βII-spectrin distribution in human iPSC-derived MN axons

(A) Representative βII-spectrin intensity profiles along four individual axons. (B) Mean normalized βII-spectrin intensity in the proximal, medial and distal portion of 10 axons. Colored traces show the mean normalized values shown in panel A. ***: p<0.001. (C) Representative reconstruction of an individual axon, immunostained for βII-spectrin, NFM and αII-tubulin. Yellow line highlights the axon being followed. Scale bar = 50 µm. Inserts: C1 dashed rectangle highlights a gap-and-patch pattern (*: patches), whereas the C2 dashed rectangle highlights axonal enlargements (E) and the axonal tip (T). Scale bar = 10 µm. (D) Representative examples (I-V) of MN axonal tips (T) immunostained for βII-spectrin, βIII-tubulin and NFH. Scale bar = 10 µm. (E) Representative examples (I-V) of MN axonal enlargements immunostained for βII-spectrin, βIII-tubulin and NFH. Scale bar = 5 µm.

Motor neurons present sharp interruptions in the βII-spectrin lattice.

(A) Confocal images of bulk cultures immunostained for βII-spectrin. Inserts a-c show examples of axons with a continuous distribution of βII-spectrin (a and b) and axons with sharp interruptions or “gaps” (asterisks in c and d). Scale bar = 10 µm (left panel) and 5 µm (zoom-in inserts). (B) Histograms showing percentual frequency of gaps length, patches length and gap frequency. (C) Top: Representative confocal images of a gap-and-patch pattern co-stained for βII-spectrin, Neurofilament M and ⍺II-tubulin. Scale bar = 2.5 µm. Bottom: Quantification of intensity ratios. For axons with a continuous βII-spectrin distribution, the ratio of intensity between two regions 5 µm apart was measured (c/c). For axons with βII-spec-gaps, the ratio of intensity between a gap and its flanking patches was measured (g/p). Mean + SEM. T-test. ns: not significant; *: p<0.05; ***: p<0.001. (D) Top panel: Representative confocal images co-stained for βII-spectrin, βIII-tubulin and Neurofilament H. Scale bar = 2.5 µm. Lower panel: Quantification of intensity ratios. For axons with a continuous βII-spectrin distribution, the ratio of intensity between two regions 5 µm apart was measured (c/c). For axons with βII-spec-gaps, the ratio of intensity between a gap and its flanking patch was measured (g/p). Mean + SEM. T-test. ns: not significant; *: p<0.05; ***: p<0.001. (E) Confocal and Differential Interference Contrast images of axons immunostained for βII-spectrin with a gap-and-patch pattern. Scale bar = 5 µm.

Staurosporine acutely induces βII-spectrin gaps-and-patches patterns.

(A) Graph showing the percentage of axons with βII-spectrin gaps in iPSC-derived MNs cultured for 1, 2 and 3 weeks, and treated for 1 h with vehicle (DMSO). One way ANOVA, *:p < 0.05. (B) Number of axons with βII-spectrin gaps (a.u.) in 2-week-old MNs treated for 1 h with arsenite (2 μM), L-glutamate (0.05 mM) and staurosporine (0.1μM), or the control vehicles, and fixed immediately afterward. One way ANOVA, *:p < 0.05. (C) Axons with βII-spectrin gaps (a.u.) of 2-week-old MNs treated for 1 h with DMSO or staurosporine (0.1μM) and fixed immediately (0), or 24 and 72 hrs later. One way ANOVA, ***:p<0.001. (D) Confocal images of 2-week-old MNs treated for 1 h with DMSO or staurosporine (0.1μM) and immunostained for βII-spectrin and Neurofilament H. Scale bar = 10 μm. (E) Histograms showing percentual frequency of gaps length, patches length and gap frequency per 100 µm, comparing DMSO and staurosporine.

Proteolytic activity is not likely related to the formation of βII-spectrin gaps.

(A) Western blot against βII-spectrin and β-actin in 2-week-old MN cultures from three independent differentiations (batches) treated with vehicle control (DMSO) or staurosporine for 1 h (stau. 1h), and then either pelleted immediately or 6 hrs later following a medium change (stau. 1h+6h). (B) Western blot against SNTF (αII-Spectrin N-Terminal Fragment) and β-actin in the same samples as in panel A. (C-D). Representative images (C) and quantification (D) of βII-spectrin and SNTF in continuous axons (c), in gaps (g) and patches (p) of staurosporine-treated 2-week-old MNs. Two way ANOVA, ns: not significant. (E-F). Representative images (E) and quantification (F) of βII-spectrin and cleaved-Caspase-3 in continuous axons (c), in gaps (g) and patches (p) of staurosporine-treated 2-week-old MNs. Two way ANOVA, ***: p<0.001. (G) Quantification of axons with βII-spectrin gaps in vehicle (DMSO) and staurosporine-treated 2-week-old MNs with or without the inhibitors for caspase-3 (z-DEVD-fmk) or calpains (ALLN1). One way ANOVA, ***: p<0.001.

