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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 the concentrations of endocytic adaptor proteins End4p and Pan1p in endocytic patches. (A and B) Raw and average time courses of the number of molecules per patch (top panels, solid lines) and distances traveled by markers associated with patches (bottom panels, dashed lines). (A) End4p. (B) Pan1p. Time courses for YFP-labeled (red, ○) and GFP-labeled (blue, ●) proteins in individual patches were aligned to initiation of patch movement at time 0 and averaged at each time point to produce averaged time courses (black, ■). Numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells. Numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells. Averages include data from three YFP-labeled and three GFP-labeled patches. (C) Comparison of average time courses for (○) clathrin heavy-chain Chc1p, (□) clathrin light-chain Clc1p, (△) End4p, (◊) Pan1p, and (●) ARPC5 subunit of Arp2/3 complex. Time courses were aligned to the initiation of patch movement (time 0). The Arp2/3 complex time course was corrected (*) to match the timing of patch assembly of fimbrin.
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Figure 4: Time courses of the concentrations of endocytic adaptor proteins End4p and Pan1p in endocytic patches. (A and B) Raw and average time courses of the number of molecules per patch (top panels, solid lines) and distances traveled by markers associated with patches (bottom panels, dashed lines). (A) End4p. (B) Pan1p. Time courses for YFP-labeled (red, ○) and GFP-labeled (blue, ●) proteins in individual patches were aligned to initiation of patch movement at time 0 and averaged at each time point to produce averaged time courses (black, ■). Numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells. Numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells. Averages include data from three YFP-labeled and three GFP-labeled patches. (C) Comparison of average time courses for (○) clathrin heavy-chain Chc1p, (□) clathrin light-chain Clc1p, (△) End4p, (◊) Pan1p, and (●) ARPC5 subunit of Arp2/3 complex. Time courses were aligned to the initiation of patch movement (time 0). The Arp2/3 complex time course was corrected (*) to match the timing of patch assembly of fimbrin.

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 the concentrations of endocytic adaptor proteins End4p and Pan1p in endocytic patches. (A and B) Raw and average time courses of the number of molecules per patch (top panels, solid lines) and distances traveled by markers associated with patches (bottom panels, dashed lines). (A) End4p. (B) Pan1p. Time courses for YFP-labeled (red, ○) and GFP-labeled (blue, ●) proteins in individual patches were aligned to initiation of patch movement at time 0 and averaged at each time point to produce averaged time courses (black, ■). Numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells. Numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells. Averages include data from three YFP-labeled and three GFP-labeled patches. (C) Comparison of average time courses for (○) clathrin heavy-chain Chc1p, (□) clathrin light-chain Clc1p, (△) End4p, (◊) Pan1p, and (●) ARPC5 subunit of Arp2/3 complex. Time courses were aligned to the initiation of patch movement (time 0). The Arp2/3 complex time course was corrected (*) to match the timing of patch assembly of fimbrin.
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Related In: Results  -  Collection

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Figure 4: Time courses of the concentrations of endocytic adaptor proteins End4p and Pan1p in endocytic patches. (A and B) Raw and average time courses of the number of molecules per patch (top panels, solid lines) and distances traveled by markers associated with patches (bottom panels, dashed lines). (A) End4p. (B) Pan1p. Time courses for YFP-labeled (red, ○) and GFP-labeled (blue, ●) proteins in individual patches were aligned to initiation of patch movement at time 0 and averaged at each time point to produce averaged time courses (black, ■). Numbers of GFP-labeled proteins were calculated from time-lapse movies of Z-sections through entire cells. Numbers of YFP-labeled proteins were calculated from time-lapse movies in single sections through the middle of cells. Averages include data from three YFP-labeled and three GFP-labeled patches. (C) Comparison of average time courses for (○) clathrin heavy-chain Chc1p, (□) clathrin light-chain Clc1p, (△) End4p, (◊) Pan1p, and (●) ARPC5 subunit of Arp2/3 complex. Time courses were aligned to the initiation of patch movement (time 0). The Arp2/3 complex time course was corrected (*) to match the timing of patch assembly of fimbrin.
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