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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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Gene editing of SUM cells to express dynamin2-EGFP. (A) Schematic representation of the gene-editing strategy used with SUM159 cells to incorporate EGFP to the C-terminus of dynamin2 based on the TALEN approach (Sanjana et al., 2012). The portions of the genomic DNA highlighted in red correspond to the sequences recognized by the DNA-binding domains constructed and then fused to two different copies of the Fok1 endonuclease; the stop codon TAG indicates the site of EGFP incorporation. The resulting sequence joined by a short linker between the C-terminus of dynamin2 and N-terminus of EGFP is indicated. (B) PCR analysis showing the biallelic integration of EGFP into the dynamin2 locus in the clonal cell line SUM-Dyn2; SUM denotes the parental cells. (C) Western blot analysis of cell lysates from brain or from SUM-Dyn2 cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that in SUM-Dyn2, ∼2% of expressed dynamin2 lacks EGFP (98% substitution) and ∼5% of dynamin1 in the total pool of dynamin1, dynamin2, and dynamin2-EGFP. Similar results were obtained with a different Western blot from a second cell lysate (unpublished data). (D) Expression of dynamin2-EGFP does not affect the receptor-mediated endocytosis of transferrin. The endocytosis of fluorescently tagged Alexa 647 transferrin was determined using a flow cytometry–based assay. Cells were first incubated with Alexa 647–transferrin for 10 min at 4°C or 10 min at 37°C and then subjected to an acid wash in ice-cold medium to remove the transferrin bound to the cell surface. The histogram shows the same amount of transferrin internalized by the parental SUM and gene-edited SUM-Dyn2 cells. The bars represent the average ± SD from duplicate determinations carried out using ∼10,000 cells/measurement. (E) Frame from a time series of coated pits from the attached surface of SUM-Dyn2 cell expressing dynamin2-EGFP (green) and clathrin mCherry-LCa (red). Consecutive images for each fluorescence channel were obtained with 30-ms exposure using spinning-disk fluorescence confocal microscopy. Channels were shifted vertically by 4 pixels to help identify pits containing both fluorescently tagged proteins. Scale bar, 5 μm.
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Figure 2: Gene editing of SUM cells to express dynamin2-EGFP. (A) Schematic representation of the gene-editing strategy used with SUM159 cells to incorporate EGFP to the C-terminus of dynamin2 based on the TALEN approach (Sanjana et al., 2012). The portions of the genomic DNA highlighted in red correspond to the sequences recognized by the DNA-binding domains constructed and then fused to two different copies of the Fok1 endonuclease; the stop codon TAG indicates the site of EGFP incorporation. The resulting sequence joined by a short linker between the C-terminus of dynamin2 and N-terminus of EGFP is indicated. (B) PCR analysis showing the biallelic integration of EGFP into the dynamin2 locus in the clonal cell line SUM-Dyn2; SUM denotes the parental cells. (C) Western blot analysis of cell lysates from brain or from SUM-Dyn2 cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that in SUM-Dyn2, ∼2% of expressed dynamin2 lacks EGFP (98% substitution) and ∼5% of dynamin1 in the total pool of dynamin1, dynamin2, and dynamin2-EGFP. Similar results were obtained with a different Western blot from a second cell lysate (unpublished data). (D) Expression of dynamin2-EGFP does not affect the receptor-mediated endocytosis of transferrin. The endocytosis of fluorescently tagged Alexa 647 transferrin was determined using a flow cytometry–based assay. Cells were first incubated with Alexa 647–transferrin for 10 min at 4°C or 10 min at 37°C and then subjected to an acid wash in ice-cold medium to remove the transferrin bound to the cell surface. The histogram shows the same amount of transferrin internalized by the parental SUM and gene-edited SUM-Dyn2 cells. The bars represent the average ± SD from duplicate determinations carried out using ∼10,000 cells/measurement. (E) Frame from a time series of coated pits from the attached surface of SUM-Dyn2 cell expressing dynamin2-EGFP (green) and clathrin mCherry-LCa (red). Consecutive images for each fluorescence channel were obtained with 30-ms exposure using spinning-disk fluorescence confocal microscopy. Channels were shifted vertically by 4 pixels to help identify pits containing both fluorescently tagged proteins. 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)

