Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Transmission EM showing that GST-Exo70 induced tubular invaginations towards the interior of the lumen of LUVs similar to the MIM I-BAR domain. GST, GST-Exo70(K571A/E572A) did not induce any membrane tubules. See EXPERIMENTAL PROCEDURES for details. Scale bar, 0.5 μm. (B) Untagged Exo70 induced membrane invaginations. The arrow shows a representative region where the tubular invagination was connected to the exterior. See also Supplemental Movie 1a for 3-D tomography of this LUV. (C) Comparison of the diameters of the membrane tubules induced by different proteins. Error bars represent standard deviation (SD). n=80; *, p<0.01. (D) Confocal microscopy of fluorescence-labeled Giant Unilamellar Vesicles (GUVs) incubated with Exo70 (upper panel) or buffer control (lower panel). Sequential frames show the inward growth of a tubule (arrowhead) from a GUV incubated with Exo70. Scale bar, 5 μm.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Transmission Assay, Membrane, Tomography, Comparison, Standard Deviation, Confocal Microscopy, Fluorescence, Labeling, Incubation, Control

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Recombinant Exo70 proteins were loaded onto a Superdex 200 10/300GL column and eluted fractions were subjected to SDS-PAGE. Exo70, but not Exo70(Δ1–75), was eluted in high MW fractions. (B) Gel-filtration fractions were lightly treated with cross-linker BM(PEG)2, which links proteins in vicinity. High MW species were observed in Exo70 but not in Exo70(Δ1–75) fractions. Arrowheads: Exo70 oligomers. (C) Lysates of HEK293T cell expressing Exo70-FLAG together with GFP, GFP-Exo70, or GFP-Exo70(Δ1–75) were incubated with anti-FLAG (M2) beads. The inputs and bound proteins were analyzed by Western blotting using anti-FLAG (upper panels) and anti-GFP monoclonal antibodies (lower panels). Exo70-FLAG was able to pull down GFP-Exo70, but to a much lesser extent, GFP-Exo70(Δ1–75). (D) Exo70 or Exo70(Δ1–75) was incubated with GUVs and subjected to confocal microscopy. Exo70(Δ1–75) induced much fewer invaginations than Exo70. (E) The contours of GUV membranes were linearized. The numbers of invaginations per 25 μm were calculated (average ± SD). Exo70(Δ1–75) and Exo70(K571A/E572A) induced much fewer invaginations than the full-length Exo70 (n=10; p<0.01). Scale bar, 5 μm.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Recombinant, SDS Page, Filtration, Expressing, Incubation, Western Blot, Bioprocessing, Confocal Microscopy

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer. Top and side views of the simulation snapshots show negative curvature formed in both directions. All snapshots were rendered with VMD (Humphrey et al., 1996). See also Supplemental Movie 2 for a more comprehensive view and Supplemental Movie 3 for analysis of negative curvature recorded in the simulations. (B) Comparison of the radius of curvature estimates (here R = 〈2Hmax〉−1) for different Exo70 variants on the membrane. Dashed line at 53.4 nm represents the threshold above which no inward membrane tubulation was detected in mesoscale simulations. (C) Snapshots from mesoscale simulations of inward growing tubules for model Exo70 domains at different surface concentration (from 20% to 100%); a0=14 nm, H0||=−1.0/a0 and εLL =1kBT. The extent of tubulation is Exo70 concentration-dependent. Exo70 proteins on the outer surface of the membrane are not shown in order to enhance the visibility of the tubules. See also Supplemental Figure 1.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Comparison, Membrane, Concentration Assay

