Research ArticleCELL BIOLOGY

Actin modulates shape and mechanics of tubular membranes

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Science Advances  22 Apr 2020:
Vol. 6, no. 17, eaaz3050
DOI: 10.1126/sciadv.aaz3050
  • Fig. 1 Effect of an actin sleeve on membrane tube stability.

    Lipids (magenta) and actin (green) are observed by spinning disk confocal imaging. (A and B) Left column: Scheme of each step toward MaAS formation. (A) (a) Preformed tube held by optical tweezer, (b) microinjection of pVCA in a sulforhodamine-B solution, (c) microinjection of monomeric actin at ti, and (d) the membrane tube is sheathed with an actin sleeve within 2 min. A quadrant photodiode (QPD) images the laser beam. (B) Escaped MaAS and (C) elongated MaAS before (top) and after (bottom) pulling; the white box indicates the location of the elongated MaAS. Scale bars, 10 μm. Dashed crosses indicate bead center.

  • Fig. 2 Actin sleeve thickness drives MaAS fate.

    (A and B) Escaped (filled circles; n = 8) and elongated (opened circles; n = 17) MaAS. Data are shown as means ± SD. (A) Quantification of actin fluorescence per lipid fluorescence depending on MaAS fate. P values are calculated using the t test. a.u., arbitrary unit. (B) Force-elongation curves for MaAS and a naked tube (light magenta filled circles; n = 15). The star symbol indicates the length at which the MaAS escapes. (C) Snapshots of two branched actin network networks for different thicknesses ℓz (top ℓz = 10.5ℓmesh; bottom ℓz = 5.5ℓmesh) under uniaxial deformation along the x axis. The two configurations correspond to a deformation of ∼150%. Colors indicate the local longitudinal stress σ̂xx at the scale of a monomer, ranging from yellow for tension to white for compression. (D) Average force F extrapolated for a cylindrical sleeve with inner radius R0 = 25 nm and variable thickness ℓz, implying an outer radius R0 + ℓz as a function of the deformation Δℓx/ℓx0 = (ℓx − ℓx0)/ℓx0, with ℓx0 the initial size, for different network thicknesses. Triangles indicate the maximum force Ftear that the network can bear before falling apart. (E) Phase diagram representing Ftear as a function of the gel thickness. The dashed black line shows the optically trapped bead force limit of about 50 pN. Actin sleeves larger than ∼10 meshsizes should, thus, display an escaped MaAS behavior.

  • Fig. 3 Membrane tube radius in elongated MaAS.

    (A) Representative confocal images before pulling for reference (top) and after elongation (bottom). Magenta and green correspond respectively to lipid and actin. The lipid image is displaced in the white rectangle right below the actin image for clarity. M and m regions are respectively defined as maximal and minimal actin intensity regions of the elongated MaAS (bottom) or before pulling (top). Graphs represent lipid and actin intensities along the elongated MaAS. Scale bar, 10 μm. Dashed crosses indicate bead center. (B and C) Relative difference in membrane tube radius between M and m regions defined in (A). (B) Values corresponding to classification schemed on the left (a, b, and c). (C) “High actin” and “Low actin” refer respectively to actin content of elongated MaAS above and below average in Fig. 2A; reference condition (Ref) is before pulling. Lines connect same MaAS before and after elongation. (D) Proportionality factor of force-elongation curve, for b-type MaAS, as a function of (L × r0)−1. In the inset, parameters used for theoretical description of b-type MaAS. Data are shown as means ± SD in (B) and (C). P values calculated using t test. **P < 0.01.

  • Fig. 4 Elongated MaAS relaxation.

    (A and B) Representative confocal images of relaxing elongated MaAS (images every 30 s). Scale bars, 10 μm. Dashed crosses indicate bead center. Dotted lines follow lipid fluorescence relaxation. (C and D) Corresponding curves as a function of time for length, force, and relative membrane tube radii associated with m and M regions defined in Fig. 3A and Materials and Methods. Time 0 corresponds to the start of MaAS pulling. In red, the fitting curve from Eq. 4 using experimental values ℓ = 26.5 μm and L = 22.1 μm. (E) Relaxation time of the force for elongated MaAS and naked tubes, using an exponential decay (Materials and Methods). Black filled circles point b-type MaAS. P values calculated using t test. *P < 0.05.

  • Fig. 5 Elongated MaAS retraction.

    (A and B) Time lapse overlay images after the trap is turned off for an elongated MaAS (A) and control (pVCA microinjection is omitted) (B). Arrowheads indicate regions devoid of actin that get thicker during retraction. Scale bars, 10 μm. Dashed crosses indicate bead center. (C to E) Empty circle, partially retracted; crosses, totally retracted. (C) Tube length as a function of time after the trap is turned off for elongated MaAS or when pVCA is omitted (gray filled circles). (D) Retraction time as a function of actin content. (E) Actin fluorescence as a function of lipid fluorescence. Full circles are escaped MaAS. Dotted lines separate sectors. We use the orthogonal residue method, where we minimize the orthogonal distance between data and the linear regression to approximate sector separations. Elongated MaAS are in the green region, and escaped MaAS are in the orange region. Totally and partially retracted MaAS are separated in sectors inside the green region. Inset: Log-log representation, previous triangular sections then become bands here.

Supplementary Materials

  • Supplementary Materials

    Actin modulates shape and mechanics of tubular membranes

    A. Allard, M. Bouzid, T. Betz, C. Simon, M. Abou-Ghali, J. Lemière, F. Valentino, J. Manzi, F. Brochard-Wyart, K. Guevorkian, J. Plastino, M. Lenz, C. Campillo, C. Sykes

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