Limits...
Inhibition of ESCRT-II-CHMP6 interactions impedes cytokinetic abscission and leads to cell death.

Goliand I, Nachmias D, Gershony O, Elia N - Mol. Biol. Cell (2014)

Bottom Line: This phenotype is abolished in a mutated version of CHMP6-N designed to prevent CHMP6-N binding to its ESCRT-II partner.Of interest, deleting the first 10 amino acids from CHMP6-N does not interfere with its arrival at the intercellular bridge but almost completely abolishes the abscission failure phenotype.Our work advances the mechanistic understanding of ESCRT-mediated membrane fission in cells and introduces an easily applicable tool for upstream inhibition of the ESCRT pathway in live mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and the National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

Show MeSH

Related in: MedlinePlus

Spatiotemporal recruitment of the ESCRT-II protein VPS36 to the intercellular bridge during cytokinesis. (A and B) Live-cell imaging of MDCK cells undergoing cytokinesis reveals acute recruitment of VPS36 to the intercellular bridge. Cells expressing low levels of GFP-VPS36 together with mCherry-tubulin were imaged using a spinning-disk confocal microscope at 5-min intervals. Shown are maximum-intensity projections of different time points throughout cytokinesis (A) or during abscission (B) from representative cells. Top, overlay of VPS36 (green) and microtubules (red). Bottom, VPS36 alone. Recruitment of VPS36 to the bridge was observed in 30 cells. Solid arrows indicate the position of the first abscission site. Dashed arrows (in B) indicate the second abscission site. Time (indicated in minutes) is relative to abscission of the first site. Plot in A, changes in VPS36 intensity at the intercellular bridge relative to abscission, as quantified from seven independent experiments. Plot in B, changes in microtubule diameter (blue) and VPS36 fluorescence intensity (green), quantified from the first abscission site (Supplemental Videos S2 and S3; bar, 2 μm). (C) Spatial organization of VPS36 in early (top) and late (bottom) intercellular bridges. MDCK cells expressing GFP-VPS36 were fixed, stained with anti–α-tubulin antibodies, and imaged by SIM. Early and late bridges were categorized based on the diameter of the intercellular bridge at the constriction site (see Materials and Methods). Each panel shows (from left to right) a 3D reconstruction of an overlay of VPS36 (green) and tubulin (white; bar, 2 μm); a zoomed-in, 3D rendered image of the protein structure alone (bar, 1 μm); a zoomed-in, 3D rendered image of the protein structure rotated 90° (bar, 1 μm); and a schematic model for VPS36 organization at the intercellular bridge based on SIM measurements. In early intercellular bridges, VPS36 concentrates in a ring located at the center of the dark zone. Ring diameter, 1.7 ± 0.25 μm; ring thickness, 0.61± 0.3 μm (n = 22). In late intercellular bridges, VPS36 distributes peripherally to the central ring, forming a structure that stretches 1.01 ± 0.23 μm (n = 6) away from the center. (D) VPS36 organizes in the area between the dark zone and the constriction sites in late intercellular bridges but does not reach the constriction site. Plot shows tubulin (gray) and VPS36 (green) line intensity profiles along the intercellular bridge (indicated by the blue arrow in C). Unlike CHMP6 and other ESCRT-III components, VPS36 signal is low at the constriction site. (E) Model for ESCRT organization at the intercellular bridge, integrating the SIM measurement obtained for ESCRT-II and CHMP6. ESCRT-I, gray; ESCRT-II, yellow; ESCRT-III, red.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230781&req=5

