www.sciencemag.org/cgi/content/full/science.1232251/DC1

Supplementary Materials for

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

Ke Xu, Guisheng Zhong, Xiaowei Zhuang*

*To whom correspondence should be addressed. E-mail: zhuang@chemistry.harvard.edu

Published 13 December 2012 on Science Express

DOI: 10.1126/science.1232251

This PDF file includes:

Materials and Methods Figs. S1 to S10 Full Reference List

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Materials and Methods

Neuron culture and hippocampal tissue slices

All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory

Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use

Committee (IACUC) of Harvard University.

Primary hippocampal cultures were prepared from neonatal (E18) rat embryos (timed pregnant Wistar rats from

Charles River Laboratories, Wilmington, MA). Hippocampi were isolated and digested with 0.05% trypsin-EDTA

(1x) at 37oC for 15 minutes. The hippocampi were transferred to Hib E solution (BrainBits HE-Ca), washed

several times with Hib E solution, and pipetted up and down until the tissues were mostly dissolved. The solution

was then passed through a cell strainer (VWR 21008-949) to remove the residual undissociated tissue and

collected in a 50 mL conical tube. Neurons were spun down to the bottom of the tube, resuspended in culture

medium consisting of 96 mL Neurobasal (Invitrogen 12349-015), 2 mL B-27 Supplement (Invitrogen 17504-044),

1 mL Penicillin-Streptomycin (Invitrogen 15140-122) and 1 mL Glutamax (Invitrogen 35050-061), and then

plated onto poly-D-lysine/laminin-coated 12-mm coverslips (BD bioscience BD354087). 5 μM cytosine-D-

arabinofuranoside was added to the culture medium to inhibit the growth of glial cells three days after plating.

The neurons were fed twice a week with freshly made culture medium until use.

For hippocampal tissue slices, brains were dissected from adult mice that were transcardially perfused with 2%

paraformaldehyde plus 2% glutaraldehyde [2% (w/v) paraformaldehyde, 2% (v/v) glutaraldehyde, 2 mM calcium

chloride, 0.1 M sodium cacodylate, pH 7.4] or with 4% paraformaldehyde [4% (w/v) paraformaldehyde in

lactated Ringer's solution)], and postfixed for 0.5-4 hours in the same fixation solution. Hippocampi were then

isolated and cryoprotected overnight at 4 ◦C by immersion in a 30% (w/v) sucrose solution for cryosectioning at

10 µm thickness, or in a 2.3 M sucrose solution for ultra-cryosectioning at 0.5-2 µm thickness. For cryosectioning,

the cryoprotected hippocampal tissue samples were allowed to equilibrate in 2:1 OCT (Tissue Tek) : 30% sucrose

solution for 30 min, and frozen with dry ice, before sectioning on a Leica cryostat. For ultra-cryosectioning, the

cryoprotected hippocampal tissue samples were mounted on copper pins and plunge-frozen in liquid nitrogen

before sectioning on a Reichert cryo-ultramicrotome.

Fluorescence labeling of neurons

Sample fixation. Cultured neuron samples were fixed at various days in vitro (DIV).For single-color imaging of

actin and two-color imaging of actin and βII-spectrin, the samples were initially fixed and extracted for 1 min

using a solution of 0.3% (v/v) glutaraldehyde and 0.25% (v/v) Triton X-100 in cytoskeleton buffer (CB, 10 mM

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MES, pH 6.1, 150 mM NaCl, 5 mM EGTA, 5 mM glucose and 5 mM MgCl2), and then post-fixed for 15 min in 2%

(v/v) glutaraldehyde in CB, a previously established protocol for maintaining actin ultrastructure (29, 38-40). The

glutaraldehyde-fixed samples were treated with freshly prepared 0.1% (w/v) sodium borohydride for 7 min to

reduce background fluorescence caused by glutaraldehyde fixation. For two-color imaging of actin and adducin,

the samples were fixed using 4% (w/v) paraformaldehyde in CB for 30 min because the epitope of adducin was

masked for immunolabeling when fixed with glutaraldehyde. Though the ultrastructure of actin is not as well

preserved in the absence of glutaraldehyde, the periodic actin structure in axons was still often observed. For

single-color and two-color imaging of molecular components not including actin (βII-spectrin, βIV-spectrin,

adducin, ankyrin-B, and sodium channels), the samples were fixed using 4% (w/v) paraformaldehyde in

phosphate buffered saline (PBS) for 10 min.

