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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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Insights regarding the structure of endocytic patches by analysis of their motions in wild-type and mutant cells. (A) Samples of tracks of patches marked with Fim1p-mEGFP in wild-type, single-deletion mutant strains acp1∆, acp2∆, and aip1∆, and double-deletion mutant strains acp1∆/acp2∆ and acp1∆/aip1∆. The points are positions at 1-s intervals connected by lines, both color-coded in time from blue (appearance of Fim1p-mEGFP) to red (disappearance of Fim1p-mEGFP). The number of points varies with the lifetime of the patch. The gray dotted line is drawn parallel to the plasma membrane to mark the initial positions of patches. Scale bar: 200 nm. (B–E) Evolution of patch parameters over time. Symbols and lines for cell types: black plain lines, wild-type; mustard +, acp1∆; olive x, acp2∆; teal ●, aip1∆; purple ◻, double mutant acp1∆/acp2∆; brown ○, acp1∆/aip1∆. (B) Average displacements over 1 s of patches estimated from mEGFP-Act1p movies. Inset, normalized efficiency plots of the numbers of actin molecules per patch (normalized to the peak value) vs. displacement of the patch. (C) Estimated Stokes' radii of moving endocytic patches extracted from the data in B. Inset, detailed view at late time points. (D) Occupancy of fimbrin on actin filaments. The occupancy was calculated as the ratio between the numbers of actin (mEGFP-Act1p/5%, Figure 4C) and Fim1p-mEGFP (Figure 4D). The schematics show the density of cross-linking in wild-type cells at −8 s and 0 s based on data from this panel and Figures 1B and 2C. (E) Average volumetric densities of actin in moving patches calculated as the ratio between the number of actin molecules (mEGFP-Act1p/5%, Figure 4C) and the volume of a sphere of the Stokes' radius (C) minus the volume of the spherical vesicle with radius of 25 nm. Inset, average radial densities of five endocytic proteins in patches of wild-type cells. Color code: black, Act1p; green, Fim1p; blue, Acp1p; purple, Acp2p; and red, Aip1p. The radial density is calculated as the ratio between the number of molecules and the Stokes' radius at each time t. The schematics are density profiles of actin in wild-type patches at three points in time based on the data in this panel and inset with the assumption that the filaments grow radially around vesicles. The blue lines illustrate how peripheral disassembly of a radially grown meshwork decreases the average distance between molecules in the meshwork, subsequently increasing its volumetric density without changing its radial density.
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Figure 5: Insights regarding the structure of endocytic patches by analysis of their motions in wild-type and mutant cells. (A) Samples of tracks of patches marked with Fim1p-mEGFP in wild-type, single-deletion mutant strains acp1∆, acp2∆, and aip1∆, and double-deletion mutant strains acp1∆/acp2∆ and acp1∆/aip1∆. The points are positions at 1-s intervals connected by lines, both color-coded in time from blue (appearance of Fim1p-mEGFP) to red (disappearance of Fim1p-mEGFP). The number of points varies with the lifetime of the patch. The gray dotted line is drawn parallel to the plasma membrane to mark the initial positions of patches. Scale bar: 200 nm. (B–E) Evolution of patch parameters over time. Symbols and lines for cell types: black plain lines, wild-type; mustard +, acp1∆; olive x, acp2∆; teal ●, aip1∆; purple ◻, double mutant acp1∆/acp2∆; brown ○, acp1∆/aip1∆. (B) Average displacements over 1 s of patches estimated from mEGFP-Act1p movies. Inset, normalized efficiency plots of the numbers of actin molecules per patch (normalized to the peak value) vs. displacement of the patch. (C) Estimated Stokes' radii of moving endocytic patches extracted from the data in B. Inset, detailed view at late time points. (D) Occupancy of fimbrin on actin filaments. The occupancy was calculated as the ratio between the numbers of actin (mEGFP-Act1p/5%, Figure 4C) and Fim1p-mEGFP (Figure 4D). The schematics show the density of cross-linking in wild-type cells at −8 s and 0 s based on data from this panel and Figures 1B and 2C. (E) Average volumetric densities of actin in moving patches calculated as the ratio between the number of actin molecules (mEGFP-Act1p/5%, Figure 4C) and the volume of a sphere of the Stokes' radius (C) minus the volume of the spherical vesicle with radius of 25 nm. Inset, average radial densities of five endocytic proteins in patches of wild-type cells. Color code: black, Act1p; green, Fim1p; blue, Acp1p; purple, Acp2p; and red, Aip1p. The radial density is calculated as the ratio between the number of molecules and the Stokes' radius at each time t. The schematics are density profiles of actin in wild-type patches at three points in time based on the data in this panel and inset with the assumption that the filaments grow radially around vesicles. The blue lines illustrate how peripheral disassembly of a radially grown meshwork decreases the average distance between molecules in the meshwork, subsequently increasing its volumetric density without changing its radial density.

Mentions: In all mutant and wild-type cells, endocytic patches were stationary at the membrane until the peak of actin accumulation at time zero and then moved in the cytoplasm with continuous changes in direction and displacement (Figure 5, A and B). Our alignment method made it possible to extract average displacements of patches over time (Figure 5B). In all mutants, the average displacement increased with time, but displacement increased more slowly and reached lower maximum rates than in wild-type cells. These defects were slightly more severe in double mutants acp1Δ/acp2Δ and acp1Δ/aip1Δ.


