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Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.

Cocucci E, Gaudin R, Kirchhausen T - Mol. Biol. Cell (2014)

Bottom Line: The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit.A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference.We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115.

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Stepwise recruitment of dynamin during the formation of coated pits detected with single-molecule sensitivity. (A) Representative plots of fluorescence intensity traces of dynamin2-EGFP recruited to four assembling coated pits in SUM-dyn2 cells imaged with TIRF microscopy every 110 ms with an exposure of 60 ms/frame; fit (black) obtained by applying a step-fitting function to estimate the average fluorescence intensity and dwell time of the steps. (B) Value of the BIC used to determine the best fit between the experimental data from 104 recruitment steps from 23 coated pits in five cells and the stepwise recruitment models of dynamin indicated at the bottom. The quality of the fit increases with more-negative BIC values. The dynamin2 substitution by dynamin2-EGFP was kept as a fixed parameter corresponding to the value estimated by Western blot analysis (98%). The best fit corresponds to a recruitment model of 8% monomers, 49% dimers, 26% tetramers, 6% hexamers, and 11% octamers. (C) Histogram of the background-corrected fluorescence intensity of the steps (gray) used to calculate the data in B; the continuous trace (dark blue) is the sum of the relative contributions calculated by the presence of one, two, three, four, and so on dynamin2-EGFP molecules (light blue; centered at 6050 ± 2200, 12,100 ± 4400, 18,150 ± 6600, 24,200 ± 8800, etc.) according to the best model presented in B. Inset, preferential recruitment of dynamin2 dimers (49%) and the less frequent recruitment of dynamin2 monomers (8%), tetramers (26%), hexamers (6%), and octamers (11%).
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Figure 9: Stepwise recruitment of dynamin during the formation of coated pits detected with single-molecule sensitivity. (A) Representative plots of fluorescence intensity traces of dynamin2-EGFP recruited to four assembling coated pits in SUM-dyn2 cells imaged with TIRF microscopy every 110 ms with an exposure of 60 ms/frame; fit (black) obtained by applying a step-fitting function to estimate the average fluorescence intensity and dwell time of the steps. (B) Value of the BIC used to determine the best fit between the experimental data from 104 recruitment steps from 23 coated pits in five cells and the stepwise recruitment models of dynamin indicated at the bottom. The quality of the fit increases with more-negative BIC values. The dynamin2 substitution by dynamin2-EGFP was kept as a fixed parameter corresponding to the value estimated by Western blot analysis (98%). The best fit corresponds to a recruitment model of 8% monomers, 49% dimers, 26% tetramers, 6% hexamers, and 11% octamers. (C) Histogram of the background-corrected fluorescence intensity of the steps (gray) used to calculate the data in B; the continuous trace (dark blue) is the sum of the relative contributions calculated by the presence of one, two, three, four, and so on dynamin2-EGFP molecules (light blue; centered at 6050 ± 2200, 12,100 ± 4400, 18,150 ± 6600, 24,200 ± 8800, etc.) according to the best model presented in B. Inset, preferential recruitment of dynamin2 dimers (49%) and the less frequent recruitment of dynamin2 monomers (8%), tetramers (26%), hexamers (6%), and octamers (11%).

Mentions: Using a TIRF configuration calibrated from single-molecule counting as previously described (Cocucci et al., 2012), we followed the accumulation of dynamin2-EGFP into coated pits at the bottom surface of SUM-Dyn2 cells. We acquired a rapid time series lasting ∼20 s, with 60-ms exposure/frame at an interval of 110 ms. This protocol minimized photobleaching to not more than 20% toward the end of the time series. We analyzed 23 traces from five cells selected to have relatively low amplitude and therefore show phase 1 of dynamin recruitment. We found consecutive stepwise intensity increments of variable magnitude and a dwell time of 1.06 ± 0.57 s (Figure 9A). The number of dynamin2-EGFP molecules recruited during each of the steps was obtained by fitting the intensity distributions with different models that took into account the dynamin2 substitution by dynamin2-EGFP (98%) and alternative contributions of monomer, dimer, tetramer, and so on of dynamin2-EGFP during the steps of recruitment (see Materials and Methods).


Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.