Hippocampal tissue slices were further post-fixed in 4% (w/v) paraformaldehyde in PBS (for STORM imaging of

βIV-spectrin) or 2% (v/v) glutaraldehyde in CB (for STORM imaging of actin) for 1 h before labeling. The

glutaraldehyde-treated samples were additional treated with freshly prepared 0.1% (w/v) sodium borohydride for

7 min to reduce background fluorescence.

Fluorescence labeling. For immunofluorescence labeling of MAP2, NrCAM, βII-spectrin, βIV-spectrin, adducin,

ankyrin-B or sodium channels, fixed samples were permeabilized and blocked in blocking buffer (3% w/v bovine

serum albumin or 10% w/v donkey serum, 0.2% v/v Triton X-100 in PBS) for 1 hr, and then stained with primary

antibodies (described below) in blocking buffer overnight at 4 ◦C. The samples were washed three times and then

stained with secondary antibodies (described below) in blocking buffer for ~1 h at room temperature.

To label actin filaments, samples were labeled with Alexa Fluor 647 (Alexa647) conjugated phalloidin

(Invitrogen A22287) overnight at 4 ◦C or ~1 h at room temperature. A concentration of ~0.5 µM phalloidin in

phosphate buffered saline (PBS) was used. To minimize the dissociation of phalloidin from actin during washing

steps, actin labeling was performed after all other labeling steps (i.e. immunofluorescence of other molecular

targets) were completed. The sample was briefly washed 2-3 times with PBS and then immediately mounted for

imaging.

Primary antibodies. To identify dendrites and axons, the samples were stained with polyclonal antibodies against

MAP2 (Synaptic Systems 188002), a marker for dendrites, or NrCAM (Abcam ab24344), a marker for axon

initial segments.

For βII-spectrin, a monoclonal antibody (Clone 42; BD Biosciences 612563 or Santa Cruz Biotechnology sc-

136074) was used as the primary antibody. This antibody targeted a sequence close to the C-terminus of βII-

spectrin, and thus labeled a position that is close to the middle of each αII-βII spectrin tetramer (9, 10).

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For βIV-spectrin, a polyclonal antibody against the N-terminus of βIV-spectrin (kindly provided by Dr. Matthew

Rasband, Baylor College of Medicine) was used as the primary antibody. The labeling position is thus at the two

ends of the spectrin tetramers (9, 10).

For adducin, a polyclonal antibody against a sequence close to the C-terminus of α-adducin was used as the

primary antibody (Abcam ab51130). The C-terminal immunogen sequence of α-adducin shares high homology

with β- and γ- adducin, and thus this antibody likely labels all three adducin subunits (α, β, γ).

For ankyrin-B, a monoclonal antibody (clone N105/17; UC Davis/NIH NeuroMab Facility) was used as the

primary antibody.

For sodium channels, a pan-Nav channel antibody (kindly provided by Dr. Matthew Rasband or Sigma S8809)

was used as the primary antibody.

Secondary antibodies. For conventional fluorescence imaging of dendrite and axon markers (MAP2, NrCAM and

βIV-spectrin), Alexa Fluor 555 (Alexa555)-labeled secondary antibodies (Invitrogen) were used.

