BetaII-spectrin gaps and patches emerge from the patterned assembly of the actin/spectrin membrane skeleton in human motor neuron axons
- Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Córdoba, Argentina
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Canada
- Faculty of Medicine and Health Sciences, McGill University, Canada
- Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
- Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina
- Instituto Universitario de Ciencias Biomédicas de Córdoba (IUCBC), Argentina
- Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Argentina
- Institute of Zoology, Biocenter Cologne, University of Cologne, Germany
Figures
Figure 1 with 1 supplement
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. Insets: 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.
Figure 1—figure supplement 1
Quantitative and qualitative analyses of βII-spectrin distribution in human iPSC-derived MN axons.
(A) Representative confocal image of a 2-week-old ‘bulk culture’, immunostained for βII-spectrin. Yellow arrows point to groups of >6 neurons. Red square shows the typical sampling performed to assess axonal features. Scale bar = 50 µm. (B) Schematic representation of the procedure to generate ‘density gradient cultures’. During seeding and the 2 h cell attachment period, culture vials containing coverslips were tilted as shown (top). Culture vials were then placed in a standard upright position for cell maintenance (2 weeks for most experiments, middle). For analyses of whole individual axons, areas toward the middle of the coverslip provided a density that supported healthy neurons while allowing the tracing of single axons (yellow squares, bottom). (C) Representative reconstruction of an individual axon, immunostained for βII-spectrin, βIII-tubulin, and NFH. Yellow line highlights the axon being followed. Scale bar = 50 µm. The 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 images (I–III) of hippocampal neurons grown for 7 DIV and stained for βII-spectrin, 4.1N, and βIII-tubulin. The dashed line contours the expanded growth cone at the tip of the axon, which was delineated using the actin-binding protein 4.1N. Scale bar = 10 µm. (E) Quantification of the number of axonal enlargements in proximal, medial, and distal sections.
Figure 2 with 1 supplement
Motor neurons present sharp interruptions in the βII-spectrin lattice.
(A) Confocal images of bulk cultures immunostained for βII-spectrin. Insets (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 insets). (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.
Figure 2—figure supplement 1
Axons of human iPSC-derived motor neurons present interruptions in αII-spectrin.
(A) Intensity (mean gray values, a.u.) of βII-spectrin and ⍺II-spectrin within regions with continuous distribution (cont.), within gaps, and within patches. (B) Confocal images of bulk cultures immunostained for ⍺II-spectrin. Insets (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 insets). (C) Histograms showing percentual frequency of gaps length, patches length, and gap frequency per 100 µm, in control (DMSO) and staurosporine-treated cells.
Figure 3 with 1 supplement
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) % of axons with βII-spectrin gaps (normalized to control) in 2-week-old MNs treated for 1 h with arsenite (2 μM), L-glutamate (0.05 mM), and staurosporine (0.1 μM), or the control vehicles, and fixed immediately afterward. One-way ANOVA, *p<0.05. (C) % of axons with βII-spectrin gaps (normalized to time 0) of 2-week-old MNs treated for 1 h with DMSO or staurosporine (0.1 μM) and fixed immediately (0), or 24 and 72 h later. One-way ANOVA, ***p<0.001. (D) Confocal images of 2-week-old MNs treated for 1 h with DMSO or staurosporine (0.1 μM) 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.
Figure 3—figure supplement 1
Interruptions in βII-spectrin increase as a function of weeks in culture and by acute treatment with staurosporine.
(A) Schematic representation of the timeline of the experiments shown in Figure 3B. (B) Total axonal length (a.u.; based on ⍺-tubulin staining) of 2-week-old MNs treated for 1 h with low doses of arsenite (2 μM), L-glutamate (0.05 mM), and staurosporine (0.1 μM), or the control vehicles. ns: not significant. (C) Schematic representation of the timeline of the experiments shown in Figure 3C. (D) Total axonal length (a.u.; based on ⍺-tubulin staining) of 2-week-old MNs treated for 1 h with DMSO or staurosporine (0.1 μM) and fixed immediately (0), or 24 and 72 h later. (E, F) Percentage of axons with βII-spectrin gaps (E) and total axonal length (F, a.u.; normalized to DMSO treatment in each line) of 2-week-old MNs treated for 1 h with DMSO or staurosporine (0.1 μM) and fixed immediately, across the control line (AIW) and lines carrying mutations in ALS-related genes: FUS (H517Q), TDP43/TARDBP (A382T), and SOD1 (G93A and D90A/G93A). *p<0.05; ***p<0.001.
