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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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Characterization of hCLTAEN/DNM2EN cells expressing dynamin2-EGFP. (A) PCR analysis confirms published results (Doyon et al., 2011) showing the biallelic integration of EGFP to the dynamin2 in the clonal cell line named hCLTAEN/DNM2EN gene-edited to express dynamin2-EGFP and LCa-RFP; SK-MEL-2 denotes the parental cell line. (B) Western blot analysis of cell lysates from brain or from hCLTAEN/DNM2EN cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that ∼50% of expressed dynamin2 lacks EGFP and that dynamin1 is undetectable in the total pool of dynamin of hCLTAEN/DNM2EN cells. Similar results were obtained using another cell lysate obtained from the hCLTAEN/DNM2EN cells (unpublished data). (C) Frame from a time series showing fluorescent spots corresponding to coated pits labeled with dynamin2-EGFP (green) and clathrin LCa-RFP. The image was acquired using spinning-disk confocal microscopy from the attached surface of hCLTAEN/DNM2EN cells. Consecutive images for each fluorescence channel were obtained with 30-ms exposure. Channels shifted vertically by four pixels. Scale bar, 5 μm.
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Figure 3: Characterization of hCLTAEN/DNM2EN cells expressing dynamin2-EGFP. (A) PCR analysis confirms published results (Doyon et al., 2011) showing the biallelic integration of EGFP to the dynamin2 in the clonal cell line named hCLTAEN/DNM2EN gene-edited to express dynamin2-EGFP and LCa-RFP; SK-MEL-2 denotes the parental cell line. (B) Western blot analysis of cell lysates from brain or from hCLTAEN/DNM2EN cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that ∼50% of expressed dynamin2 lacks EGFP and that dynamin1 is undetectable in the total pool of dynamin of hCLTAEN/DNM2EN cells. Similar results were obtained using another cell lysate obtained from the hCLTAEN/DNM2EN cells (unpublished data). (C) Frame from a time series showing fluorescent spots corresponding to coated pits labeled with dynamin2-EGFP (green) and clathrin LCa-RFP. The image was acquired using spinning-disk confocal microscopy from the attached surface of hCLTAEN/DNM2EN cells. Consecutive images for each fluorescence channel were obtained with 30-ms exposure. Channels shifted vertically by four pixels. Scale bar, 5 μm.

Mentions: To study the molecular events associated with dynamin-mediated membrane fission at the neck of clathrin-coated pits, we established a gene-edited cell line in which we replaced the ubiquitous dynamin2 with a dynamin2-EGFP chimera. We selected the human cell line SUM159 (Forozan et al., 1999), breast cancer–derived, mostly diploid cells, and used transcription activator-like effector nuclease (TALEN)–mediated editing to create the chimera-encoding gene (Sanjana et al., 2012). We chose to connect EGFP with a linker of six amino acids to the C-terminus of dynamin2 (Figure 2A). We isolated clone SUM-Dyn2, bearing the insertion at both alleles. Analysis of the genomic DNA by PCR showed that both alleles were indeed substituted and that there was no DNA encoding wild-type dynamin2 (Figure 2B). Consistent with this result, Western blot analysis using a monoclonal antibody specific for dynamin2 showed ∼98% substitution of dynamin2 by the expressed dynamin2-EGFP (Figure 2C). A different linker, used previously to express dynamin2-EGFP in the gene-edited SK-MEL-2 cell line hCLTAEN/DNM2EN (Figure 3A), does appear to be susceptible to proteolysis, as the apparent level of substitution is only ∼50% (Figure 3B; Doyon et al., 2011), and these cells also have free EGFP (Doyon et al., 2011; unpublished data). Western blot analysis with a monoclonal antibody specific for dynamin1 showed a very low level of dynamin1 in the SUM-Dyn2 cells (∼5% of dynamin2-EGFP) and undetectable levels in the hCLTAEN/DNM2EN cells.


