fascin (human, recombinant  (Cytoskeleton Inc)

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration of the GUV content and the two macromolecular reactions at membrane level: the MinDE self-assembly mechanism behind pattern formation and the diffusiophoresis-mediated transport of neutravidin-bound actomyosin bundles by Min proteins. The active flux of MinDE proteins on the vesicle membrane interacts non-specifically via frictional forces with membrane-bound neutravidin inducing the transport and positioning of these molecules, and consequently the actomyosin bundles linked to them, towards areas of low MinD density. b 3D projections of confocal images showing the 4 phenotypes of actin architectures obtained after encapsulating 2.4 µM actin, 0.6 µM fascin (fascin/actin molar ratio = 0.25), 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 10 µm. c Bar graphs with the frequencies of the four actomyosin phenotypes observed at different vesicle diameters when encapsulation experiments were performed at 0.25 and 0.5 fascin/actin molar (M/M) ratio in the presence and absence of Min proteins and protein/crowding conditions specified in b. Experiments performed per condition n = 3, total number of GUVs analysed per condition = 150. d 3D projections of time-lapse confocal images depicting the reorganization and stacking of actomyosin bundles towards the vesicle equator driven by the diffusiophoretic transport of Min pole-to-pole oscillations. Yellow arrows indicate the perpendicular orientation of MinDE oscillations with respect to actomyosin bundles, which get antagonistically positioned at mid-cell. Kymographs generated at the vesicle equator (blue dashed circle) are meant to visually define the position of fluorescent features at this region over time. Orange dotted lines depict the approximate distribution of actin bundles on the membrane at two time points. Vesicle content as specified in b. Scale bars: 10 µm.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane, Encapsulation, Generated

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration behind the mechanism of membrane deformation. Contractile actomyosin bundles positioned by MinDE proteins at mid-cell induce furrow-like membrane invaginations. 3D projections and 2D confocal images show an actomyosin ring constricting the vesicle at its equator. Orange lines indicate the major (a) and minor (b) axes measured to calculate the aspect ratio of the deformed vesicle (for spherical vesicles: aspect ratio = 1). Inner solution mix: 4 µM actin, 2 µM fascin (fascin/actin molar ratio = 0.5), 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bar: 10µm. b Schematic illustration, 3D projections and 2D confocal images of a vesicle containing a soft web of actomyosin bundles at the vesicle centre being positioned by pole-to-pole Min oscillations. The contractile actomyosin band formed causes the deformation of the vesicle (aspect ratio < 1). Inner solution mix: 2.4 µM actin, 0.6 µM fascin (fascin/actin molar ratio = 0.25), 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bar: 10µm. c Schematic illustration, 3D projection and 2D confocal image of a vesicle with a non-positioned contractile actomyosin assembly due to the loss in pole-to-pole MinDE oscillations. Constriction of the actomyosin bundles results in the deformation of the vesicle membrane into an asymmetric dumbbell shape. Scatter plot depicts the aspect ratio of the vesicle at different time points. Inner reaction mix: 4 µM actin, 2 µM fascin (fascin/actin molar ratio = 0.5), 0.05 µM myosin II, 20 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bar: 10 µm.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration depicting the change in vesicle shape due to MinDE chaotic oscillations. Min proteins attach to areas delimited by soft actomyosin bundles and deform the membrane generating dynamic bleb-like protrusions. Fluorescence and brightfield confocal time-series show a blebbing vesicle. After bleb retraction, the reduction in bilayer tension generates an outward lipid bud (blue arrows). Encapsulation conditions: 2.4 µM actin, 0.6 µM fascin, 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 10 µm. b Confocal cross-section images at two time points of the vesicle in section a. Peripheral actomyosin anchoring creates a delimiting area which deforms upon MinDE binding. Additionally, MinDE diffusiophoretic transport changes the position of actomyosin bundles and the shape of the membrane area available for Min protein recruitment (blue arrows). Fluorescence intensity line plots of EGFP-MinD (green) and ATTO647-actin (magenta) demonstrate the demixing of both protein systems at the membrane perimeter (orange dotted line). Scale bars: 10 µm. c Schematic illustration of the proposed mechanism behind MinDE-induced blebbing. The recruitment of MinDE proteins to the compartmentalized inner leaflet of the bilayer generates the effect of a membrane outward protrusion in bleb form. d Schematic illustration depicting the radius of curvature R C used to calculate the curvature (Κ = 1/R C ) of the blebs. 3D projection and 2D time-lapse confocal images show a vesicle with diverse bleb-like deformations emerging over time. Orange arrow points at a bleb with Κ = 0.73 µm -1 . Blue arrow, Κ = 0.27 µm -1 . Magenta arrow, Κ = 0.10 µm -1 . Encapsulation mix: 4 µM actin, 2 µM fascin, 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 20 µm.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane, Fluorescence, Encapsulation, Binding Assay

