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Quantitative analysis of the mechanism of endocytic actin patch assembly and disassembly in fission yeast.

Sirotkin V, Berro J, Macmillan K, Zhao L, Pollard TD - Mol. Biol. Cell (2010)

Bottom Line: Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s.Patch internalization occurs without recruitment of dynamins.Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

ABSTRACT
We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30-50% between individual patches. The pathway begins with accumulation of 30-40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (approximately 120 End4p and approximately 230 Pan1p), activators of Arp2/3 complex (approximately 200 Wsp1p and approximately 340 Myo1p) and approximately 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (approximately 7000 actins, approximately 200 capping proteins, and approximately 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

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Time courses of protein accumulation and loss in endocytic patches. (A) Time series of micrographs showing the accumulation and loss of YFP-labeled Pan1p, Wsp1p, Myo1p, ARPC5, Fim1p, and Crn1p in individual patches. Confocal fluorescence images through the middle of cells were collected every 3 s. Arrowheads mark the appearance, movement (except for Myo1p) and disappearance of each component. (B) A micrograph of a single confocal section through the middle of a cell expressing Fim1p-mYFP showing areas selected as patches (blue circles) and cytoplasmic background (yellow circles). Solid white line shows cell outline, and dashed white line outlines the nucleus. Scale bars, 1 μm. (C) Comparison and averaging of raw time courses for the number of molecules of YFP-labeled (red, ○) and GFP-labeled (blue, ●) fimbrin Fim1p in individual patches. Averaged time courses (black, ■) were produced by aligning individual time courses to time 0, when patch initiated movement, and averaging at each time point the molecules per patch (top panel, solid lines) and distances traveled by patches (bottom panel, dashed lines). (D) Comparison of average time courses obtained with YFP-labeled proteins (open symbols) and GFP-labeled proteins (closed symbols). Solid lines in the top panel are the average number of molecules per patch, and dashed lines in the bottom panel are distances traveled for Pan1p (blue squares), Wsp1p (red circles), ARPC5 (black triangles), and Crn1p (green diamonds) associated with patches. Each time course is an average of data from three to seven patches. In C and D the numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells, and the numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells.
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Figure 1: Time courses of protein accumulation and loss in endocytic patches. (A) Time series of micrographs showing the accumulation and loss of YFP-labeled Pan1p, Wsp1p, Myo1p, ARPC5, Fim1p, and Crn1p in individual patches. Confocal fluorescence images through the middle of cells were collected every 3 s. Arrowheads mark the appearance, movement (except for Myo1p) and disappearance of each component. (B) A micrograph of a single confocal section through the middle of a cell expressing Fim1p-mYFP showing areas selected as patches (blue circles) and cytoplasmic background (yellow circles). Solid white line shows cell outline, and dashed white line outlines the nucleus. Scale bars, 1 μm. (C) Comparison and averaging of raw time courses for the number of molecules of YFP-labeled (red, ○) and GFP-labeled (blue, ●) fimbrin Fim1p in individual patches. Averaged time courses (black, ■) were produced by aligning individual time courses to time 0, when patch initiated movement, and averaging at each time point the molecules per patch (top panel, solid lines) and distances traveled by patches (bottom panel, dashed lines). (D) Comparison of average time courses obtained with YFP-labeled proteins (open symbols) and GFP-labeled proteins (closed symbols). Solid lines in the top panel are the average number of molecules per patch, and dashed lines in the bottom panel are distances traveled for Pan1p (blue squares), Wsp1p (red circles), ARPC5 (black triangles), and Crn1p (green diamonds) associated with patches. Each time course is an average of data from three to seven patches. In C and D the numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells, and the numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells.

Mentions: The intensity of YFP or GFP fluorescence detected by microscopy is directly proportional to the number of fluorescent protein molecules (Wu and Pollard, 2005), so we used fluorescence intensity to measure the cytoplasmic concentrations of the tagged proteins in interphase cells and protein numbers in actin patches (Table 1, Tables S2 and S3). To track protein numbers in actin patches over time (Figures 1 and 2A). see also Figures 4–7), we optimized image acquisition, improved methods to correct for cytoplasmic background and photobleaching, and developed a strategy for aligning data on the same time scale.