Cocucci E, Gaudin R, Kirchhausen T - Mol. Biol. Cell (2014)

Stepwise recruitment of dynamin during the formation of coated pits detected with single-molecule sensitivity. (A) Representative plots of fluorescence intensity traces of dynamin2-EGFP recruited to four assembling coated pits in SUM-dyn2 cells imaged with TIRF microscopy every 110 ms with an exposure of 60 ms/frame; fit (black) obtained by applying a step-fitting function to estimate the average fluorescence intensity and dwell time of the steps. (B) Value of the BIC used to determine the best fit between the experimental data from 104 recruitment steps from 23 coated pits in five cells and the stepwise recruitment models of dynamin indicated at the bottom. The quality of the fit increases with more-negative BIC values. The dynamin2 substitution by dynamin2-EGFP was kept as a fixed parameter corresponding to the value estimated by Western blot analysis (98%). The best fit corresponds to a recruitment model of 8% monomers, 49% dimers, 26% tetramers, 6% hexamers, and 11% octamers. (C) Histogram of the background-corrected fluorescence intensity of the steps (gray) used to calculate the data in B; the continuous trace (dark blue) is the sum of the relative contributions calculated by the presence of one, two, three, four, and so on dynamin2-EGFP molecules (light blue; centered at 6050 ± 2200, 12,100 ± 4400, 18,150 ± 6600, 24,200 ± 8800, etc.) according to the best model presented in B. Inset, preferential recruitment of dynamin2 dimers (49%) and the less frequent recruitment of dynamin2 monomers (8%), tetramers (26%), hexamers (6%), and octamers (11%).
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Related In: Results  -  Collection

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Figure 9: Stepwise recruitment of dynamin during the formation of coated pits detected with single-molecule sensitivity. (A) Representative plots of fluorescence intensity traces of dynamin2-EGFP recruited to four assembling coated pits in SUM-dyn2 cells imaged with TIRF microscopy every 110 ms with an exposure of 60 ms/frame; fit (black) obtained by applying a step-fitting function to estimate the average fluorescence intensity and dwell time of the steps. (B) Value of the BIC used to determine the best fit between the experimental data from 104 recruitment steps from 23 coated pits in five cells and the stepwise recruitment models of dynamin indicated at the bottom. The quality of the fit increases with more-negative BIC values. The dynamin2 substitution by dynamin2-EGFP was kept as a fixed parameter corresponding to the value estimated by Western blot analysis (98%). The best fit corresponds to a recruitment model of 8% monomers, 49% dimers, 26% tetramers, 6% hexamers, and 11% octamers. (C) Histogram of the background-corrected fluorescence intensity of the steps (gray) used to calculate the data in B; the continuous trace (dark blue) is the sum of the relative contributions calculated by the presence of one, two, three, four, and so on dynamin2-EGFP molecules (light blue; centered at 6050 ± 2200, 12,100 ± 4400, 18,150 ± 6600, 24,200 ± 8800, etc.) according to the best model presented in B. Inset, preferential recruitment of dynamin2 dimers (49%) and the less frequent recruitment of dynamin2 monomers (8%), tetramers (26%), hexamers (6%), and octamers (11%).
Mentions: Using a TIRF configuration calibrated from single-molecule counting as previously described (Cocucci et al., 2012), we followed the accumulation of dynamin2-EGFP into coated pits at the bottom surface of SUM-Dyn2 cells. We acquired a rapid time series lasting ∼20 s, with 60-ms exposure/frame at an interval of 110 ms. This protocol minimized photobleaching to not more than 20% toward the end of the time series. We analyzed 23 traces from five cells selected to have relatively low amplitude and therefore show phase 1 of dynamin recruitment. We found consecutive stepwise intensity increments of variable magnitude and a dwell time of 1.06 ± 0.57 s (Figure 9A). The number of dynamin2-EGFP molecules recruited during each of the steps was obtained by fitting the intensity distributions with different models that took into account the dynamin2 substitution by dynamin2-EGFP (98%) and alternative contributions of monomer, dimer, tetramer, and so on of dynamin2-EGFP during the steps of recruitment (see Materials and Methods).

Bottom Line: The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit.A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference.We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115.

Show MeSH
Related in: MedlinePlus