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Overview of a B16F1 cell co-expressing GFP-Exo70 and mCherry-Lifeact (labeling F-actin). (B-D) Time-lapse microscopy showing dynamics of individual filopodia in Box b, c, d, of (A). Time interval between frames is 42 seconds. The lines mark the most distal points of GFP-Exo70 (green) and actin (red) signals in the filopodia over time; the yellow lines indicate the coincidence of these signals. (B) For a regular filopodium, the dynamics of GFP-Exo70 and F-actin were indistinguishable. (C) While GFP-Exo70 signal was stationary, F-actin lagged behind and gradually filled the filopodium. (D) GFP-Exo70 and F-actin co-localized in the long stationary filopodium, whereas the nascent GFP-Exo70-positive filopodium (green arrow) was actin-free. In the same field, a new membrane protrusion (yellow arrowhead) was emerging and gradually filled with F-actin. (E) The percentage of filopodia completely filled with F-actin (red) and lacking F-actin (blue) in the interior. A total of 1687 filopodia in 31 cells were counted. (F) B16F1 cells expressing GFP-Exo70 variants were stained for F-actin. GFP-Exo70 induced significantly more filopodia than GFP or GFP-Exo70 mutants. In addition, approximately 20% of the GFP-Exo70-induced membrane protrusions were actin-free. Much lower percentages of the protrusions in cells expressing GFP or GFP-Exo70 mutants (see text for description of the mutants) were actin-free. Error bars, SD. n=10; *, p<0.01. See also Supplemental Figure 2E. (G) B16F1 cells treated with siRNA targeting Exo70, Sec8 or Sec15 were transfected with GFP or GFP-Cdc42L. The expression of GFP-Cdc42L induced more filopodia comparing to GFP cells. The number of filopodia was significantly reduced upon Exo70 knockdown. Knockdown of Sec8 or Sec15 did not affect the filopodia formation in GFP-Cdc42L or GFP cells. Error bars, SD. n=10; *, p<0.01. See also Supplemental Figure 3B for Western blot showing the knockdown efficiency.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Expressing, Labeling, Time-lapse Microscopy, Membrane, Staining, Transfection, Knockdown, Western Blot

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A-C) Correlative fluorescence and platinum replica EM showing the organization of actin in protrusive structures in a GFP-Exo70-expressing cell. (A) Overview of a B16F1 cell expressing GFP-Exo70. Scale bar, 10 μm. (B) Frames from the time-lapse sequence showing dynamics of a protruding filopodium (corresponding to box b in A). Correlative EM of region B is shown in C or with overlaid GFP-Exo70 fluorescence (green) (C′). The red arrows show a newly generated membrane protrusion that was devoid of actin. (D-H) Correlative fluorescence and platinum replica EM of a GFP-Exo70-expressing cell shown by GFP fluorescence (D-F) or EM (G) at the beginning (D) and the end (E and F) of the 220 sec time-lapse sequence and after detergent extraction (“Ext”, with 1% Triton X-100 in PEM buffer containing 2 μM phalloidin without 2% PEG) (F, G). Inset in E is an overlay of boxed regions from D and E, showing that most of filopodial protrusions marked by GFP-Exo70 was barely changed. Inset in F is an overlay of boxed regions from E and F showing that half of membrane protrusions were removed by detergent extraction, while others remained (shown in red), which contained actin filaments as shown by EM (inset in G). (H) Correlative EM showing that each detergent-resistant filopodia contained only 2–4 actin filaments, which are superimposed with GFP-Exo70 signals (colored red).

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Fluorescence, Expressing, Sequencing, Generated, Membrane, Extraction

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) MDA-MB-231 cells expressing indicated siRNA and GFP-tagged rat Exo70 variants were stained for Arp3 (red) and F-actin (blue) to detect lamellipodia. Control siRNA-treated cells (Panel 1) had clear and extended lamellipodia. In Exo70-knockdown cells (Panel 2), the formation of lamellipodia was impaired. Expression of GFP-rExo70 (Panel 3) rescued lamellipodia formation, whereas the expression of Exo70 mutants failed to rescue the defect (Panel 4–5). Scale bar, 5 μm. (B) The ratios between the length of the lamellipodia and the total cell perimeters (“lamellipodia ratio”) were compared for each group. The mutant groups have lower lamellipodia ratios. Error bars, SD. n=25; *, p<0.01.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Expressing, Staining, Control, Knockdown, Mutagenesis

