Fig 1.
(A) Illustration of the axon membrane skeleton based on super-resolution microscopy results [3] exhibiting actin rings connected by spectrin tetramers. Ankyrin associated Nav channels anchor the lipid bilayer to the membrane skeleton. Adducin has also been observed to colocalize with the actin rings possibly capping actin filaments. (B) A coarse-grain membrane skeleton dynamics model comprising representation of actin rings, spectrin filaments, and ankyrin. The insert shows the dimensions of the considered particles. (S2 Fig)
Fig 2.
Young's moduli of rat hippocampal neuronal subcompartments determined by AFM.
Histograms of Young’s moduli of rat hippocampal (A) soma, (B) dendrites, (C) axons, and (D) axons treated with 20μm Latrunculin B. The median Young's modulus of the soma is 0.7 ± 0.2 kPa (A), of dendrites is 2.5 ± 0.7 kPa (B). For the axon plasma membrane, the median Young’s modulus is 4.6 ± 1.5 kPa (C). When axons were treated with Latrunculin B (20μm, 1 hour) the median value of the axon plasma membrane Young’s modulus was reduced to 2.2 ± 0.6 kPa. Number of samples (N = 2), total number of tested neurons (n = 8). N = 1 and n = 6 for axon + Latrunculin B. (E) Box-whisker plots of mean Young's moduli of the soma, dendrites, axon, and axon treated with Latrunculin B. *** indicates statistical significance of p < 0.001 (Kruskal-Wallis test).
Fig 3.
Measurement of the axon plasma membrane Young’s modulus.
(A) We expand the radius of the axon by 10 nm, in sequential steps of 0.5 nm, by applying a cylindrical Lennard-Jones potential with equilibrium distance measured from the center-line of the axon. At each radius, we measure the total force applied on all the membrane skeleton particles that belong (i) to stripes of 80 nm diameter around the actin rings (red stripes), (ii) to stripes of 105 nm diameter located between actin rings (green stripes), and (iii) to the entire membrane skeleton along the axon. By dividing the total force with the corresponding total area, we compute the pressure on the region between actin rings, on the region around the actin rings and the average pressure on the entire membrane skeleton. Every 0.5 nm increase occurs in 1000 time steps. At each radius, the system is equilibrated for another 1000 time steps while the actual measurements occur every time step for 8000 time steps. Labels I-IV indicate the boundaries of the areas on which we measure the pressure. (B) The mean pressure values obtained for each stripe type are plotted vs the corresponding radii values. From the slope of the fitted straight line and using the linear elastic cylindrical shell theory, the Young’s moduli for each region and the average value are computed. The probability distributions of the pressures measured for the two stripes and for the entire membrane skeleton at 1.5 nm radius are shown in S16 Fig.
Fig 4.
Membrane skeleton dynamics simulations.
(A) The membrane skeleton was equilibrated at a distance of approximately 185 nm between actin rings while the trajectory of ankyrin particles (insert) was recorded for 10×106 time steps every 105 time steps. (B) The skeleton was equilibrated at a distance of approximately 110 nm between actin rings while the trajectory of ankyrin particles (insert) was recorded for 10×106 105 time steps. (C, D) Normalized probability distribution of the ratio d(z)/Lc, where d(z) is the deviation of an ankyrin point from its mean position during its thermal motion along the z-direction and Lc is the mean distance between two consecutive ankyrin points along the z-direction when the spectrin is under tension Lc = 185.78 nm (C) and when the spectrin is almost at equilibrium Lc = 112.32 nm (D). The longitudinal and circumferential separations of the trajectories of neighboring ankyrin particles, and consequently of the corresponding Nav channels, are well-defined in (A) but not in (B).
Fig 5.
Laceration of spectrin filaments.
(A) Axon membrane skeleton with severed spectrin filaments in the marked area between two consecutive rings. (B) None of the severed spectrin filaments (blue color) were reconnected to their initial junction points.