For single-color STORM imaging, secondary antibodies were custom labeled with a photoswitchable reporter dye,

Alexa647, and an activator dye, either Cy3 or Alexa Flour 405 (Alexa405), which facilitates the photoswitching

of the reporter dye. Donkey anti-mouse and donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch)

were each labeled with a mixture of amine-reactive activator and reporter dyes in a one-step reaction, as described

previously (26).

For two-color STORM imaging involving actin, where actin was labeled with Alexa647-phalloidin, the other

target [βII-spectrin (Fig. 4A) or adducin (Fig. 4B)] was immunostained with a secondary antibody labeled with

Cy3B. Cy3B is a photoswitchable dye that has spectrally distinct emission from Alexa647, allowing two-color

imaging to be conducted with these two dyes (41).

For two-color imaging of two immunolabeled targets [i.e. βII-spectrin and adducin (Fig. 4C) or sodium channels

and βIV-spectrin (Fig. 4D)], secondary antibodies labeled with Alexa405 (activator) and Alexa647 (reporter) and

secondary antibodies labeled with Cy3 (activator) and Alexa647 (reporter) were used to label the two different

targets respectively. In this case, the same photoswitchable reporter Alexa647 was imaged for both color channels,

and the two color channels are distinguished by which wavelength light was used to activate the photoswitchable

probes (26).

Imaging buffer

The STORM imaging buffer was PBS containing 100 mM cysteamine, 5% glucose, 0.8 mg/mL glucose oxidase

(Sigma-Aldrich), and 40 μg/mL catalase (Roche Applied Science). Approximately 4 μL of imaging buffer was

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dropped at the center of a freshly-cleaned, #1.5 rectangular coverslip (22 mm by 60 mm), and the sample on the

12-mm coverslip was mounted on the rectangular coverslip and sealed with nail polish or Cytoseal.

Dual-objective STORM imaging

Single-color STORM imaging of Alexa647-labeled actin (Figs. 1, 2, S1, S2, S3, and S10A, B), βII-spectrin (Figs.

3A, S4, S5, and S10D), adducin (Fig. 3E), and βIV-spectrin in brain tissue slices (Fig. S8AB), as well as two-

color STORM imaging of Alexa647- and Cy3B-labeled samples [actin and βII-spectrin (Fig. 4A) and actin and

adducin (Fig. 4B)] were carried out on a dual-objective STORM setup (29), with modifications to accommodate

two-color imaging.

In the a dual-objective STORM setup, two infinity-corrected microscope objectives (Olympus Super Apochromat

UPLSAPO 100x, oil immersion, NA 1.40) were placed opposing each other and aligned so that they focused on

the same spot of the sample. A customized dichroic mirror (Chroma) that allowed the 400-570 nm and 640-

653 nm light to pass through at an incident angle of 22.5° was used to introduce 405-nm (CUBE 405-50C;

Coherent), 532-nm (Compass 315m-150; Coherent), 560-nm (VFL-P-2000-560; MPB), and 647-nm (Innova 70C

Spectrum; Coherent) lasers into the sample through the back focal plane of the first objective. A translation stage

allowed the laser beams to be shifted towards the edge of the objective so that the emerging light reached the

sample at incidence angles slightly smaller than the critical angle of the glass-water interface, thus illuminating

only the fluorophores within a few micrometers of the coverslip surface. The sealed sample was mounted between

the two opposing objectives. The fluorescence emission was collected by both objectives. Astigmatism was

introduced into the imaging paths of both objectives using a cylindrical lens so that the single-molecule images

obtained by each objective were elongated in x and y for molecules on the proximal and distal sides of the focal

plane (relative to the objective), respectively.

For single-color STORM imaging of Alexa647-labeled samples, the 647-nm laser was used to excite fluorescence

from Alexa647 molecules and switch them into the dark state. A pair of 647-nm notch filters (Semrock NF01-

543/647), which reject the 647-nm excitation light, were inserted into the imaging paths of the two objectives.