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Transmission EM showing that GST-Exo70 induced tubular invaginations towards the interior of the lumen of LUVs similar to the MIM I-BAR domain. GST, GST-Exo70(K571A/E572A) did not induce any membrane tubules. See EXPERIMENTAL PROCEDURES for details. Scale bar, 0.5 μm. (B) Untagged Exo70 induced membrane invaginations. The arrow shows a representative region where the tubular invagination was connected to the exterior. See also Supplemental Movie 1a for 3-D tomography of this LUV. (C) Comparison of the diameters of the membrane tubules induced by different proteins. Error bars represent standard deviation (SD). n=80; *, p<0.01. (D) Confocal microscopy of fluorescence-labeled Giant Unilamellar Vesicles (GUVs) incubated with Exo70 (upper panel) or buffer control (lower panel). Sequential frames show the inward growth of a tubule (arrowhead) from a GUV incubated with Exo70. Scale bar, 5 μm.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Transmission Assay, Membrane, Tomography, Comparison, Standard Deviation, Confocal Microscopy, Fluorescence, Labeling, Incubation, Control

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Recombinant Exo70 proteins were loaded onto a Superdex 200 10/300GL column and eluted fractions were subjected to SDS-PAGE. Exo70, but not Exo70(Δ1–75), was eluted in high MW fractions. (B) Gel-filtration fractions were lightly treated with cross-linker BM(PEG)2, which links proteins in vicinity. High MW species were observed in Exo70 but not in Exo70(Δ1–75) fractions. Arrowheads: Exo70 oligomers. (C) Lysates of HEK293T cell expressing Exo70-FLAG together with GFP, GFP-Exo70, or GFP-Exo70(Δ1–75) were incubated with anti-FLAG (M2) beads. The inputs and bound proteins were analyzed by Western blotting using anti-FLAG (upper panels) and anti-GFP monoclonal antibodies (lower panels). Exo70-FLAG was able to pull down GFP-Exo70, but to a much lesser extent, GFP-Exo70(Δ1–75). (D) Exo70 or Exo70(Δ1–75) was incubated with GUVs and subjected to confocal microscopy. Exo70(Δ1–75) induced much fewer invaginations than Exo70. (E) The contours of GUV membranes were linearized. The numbers of invaginations per 25 μm were calculated (average ± SD). Exo70(Δ1–75) and Exo70(K571A/E572A) induced much fewer invaginations than the full-length Exo70 (n=10; p<0.01). Scale bar, 5 μm.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Recombinant, SDS Page, Filtration, Expressing, Incubation, Western Blot, Bioprocessing, Confocal Microscopy

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer. Top and side views of the simulation snapshots show negative curvature formed in both directions. All snapshots were rendered with VMD (Humphrey et al., 1996). See also Supplemental Movie 2 for a more comprehensive view and Supplemental Movie 3 for analysis of negative curvature recorded in the simulations. (B) Comparison of the radius of curvature estimates (here R = 〈2Hmax〉−1) for different Exo70 variants on the membrane. Dashed line at 53.4 nm represents the threshold above which no inward membrane tubulation was detected in mesoscale simulations. (C) Snapshots from mesoscale simulations of inward growing tubules for model Exo70 domains at different surface concentration (from 20% to 100%); a0=14 nm, H0||=−1.0/a0 and εLL =1kBT. The extent of tubulation is Exo70 concentration-dependent. Exo70 proteins on the outer surface of the membrane are not shown in order to enhance the visibility of the tubules. See also Supplemental Figure 1.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Comparison, Membrane, Concentration Assay

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) Overview of a B16F1 cell co-expressing GFP-Exo70 and mCherry-Lifeact (labeling F-actin). (B-D) Time-lapse microscopy showing dynamics of individual filopodia in Box b, c, d, of (A). Time interval between frames is 42 seconds. The lines mark the most distal points of GFP-Exo70 (green) and actin (red) signals in the filopodia over time; the yellow lines indicate the coincidence of these signals. (B) For a regular filopodium, the dynamics of GFP-Exo70 and F-actin were indistinguishable. (C) While GFP-Exo70 signal was stationary, F-actin lagged behind and gradually filled the filopodium. (D) GFP-Exo70 and F-actin co-localized in the long stationary filopodium, whereas the nascent GFP-Exo70-positive filopodium (green arrow) was actin-free. In the same field, a new membrane protrusion (yellow arrowhead) was emerging and gradually filled with F-actin. (E) The percentage of filopodia completely filled with F-actin (red) and lacking F-actin (blue) in the interior. A total of 1687 filopodia in 31 cells were counted. (F) B16F1 cells expressing GFP-Exo70 variants were stained for F-actin. GFP-Exo70 induced significantly more filopodia than GFP or GFP-Exo70 mutants. In addition, approximately 20% of the GFP-Exo70-induced membrane protrusions were actin-free. Much lower percentages of the protrusions in cells expressing GFP or GFP-Exo70 mutants (see text for description of the mutants) were actin-free. Error bars, SD. n=10; *, p<0.01. See also Supplemental Figure 2E. (G) B16F1 cells treated with siRNA targeting Exo70, Sec8 or Sec15 were transfected with GFP or GFP-Cdc42L. The expression of GFP-Cdc42L induced more filopodia comparing to GFP cells. The number of filopodia was significantly reduced upon Exo70 knockdown. Knockdown of Sec8 or Sec15 did not affect the filopodia formation in GFP-Cdc42L or GFP cells. Error bars, SD. n=10; *, p<0.01. See also Supplemental Figure 3B for Western blot showing the knockdown efficiency.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Expressing, Labeling, Time-lapse Microscopy, Membrane, Staining, Transfection, Knockdown, Western Blot

