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Local and global analysis of endocytic patch dynamics in fission yeast using a new "temporal superresolution" realignment method.

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

Bottom Line: These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP.We show that the time course of actin assembly and disassembly varies <600 ms between patches.Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology Department of Molecular Biophysics and Biochemistry 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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Polymerization efficiency plots of the number of molecules per patch vs. (A) the distance from origin or (B) the displacement of the patch. The data points in the lower left are the first measured on individual patches (colored lines) or averaged from 24 patches (black lines). The successive data points are at 1-s intervals for the individual patches (colored lines) and 0.5-s intervals for the averaged data (black line). Black lines: data based on the average for 24 patches after continuous alignment in time using intensities; blue, red, and green are representative data for individual patches from Figure 3A. The rate of patch movements increases only after the maximum fimbrin accumulation, as seen for individual tracks (colors) and for averaged data (black).
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Figure 6: Polymerization efficiency plots of the number of molecules per patch vs. (A) the distance from origin or (B) the displacement of the patch. The data points in the lower left are the first measured on individual patches (colored lines) or averaged from 24 patches (black lines). The successive data points are at 1-s intervals for the individual patches (colored lines) and 0.5-s intervals for the averaged data (black line). Black lines: data based on the average for 24 patches after continuous alignment in time using intensities; blue, red, and green are representative data for individual patches from Figure 3A. The rate of patch movements increases only after the maximum fimbrin accumulation, as seen for individual tracks (colors) and for averaged data (black).

Mentions: To characterize the role of actin polymerization in patches, we plotted the number of fimbrin molecules versus displacement of the patch (Figure 6A; or distance from its origin, Figure 6B). Owing to their random movements, plots of individual patches were noisy, but average data from 24 patches made smooth curves (black lines). These plots revealed two phases separated by a sharp transition. The first phase began with the data point in the lower left corner at time −6 s, when displacement was slow (<50 nm/s) and only 200 fimbrin molecules associated with a patch. Over the next 6 s, ∼800 molecules of fimbrin accumulated, but the displacement remained low (nearly vertical black line). The second phase began at 0 s, when the number of fimbrin molecules peaked and the displacement increased abruptly to ∼130 nm/s (horizontal black line). Thereafter the displacement increased slowly to up to 200 nm/s as fimbrin disassociated. Thus actin polymerizes during the formation of clathrin-coated pits but disassembles as the vesicle moves.


Local and global analysis of endocytic patch dynamics in fission yeast using a new "temporal superresolution" realignment method.

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

Polymerization efficiency plots of the number of molecules per patch vs. (A) the distance from origin or (B) the displacement of the patch. The data points in the lower left are the first measured on individual patches (colored lines) or averaged from 24 patches (black lines). The successive data points are at 1-s intervals for the individual patches (colored lines) and 0.5-s intervals for the averaged data (black line). Black lines: data based on the average for 24 patches after continuous alignment in time using intensities; blue, red, and green are representative data for individual patches from Figure 3A. The rate of patch movements increases only after the maximum fimbrin accumulation, as seen for individual tracks (colors) and for averaged data (black).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230612&req=5

Figure 6: Polymerization efficiency plots of the number of molecules per patch vs. (A) the distance from origin or (B) the displacement of the patch. The data points in the lower left are the first measured on individual patches (colored lines) or averaged from 24 patches (black lines). The successive data points are at 1-s intervals for the individual patches (colored lines) and 0.5-s intervals for the averaged data (black line). Black lines: data based on the average for 24 patches after continuous alignment in time using intensities; blue, red, and green are representative data for individual patches from Figure 3A. The rate of patch movements increases only after the maximum fimbrin accumulation, as seen for individual tracks (colors) and for averaged data (black).
Mentions: To characterize the role of actin polymerization in patches, we plotted the number of fimbrin molecules versus displacement of the patch (Figure 6A; or distance from its origin, Figure 6B). Owing to their random movements, plots of individual patches were noisy, but average data from 24 patches made smooth curves (black lines). These plots revealed two phases separated by a sharp transition. The first phase began with the data point in the lower left corner at time −6 s, when displacement was slow (<50 nm/s) and only 200 fimbrin molecules associated with a patch. Over the next 6 s, ∼800 molecules of fimbrin accumulated, but the displacement remained low (nearly vertical black line). The second phase began at 0 s, when the number of fimbrin molecules peaked and the displacement increased abruptly to ∼130 nm/s (horizontal black line). Thereafter the displacement increased slowly to up to 200 nm/s as fimbrin disassociated. Thus actin polymerizes during the formation of clathrin-coated pits but disassembles as the vesicle moves.

Bottom Line: These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP.We show that the time course of actin assembly and disassembly varies <600 ms between patches.Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology Department of Molecular Biophysics and Biochemistry 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