After passing through these filters, fluorescence from Alexa647 collected by the two objectives were each focused

by a 20 cm achromatic lens, cropped by a slit at the focal plane, and then separately projected onto two different

areas of the same EMCCD camera (Andor iXon DU-897) using two pairs of relay lenses. A single-band band-

pass filter (Chroma ET700/75m) was placed in front of the camera. Prior to acquiring STORM images, we first

used relatively weak 647-nm light (~0.05 W/cm2) to illuminate the sample and recorded the conventional

fluorescence image before a substantial fraction of the dye molecules were switched off. We then increased the

647-nm light intensity (to ~2 kW/cm2) to rapidly switch the dyes off for STORM imaging. The 405-nm laser

(when Alexa405 was used as the activator dye or no activator dye was used) or 532-nm laser (when Cy3 was used

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as the activator dye) was used to reactivate the fluorophores from the dark state back to the emitting state. The

power of the activation lasers (typical range 0-1 W/cm2) was adjusted during image acquisition so that at any

given instant, only a small, optically resolvable fraction of the fluorophores in the sample were activated. The

EMCCD camera acquired images from both objectives simultaneously at a frame rate of 60 Hz. Approximately

80,000 frames were typically recorded to generate the super-resolution images for Alexa647.

For two-color STORM imaging of Alexa647- and Cy3B-labeled samples, the filter on the camera was changed to

a dual-band band-pass filter (Semrock FF01-594/730), which allowed both Alexa647 and Cy3B emission to pass

through. No activator was used in this case. Alexa647 and Cy3B are both reversible photoswitchable dyes, which

can be activated by the 405-nm light. The two fluorophores were imaged sequentially, with Alexa647 being

imaged first as described above. For Cy3B imaging, two additional band-pass filters (Semrock FF01-593/40)

were then inserted into the light paths of the two objectives to reject both the 560-nm excitation laser and the

emission from Alexa647 excited by the 560-nm laser. The 560-nm laser (~2 kW/cm2) was used in lieu of the 647-

nm laser to excite fluorescence from Cy3B molecules and switch them into the dark state. The 405-nm laser

(typical range 0-50 W/cm2) was used to reactivate the fluorophores to the emitting state. Approximately 50,000

frames were typically recorded to generate the super-resolution images for Cy3B.

The recorded STORM data were first split into two movies, each of which comprises a sequence of images

obtained by one of the two objectives. Each movie was first analyzed separately according to previously described

methods (27). The centroid positions and ellipticities of the single-molecule images provided lateral and axial

positions of each activated fluorescent molecule, respectively (27). The molecular positions obtained by the

second objective were mapped to the coordinates of the first objective, and the final coordinates for each activated

fluorescent molecule were determined as a weighted averaged of the coordinates from each objective, as

described previously (29). The final super-resolution images were reconstructed from these molecular coordinates

by depicting each location as a 2D Gaussian peak. For Alexa647, localization precisions were ~4 nm for lateral

(xy) and ~8 nm for axial (z) directions measured in standard deviation (SD), or ~9 nm for lateral and ~19 nm for

axial directions measured in full width at half maximum (FWHM) (29). For Cy3B, localization precisions were

~5 nm in SD or ~12 nm in FWHM in the lateral directions. For two-color STORM imaging, 2D STORM images

obtained from the two color channels were aligned using fluorescent beads added into the sample. The residual

alignment error between the two color channels was ~7 nm.

Single-objective STORM imaging

Single-color STORM imaging of βIV-spectrin in cultured neurons (Fig. 3B) and actin in brain tissue slices (Fig.

S8G, I), as well as two-color STORM imaging of Alexa405-Alexa647-labeled and Cy3-Alexa647-labeled

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samples [βII-spectrin and adducin (Fig. 4C) and sodium channels and βIV-spectrin (Fig. 4D)] were carried out on

a single-objective STORM setup (27).