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

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

Characterization of hCLTAEN/DNM2EN cells expressing dynamin2-EGFP. (A) PCR analysis confirms published results (Doyon et al., 2011) showing the biallelic integration of EGFP to the dynamin2 in the clonal cell line named hCLTAEN/DNM2EN gene-edited to express dynamin2-EGFP and LCa-RFP; SK-MEL-2 denotes the parental cell line. (B) Western blot analysis of cell lysates from brain or from hCLTAEN/DNM2EN cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that ∼50% of expressed dynamin2 lacks EGFP and that dynamin1 is undetectable in the total pool of dynamin of hCLTAEN/DNM2EN cells. Similar results were obtained using another cell lysate obtained from the hCLTAEN/DNM2EN cells (unpublished data). (C) Frame from a time series showing fluorescent spots corresponding to coated pits labeled with dynamin2-EGFP (green) and clathrin LCa-RFP. The image was acquired using spinning-disk confocal microscopy from the attached surface of hCLTAEN/DNM2EN cells. Consecutive images for each fluorescence channel were obtained with 30-ms exposure. Channels shifted vertically by four pixels. Scale bar, 5 μm.
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Related In: Results  -  Collection

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Figure 3: Characterization of hCLTAEN/DNM2EN cells expressing dynamin2-EGFP. (A) PCR analysis confirms published results (Doyon et al., 2011) showing the biallelic integration of EGFP to the dynamin2 in the clonal cell line named hCLTAEN/DNM2EN gene-edited to express dynamin2-EGFP and LCa-RFP; SK-MEL-2 denotes the parental cell line. (B) Western blot analysis of cell lysates from brain or from hCLTAEN/DNM2EN cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that ∼50% of expressed dynamin2 lacks EGFP and that dynamin1 is undetectable in the total pool of dynamin of hCLTAEN/DNM2EN cells. Similar results were obtained using another cell lysate obtained from the hCLTAEN/DNM2EN cells (unpublished data). (C) Frame from a time series showing fluorescent spots corresponding to coated pits labeled with dynamin2-EGFP (green) and clathrin LCa-RFP. The image was acquired using spinning-disk confocal microscopy from the attached surface of hCLTAEN/DNM2EN cells. Consecutive images for each fluorescence channel were obtained with 30-ms exposure. Channels shifted vertically by four pixels. Scale bar, 5 μm.
Mentions: To study the molecular events associated with dynamin-mediated membrane fission at the neck of clathrin-coated pits, we established a gene-edited cell line in which we replaced the ubiquitous dynamin2 with a dynamin2-EGFP chimera. We selected the human cell line SUM159 (Forozan et al., 1999), breast cancer–derived, mostly diploid cells, and used transcription activator-like effector nuclease (TALEN)–mediated editing to create the chimera-encoding gene (Sanjana et al., 2012). We chose to connect EGFP with a linker of six amino acids to the C-terminus of dynamin2 (Figure 2A). We isolated clone SUM-Dyn2, bearing the insertion at both alleles. Analysis of the genomic DNA by PCR showed that both alleles were indeed substituted and that there was no DNA encoding wild-type dynamin2 (Figure 2B). Consistent with this result, Western blot analysis using a monoclonal antibody specific for dynamin2 showed ∼98% substitution of dynamin2 by the expressed dynamin2-EGFP (Figure 2C). A different linker, used previously to express dynamin2-EGFP in the gene-edited SK-MEL-2 cell line hCLTAEN/DNM2EN (Figure 3A), does appear to be susceptible to proteolysis, as the apparent level of substitution is only ∼50% (Figure 3B; Doyon et al., 2011), and these cells also have free EGFP (Doyon et al., 2011; unpublished data). Western blot analysis with a monoclonal antibody specific for dynamin1 showed a very low level of dynamin1 in the SUM-Dyn2 cells (∼5% of dynamin2-EGFP) and undetectable levels in the hCLTAEN/DNM2EN cells.

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