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration (top) and 3D confocal image (bottom) show the membrane composition employed to generate phase-separated vesicles and the domains obtained. Scale bar: 10 µm. b 3D projections and 2D confocal images depict a blebbing phase-separated vesicle. MinDE proteins bind and oscillate on Ld domains. Actomyosin bundles remain at lipid-phase boundaries as Min proteins transiently deform Ld domains (orange arrows). Inner encapsulation mix: 2.4 µM actin, 0.6 µM fascin (fascin/actin molar ratio = 0.25), 0.05 µM myosin II, 20 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 20 µm. c Schematic illustration of the proposed mechanism behind the dynamic deformation of Ld domains by MinDE protein oscillations.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane, Encapsulation

Journal: FEBS letters

Article Title: Crowding tunes the organization and mechanics of actin bundles formed by crosslinking proteins.

doi: 10.1002/1873-3468.13949

Figure Lengend Snippet: Fig. 1. Effects of crowding on fascin-induced bundles. (A) Representative TIRF microscopy images of fascin-induced bundles (the molar ratio of actin to fascin = 2 : 1). 1 μM of actin filaments (50% rhodamine-labeled) and 0.5 μM of fascin were incubated in dilute polymerization buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 2 mM MgCl2, 1 mM ATP, and 1 mM DTT) or buffers containing crowding agents (1–5%w/w PEG, 10–50% w/w sucrose, or 5–20% w/w Ficoll) for 1 h at room temperature prior to imaging (scale bar, 10 μm). The insets are zoom-ins of each TIRF image (scale bar, 5 μm). (B) The number of filaments per bundle analyzed from cross-sectional fluorescence intensities without or with crowders. The box represents the 25–75% of data, whiskers indicate standard deviation (SD), and the middle square is the mean. (C) Bending persistence length (Lp) of fascin bundles without or with crowders. Sample size: Ncontrol = 291, N1%w/w PEG = 222, N3%w/w PEG = 241, N5%w/w PEG = 240, N10%w/w sucrose = 323, N30%w/w sucrose = 299, N50%w/w sucrose = 357, N5%w/w Ficoll = 389, N10%w/w Ficoll = 414, N20%w/w Ficoll = 502. P values were determined with one-way ANOVA and post hoc Tukey’s test. (n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: Bundle formation was induced by adding human recombinant protein fascin with a His-tag (Novus Biologicals, Littleton, CO, USA) or rabbit skeletal muscle α-actinin (Cytoskeleton, Inc.) in the polymerization buffer containing crowding agents: polyethylene glycol (PEG) (MW = 8000 Da) (Fisher, Waltham MA, USA), sucrose (MW = 342 Da) (Sigma, Saint Louis, MO, USA), or Ficoll 70 (MW = 70000 Da) (GE Healthcare, Chicago, IL, USA) at varying concentrations.

Techniques: Microscopy, Labeling, Incubation, Imaging, Standard Deviation

Journal: FEBS letters

Article Title: Crowding tunes the organization and mechanics of actin bundles formed by crosslinking proteins.