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A-C) Correlative fluorescence and platinum replica EM showing the organization of actin in protrusive structures in a GFP-Exo70-expressing cell. (A) Overview of a B16F1 cell expressing GFP-Exo70. Scale bar, 10 μm. (B) Frames from the time-lapse sequence showing dynamics of a protruding filopodium (corresponding to box b in A). Correlative EM of region B is shown in C or with overlaid GFP-Exo70 fluorescence (green) (C′). The red arrows show a newly generated membrane protrusion that was devoid of actin. (D-H) Correlative fluorescence and platinum replica EM of a GFP-Exo70-expressing cell shown by GFP fluorescence (D-F) or EM (G) at the beginning (D) and the end (E and F) of the 220 sec time-lapse sequence and after detergent extraction (“Ext”, with 1% Triton X-100 in PEM buffer containing 2 μM phalloidin without 2% PEG) (F, G). Inset in E is an overlay of boxed regions from D and E, showing that most of filopodial protrusions marked by GFP-Exo70 was barely changed. Inset in F is an overlay of boxed regions from E and F showing that half of membrane protrusions were removed by detergent extraction, while others remained (shown in red), which contained actin filaments as shown by EM (inset in G). (H) Correlative EM showing that each detergent-resistant filopodia contained only 2–4 actin filaments, which are superimposed with GFP-Exo70 signals (colored red).

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Fluorescence, Expressing, Sequencing, Generated, Membrane, Extraction

Journal: Developmental cell

Article Title: Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

doi: 10.1016/j.devcel.2013.07.007

Figure Lengend Snippet: (A) MDA-MB-231 cells expressing indicated siRNA and GFP-tagged rat Exo70 variants were stained for Arp3 (red) and F-actin (blue) to detect lamellipodia. Control siRNA-treated cells (Panel 1) had clear and extended lamellipodia. In Exo70-knockdown cells (Panel 2), the formation of lamellipodia was impaired. Expression of GFP-rExo70 (Panel 3) rescued lamellipodia formation, whereas the expression of Exo70 mutants failed to rescue the defect (Panel 4–5). Scale bar, 5 μm. (B) The ratios between the length of the lamellipodia and the total cell perimeters (“lamellipodia ratio”) were compared for each group. The mutant groups have lower lamellipodia ratios. Error bars, SD. n=25; *, p<0.01.

Article Snippet: Analysis of the protein and lipid conformations in our simulations revealed that the induction of the curvature was mediated by the positively charged residues throughout the surface of the Exo70 dimers that interact with the negatively charged phospholipids, which suggests that curvature induction requires a scaffold of at least two linked Exo70 monomers that act in concert to remodel the associated lipid bilayer from its native planar state; a representation of the electrostatic map depicting this charge complementarity between the protein and the lipid interfaces is shown in Supplemental Figure 1D and E . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 Molecular dynamics and mesoscale simulations demonstrate that Exo70 induces negative membrane curvature (A) Molecular dynamics simulation of modeled Exo70 anti-parallel (left) and parallel (right) dimers on a DOPC/DOPS bilayer.

Techniques: Expressing, Staining, Control, Knockdown, Mutagenesis