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Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons

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Science  25 Jan 2013:
Vol. 339, Issue 6118, pp. 452-456
DOI: 10.1126/science.1232251

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  1. Fig. 1

    STORM imaging reveals distinct organization of actin filaments in the axons and dendrites of neurons. (A) Conventional fluorescence image of actin (green) and a dendritic marker, MAP2 (magenta), in a cultured hippocampal neuron fixed at 7 DIV. (B) Three-dimensional STORM image of actin in a dendritic region corresponding to the white box in (A). The z positions in the STORM image are color-coded according to the color scale, with violet and red indicating the positions closest to and farthest from the substratum, respectively. (C) Magnification of the region inside the red box in (B). The yz cross section corresponding to the white-boxed region is shown in the inset. (D) Conventional fluorescence image of actin (green) and MAP2 (magenta) in a neuron fixed at 12 DIV. (E) Three-dimensional STORM image of actin in a region containing axons (devoid of the dendritic marker MAP2), corresponding to the yellow box in (D). The yz cross sections corresponding to the white-boxed regions are shown in the insets. The 3D STORM image of a region containing a dendrite of this neuron is shown in fig. S1B (28). (F) Conventional fluorescence image of actin (green) and an axon initial segment marker, NrCAM (magenta), in a neuron fixed at 9 DIV. (G) Three-dimensional STORM image of actin in a region containing the axon initial segments, corresponding to the yellow box in (F).

  2. Fig. 2

    Actin filaments in axons form a quasi-1D, periodic structure with a uniform spacing of ~180 to 190 nm. (A) Three-dimensional STORM image of a segment of axon (top) and the distribution of localized molecules after the 3D image was projected to one dimension along the axon long axis (bottom). (B) Fourier transform of the 1D localization distribution shown in (A). The Fourier transform shows a fundamental frequency of (190 nm)−1 and an overtone. (C) Histogram of the spacings between adjacent actin ringlike structures (N = 204 spacings). The red line is a Gaussian fit with a mean of 182 nm and a SD of 16 nm.

  3. Fig. 3

    Spectrin and adducin exhibit quasi-1D, periodic patterns in axons, quantitatively similar to that observed for actin. (A) Three-dimensional STORM image of βII-spectrin in axons. βII-spectrin is immunostained against its C-terminal region, which is situated at the center of the rodlike αII-βII spectrin tetramer. (Inset) The yz cross section of the boxed region showing the ringlike structure. The smaller white box denotes the position of the inset image. (B) Histogram of the spacings between adjacent spectrin rings (N = 340 spacings). The red line is a Gaussian fit with a mean of 182 nm and a SD of 18 nm. (C and D) Same as (A) and (B) but for βIV-spectrin, which is specifically located in the initial segments of axons. βIV-spectrin is immunostained against its N-terminal region, which corresponds to the ends of the spectrin tetramer. The red line superimposed on the histogram is a Gaussian fit with a mean of 194 nm and a SD of 15 nm (N = 88 spacings). (E and F) Same as (A) and (B) but for adducin, an actin-capping protein. The red line superimposed on the histogram is a Gaussian fit with a mean of 187 nm and a SD of 16 nm (N = 216 spacings).

  4. Fig. 4

    Actin, spectrin, and adducin form a coordinated, quasi-1D lattice structure in axons, and sodium channels are distributed in a periodic pattern in coordination with the actin-spectrin–based submembrane cytoskeleton. (A) Two-color STORM image of actin (green) and βII-spectrin (magenta). βII-spectrin is immunostained against its C-terminal region, which is situated at the center of the spectrin tetramer. (B) Two-color STORM image of actin (green) and adducin (magenta). (C) Two-color STORM image of βII-spectrin (green) and adducin (magenta). (D) Two-color STORM image of sodium channels (Nav, green) and βIV-spectrin (magenta). βIV-spectrin is immunostained against its N-terminal region, which is situated at the two ends of the spectrin tetramer. The distributions of the localized molecules along the axon shafts are shown in fig. S9 (28). (E) Spatial correlations between actin and the βII-spectrin C terminus [(A), black], between actin and adducin [(B), blue], between adducin and the βII-spectrin C terminus [(C), red], and between sodium channels and the βIV-spectrin N terminus [(D), green]. The correlation function is calculated for varying relative shifts between the two color channels along the axons. (F) A model for the cortical cytoskeleton in axons. Short actin filaments (green), capped by adducin (blue) at one end, form ringlike structures wrapping around the circumference of the axon. Spectrin tetramers (magenta) connect the adjacent actin/adducin rings along the axon, creating a quasi-1D lattice structure with a periodicity of ~180 to 190 nm. The letters "C" and "N" denote the C terminus (magenta triangles) and N terminus (magenta squares) of β-spectrin, respectively. Ankyrin and sodium channels, not shown in the model, also form semiperiodic patterns in coordination with the periodic cytoskeletal structure.

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