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

Diffusive movements of the same sample of 24 endocytic patches as Figure 3D. (A) Endocytic patch displacements and (B) distances from their origins (i.e., position of first appearance). Black lines are average displacements and distances after continuous alignment; gray lines are ± 1 SD; gray shading is ± 1 SEM. Both figures feature three patches shown in inset in A and identified as red, green, and blue. Dots in the inset of A show the positions of the centers of these three patches in the x,y-plane at successive 1-s time intervals. All three tracks are aligned at a common origin at time zero. The brightness of the color decreases over time from dark at the beginning to light at the end. Gray horizontal line: position of the plasma membrane. Scale bar: 200 nm. (C) Changes in direction of 24 endocytic patches over time. Each dot is a different time point and each color corresponds to one of the 24 patches. (D) Histogram of the frequency of changes in direction angles for the 24 patches. (E) Cumulative frequency of changes in direction angles of the 24 patches. The distribution does not differ significantly from a uniform distribution, as expected for a diffusive motion. Blue: same data as C; red: a uniform distribution between 0 and 180°. (F) Plot of displacement vs. direction change of the 24 patches shows no obvious correlation. Only data after time zero are represented.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230612&req=5

Figure 4: Diffusive movements of the same sample of 24 endocytic patches as Figure 3D. (A) Endocytic patch displacements and (B) distances from their origins (i.e., position of first appearance). Black lines are average displacements and distances after continuous alignment; gray lines are ± 1 SD; gray shading is ± 1 SEM. Both figures feature three patches shown in inset in A and identified as red, green, and blue. Dots in the inset of A show the positions of the centers of these three patches in the x,y-plane at successive 1-s time intervals. All three tracks are aligned at a common origin at time zero. The brightness of the color decreases over time from dark at the beginning to light at the end. Gray horizontal line: position of the plasma membrane. Scale bar: 200 nm. (C) Changes in direction of 24 endocytic patches over time. Each dot is a different time point and each color corresponds to one of the 24 patches. (D) Histogram of the frequency of changes in direction angles for the 24 patches. (E) Cumulative frequency of changes in direction angles of the 24 patches. The distribution does not differ significantly from a uniform distribution, as expected for a diffusive motion. Blue: same data as C; red: a uniform distribution between 0 and 180°. (F) Plot of displacement vs. direction change of the 24 patches shows no obvious correlation. Only data after time zero are represented.

Mentions: The highly reproducible temporal evolution of the number of fimbrin molecules in the endocytic patches makes the full time course a better criterion for aligning data from multiple patches than any measurement of patch movement. Once the data sets are aligned in time, one may calculate other parameters, such as displacement (Figure 4A) and distance from the origin (Figure 4B). Without precise alignment, it is impossible to accurately extract these other features from the data.


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)

Diffusive movements of the same sample of 24 endocytic patches as Figure 3D. (A) Endocytic patch displacements and (B) distances from their origins (i.e., position of first appearance). Black lines are average displacements and distances after continuous alignment; gray lines are ± 1 SD; gray shading is ± 1 SEM. Both figures feature three patches shown in inset in A and identified as red, green, and blue. Dots in the inset of A show the positions of the centers of these three patches in the x,y-plane at successive 1-s time intervals. All three tracks are aligned at a common origin at time zero. The brightness of the color decreases over time from dark at the beginning to light at the end. Gray horizontal line: position of the plasma membrane. Scale bar: 200 nm. (C) Changes in direction of 24 endocytic patches over time. Each dot is a different time point and each color corresponds to one of the 24 patches. (D) Histogram of the frequency of changes in direction angles for the 24 patches. (E) Cumulative frequency of changes in direction angles of the 24 patches. The distribution does not differ significantly from a uniform distribution, as expected for a diffusive motion. Blue: same data as C; red: a uniform distribution between 0 and 180°. (F) Plot of displacement vs. direction change of the 24 patches shows no obvious correlation. Only data after time zero are represented.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Diffusive movements of the same sample of 24 endocytic patches as Figure 3D. (A) Endocytic patch displacements and (B) distances from their origins (i.e., position of first appearance). Black lines are average displacements and distances after continuous alignment; gray lines are ± 1 SD; gray shading is ± 1 SEM. Both figures feature three patches shown in inset in A and identified as red, green, and blue. Dots in the inset of A show the positions of the centers of these three patches in the x,y-plane at successive 1-s time intervals. All three tracks are aligned at a common origin at time zero. The brightness of the color decreases over time from dark at the beginning to light at the end. Gray horizontal line: position of the plasma membrane. Scale bar: 200 nm. (C) Changes in direction of 24 endocytic patches over time. Each dot is a different time point and each color corresponds to one of the 24 patches. (D) Histogram of the frequency of changes in direction angles for the 24 patches. (E) Cumulative frequency of changes in direction angles of the 24 patches. The distribution does not differ significantly from a uniform distribution, as expected for a diffusive motion. Blue: same data as C; red: a uniform distribution between 0 and 180°. (F) Plot of displacement vs. direction change of the 24 patches shows no obvious correlation. Only data after time zero are represented.
Mentions: The highly reproducible temporal evolution of the number of fimbrin molecules in the endocytic patches makes the full time course a better criterion for aligning data from multiple patches than any measurement of patch movement. Once the data sets are aligned in time, one may calculate other parameters, such as displacement (Figure 4A) and distance from the origin (Figure 4B). Without precise alignment, it is impossible to accurately extract these other features from the data.

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