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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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Evolution of patch distribution over time in individual living cells. (A) Montage of four different cells expressing Fim1p-mEGFP (inverted contrast) over 215 min, imaged every 5 min. Vertical bars are 5 μm, colored to identify these cells in the other panels. Micrographs are sum projections of 18 consecutive z-slices spaced by 360 nm. (B) Kymographs of the temporal evolution of the patch density along the lengths of the four cells in A. In each subpanel, each vertical strip corresponds to the density of patches along the long axis of the cell at a given time point. The densities are color coded from dark blue for the regions of the cell with the lowest density of patches to dark red for the regions with highest densities of patches. Vertical black bar: 5 μm. Colored horizontal bars: 60 min. (C–E) Changes in the distributions of patches in cells in A over time using their lengths as a proxy for time. (C) Evolution of the number of patches over time with each point corresponding to one image from A. (D) Evolution of tip symmetry with time. (E) Evolution of the proportion of patches in the middle of the cell with time. Points, cells in interphase; crosses, cells in mitosis. The bars in B and the dots and crosses in C, D, and E have the color assigned in A.
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Figure 8: Evolution of patch distribution over time in individual living cells. (A) Montage of four different cells expressing Fim1p-mEGFP (inverted contrast) over 215 min, imaged every 5 min. Vertical bars are 5 μm, colored to identify these cells in the other panels. Micrographs are sum projections of 18 consecutive z-slices spaced by 360 nm. (B) Kymographs of the temporal evolution of the patch density along the lengths of the four cells in A. In each subpanel, each vertical strip corresponds to the density of patches along the long axis of the cell at a given time point. The densities are color coded from dark blue for the regions of the cell with the lowest density of patches to dark red for the regions with highest densities of patches. Vertical black bar: 5 μm. Colored horizontal bars: 60 min. (C–E) Changes in the distributions of patches in cells in A over time using their lengths as a proxy for time. (C) Evolution of the number of patches over time with each point corresponding to one image from A. (D) Evolution of tip symmetry with time. (E) Evolution of the proportion of patches in the middle of the cell with time. Points, cells in interphase; crosses, cells in mitosis. The bars in B and the dots and crosses in C, D, and E have the color assigned in A.

Mentions: We confirmed these measurements by following individual cells over time (Figure 8, A and B). Indeed, the number of patches in individual cells increased in proportion to length (Figure 8C). Cells stopped growing when they entered mitosis, and the number of patches remained roughly constant (Figure 8C, olive and purple crosses).


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)

Evolution of patch distribution over time in individual living cells. (A) Montage of four different cells expressing Fim1p-mEGFP (inverted contrast) over 215 min, imaged every 5 min. Vertical bars are 5 μm, colored to identify these cells in the other panels. Micrographs are sum projections of 18 consecutive z-slices spaced by 360 nm. (B) Kymographs of the temporal evolution of the patch density along the lengths of the four cells in A. In each subpanel, each vertical strip corresponds to the density of patches along the long axis of the cell at a given time point. The densities are color coded from dark blue for the regions of the cell with the lowest density of patches to dark red for the regions with highest densities of patches. Vertical black bar: 5 μm. Colored horizontal bars: 60 min. (C–E) Changes in the distributions of patches in cells in A over time using their lengths as a proxy for time. (C) Evolution of the number of patches over time with each point corresponding to one image from A. (D) Evolution of tip symmetry with time. (E) Evolution of the proportion of patches in the middle of the cell with time. Points, cells in interphase; crosses, cells in mitosis. The bars in B and the dots and crosses in C, D, and E have the color assigned in A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: Evolution of patch distribution over time in individual living cells. (A) Montage of four different cells expressing Fim1p-mEGFP (inverted contrast) over 215 min, imaged every 5 min. Vertical bars are 5 μm, colored to identify these cells in the other panels. Micrographs are sum projections of 18 consecutive z-slices spaced by 360 nm. (B) Kymographs of the temporal evolution of the patch density along the lengths of the four cells in A. In each subpanel, each vertical strip corresponds to the density of patches along the long axis of the cell at a given time point. The densities are color coded from dark blue for the regions of the cell with the lowest density of patches to dark red for the regions with highest densities of patches. Vertical black bar: 5 μm. Colored horizontal bars: 60 min. (C–E) Changes in the distributions of patches in cells in A over time using their lengths as a proxy for time. (C) Evolution of the number of patches over time with each point corresponding to one image from A. (D) Evolution of tip symmetry with time. (E) Evolution of the proportion of patches in the middle of the cell with time. Points, cells in interphase; crosses, cells in mitosis. The bars in B and the dots and crosses in C, D, and E have the color assigned in A.
Mentions: We confirmed these measurements by following individual cells over time (Figure 8, A and B). Indeed, the number of patches in individual cells increased in proportion to length (Figure 8C). Cells stopped growing when they entered mitosis, and the number of patches remained roughly constant (Figure 8C, olive and purple crosses).

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