doi: 10.1002/1873-3468.13949

Figure Lengend Snippet: Fig. 3. The structure of fascin- and α-actinin-induced bundles in crowded environments. (A) AFM height images of fascin- and α-actinin-induced bundles with various crowding conditions. The molar ratio of unlabeled actin to crosslinking proteins was 2 : 1 except for control of α-actinin-induced bundle (5 : 1) ([actin] = 5 μM). Actin filaments were incubated with fascin or α-actinin in dilute polymerization buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 2 mM MgCl2, 1 mM ATP, and 1 mM DTT) or buffers containing crowding agents (5% w/w PEG, 10% w/w sucrose, or 20% w/w Ficoll) for 1 h at room temperature prior to imaging. All scales bars = 500 nm except α-actinin control scale bar = 1 μm. Diameters of (B) fascin-induced actin bundles and (C) α- actinin-induced bundles under varying crowding conditions. Bundle diameters were determined by the full width at half maximum (FWHM) on the AFM images. The box represents the 25–75% of data, whiskers indicate SD, and the middle square is the mean. P values were determined with one-way ANOVA and post hoc Tukey’s test (n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: Bundle formation was induced by adding human recombinant protein fascin with a His-tag (Novus Biologicals, Littleton, CO, USA) or rabbit skeletal muscle α-actinin (Cytoskeleton, Inc.) in the polymerization buffer containing crowding agents: polyethylene glycol (PEG) (MW = 8000 Da) (Fisher, Waltham MA, USA), sucrose (MW = 342 Da) (Sigma, Saint Louis, MO, USA), or Ficoll 70 (MW = 70000 Da) (GE Healthcare, Chicago, IL, USA) at varying concentrations.

Techniques: Control, Incubation, Imaging

Journal: FEBS letters

Article Title: Crowding tunes the organization and mechanics of actin bundles formed by crosslinking proteins.

doi: 10.1002/1873-3468.13949

Figure Lengend Snippet: Fig. 5. Interaction energy and interhydrogen bond analysis. (A) Average interaction energy (SD) and (B) the number of hydrogen bonds (SD) between filaments and fascin or CH1 domain of α-actinin in the absence or presence of crowders, for the last 10 ns of the 20-ns equilibrium all-atom molecular dynamics (MD) simulations.

Article Snippet: Bundle formation was induced by adding human recombinant protein fascin with a His-tag (Novus Biologicals, Littleton, CO, USA) or rabbit skeletal muscle α-actinin (Cytoskeleton, Inc.) in the polymerization buffer containing crowding agents: polyethylene glycol (PEG) (MW = 8000 Da) (Fisher, Waltham MA, USA), sucrose (MW = 342 Da) (Sigma, Saint Louis, MO, USA), or Ficoll 70 (MW = 70000 Da) (GE Healthcare, Chicago, IL, USA) at varying concentrations.

Techniques:

Journal: FEBS letters

Article Title: Crowding tunes the organization and mechanics of actin bundles formed by crosslinking proteins.

doi: 10.1002/1873-3468.13949

Figure Lengend Snippet: Fig. 4. Conformations of actin filament (F-actin) with fascin or CH1 domain of α-actinin without or with crowders.

Article Snippet: Bundle formation was induced by adding human recombinant protein fascin with a His-tag (Novus Biologicals, Littleton, CO, USA) or rabbit skeletal muscle α-actinin (Cytoskeleton, Inc.) in the polymerization buffer containing crowding agents: polyethylene glycol (PEG) (MW = 8000 Da) (Fisher, Waltham MA, USA), sucrose (MW = 342 Da) (Sigma, Saint Louis, MO, USA), or Ficoll 70 (MW = 70000 Da) (GE Healthcare, Chicago, IL, USA) at varying concentrations.

Techniques:

Journal: FEBS letters

Article Title: Crowding tunes the organization and mechanics of actin bundles formed by crosslinking proteins.

doi: 10.1002/1873-3468.13949

Figure Lengend Snippet: Fig. 6. Proposed scheme of bundling behavior of fascin and α-actinin in crowded condition. (A) Crowding induces densely packed fascin-induced bundles with increased number of filaments per bundle and decreased bundle diameter (D). Binding of fascin to actin filaments is reduced in crowded conditions. (B) Crowding reduces the binding of α-actinin to actin filaments, potentially leading to the formation of short and thin bundles. The diameter (D) and the number of filaments per bundle of α-actinin- crosslinked bundles decrease in the presence of crowders. Crowded conditions may decrease the angle between α-actinin and filaments.

Article Snippet: Bundle formation was induced by adding human recombinant protein fascin with a His-tag (Novus Biologicals, Littleton, CO, USA) or rabbit skeletal muscle α-actinin (Cytoskeleton, Inc.) in the polymerization buffer containing crowding agents: polyethylene glycol (PEG) (MW = 8000 Da) (Fisher, Waltham MA, USA), sucrose (MW = 342 Da) (Sigma, Saint Louis, MO, USA), or Ficoll 70 (MW = 70000 Da) (GE Healthcare, Chicago, IL, USA) at varying concentrations.

Techniques: Binding Assay