The STORM setup was based on a Nikon Eclipse Ti-U inverted optical microscope. 405-nm (CUBE 405-50C;

Coherent), 532-nm (Compass 315m-150; Coherent), and 656-nm (CL656-300; CrystaLaser) lasers were

introduced into the sample through the back focal plane of the microscope. A translation stage allowed the laser

beams to be shifted towards the edge of the objective so that the emerging light reached the sample at incidence

angles slightly smaller than the critical angle of the glass-water interface, thus illuminating only the fluorophores

within a few micrometers of the coverslip surface. A 660dcxr (extended reflectivity dichroic, Chroma) was used

as the dichroic mirror and an ET705/72M band-pass filter (Chroma) was used as the emission filter. For 3D

STORM imaging, a cylindrical lens was inserted into the imaging path so that images of single molecules were

elongated in x and y for molecules on the proximal and distal sides of the focal plane (relative to the objective),

respectively (27).

For single-color imaging, continuous illumination of 656-nm laser (~2 kW/cm2) was used to excite fluorescence

from Alexa647 molecules and switch them into the dark state. Continuous illumination of the 405-nm laser (when

Alexa405 was used as the activator dye) or 532-nm laser (when Cy3 was used as the activator dye) was used to

reactivate the fluorophores to the emitting state. The power of the activation lasers (typical range 0-1 W/cm2) was

adjusted during image acquisition so that at any given instant, only a small, optically resolvable fraction of the

fluorophores in the sample were in the emitting state.

For two-color imaging of Alexa405-Alexa647-labeled and Cy3-Alexa647-labeled samples, the two activation

lasers (405 and 532 nm) and the excitation laser (656 nm) were switched in an alternated manner, as described

previously (26). The illumination sequence was 532 nm × 1 frame (1 frame of 532-nm laser), 656 nm × 3 frame,

405 nm × 1 frame, and 656 nm × 3 frame, before repeating. Photoactivation events detected in the first imaging

frames following the 532-nm and 405-nm activation frames were assigned to the Cy3-Alexa647 and Alexa405-

Alexa647 pairs, respectively.

A typical STORM image was generated from a sequence of about 60,000 image frames at a frame rate of 60 Hz.

The recorded STORM movie was analyzed according to previously described methods (27, 42). The centroid

positions and ellipticities of the single-molecule images provided lateral and axial positions of each activated

fluorescent molecule, respectively (27). For two-color imaging, each localization was assigned to one of the two

colors according to the distinct activation laser pulse used as described above. An automated algorithm was used

to correct for the crosstalk between the two activator imaging channels (42). Super-resolution images were

reconstructed from the molecular coordinates by depicting each location as a 2D Gaussian peak. Localization

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precisions were ~10 nm for lateral (xy) and ~22 nm for axial (z) directions measured in SD, or ~25 nm for lateral

and ~50 nm for axial directions measured in FWHM (27).

References

38. J. V. Small, K. Rottner, P. Hahne, K. I. Anderson, Microsc. Res. Tech. 47, 3 (1999). 39. S. A. Koestler, S. Auinger, M. Vinzenz, K. Rottner, J. V. Small, Nat. Cell Biol. 10, 306 (2008). 40. S. Auinger, J. V. Small, Methods in Cell Biology 88, 257 (2008). 41. G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, X. Zhuang, Nat. Methods 8, 1027 (2011). 42. A. Dani, B. Huang, J. Bergan, C. Dulac, X. W. Zhuang, Neuron 68, 843 (2010).

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Supplementary Figures

Fig. S1. Dendrites contain long actin filaments running largely along the dendritic shaft. (A) Conventional fluorescence image of actin (green) and MAP2 (magenta) in a neuron (same image as Fig. 1D). (B) 3D STORM image of actin in the region corresponding to the white box in (A). The main feature in this region is a segment of a MAP2-positive dendrite. Two distal axons devoid of the MAP2 signal can also be seen here, which shows the quasi-1D, periodic arrangement of actin. All z-positions are color-coded according to the colored scale bar in Fig. 1B.