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)

Insights regarding the structure of endocytic patches by analysis of their motions in wild-type and mutant cells. (A) Samples of tracks of patches marked with Fim1p-mEGFP in wild-type, single-deletion mutant strains acp1∆, acp2∆, and aip1∆, and double-deletion mutant strains acp1∆/acp2∆ and acp1∆/aip1∆. The points are positions at 1-s intervals connected by lines, both color-coded in time from blue (appearance of Fim1p-mEGFP) to red (disappearance of Fim1p-mEGFP). The number of points varies with the lifetime of the patch. The gray dotted line is drawn parallel to the plasma membrane to mark the initial positions of patches. Scale bar: 200 nm. (B–E) Evolution of patch parameters over time. Symbols and lines for cell types: black plain lines, wild-type; mustard +, acp1∆; olive x, acp2∆; teal ●, aip1∆; purple ◻, double mutant acp1∆/acp2∆; brown ○, acp1∆/aip1∆. (B) Average displacements over 1 s of patches estimated from mEGFP-Act1p movies. Inset, normalized efficiency plots of the numbers of actin molecules per patch (normalized to the peak value) vs. displacement of the patch. (C) Estimated Stokes' radii of moving endocytic patches extracted from the data in B. Inset, detailed view at late time points. (D) Occupancy of fimbrin on actin filaments. The occupancy was calculated as the ratio between the numbers of actin (mEGFP-Act1p/5%, Figure 4C) and Fim1p-mEGFP (Figure 4D). The schematics show the density of cross-linking in wild-type cells at −8 s and 0 s based on data from this panel and Figures 1B and 2C. (E) Average volumetric densities of actin in moving patches calculated as the ratio between the number of actin molecules (mEGFP-Act1p/5%, Figure 4C) and the volume of a sphere of the Stokes' radius (C) minus the volume of the spherical vesicle with radius of 25 nm. Inset, average radial densities of five endocytic proteins in patches of wild-type cells. Color code: black, Act1p; green, Fim1p; blue, Acp1p; purple, Acp2p; and red, Aip1p. The radial density is calculated as the ratio between the number of molecules and the Stokes' radius at each time t. The schematics are density profiles of actin in wild-type patches at three points in time based on the data in this panel and inset with the assumption that the filaments grow radially around vesicles. The blue lines illustrate how peripheral disassembly of a radially grown meshwork decreases the average distance between molecules in the meshwork, subsequently increasing its volumetric density without changing its radial density.
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Related In: Results  -  Collection

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Figure 5: Insights regarding the structure of endocytic patches by analysis of their motions in wild-type and mutant cells. (A) Samples of tracks of patches marked with Fim1p-mEGFP in wild-type, single-deletion mutant strains acp1∆, acp2∆, and aip1∆, and double-deletion mutant strains acp1∆/acp2∆ and acp1∆/aip1∆. The points are positions at 1-s intervals connected by lines, both color-coded in time from blue (appearance of Fim1p-mEGFP) to red (disappearance of Fim1p-mEGFP). The number of points varies with the lifetime of the patch. The gray dotted line is drawn parallel to the plasma membrane to mark the initial positions of patches. Scale bar: 200 nm. (B–E) Evolution of patch parameters over time. Symbols and lines for cell types: black plain lines, wild-type; mustard +, acp1∆; olive x, acp2∆; teal ●, aip1∆; purple ◻, double mutant acp1∆/acp2∆; brown ○, acp1∆/aip1∆. (B) Average displacements over 1 s of patches estimated from mEGFP-Act1p movies. Inset, normalized efficiency plots of the numbers of actin molecules per patch (normalized to the peak value) vs. displacement of the patch. (C) Estimated Stokes' radii of moving endocytic patches extracted from the data in B. Inset, detailed view at late time points. (D) Occupancy of fimbrin on actin filaments. The occupancy was calculated as the ratio between the numbers of actin (mEGFP-Act1p/5%, Figure 4C) and Fim1p-mEGFP (Figure 4D). The schematics show the density of cross-linking in wild-type cells at −8 s and 0 s based on data from this panel and Figures 1B and 2C. (E) Average volumetric densities of actin in moving patches calculated as the ratio between the number of actin molecules (mEGFP-Act1p/5%, Figure 4C) and the volume of a sphere of the Stokes' radius (C) minus the volume of the spherical vesicle with radius of 25 nm. Inset, average radial densities of five endocytic proteins in patches of wild-type cells. Color code: black, Act1p; green, Fim1p; blue, Acp1p; purple, Acp2p; and red, Aip1p. The radial density is calculated as the ratio between the number of molecules and the Stokes' radius at each time t. The schematics are density profiles of actin in wild-type patches at three points in time based on the data in this panel and inset with the assumption that the filaments grow radially around vesicles. The blue lines illustrate how peripheral disassembly of a radially grown meshwork decreases the average distance between molecules in the meshwork, subsequently increasing its volumetric density without changing its radial density.
Mentions: In all mutant and wild-type cells, endocytic patches were stationary at the membrane until the peak of actin accumulation at time zero and then moved in the cytoplasm with continuous changes in direction and displacement (Figure 5, A and B). Our alignment method made it possible to extract average displacements of patches over time (Figure 5B). In all mutants, the average displacement increased with time, but displacement increased more slowly and reached lower maximum rates than in wild-type cells. These defects were slightly more severe in double mutants acp1Δ/acp2Δ and acp1Δ/aip1Δ.

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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