Gene editing of SUM cells to express dynamin2-EGFP. (A) Schematic representation of the gene-editing strategy used with SUM159 cells to incorporate EGFP to the C-terminus of dynamin2 based on the TALEN approach (Sanjana et al., 2012). The portions of the genomic DNA highlighted in red correspond to the sequences recognized by the DNA-binding domains constructed and then fused to two different copies of the Fok1 endonuclease; the stop codon TAG indicates the site of EGFP incorporation. The resulting sequence joined by a short linker between the C-terminus of dynamin2 and N-terminus of EGFP is indicated. (B) PCR analysis showing the biallelic integration of EGFP into the dynamin2 locus in the clonal cell line SUM-Dyn2; SUM denotes the parental cells. (C) Western blot analysis of cell lysates from brain or from SUM-Dyn2 cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that in SUM-Dyn2, ∼2% of expressed dynamin2 lacks EGFP (98% substitution) and ∼5% of dynamin1 in the total pool of dynamin1, dynamin2, and dynamin2-EGFP. Similar results were obtained with a different Western blot from a second cell lysate (unpublished data). (D) Expression of dynamin2-EGFP does not affect the receptor-mediated endocytosis of transferrin. The endocytosis of fluorescently tagged Alexa 647 transferrin was determined using a flow cytometry–based assay. Cells were first incubated with Alexa 647–transferrin for 10 min at 4°C or 10 min at 37°C and then subjected to an acid wash in ice-cold medium to remove the transferrin bound to the cell surface. The histogram shows the same amount of transferrin internalized by the parental SUM and gene-edited SUM-Dyn2 cells. The bars represent the average ± SD from duplicate determinations carried out using ∼10,000 cells/measurement. (E) Frame from a time series of coated pits from the attached surface of SUM-Dyn2 cell expressing dynamin2-EGFP (green) and clathrin mCherry-LCa (red). Consecutive images for each fluorescence channel were obtained with 30-ms exposure using spinning-disk fluorescence confocal microscopy. Channels were shifted vertically by 4 pixels to help identify pits containing both fluorescently tagged proteins. Scale bar, 5 μm.
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Related In: Results  -  Collection

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Figure 2: Gene editing of SUM cells to express dynamin2-EGFP. (A) Schematic representation of the gene-editing strategy used with SUM159 cells to incorporate EGFP to the C-terminus of dynamin2 based on the TALEN approach (Sanjana et al., 2012). The portions of the genomic DNA highlighted in red correspond to the sequences recognized by the DNA-binding domains constructed and then fused to two different copies of the Fok1 endonuclease; the stop codon TAG indicates the site of EGFP incorporation. The resulting sequence joined by a short linker between the C-terminus of dynamin2 and N-terminus of EGFP is indicated. (B) PCR analysis showing the biallelic integration of EGFP into the dynamin2 locus in the clonal cell line SUM-Dyn2; SUM denotes the parental cells. (C) Western blot analysis of cell lysates from brain or from SUM-Dyn2 cells expressing dynamin2-EGFP probed with antibodies specific for dynamin1 or dynamin2. Quantification of band intensities indicates that in SUM-Dyn2, ∼2% of expressed dynamin2 lacks EGFP (98% substitution) and ∼5% of dynamin1 in the total pool of dynamin1, dynamin2, and dynamin2-EGFP. Similar results were obtained with a different Western blot from a second cell lysate (unpublished data). (D) Expression of dynamin2-EGFP does not affect the receptor-mediated endocytosis of transferrin. The endocytosis of fluorescently tagged Alexa 647 transferrin was determined using a flow cytometry–based assay. Cells were first incubated with Alexa 647–transferrin for 10 min at 4°C or 10 min at 37°C and then subjected to an acid wash in ice-cold medium to remove the transferrin bound to the cell surface. The histogram shows the same amount of transferrin internalized by the parental SUM and gene-edited SUM-Dyn2 cells. The bars represent the average ± SD from duplicate determinations carried out using ∼10,000 cells/measurement. (E) Frame from a time series of coated pits from the attached surface of SUM-Dyn2 cell expressing dynamin2-EGFP (green) and clathrin mCherry-LCa (red). Consecutive images for each fluorescence channel were obtained with 30-ms exposure using spinning-disk fluorescence confocal microscopy. Channels were shifted vertically by 4 pixels to help identify pits containing both fluorescently tagged proteins. 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