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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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Time course of protein appearance, disappearance, and movements in actin patches. Time zero corresponds to the peak of actin and the onset of movements. Dark lines are the average values over time; light lines are ± 1 SD of the means. Color code: green, Fim1p-mEGFP; black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; and red, mEGFP-Aip1p. Data sets were aligned according to the two-color data in Figures S1 and S2. (A) Numbers of molecules over time. Fim1p, Acp1p, Acp2p, and Aip1p were tagged in the genome, so the numbers are the total numbers of each protein in patches. mEGFP-actin was expressed from the leu1 locus under the control of the 41xnmt promoter and represents 5% of the total actin. (B) Occupancy of endocytic proteins on actin filaments. The occupancy was calculated as the ratio between the numbers of actin subunits (number of mEGFP-Act1p/5%) and Fim1p-mEGFP, Acp1p-mEGFP, Acp2p-mEGFP, or mEGFP-Aip1p measured in A. (B) Inset, ratio between the numbers of mEGFP-Aip1p and Acp1p-mEGFP. (C) Average displacements over 1-s intervals of patches marked by each tagged protein.
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Figure 1: Time course of protein appearance, disappearance, and movements in actin patches. Time zero corresponds to the peak of actin and the onset of movements. Dark lines are the average values over time; light lines are ± 1 SD of the means. Color code: green, Fim1p-mEGFP; black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; and red, mEGFP-Aip1p. Data sets were aligned according to the two-color data in Figures S1 and S2. (A) Numbers of molecules over time. Fim1p, Acp1p, Acp2p, and Aip1p were tagged in the genome, so the numbers are the total numbers of each protein in patches. mEGFP-actin was expressed from the leu1 locus under the control of the 41xnmt promoter and represents 5% of the total actin. (B) Occupancy of endocytic proteins on actin filaments. The occupancy was calculated as the ratio between the numbers of actin subunits (number of mEGFP-Act1p/5%) and Fim1p-mEGFP, Acp1p-mEGFP, Acp2p-mEGFP, or mEGFP-Aip1p measured in A. (B) Inset, ratio between the numbers of mEGFP-Aip1p and Acp1p-mEGFP. (C) Average displacements over 1-s intervals of patches marked by each tagged protein.

Mentions: We used the temporal superresolution method (Berro and Pollard, 2014) to align the temporal evolution of the numbers of molecules in samples of individual patches from each strain (Figure 1A). This method improved the time resolution of the averaged data and reduced the artificial variability created by discrete alignment of data collected at time intervals of 1 s. We realigned the averaged data sets using two-color data with Fim1p-mCherry as the reference (Supplemental Figures S1 and S2 and Supplemental Tables S4 and S5). Using this objective internal standard was essential, because mEGFP-Aip1p arrives after patches start moving (the temporal benchmark used previously). It also allowed for meaningful calculations of molar ratios (Figure 1B) and displacements (Figure 1C).


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)

Time course of protein appearance, disappearance, and movements in actin patches. Time zero corresponds to the peak of actin and the onset of movements. Dark lines are the average values over time; light lines are ± 1 SD of the means. Color code: green, Fim1p-mEGFP; black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; and red, mEGFP-Aip1p. Data sets were aligned according to the two-color data in Figures S1 and S2. (A) Numbers of molecules over time. Fim1p, Acp1p, Acp2p, and Aip1p were tagged in the genome, so the numbers are the total numbers of each protein in patches. mEGFP-actin was expressed from the leu1 locus under the control of the 41xnmt promoter and represents 5% of the total actin. (B) Occupancy of endocytic proteins on actin filaments. The occupancy was calculated as the ratio between the numbers of actin subunits (number of mEGFP-Act1p/5%) and Fim1p-mEGFP, Acp1p-mEGFP, Acp2p-mEGFP, or mEGFP-Aip1p measured in A. (B) Inset, ratio between the numbers of mEGFP-Aip1p and Acp1p-mEGFP. (C) Average displacements over 1-s intervals of patches marked by each tagged protein.
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Related In: Results  -  Collection

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Figure 1: Time course of protein appearance, disappearance, and movements in actin patches. Time zero corresponds to the peak of actin and the onset of movements. Dark lines are the average values over time; light lines are ± 1 SD of the means. Color code: green, Fim1p-mEGFP; black, mEGFP-actin; blue, capping protein subunit Acp1p-mEGFP; purple, capping protein subunit Acp2p-mEGFP; and red, mEGFP-Aip1p. Data sets were aligned according to the two-color data in Figures S1 and S2. (A) Numbers of molecules over time. Fim1p, Acp1p, Acp2p, and Aip1p were tagged in the genome, so the numbers are the total numbers of each protein in patches. mEGFP-actin was expressed from the leu1 locus under the control of the 41xnmt promoter and represents 5% of the total actin. (B) Occupancy of endocytic proteins on actin filaments. The occupancy was calculated as the ratio between the numbers of actin subunits (number of mEGFP-Act1p/5%) and Fim1p-mEGFP, Acp1p-mEGFP, Acp2p-mEGFP, or mEGFP-Aip1p measured in A. (B) Inset, ratio between the numbers of mEGFP-Aip1p and Acp1p-mEGFP. (C) Average displacements over 1-s intervals of patches marked by each tagged protein.
Mentions: We used the temporal superresolution method (Berro and Pollard, 2014) to align the temporal evolution of the numbers of molecules in samples of individual patches from each strain (Figure 1A). This method improved the time resolution of the averaged data and reduced the artificial variability created by discrete alignment of data collected at time intervals of 1 s. We realigned the averaged data sets using two-color data with Fim1p-mCherry as the reference (Supplemental Figures S1 and S2 and Supplemental Tables S4 and S5). Using this objective internal standard was essential, because mEGFP-Aip1p arrives after patches start moving (the temporal benchmark used previously). It also allowed for meaningful calculations of molar ratios (Figure 1B) and displacements (Figure 1C).

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