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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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Summary time course of the assembly and disassembly of actin patch proteins at the site of endocytosis. (A) Summary time course of the number of molecules (solid lines) and distance traveled (dashed lines) of 12 endocytic actin patch components. Each time course is an average of data for three YFP-labeled patches for clathrin, seven YFP-labeled patches for Wsp1p, six GFP-labeled patches for Act1p, and three YFP- and three GFP-labeled patches for all other components. Numbers of GFP-labeled proteins were calculated from time-lapse 3D image series and numbers of YFP-labeled proteins were calculated from time-lapse series in one confocal section. All time courses were aligned to time 0, which marks initiation of patch movement, except for Myo1p, which was aligned with the Wsp1p peak. The ARPC5 time course was corrected so that the timing of patch assembly matched that of fimbrin. mGFP-Act1p is estimated to represent 6% of total actin. (B) Schematic diagram illustrating counts of proteins associated with an actin patch at the site of endocytic invagination at times −10, −3, and 0 s. Colors of fonts correspond to the colors in A. Numbers in parenthesis indicate the peak numbers of each protein, except for End4p and Pan1p at −10 s (asterisks). All of the proteins listed below Arp2/3 complex are presumed to be distributed throughout the actin network (shaded teal). The approximate positions of other proteins are adapted from Idrissi et al. (2008).
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Figure 7: Summary time course of the assembly and disassembly of actin patch proteins at the site of endocytosis. (A) Summary time course of the number of molecules (solid lines) and distance traveled (dashed lines) of 12 endocytic actin patch components. Each time course is an average of data for three YFP-labeled patches for clathrin, seven YFP-labeled patches for Wsp1p, six GFP-labeled patches for Act1p, and three YFP- and three GFP-labeled patches for all other components. Numbers of GFP-labeled proteins were calculated from time-lapse 3D image series and numbers of YFP-labeled proteins were calculated from time-lapse series in one confocal section. All time courses were aligned to time 0, which marks initiation of patch movement, except for Myo1p, which was aligned with the Wsp1p peak. The ARPC5 time course was corrected so that the timing of patch assembly matched that of fimbrin. mGFP-Act1p is estimated to represent 6% of total actin. (B) Schematic diagram illustrating counts of proteins associated with an actin patch at the site of endocytic invagination at times −10, −3, and 0 s. Colors of fonts correspond to the colors in A. Numbers in parenthesis indicate the peak numbers of each protein, except for End4p and Pan1p at −10 s (asterisks). All of the proteins listed below Arp2/3 complex are presumed to be distributed throughout the actin network (shaded teal). The approximate positions of other proteins are adapted from Idrissi et al. (2008).

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)

Summary time course of the assembly and disassembly of actin patch proteins at the site of endocytosis. (A) Summary time course of the number of molecules (solid lines) and distance traveled (dashed lines) of 12 endocytic actin patch components. Each time course is an average of data for three YFP-labeled patches for clathrin, seven YFP-labeled patches for Wsp1p, six GFP-labeled patches for Act1p, and three YFP- and three GFP-labeled patches for all other components. Numbers of GFP-labeled proteins were calculated from time-lapse 3D image series and numbers of YFP-labeled proteins were calculated from time-lapse series in one confocal section. All time courses were aligned to time 0, which marks initiation of patch movement, except for Myo1p, which was aligned with the Wsp1p peak. The ARPC5 time course was corrected so that the timing of patch assembly matched that of fimbrin. mGFP-Act1p is estimated to represent 6% of total actin. (B) Schematic diagram illustrating counts of proteins associated with an actin patch at the site of endocytic invagination at times −10, −3, and 0 s. Colors of fonts correspond to the colors in A. Numbers in parenthesis indicate the peak numbers of each protein, except for End4p and Pan1p at −10 s (asterisks). All of the proteins listed below Arp2/3 complex are presumed to be distributed throughout the actin network (shaded teal). The approximate positions of other proteins are adapted from Idrissi et al. (2008).
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Figure 7: Summary time course of the assembly and disassembly of actin patch proteins at the site of endocytosis. (A) Summary time course of the number of molecules (solid lines) and distance traveled (dashed lines) of 12 endocytic actin patch components. Each time course is an average of data for three YFP-labeled patches for clathrin, seven YFP-labeled patches for Wsp1p, six GFP-labeled patches for Act1p, and three YFP- and three GFP-labeled patches for all other components. Numbers of GFP-labeled proteins were calculated from time-lapse 3D image series and numbers of YFP-labeled proteins were calculated from time-lapse series in one confocal section. All time courses were aligned to time 0, which marks initiation of patch movement, except for Myo1p, which was aligned with the Wsp1p peak. The ARPC5 time course was corrected so that the timing of patch assembly matched that of fimbrin. mGFP-Act1p is estimated to represent 6% of total actin. (B) Schematic diagram illustrating counts of proteins associated with an actin patch at the site of endocytic invagination at times −10, −3, and 0 s. Colors of fonts correspond to the colors in A. Numbers in parenthesis indicate the peak numbers of each protein, except for End4p and Pan1p at −10 s (asterisks). All of the proteins listed below Arp2/3 complex are presumed to be distributed throughout the actin network (shaded teal). The approximate positions of other proteins are adapted from Idrissi et al. (2008).
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