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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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(A) Diffusion coefficients and (B) Stokes' radii of 24 moving actin patches over time estimated from the average displacements in Figure 4A. Gray area: confidence interval at 95% for the diffusion coefficient and the Stokes' radius. In B, the extremities of the horizontal lines represent time points where Stokes' radii are significantly different (z-test, 5%). More statistical tests are available in Figure S4. Cartoons represent the typical size of an endocytic vesicle with or without a typical actin network (teal). (C) Stokes' radii of actin patches in cof1-M2 mutant cells with deficient actin-filament severing. Each dot corresponds to one patch tracked over 20–60 s. Stokes' radii were estimated as a temporal average of individual patches, because their fluorescence intensity did not change over this time interval. Intensities are relative to the intensity of the brightest nonmotile isolated patch measured in the field. Only the patches with average displacement above 50 nm/s are represented to assure that these vesicles were released from the plasma membrane.
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Figure 5: (A) Diffusion coefficients and (B) Stokes' radii of 24 moving actin patches over time estimated from the average displacements in Figure 4A. Gray area: confidence interval at 95% for the diffusion coefficient and the Stokes' radius. In B, the extremities of the horizontal lines represent time points where Stokes' radii are significantly different (z-test, 5%). More statistical tests are available in Figure S4. Cartoons represent the typical size of an endocytic vesicle with or without a typical actin network (teal). (C) Stokes' radii of actin patches in cof1-M2 mutant cells with deficient actin-filament severing. Each dot corresponds to one patch tracked over 20–60 s. Stokes' radii were estimated as a temporal average of individual patches, because their fluorescence intensity did not change over this time interval. Intensities are relative to the intensity of the brightest nonmotile isolated patch measured in the field. Only the patches with average displacement above 50 nm/s are represented to assure that these vesicles were released from the plasma membrane.

Mentions: First, we used the displacement of patches (Figure 4A) to calculate their Stokes' radius—the radius of an equivalent, freely diffusing hard sphere (Figure 5 and Materials and Methods). The calculated Stokes' radii declined from ∼150 nm at the onset of movement to ∼50 nm when fimbrin becomes barely detectable. These values are remarkably similar to the 150-nm radius of a newly released actin-covered vesicle measured by electron microscopy (Kanbe et al., 1989; Takagi et al., 2003; Kukulski et al., 2012) and the 30-nm radius of a naked vesicle.


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)

(A) Diffusion coefficients and (B) Stokes' radii of 24 moving actin patches over time estimated from the average displacements in Figure 4A. Gray area: confidence interval at 95% for the diffusion coefficient and the Stokes' radius. In B, the extremities of the horizontal lines represent time points where Stokes' radii are significantly different (z-test, 5%). More statistical tests are available in Figure S4. Cartoons represent the typical size of an endocytic vesicle with or without a typical actin network (teal). (C) Stokes' radii of actin patches in cof1-M2 mutant cells with deficient actin-filament severing. Each dot corresponds to one patch tracked over 20–60 s. Stokes' radii were estimated as a temporal average of individual patches, because their fluorescence intensity did not change over this time interval. Intensities are relative to the intensity of the brightest nonmotile isolated patch measured in the field. Only the patches with average displacement above 50 nm/s are represented to assure that these vesicles were released from the plasma membrane.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: (A) Diffusion coefficients and (B) Stokes' radii of 24 moving actin patches over time estimated from the average displacements in Figure 4A. Gray area: confidence interval at 95% for the diffusion coefficient and the Stokes' radius. In B, the extremities of the horizontal lines represent time points where Stokes' radii are significantly different (z-test, 5%). More statistical tests are available in Figure S4. Cartoons represent the typical size of an endocytic vesicle with or without a typical actin network (teal). (C) Stokes' radii of actin patches in cof1-M2 mutant cells with deficient actin-filament severing. Each dot corresponds to one patch tracked over 20–60 s. Stokes' radii were estimated as a temporal average of individual patches, because their fluorescence intensity did not change over this time interval. Intensities are relative to the intensity of the brightest nonmotile isolated patch measured in the field. Only the patches with average displacement above 50 nm/s are represented to assure that these vesicles were released from the plasma membrane.
Mentions: First, we used the displacement of patches (Figure 4A) to calculate their Stokes' radius—the radius of an equivalent, freely diffusing hard sphere (Figure 5 and Materials and Methods). The calculated Stokes' radii declined from ∼150 nm at the onset of movement to ∼50 nm when fimbrin becomes barely detectable. These values are remarkably similar to the 150-nm radius of a newly released actin-covered vesicle measured by electron microscopy (Kanbe et al., 1989; Takagi et al., 2003; Kukulski et al., 2012) and the 30-nm radius of a naked vesicle.

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