-type and acp1Δ/aip1Δ strains over time.

Mentions: Although the only documented function of Acp1p and Acp2p is to form a heterodimer to cap filaments, deletion of both genes increased the numbers of actins in patches less than deletion of either acp1+ or acp2+ (Figure 3, C–E, and Table 1). Assembly of actin in endocytic patches was prolonged in all three mutants, but the initial assembly rate was lower in the acp1Δ/acp2Δ strain than the single mutants and wild-type cells (Figure 3B). Furthermore, the peak of Fim1p was 25% smaller in acp2Δ and acp1Δ/acp2Δ mutants than in wild-type and acp1Δ cells (Figure 4D and Table 1). Slightly less Aip1p accumulated in acp1Δ/acp2Δ cells than wild-type and single-deletion strains (Table 1), and Aip1p accumulation took longer in acp1Δ and acp1Δ/acp2Δ (Figure 3, C and E) than in wild-type and acp2Δ cells (Figures 3D and S4). These differences reveal that acp1+ and acp2+ may have undocumented independent functions beyond forming heterodimeric capping protein.


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)

Evidence for functional differences between capping protein subunits. (A) Nuclear, cytoplasmic, and total cell concentrations of Acp1p-mEGFP and Acp2p-mEGFP in wild-type cells and single mutants acp1Δ and acp2Δ, and double mutants acp1Δ/aip1Δ. Color code: blue, Acp1p-mEGFP; purple, Acp2p-mEGFP. Empty bars with a filled circle, nuclear concentration; filled bar with an empty circle, cytoplasmic concentration; filled bar, total cell concentration. (B) Western blots with antibodies against GFP for Acp2p-mEGFP in wild-type and acp1Δ cells, and Acp1p-mEGFP in wild-type and acp2Δ cells. Intensities of the bands are proportional to total cell concentrations for comparison with the filled bars in B. Numbers represent the relative intensities of bands in corresponding mutants. (C–E) Time courses with time 0 s at the peak of actin assembly. 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; ●, aip1∆ cells; ◻, double mutant acp1∆/acp2∆ cells; ○, double mutant acp1∆/aip1∆ cells. Color code: black, mEGFP-actin; and green, fimbrin Fim1p-mEGFP. Time zero is the peak of actin. (C) Numbers of mEGFP-actin molecules in all mutants over time. (D) Number of molecules of Fim1p-mEGFP in all mutants over time. (E) Net actin assembly rates in wild-type and acp1Δ/aip1Δ strains over time.
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Related In: Results  -  Collection

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Figure 4: Evidence for functional differences between capping protein subunits. (A) Nuclear, cytoplasmic, and total cell concentrations of Acp1p-mEGFP and Acp2p-mEGFP in wild-type cells and single mutants acp1Δ and acp2Δ, and double mutants acp1Δ/aip1Δ. Color code: blue, Acp1p-mEGFP; purple, Acp2p-mEGFP. Empty bars with a filled circle, nuclear concentration; filled bar with an empty circle, cytoplasmic concentration; filled bar, total cell concentration. (B) Western blots with antibodies against GFP for Acp2p-mEGFP in wild-type and acp1Δ cells, and Acp1p-mEGFP in wild-type and acp2Δ cells. Intensities of the bands are proportional to total cell concentrations for comparison with the filled bars in B. Numbers represent the relative intensities of bands in corresponding mutants. (C–E) Time courses with time 0 s at the peak of actin assembly. 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; ●, aip1∆ cells; ◻, double mutant acp1∆/acp2∆ cells; ○, double mutant acp1∆/aip1∆ cells. Color code: black, mEGFP-actin; and green, fimbrin Fim1p-mEGFP. Time zero is the peak of actin. (C) Numbers of mEGFP-actin molecules in all mutants over time. (D) Number of molecules of Fim1p-mEGFP in all mutants over time. (E) Net actin assembly rates in wild-type and acp1Δ/aip1Δ strains over time.
Mentions: Although the only documented function of Acp1p and Acp2p is to form a heterodimer to cap filaments, deletion of both genes increased the numbers of actins in patches less than deletion of either acp1+ or acp2+ (Figure 3, C–E, and Table 1). Assembly of actin in endocytic patches was prolonged in all three mutants, but the initial assembly rate was lower in the acp1Δ/acp2Δ strain than the single mutants and wild-type cells (Figure 3B). Furthermore, the peak of Fim1p was 25% smaller in acp2Δ and acp1Δ/acp2Δ mutants than in wild-type and acp1Δ cells (Figure 4D and Table 1). Slightly less Aip1p accumulated in acp1Δ/acp2Δ cells than wild-type and single-deletion strains (Table 1), and Aip1p accumulation took longer in acp1Δ and acp1Δ/acp2Δ (Figure 3, C and E) than in wild-type and acp2Δ cells (Figures 3D and S4). These differences reveal that acp1+ and acp2+ may have undocumented independent functions beyond forming heterodimeric capping protein.

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