Limits...
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.

Show MeSH

Related in: MedlinePlus

Distribution of patches along the long axis of asynchronous fission yeast cells imaged at single points in time. (A) Distributions of patches in small-sized (olive) and medium-sized (teal) cells in interphase and a cell in mitosis (purple). The images are sum projections of cells expressing Fim1p-mEGFP at their native locus from 18 consecutive confocal z-slices spaced at 360-nm intervals. Blue lines: distributions along the long axis measured from the fluorescence intensity and the mean fluorescence per patch. Black numbers: direct manual counts of patches in the zones between two red vertical dashed lines from stacks of confocal images. Blue numbers: count of patches in the same zone estimated from the patch density distribution. Scale bars: 5 μm. (B) Schematic explaining our definitions for polarization and dispersion and how the OP50 index changes accordingly. (C) Numbers of patches in 47 cells vs. their lengths, a proxy for stage of the cell cycle. Points colored olive, teal, and purple are data from the cells in A. (D) Distribution of patches in the cell vs. cell length. Red, left third of the cell; green, middle third of the cell; blue, right third of the cell. (E) Tip symmetry index, the ratio of the number of patches in each tip, vs. cell length. A perfectly symmetrical distribution would have a symmetry index of 1. (F) Dispersion index vs. cell length. The OP50 index represents the percentage of the length of a cell containing 50% of total patches (see Materials and Methods).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230612&req=5

Figure 7: Distribution of patches along the long axis of asynchronous fission yeast cells imaged at single points in time. (A) Distributions of patches in small-sized (olive) and medium-sized (teal) cells in interphase and a cell in mitosis (purple). The images are sum projections of cells expressing Fim1p-mEGFP at their native locus from 18 consecutive confocal z-slices spaced at 360-nm intervals. Blue lines: distributions along the long axis measured from the fluorescence intensity and the mean fluorescence per patch. Black numbers: direct manual counts of patches in the zones between two red vertical dashed lines from stacks of confocal images. Blue numbers: count of patches in the same zone estimated from the patch density distribution. Scale bars: 5 μm. (B) Schematic explaining our definitions for polarization and dispersion and how the OP50 index changes accordingly. (C) Numbers of patches in 47 cells vs. their lengths, a proxy for stage of the cell cycle. Points colored olive, teal, and purple are data from the cells in A. (D) Distribution of patches in the cell vs. cell length. Red, left third of the cell; green, middle third of the cell; blue, right third of the cell. (E) Tip symmetry index, the ratio of the number of patches in each tip, vs. cell length. A perfectly symmetrical distribution would have a symmetry index of 1. (F) Dispersion index vs. cell length. The OP50 index represents the percentage of the length of a cell containing 50% of total patches (see Materials and Methods).

Mentions: Figure 7A illustrates how one may estimate the total number of patches in a cell by measuring the total fluorescence intensity of an entire cell, subtracting the total fluorescence intensity of the cytoplasm and the surrounding media, then dividing the difference by the temporal average intensity of a patch (calculated from the data in Figure 3D). This process is valid in any part of a cell, so one can estimate the local density of patches along the long axis of a cell using the same formula at each position (Figure 7A). The precision of this measurement can be estimated formally and the relative error depends on the ratio between the temporal average intensity of a patch and its SD and on the inverse of the square root of the number of patches (see Materials and Methods). More practically, the error in the calculated number of patches is around 25% for regions with a small number of patches (i.e., ∼5 patches) and < 5% for the total number of patches in a whole cell.


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)

Distribution of patches along the long axis of asynchronous fission yeast cells imaged at single points in time. (A) Distributions of patches in small-sized (olive) and medium-sized (teal) cells in interphase and a cell in mitosis (purple). The images are sum projections of cells expressing Fim1p-mEGFP at their native locus from 18 consecutive confocal z-slices spaced at 360-nm intervals. Blue lines: distributions along the long axis measured from the fluorescence intensity and the mean fluorescence per patch. Black numbers: direct manual counts of patches in the zones between two red vertical dashed lines from stacks of confocal images. Blue numbers: count of patches in the same zone estimated from the patch density distribution. Scale bars: 5 μm. (B) Schematic explaining our definitions for polarization and dispersion and how the OP50 index changes accordingly. (C) Numbers of patches in 47 cells vs. their lengths, a proxy for stage of the cell cycle. Points colored olive, teal, and purple are data from the cells in A. (D) Distribution of patches in the cell vs. cell length. Red, left third of the cell; green, middle third of the cell; blue, right third of the cell. (E) Tip symmetry index, the ratio of the number of patches in each tip, vs. cell length. A perfectly symmetrical distribution would have a symmetry index of 1. (F) Dispersion index vs. cell length. The OP50 index represents the percentage of the length of a cell containing 50% of total patches (see Materials and Methods).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230612&req=5

Figure 7: Distribution of patches along the long axis of asynchronous fission yeast cells imaged at single points in time. (A) Distributions of patches in small-sized (olive) and medium-sized (teal) cells in interphase and a cell in mitosis (purple). The images are sum projections of cells expressing Fim1p-mEGFP at their native locus from 18 consecutive confocal z-slices spaced at 360-nm intervals. Blue lines: distributions along the long axis measured from the fluorescence intensity and the mean fluorescence per patch. Black numbers: direct manual counts of patches in the zones between two red vertical dashed lines from stacks of confocal images. Blue numbers: count of patches in the same zone estimated from the patch density distribution. Scale bars: 5 μm. (B) Schematic explaining our definitions for polarization and dispersion and how the OP50 index changes accordingly. (C) Numbers of patches in 47 cells vs. their lengths, a proxy for stage of the cell cycle. Points colored olive, teal, and purple are data from the cells in A. (D) Distribution of patches in the cell vs. cell length. Red, left third of the cell; green, middle third of the cell; blue, right third of the cell. (E) Tip symmetry index, the ratio of the number of patches in each tip, vs. cell length. A perfectly symmetrical distribution would have a symmetry index of 1. (F) Dispersion index vs. cell length. The OP50 index represents the percentage of the length of a cell containing 50% of total patches (see Materials and Methods).
Mentions: Figure 7A illustrates how one may estimate the total number of patches in a cell by measuring the total fluorescence intensity of an entire cell, subtracting the total fluorescence intensity of the cytoplasm and the surrounding media, then dividing the difference by the temporal average intensity of a patch (calculated from the data in Figure 3D). This process is valid in any part of a cell, so one can estimate the local density of patches along the long axis of a cell using the same formula at each position (Figure 7A). The precision of this measurement can be estimated formally and the relative error depends on the ratio between the temporal average intensity of a patch and its SD and on the inverse of the square root of the number of patches (see Materials and Methods). More practically, the error in the calculated number of patches is around 25% for regions with a small number of patches (i.e., ∼5 patches) and < 5% for the total number of patches in a whole cell.

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.

Show MeSH
Related in: MedlinePlus