Journal: Nature

Article Title: In situ structural mechanism of epothilone-B-induced CNS axon regeneration

doi: 10.1038/s41586-025-09654-z

Figure Lengend Snippet: a , Colocalization of microtubule (SiR-tubulin) and membrane (CellMask) signals at regenerating axon tips 1 h after axotomy and EpoB treatment. Scale bar, 10 µm. b , Actin organization (SPY555-FastAct) at the tip of axons under control (Ax − EpoB − ) and regenerating (Ax + EpoB + ) conditions. Scale bars, 3 µm. c – e , Membrane tension analysis by FLIM. c , Membrane tension is increased at tips relative to shafts (>15 µm downstream from the tip) 1 h after axotomy ( n = 12). d , Tension normalizes at 4 h ( n = 14). e , Control neurons show no difference between tip and shaft ( n = 20). Data are mean ± s.e.m.; two-tailed Mann–Whitney test. f , Cryo-EM images of regenerating axons. The grid view gives an overview of the location (left; scale bar, 200 µm), with a magnified view showing an axotomy site (right; scale bar, 5 µm). Arrow indicates axonal growth direction and dashed line and scissors indicate the axotomy site. g , Distribution of microtubule lengths in cryo-EM snapshots 1 h after axotomy. Ax + EpoB + : n = 17, median = 19.1; Ax + EpoB − : n = 21, median = 0. The centre line shows the median; two-tailed Mann–Whitney test. h , Time course of axonal extension by cryo-EM snapshots, in the presence of EpoB after axotomy. n = 6 (15 min), n = 17 (1 h), n = 6 (3 h) and n = 12 (6 h) axons. Data are mean ± s.e.m. i , High-magnification montage of a regenerating axon 1 h after axotomy with EpoB. Top, cryo-EM montage; dashed line indicates the axotomy site. Scale bar, 400 nm. Bottom, segmentation depicting microtubules (green), membranes (vesicles and endoplasmic reticulum (ER)) (orange) and actin (magenta). Arrow depicts axonal growth direction.

Article Snippet: For imaging actin, microtubules, and plasma membrane together (Extended Data Fig. ), 50 nM of SPY555-FastAct (Cytoskeleton Spirochrome CY-SC205), 50 nM of SPY650-Tubulin (CY-SC503), and a MemGlow 488 (1:200,000) (Cytoskeleton MG01-10) were incubated with the neurons for 30 min prior to imaging.

Techniques: Membrane, Control, Two Tailed Test, MANN-WHITNEY, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Matrix viscoelasticity controls epithelial cell mechanobiology through dimensionality

doi: 10.1101/2024.03.05.583526

Figure Lengend Snippet: (a) Representative x-y trajectories of MCF-10A cells on elastic and viscoelastic polyacrylamide hydrogels over 5 hours (n = 48 trajectories for soft E, n = 43 trajectories for Soft V, n = 49 trajectories for Stiff E, n = 34 trajectories for Stiff V). (b) MCF-10A cell migration speed on soft elastic (Soft E, n=56 cells) and viscoelastic (Soft V, n = 61 cells) matrices obtained from three independent experiments. *** p = 0.0007, unpaired two-tailed t-test. (c) Average mean square displacement (MSD) vs lag time for MCF-10A cells on soft elastic (Soft E) and viscoelastic (Soft V) matrices. The diffusion exponent, α, is shown in the graph. Data is shown as mean ±SEM (n = 48 cells for Soft E, n = 43 cells for Soft V) from three independent experiments. (d) MCF-10A cell migration speed on stiff elastic (Stiff E, n=55 cells) and viscoelastic (Stiff V, n = 35 cells) matrices obtained from at least two independent experiments. *** p = 0.0007, unpaired two-tailed t-test. **** p < 0.0001, unpaired two-tailed t-test. (e) Average mean square displacement (MSD) vs lag time for cells on stiff elastic (Stiff E) and viscoelastic (Stiff V) matrices. The diffusion exponent, α, is shown in the graph. Data is shown as mean ±SEM (n = 49 cells for Stiff E, n = 34 cells for Stiff V) from at least two independent experiments. (f) Diffusion exponent, α, plotted against average cell migration speed. Data is shown as mean ±SEM (R 2 = 0.8414) for the same number of cells and independent experiments as in b-d (for cell migration speed) and c-e (for MSD). (g) Representative images of MCF-10A tagged with live Spy555-FastAct on elastic and viscoelastic polyacrylamide hydrogels. Yellow line shows location where kymographs were computed, on average. Insets show representative kymographs for each condition, with yellow line indicating the slope from which the actin retrograde flow speed is computed. The spatial scale bar in the inset is 2 μm, whereas the temporal scale bar is 2 minutes. (h) Quantification of actin retrograde flow speed for MCF-10A cells cultured on Soft E (n = 19 kymographs), Soft V (n=48 kymographs), Stiff E (n=44 kymographs) and Stiff V (n=52 kymographs) hydrogels from at least two independent experiments. **** p < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test. (i) Diffusion exponent, α, vs actin retrograde flow speed (left, R 2 = 0.9711); and migration speed vs actin retrograde flow speed (right, R 2 =0.8322). Data is shown as mean ±SEM for the same number of cells/kymographs as in c-e and h, respectively. (j) Schematic summary of experimental findings on how matrix viscoelasticity modulates MCF-10A cell migration speed and persistence via actin retrograde flow speed. Green arrows inside the cells represent actin retrograde flow, whereas colourful arrows outside the cell depict schematic migration trajectories.

Article Snippet: Prior to imaging, cells were incubated with SPY555-FastAct™ (Spirochrome) for 2 hours at 37°C following the manufacturer’s protocol (dilution 1:1000 in culture medium).

Techniques: Migration, Two Tailed Test, Diffusion-based Assay, Cell Culture