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Fig. S2. The quasi-1D, periodic actin pattern typically starts to appear at ~5 DIV, and become clearly visible in axons at 7 DIV. (A-C, E) Representative 3D STORM images of cultured neuron samples fixed at 1, 3, 5 and 7 DIV, respectively. (D) A close-up of the boxed region in (C), showing an emerging periodic pattern. No such pattern was observed for 1- and 3-DIV neurons. All z-positions are color-coded according to the colored scale bar in (E).

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Fig. S3. The quasi-1D, periodic actin structure is present in the axons of mature cultured neurons. (A) Conventional fluorescence image of actin (green) and a dendritic marker, MAP2 (magenta) in a cultured neuron sample fixed at 28 DIV. (B) 3D STORM image of actin in the same region. The periodic, ladder-like structure is found in nearly all axons devoid of the MAP2 signal. Some filopodia-like structures branching from axon shafts do not appear to have periodic patterns. The z-positions are color-coded according to the colored scale bar.

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Fig. S4. The quasi-1D, periodic spectrin structure is present in the axons of mature cultured neurons. (A) Conventional fluorescence image of βII-spectrin in a cultured neuron sample fixed at 25 DIV. (B) 3D STORM image of the same region. The z-positions are color-coded according to the colored scale bar.

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Fig. S5. Distinct spatial distributions of βII-spectrin in axons and dendrites. (A) Conventional fluorescence image of βII-spectrin (green) and a dendritic marker, MAP2 (magenta) in a cultured neuron sample fixed at 10 DIV. (B) 3D STORM image of βII-spectrin in the same region. Arrows point to dendrites enriched with MAP2. The inset shows the zoom-in image of the region in the dashed box. βII-spectrin is enriched in axons devoid of the MAP2 signal and less abundant in dendrites enriched with MAP2. The periodic, ladder-like structures are only found in axons but not in dendrites. The z-positions are color-coded according to the colored scale bar.

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Fig. S6. βIV-spectrin was observed specifically in the initial segment of axons. The two-color image of βIV-spectrin (magenta) and MAP2 (green) shows that βIV-spectrin was only observed in the initial segment of the axon and MAP2 was enriched in the cell body and dendrites.

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Fig. S7. Semi-periodic distribution of ankyrin-B in axons. (A) 3D STORM image of ankyrin-B in a cultured neuron sample fixed at 10 DIV. The z-positions are color-coded according to the colored scale bar. (B) Fourier transform of the 1D localization distribution of ankyrin B averaged over 30 axon segments of 90 µm total length. The peak in the Fourier transform corresponds to a spatial period of ~197 nm.

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Fig. S8. The quasi-1D, periodic actin-spectrin-based cytoskeleton structure is present in axons in brain tissue slices from adult mice. Here we use βIV-spectrin, which is specifically localized in the initial segment of axon, to label this region. (A, B) 3D STORM images of βIV-spectrin immunolabeled with the photoswitchable dye Alexa 647 in hippocampal tissue slices. βIV-spectrin is labeled against its N-terminal region, which corresponds to the ends of the spectrin tetramer. Some parts of the axons appear missing because they are either outside the thin tissue slices or out of focus. (C) The 1D localization histogram along the dashed line in (A). (D) Fourier transform of the 1D localization distribution shown in (C). The main peak in the Fourier transform corresponds to a spatial period of ~195 nm. (E) Histogram of the spacings between adjacent βIV-spectrin stripes in the periodic structure. The distribution was constructed from multiple axons. The red line is a Gaussian fit with mean = 189 nm and standard deviation = 22 nm (N = 80). We note that nearly all of the βIV-spectrin-labeled axon segments (43 out of the 46 imaged) unambiguously exhibit a quasi-1D, periodic, ladder-like distribution of βIV-spectrin. (F, H) Conventional fluorescence images of actin and βIV-spectrin in hippocampal tissue slices. Here, actin is phalloidin-labeled with photoswitchable Alexa 647 for STORM imaging and βIV-spectrin is immunolabeled with non-switchable Alexa555 for marking axon initial segments in the conventional images. (G, I) 3D STORM