The MPS is absent in βII-spectrin gaps, but it is well organized within patches

(A) Representative images of βII-spectrin acquired using confocal and STED microscopy in axons with continuous βII-spectrin distribution and in axons with βII-spectrin gaps-and-patches pattern, under both control conditions and acute staurosporine treatment. Scale bars = 1 μm. (B) Autocorrelation amplitude of the intensity profiles along the different regions of interest and treatments. Mean ± SEM. ns: not significant. (C) MPS correlation analyses by Gollum within the different regions of interest and treatments. Mean ± SEM. Two way ANOVA, ***: p<0.001. (D) STED images of double staining for βII-spectrin and F-actin showed the expected anti-phase organization in both continuous axons and patches. Representative images (left) and corresponding intensity profiles between the arrowheads (right). Scale bar = 1 μm. (E) Autocorrelation analyses of βII-spectrin and F-actin in patches and gaps. The traces represent the mean autocorrelation from 17 pairs of patches and gaps.

Gaps and patches preferentially occur in the medial portion of axons

(A) Schematic representation of the location of the gaps and patches pattern along individual axons (left) and its observed frequency (right). (B) Normalized number of patches per 100 μm in the proximal, medial and distal portions. Each trace represents an individual axon. Most axons showed more gaps and patches in the medial portion (light gray traces and green dots), while only a few showed more in the proximal region (dark gray traces and red dots). (C) Schematic (top) and representative confocal images of three βII-spectrin-stained axons with a gap-and-patch pattern confined to the medial sections, showing proximal, medial and distal sections (bottom). Scale bar = 1 μm. (D) Mean normalized βII-spectrin intensity in the proximal, medial (with gaps and patches pattern), and distal sections. Values were normalized to the proximal intensity of each axon. Colored dots correspond to the three examples shown in panel C. ns: not significant; *: p<0.05; ***: p<0.001.

Patches may represent nascent MPS assemblages

(A) Representative STED images of four individual axons immunostained against βII-spectrin, showing continuous segments in the proximal and distal portions, and patch segments in the medial portions. Scale bar = 1 μm. (B) MPS correlation values of 12 individual axons in the continuous segments of the proximal and distal portions, and patch segments in the medial portion. Colored traces correspond to the axons shown in panel A. The dashed line at 0.12 indicates the threshold above which a periodic distribution is visually discernible. One way ANOVA, ns: not significant; ***: p<0.001. (C) Representative confocal and STED images of four individual gap-patch borders immunostained for βII-spectrin. White arrowheads indicate the locations of the intensity profiles. Red arrows marked the regions used to calculate the mean period for each section, as shown below. Pink stripes in the background are separated by 200 nm and were drawn for visual reference. Scale bars = 1 μm. (D) Mean autocorrelation curves of longitudinal βII-spectrin structures across sample groups with varying βII-spectrin intensity in the gaps, expressed as a percentage of the intensity found in the neighboring patches. A mean AC curve of patches (grey) is shown for reference.

Latrunculin A prevents staurosporine-induced gaps-and-patches pattern formation.

(A) Representative confocal images of 2-week-old MNs treated with vehicle (DMSO or EtOH+DMSO), staurosporine (Stauro), or latrunculin A + staurosporine (LatA+Stauro). Cells were immunostained for βII-spectrin, α-tubulin and F-actin (phalloidin). Scale bar = 20 μm. (B) Percentage of axons with βII-spectrin gaps in 2-week-old MNs treated with vehicle (DMSO or EtOH+DMSO), (Stauro), or latrunculin A + staurosporine (LatA+Stauro). Mean ± SEM. *: p<0.05. Tukey post hoc test. (C) Normalized percentage of F-actin intensity in 2-week-old MNs treated with vehicle (DMSO or EtOH+DMSO), (Stauro), or latrunculin A + staurosporine (LatA+Stauro). *: p<0.05. Tukey post hoc test. (D) Representative STED images of MNs treated with vehicle (DMSO), staurosporine alone (Stauro), latrunculin A alone (LatA) or latrunculin-A + staurosporine (LatA+Stauro), and immunostained for βII-spectrin. Two examples are shown for continuous axonal sections (top rows) and for patches (bottom rows). Scale bar = 1 μm. (E) MPS correlation analyses by Gollum within the different regions of interest and treatments shown in panel D (C: continuous, P: patches). ns: not significant. Mean ± SEM.