Figure 2: Spatiotemporal recruitment of the ESCRT-II protein VPS36 to the intercellular bridge during cytokinesis. (A and B) Live-cell imaging of MDCK cells undergoing cytokinesis reveals acute recruitment of VPS36 to the intercellular bridge. Cells expressing low levels of GFP-VPS36 together with mCherry-tubulin were imaged using a spinning-disk confocal microscope at 5-min intervals. Shown are maximum-intensity projections of different time points throughout cytokinesis (A) or during abscission (B) from representative cells. Top, overlay of VPS36 (green) and microtubules (red). Bottom, VPS36 alone. Recruitment of VPS36 to the bridge was observed in 30 cells. Solid arrows indicate the position of the first abscission site. Dashed arrows (in B) indicate the second abscission site. Time (indicated in minutes) is relative to abscission of the first site. Plot in A, changes in VPS36 intensity at the intercellular bridge relative to abscission, as quantified from seven independent experiments. Plot in B, changes in microtubule diameter (blue) and VPS36 fluorescence intensity (green), quantified from the first abscission site (Supplemental Videos S2 and S3; bar, 2 μm). (C) Spatial organization of VPS36 in early (top) and late (bottom) intercellular bridges. MDCK cells expressing GFP-VPS36 were fixed, stained with anti–α-tubulin antibodies, and imaged by SIM. Early and late bridges were categorized based on the diameter of the intercellular bridge at the constriction site (see Materials and Methods). Each panel shows (from left to right) a 3D reconstruction of an overlay of VPS36 (green) and tubulin (white; bar, 2 μm); a zoomed-in, 3D rendered image of the protein structure alone (bar, 1 μm); a zoomed-in, 3D rendered image of the protein structure rotated 90° (bar, 1 μm); and a schematic model for VPS36 organization at the intercellular bridge based on SIM measurements. In early intercellular bridges, VPS36 concentrates in a ring located at the center of the dark zone. Ring diameter, 1.7 ± 0.25 μm; ring thickness, 0.61± 0.3 μm (n = 22). In late intercellular bridges, VPS36 distributes peripherally to the central ring, forming a structure that stretches 1.01 ± 0.23 μm (n = 6) away from the center. (D) VPS36 organizes in the area between the dark zone and the constriction sites in late intercellular bridges but does not reach the constriction site. Plot shows tubulin (gray) and VPS36 (green) line intensity profiles along the intercellular bridge (indicated by the blue arrow in C). Unlike CHMP6 and other ESCRT-III components, VPS36 signal is low at the constriction site. (E) Model for ESCRT organization at the intercellular bridge, integrating the SIM measurement obtained for ESCRT-II and CHMP6. ESCRT-I, gray; ESCRT-II, yellow; ESCRT-III, red.

Mentions: We next examined the spatiotemporal organization of the ESCRT-II complex during cytokinesis. Live-cell imaging revealed that the ESCRT-II components VPS36 and VPS22 localize to the intercellular bridge during late cytokinesis (Figure 2, A and B, Supplemental Figure S1A, and Supplemental Videos S2–S4, respectively). Quantitative analysis of the recruitment pattern of VPS36 to the intercellular bridge revealed that VPS36 begins accumulating at the bridge only ∼20 min before final scission and reaches its peak levels at the time of scission (Figure 2A and Supplemental Video S2). The increase in VPS36 levels is temporally correlated with acute constriction of the microtubules on either side of the intercellular bridge (Figure 2B and Supplemental Video S3). VPS36 was also clearly observed in postabscission remnants (Figure 2B and Supplemental Video S3).


Inhibition of ESCRT-II-CHMP6 interactions impedes cytokinetic abscission and leads to cell death.

Goliand I, Nachmias D, Gershony O, Elia N - Mol. Biol. Cell (2014)

Spatiotemporal recruitment of the ESCRT-II protein VPS36 to the intercellular bridge during cytokinesis. (A and B) Live-cell imaging of MDCK cells undergoing cytokinesis reveals acute recruitment of VPS36 to the intercellular bridge. Cells expressing low levels of GFP-VPS36 together with mCherry-tubulin were imaged using a spinning-disk confocal microscope at 5-min intervals. Shown are maximum-intensity projections of different time points throughout cytokinesis (A) or during abscission (B) from representative cells. Top, overlay of VPS36 (green) and microtubules (red). Bottom, VPS36 alone. Recruitment of VPS36 to the bridge was observed in 30 cells. Solid arrows indicate the position of the first abscission site. Dashed arrows (in B) indicate the second abscission site. Time (indicated in minutes) is relative to abscission of the first site. Plot in A, changes in VPS36 intensity at the intercellular bridge relative to abscission, as quantified from seven independent experiments. Plot in B, changes in microtubule diameter (blue) and VPS36 fluorescence intensity (green), quantified from the first abscission site (Supplemental Videos S2 and S3; bar, 2 μm). (C) Spatial organization of VPS36 in early (top) and late (bottom) intercellular bridges. MDCK cells expressing GFP-VPS36 were fixed, stained with anti–α-tubulin antibodies, and imaged by SIM. Early and late bridges were categorized based on the diameter of the intercellular bridge at the constriction site (see Materials and Methods). Each panel shows (from left to right) a 3D reconstruction of an overlay of VPS36 (green) and tubulin (white; bar, 2 μm); a zoomed-in, 3D rendered image of the protein structure alone (bar, 1 μm); a zoomed-in, 3D rendered image of the protein structure rotated 90° (bar, 1 μm); and a schematic model for VPS36 organization at the intercellular bridge based on SIM measurements. In early intercellular bridges, VPS36 concentrates in a ring located at the center of the dark zone. Ring diameter, 1.7 ± 0.25 μm; ring thickness, 0.61± 0.3 μm (n = 22). In late intercellular bridges, VPS36 distributes peripherally to the central ring, forming a structure that stretches 1.01 ± 0.23 μm (n = 6) away from the center. (D) VPS36 organizes in the area between the dark zone and the constriction sites in late intercellular bridges but does not reach the constriction site. Plot shows tubulin (gray) and VPS36 (green) line intensity profiles along the intercellular bridge (indicated by the blue arrow in C). Unlike CHMP6 and other ESCRT-III components, VPS36 signal is low at the constriction site. (E) Model for ESCRT organization at the intercellular bridge, integrating the SIM measurement obtained for ESCRT-II and CHMP6. ESCRT-I, gray; ESCRT-II, yellow; ESCRT-III, red.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230781&req=5