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images of actin in the same regions shown in (F) and (H) respectively. The z-positions are color-coded according to the colored scale bar in (I). (J) The 1D localization histogram along the dashed line in (G). (K) Fourier transform of the 1D localization distribution shown in (J). The peak in the Fourier transform corresponds to a spatial period of ~198 nm. (L) Histogram of the spacings between adjacent actin stripes in the periodic structure. The distribution was constructed from multiple axons. The red line is a Gaussian fit with mean = 193 nm and standard deviation = 16 nm (N = 50). We note that resolving actin structures in tissue slices is in general more difficult than resolving the structures of βIV-spectrin because actin is present in all cell bodies, dendrites and axons, which are densely packed in three dimensions in tissues, generating high background signals. Since the membrane cytoskeleton consists of only a thin layer of actin beneath the axonal membrane, its structure can be easily overwhelmed by the background actin signals from other neurites and cell bodies. However, in those axon initial segments that are sufficiently separated from other neurites and cell bodies in the tissue preparation, the periodic, ladder-like structure of actin is clearly visible, as marked by the arrowheads in (G, I).

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Fig. S9. Two-color STORM images of various combinations of actin, spectrin, adducin and sodium channels in axons, and histograms of the localized molecules along the axon shafts. (A) Left: Two-color images 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. Two examples are shown here and the image in the upper panel is the same as that in Fig. 4A. Right: the histograms of localized molecules along the dashed lines in the left panels, after the images were projected to one dimension along the axon long axes. Actin and βII-spectrin (C-terminus) exhibit alternating patterns along the axon, as evident from both the images and the alternating green and magenta peaks in the histograms. (B) Same as (A) except that actin was co-imaged with adducin instead of βII-spectrin. Actin (green) and adducin (magenta) patterns colocalize along the axon, as evident from both the image and the colocalizing green and magenta peaks in the histograms. (C) Same as (B) except that βII-spectrin (C-terminus), instead of actin, was co-imaged with adducin. βII-spectrin (C-terminus, green) and adducin (magenta) clearly exhibit alternating patterns along the axon. (D) Two-color image of sodium channels (green) and βIV-spectrin (magenta) and the histogram of localized molecules along the dashed line. βIV-spectrin was immunostained against its N-terminal region, which is situated at the two ends of the spectrin tetramer. Both the image and the alternating green and magenta peaks in the histograms show that sodium channels and βIV-spectrin N-terminus alternate along the axon. The images shown in (B-D) here are the same as those shown in Fig. 4B-D.

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Fig. S10. Effect of latrunculin A on the actin-spectrin-based membrane cytoskeleton. (A, B) STORM images of actin in cultured neurons treated for 1 hour with 2 µM (A) and 5 µM (B) latrunculin A, an actin-monomer-sequestering drug. The periodic, ladder-like structure appears to be somewhat more stable than the long actin filaments: upon addition of 2 µM latrunculin A, the long actin filaments running along the dendrites entirely disappeared, but some of the periodic, ladder-like structures in the axons remained. When the latrunculin A concentration was increase to 5 µM, the periodic, ladder-like structures also disappeared. The conventional fluorescence images of actin (green) and MAP2 (magenta) of the same regions are shown in the insets. (C) Conventional fluorescence image of βII-spectrin (green) and MAP2 (magenta) in cultured neurons treated with 5 µM latrunculin A for 1 hour. (D) STORM image of βII-spectrin of the same region as shown in (C). The periodic, ladder-like spectrin structures were largely disrupted under this condition. The z-positions are color-coded according to the colored scale bar.

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