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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 actin and actin-binding proteins in endocytic patches. Comparison of raw and average time courses of the number of molecules per patch (solid lines, top panels) and distance traveled by patches (dashed lines, bottom panels) for Arp2/3 complex, actin and actin-binding proteins. (A–C) Time courses for actin Act1p, fimbrin Fim1p, and App1p. (A) Comparison of average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) fimbrin Fim1p, and (▵) App1p versus (•) ARPC5* time course that was corrected to match the timing of assembly of fimbrin into patches. (B and C) Raw and average time courses. (B) mGFP-Act1p. (C) App1p. (D–F) Time courses for actin-capping protein Acp2p and twinfilin Twf1p. (D) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) capping protein Acp2p, (□) twinfilin Twf1p, and (▴) the maximum number of growing barbed actin filament ends. The maximum number of growing ends is the difference between the number of ARPC5 and Acp2p molecules. (E and F) Raw and average time courses. (E) Acp2p. (F) Twf1p. (G and H) Time courses for coronin Crn1p. (G) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) coronin Crn1p, and (▵) Abp1p homolog App1p. Note that Crn1p appears and peaks 3–5 s after the other proteins. (H) Raw and average time courses for Crn1p. In B, C, E, F, and H, raw time courses for YFP-labeled (red, ○) and GFP-labeled proteins (blue, •) in individual patches were aligned to the time of initiation of patch movement at time 0 and the molecules per patch (top panels, solid lines) and distance (bottom panels, dashed line) traveled by markers associated with patches were averaged at each time point to produce average 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 six GFP-labeled patches for Act1p and Twf1p, and three YFP- and three GFP-labeled patches for all others.
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Figure 6: Time courses of the concentrations of actin and actin-binding proteins in endocytic patches. Comparison of raw and average time courses of the number of molecules per patch (solid lines, top panels) and distance traveled by patches (dashed lines, bottom panels) for Arp2/3 complex, actin and actin-binding proteins. (A–C) Time courses for actin Act1p, fimbrin Fim1p, and App1p. (A) Comparison of average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) fimbrin Fim1p, and (▵) App1p versus (•) ARPC5* time course that was corrected to match the timing of assembly of fimbrin into patches. (B and C) Raw and average time courses. (B) mGFP-Act1p. (C) App1p. (D–F) Time courses for actin-capping protein Acp2p and twinfilin Twf1p. (D) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) capping protein Acp2p, (□) twinfilin Twf1p, and (▴) the maximum number of growing barbed actin filament ends. The maximum number of growing ends is the difference between the number of ARPC5 and Acp2p molecules. (E and F) Raw and average time courses. (E) Acp2p. (F) Twf1p. (G and H) Time courses for coronin Crn1p. (G) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) coronin Crn1p, and (▵) Abp1p homolog App1p. Note that Crn1p appears and peaks 3–5 s after the other proteins. (H) Raw and average time courses for Crn1p. In B, C, E, F, and H, raw time courses for YFP-labeled (red, ○) and GFP-labeled proteins (blue, •) in individual patches were aligned to the time of initiation of patch movement at time 0 and the molecules per patch (top panels, solid lines) and distance (bottom panels, dashed line) traveled by markers associated with patches were averaged at each time point to produce average 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 six GFP-labeled patches for Act1p and Twf1p, and three YFP- and three GFP-labeled patches for all others.

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 actin and actin-binding proteins in endocytic patches. Comparison of raw and average time courses of the number of molecules per patch (solid lines, top panels) and distance traveled by patches (dashed lines, bottom panels) for Arp2/3 complex, actin and actin-binding proteins. (A–C) Time courses for actin Act1p, fimbrin Fim1p, and App1p. (A) Comparison of average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) fimbrin Fim1p, and (▵) App1p versus (•) ARPC5* time course that was corrected to match the timing of assembly of fimbrin into patches. (B and C) Raw and average time courses. (B) mGFP-Act1p. (C) App1p. (D–F) Time courses for actin-capping protein Acp2p and twinfilin Twf1p. (D) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) capping protein Acp2p, (□) twinfilin Twf1p, and (▴) the maximum number of growing barbed actin filament ends. The maximum number of growing ends is the difference between the number of ARPC5 and Acp2p molecules. (E and F) Raw and average time courses. (E) Acp2p. (F) Twf1p. (G and H) Time courses for coronin Crn1p. (G) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) coronin Crn1p, and (▵) Abp1p homolog App1p. Note that Crn1p appears and peaks 3–5 s after the other proteins. (H) Raw and average time courses for Crn1p. In B, C, E, F, and H, raw time courses for YFP-labeled (red, ○) and GFP-labeled proteins (blue, •) in individual patches were aligned to the time of initiation of patch movement at time 0 and the molecules per patch (top panels, solid lines) and distance (bottom panels, dashed line) traveled by markers associated with patches were averaged at each time point to produce average 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 six GFP-labeled patches for Act1p and Twf1p, and three YFP- and three GFP-labeled patches for all others.
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Figure 6: Time courses of the concentrations of actin and actin-binding proteins in endocytic patches. Comparison of raw and average time courses of the number of molecules per patch (solid lines, top panels) and distance traveled by patches (dashed lines, bottom panels) for Arp2/3 complex, actin and actin-binding proteins. (A–C) Time courses for actin Act1p, fimbrin Fim1p, and App1p. (A) Comparison of average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) fimbrin Fim1p, and (▵) App1p versus (•) ARPC5* time course that was corrected to match the timing of assembly of fimbrin into patches. (B and C) Raw and average time courses. (B) mGFP-Act1p. (C) App1p. (D–F) Time courses for actin-capping protein Acp2p and twinfilin Twf1p. (D) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) capping protein Acp2p, (□) twinfilin Twf1p, and (▴) the maximum number of growing barbed actin filament ends. The maximum number of growing ends is the difference between the number of ARPC5 and Acp2p molecules. (E and F) Raw and average time courses. (E) Acp2p. (F) Twf1p. (G and H) Time courses for coronin Crn1p. (G) Comparison of (•) adjusted average ARPC5* time course with average time courses for (○) mGFP-Act1p estimated to represent 6% of total actin, (□) coronin Crn1p, and (▵) Abp1p homolog App1p. Note that Crn1p appears and peaks 3–5 s after the other proteins. (H) Raw and average time courses for Crn1p. In B, C, E, F, and H, raw time courses for YFP-labeled (red, ○) and GFP-labeled proteins (blue, •) in individual patches were aligned to the time of initiation of patch movement at time 0 and the molecules per patch (top panels, solid lines) and distance (bottom panels, dashed line) traveled by markers associated with patches were averaged at each time point to produce average 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 six GFP-labeled patches for Act1p and Twf1p, and three YFP- and three GFP-labeled patches for all others.
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