Figure 2: Spatiotemporal recruitment of the ESCRT-II protein VPS36 to the intercellular bridge during cytokinesis. (A and B) Live-cell imaging of MDCK cells undergoing cytokinesis reveals acute recruitment of VPS36 to the intercellular bridge. Cells expressing low levels of GFP-VPS36 together with mCherry-tubulin were imaged using a spinning-disk confocal microscope at 5-min intervals. Shown are maximum-intensity projections of different time points throughout cytokinesis (A) or during abscission (B) from representative cells. Top, overlay of VPS36 (green) and microtubules (red). Bottom, VPS36 alone. Recruitment of VPS36 to the bridge was observed in 30 cells. Solid arrows indicate the position of the first abscission site. Dashed arrows (in B) indicate the second abscission site. Time (indicated in minutes) is relative to abscission of the first site. Plot in A, changes in VPS36 intensity at the intercellular bridge relative to abscission, as quantified from seven independent experiments. Plot in B, changes in microtubule diameter (blue) and VPS36 fluorescence intensity (green), quantified from the first abscission site (Supplemental Videos S2 and S3; bar, 2 μm). (C) Spatial organization of VPS36 in early (top) and late (bottom) intercellular bridges. MDCK cells expressing GFP-VPS36 were fixed, stained with anti–α-tubulin antibodies, and imaged by SIM. Early and late bridges were categorized based on the diameter of the intercellular bridge at the constriction site (see Materials and Methods). Each panel shows (from left to right) a 3D reconstruction of an overlay of VPS36 (green) and tubulin (white; bar, 2 μm); a zoomed-in, 3D rendered image of the protein structure alone (bar, 1 μm); a zoomed-in, 3D rendered image of the protein structure rotated 90° (bar, 1 μm); and a schematic model for VPS36 organization at the intercellular bridge based on SIM measurements. In early intercellular bridges, VPS36 concentrates in a ring located at the center of the dark zone. Ring diameter, 1.7 ± 0.25 μm; ring thickness, 0.61± 0.3 μm (n = 22). In late intercellular bridges, VPS36 distributes peripherally to the central ring, forming a structure that stretches 1.01 ± 0.23 μm (n = 6) away from the center. (D) VPS36 organizes in the area between the dark zone and the constriction sites in late intercellular bridges but does not reach the constriction site. Plot shows tubulin (gray) and VPS36 (green) line intensity profiles along the intercellular bridge (indicated by the blue arrow in C). Unlike CHMP6 and other ESCRT-III components, VPS36 signal is low at the constriction site. (E) Model for ESCRT organization at the intercellular bridge, integrating the SIM measurement obtained for ESCRT-II and CHMP6. ESCRT-I, gray; ESCRT-II, yellow; ESCRT-III, red.
Mentions: We next examined the spatiotemporal organization of the ESCRT-II complex during cytokinesis. Live-cell imaging revealed that the ESCRT-II components VPS36 and VPS22 localize to the intercellular bridge during late cytokinesis (Figure 2, A and B, Supplemental Figure S1A, and Supplemental Videos S2–S4, respectively). Quantitative analysis of the recruitment pattern of VPS36 to the intercellular bridge revealed that VPS36 begins accumulating at the bridge only ∼20 min before final scission and reaches its peak levels at the time of scission (Figure 2A and Supplemental Video S2). The increase in VPS36 levels is temporally correlated with acute constriction of the microtubules on either side of the intercellular bridge (Figure 2B and Supplemental Video S3). VPS36 was also clearly observed in postabscission remnants (Figure 2B and Supplemental Video S3).

Bottom Line: This phenotype is abolished in a mutated version of CHMP6-N designed to prevent CHMP6-N binding to its ESCRT-II partner.Of interest, deleting the first 10 amino acids from CHMP6-N does not interfere with its arrival at the intercellular bridge but almost completely abolishes the abscission failure phenotype.Our work advances the mechanistic understanding of ESCRT-mediated membrane fission in cells and introduces an easily applicable tool for upstream inhibition of the ESCRT pathway in live mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and the National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

Show MeSH
Related in: MedlinePlus