Quantitative analysis of the mechanism of endocytic actin patch assembly and disassembly in fission yeast.

Sirotkin V, Berro J, Macmillan K, Zhao L, Pollard TD - Mol. Biol. Cell (2010)

Time courses of protein accumulation and loss in endocytic patches. (A) Time series of micrographs showing the accumulation and loss of YFP-labeled Pan1p, Wsp1p, Myo1p, ARPC5, Fim1p, and Crn1p in individual patches. Confocal fluorescence images through the middle of cells were collected every 3 s. Arrowheads mark the appearance, movement (except for Myo1p) and disappearance of each component. (B) A micrograph of a single confocal section through the middle of a cell expressing Fim1p-mYFP showing areas selected as patches (blue circles) and cytoplasmic background (yellow circles). Solid white line shows cell outline, and dashed white line outlines the nucleus. Scale bars, 1 μm. (C) Comparison and averaging of raw time courses for the number of molecules of YFP-labeled (red, ○) and GFP-labeled (blue, ●) fimbrin Fim1p in individual patches. Averaged time courses (black, ■) were produced by aligning individual time courses to time 0, when patch initiated movement, and averaging at each time point the molecules per patch (top panel, solid lines) and distances traveled by patches (bottom panel, dashed lines). (D) Comparison of average time courses obtained with YFP-labeled proteins (open symbols) and GFP-labeled proteins (closed symbols). Solid lines in the top panel are the average number of molecules per patch, and dashed lines in the bottom panel are distances traveled for Pan1p (blue squares), Wsp1p (red circles), ARPC5 (black triangles), and Crn1p (green diamonds) associated with patches. Each time course is an average of data from three to seven patches. In C and D the numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells, and the numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells.
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Related In: Results  -  Collection

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Figure 1: Time courses of protein accumulation and loss in endocytic patches. (A) Time series of micrographs showing the accumulation and loss of YFP-labeled Pan1p, Wsp1p, Myo1p, ARPC5, Fim1p, and Crn1p in individual patches. Confocal fluorescence images through the middle of cells were collected every 3 s. Arrowheads mark the appearance, movement (except for Myo1p) and disappearance of each component. (B) A micrograph of a single confocal section through the middle of a cell expressing Fim1p-mYFP showing areas selected as patches (blue circles) and cytoplasmic background (yellow circles). Solid white line shows cell outline, and dashed white line outlines the nucleus. Scale bars, 1 μm. (C) Comparison and averaging of raw time courses for the number of molecules of YFP-labeled (red, ○) and GFP-labeled (blue, ●) fimbrin Fim1p in individual patches. Averaged time courses (black, ■) were produced by aligning individual time courses to time 0, when patch initiated movement, and averaging at each time point the molecules per patch (top panel, solid lines) and distances traveled by patches (bottom panel, dashed lines). (D) Comparison of average time courses obtained with YFP-labeled proteins (open symbols) and GFP-labeled proteins (closed symbols). Solid lines in the top panel are the average number of molecules per patch, and dashed lines in the bottom panel are distances traveled for Pan1p (blue squares), Wsp1p (red circles), ARPC5 (black triangles), and Crn1p (green diamonds) associated with patches. Each time course is an average of data from three to seven patches. In C and D the numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells, and the numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells.
Mentions: The intensity of YFP or GFP fluorescence detected by microscopy is directly proportional to the number of fluorescent protein molecules (Wu and Pollard, 2005), so we used fluorescence intensity to measure the cytoplasmic concentrations of the tagged proteins in interphase cells and protein numbers in actin patches (Table 1, Tables S2 and S3). To track protein numbers in actin patches over time (Figures 1 and 2A). see also Figures 4–7), we optimized image acquisition, improved methods to correct for cytoplasmic background and photobleaching, and developed a strategy for aligning data on the same time scale.

Bottom Line: Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s.Patch internalization occurs without recruitment of dynamins.Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

ABSTRACT
We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30-50% between individual patches. The pathway begins with accumulation of 30-40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (approximately 120 End4p and approximately 230 Pan1p), activators of Arp2/3 complex (approximately 200 Wsp1p and approximately 340 Myo1p) and approximately 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (approximately 7000 actins, approximately 200 capping proteins, and approximately 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

Show MeSH
Related in: MedlinePlus