Figure 4 with 1 supplement
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. 1 h), and then either pelleted immediately or 6 h 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.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108021/elife-108021-fig4-data1-v1.pdf
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4.
- https://cdn.elifesciences.org/articles/108021/elife-108021-fig4-data2-v1.zip
Figure 4—figure supplement 1
Proteolytic cleavage of spectrins is not a mechanism that forms gaps in the MPS lattice.
(A) Schematic representation of the experimental design and treatment timeline analyzed by western blot (results shown in Figure 4 and in this figure). (B) Quantification of the densitometry of βII-spectrin reactive bands indicated in Figure 4A (*). (C) Quantification of the densitometry of SNTF (αII-Spectrin N-Terminal Fragment) reactive bands indicated in Figure 4B (*). (D) Western blot image against αII-spectrin and β-actin (left) and quantification of the densitometry of reactive bands (*, right). (E) Western blot image against Caspase-3 and β-actin (left) and quantification of the densitometry of reactive bands (*, right). (F) Western blot image against cleaved-Caspase-3 and β-actin (left) and quantification of the densitometry of reactive bands (*, right).
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Figure 4—figure supplement 1—source data 1
PDF file containing original western blots for Figure 4—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108021/elife-108021-fig4-figsupp1-data1-v1.pdf
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/108021/elife-108021-fig4-figsupp1-data2-v1.zip
Figure 5 with 1 supplement
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.
Figure 5—figure supplement 1
The MPS is absent in βII-spectrin gaps, but it is well organized within patches.
(A) Representative STED images of αII-spectrin acquired using STED microscopy in axons with continuous αII-spectrin distribution (I–IV) and in axons with αII-spectrin-gaps (V–VIII). Scale bar = 1 μm. (B) Analyses of the percentage of axonal sections that presented a MPS in 2-week-old MNs treated for 1 h with DMSO or staurosporine (0.1 μM) and fixed immediately, across the control line (AIW) and lines carrying mutations in ALS-related genes: SOD1 (D90A/G93A) and TDP43/TARDBP (A382T). Above each bar, the total number of sections analyzed per group is shown. (C, D) Period (C) and autocorrelation amplitude values (D) of MPS axonal sections shown in (B). In (C), values inserted in the graph are the mean value of each group.
Figure 6 with 1 supplement
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.
Figure 6—figure supplement 1
Gaps and patches preferentially occur in the middle section of axons.
(A) Representative axonal reconstructions of two neurons. Numbers along axons indicate approximate distance to the cell soma (μm). Insets show confocal images of proximal, medial, and distal regions, indicated as gray rectangles in the axonal reconstruction, immunostained for βII-spectrin (cyan) and βIII-tubulin (magenta). Scale bar = 1 μm. (B) Quantification of patches length found in vehicle-treated axons (DMSO) and staurosporine-treated 2-week-old MNs with or without the inhibitors for caspase-3 (z-DEVD-fmk) or calpains (ALLN1). In the graph, the values above each bar are the mean length (μm) of each group. One-way ANOVA, *p<0.05, **p<0.01.
Figure 7
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 (gray) is shown for reference.
Figure 8
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), latrunculin A (LatA), 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 groups shown in panel (A). Mean ± SEM. *p<0.05. Tukey post hoc test. (C) Normalized percentage of F-actin intensity in 2-week-old MNs treated groups shown in panel (A). Mean ± SEM. *p<0.05, ***p<0.001. 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.
Tables
Appendix 1—table 1
List of reagents for neuronal treatments.
| Reagent | Working concentration | Solvent (vehicle) | Time of treatment | Reference |
|---|---|---|---|---|
| Staurosporine | 0.1 µM | DMSO | 1 or 2 h | Tocris, cat. #1285 |
| L-glutamate | 0.5 mM | DMEM | 1 h | Sigma-Aldrich, cat. #G1626 |
| Sodium arsenite | 0.25 µM | DMEM | 1 h | Sigma-Aldrich, cat. #S7400 |
| Latrunculin A | 0.2, 1, 5 µM | Ethanol | 1 or 2 h | Cayman, cat. #10010630 |
| ALLN-1 | 5 µM | DMSO | 2 h | Cayman, cat. #14921 |
| Caspase-3 inhibitor (zDEVD-fmk) | 50 µM | DMSO | 2 h | R&D Systems, cat. #FMK004 |
Appendix 1—table 2
List of primary and secondary antibodies for immunofluorescence staining.
| Primary antibodies | |||
|---|---|---|---|
| Antibody | Host species | Working dilution | Reference |
| βII-spectrin | Mouse | 1:400 (ICC), 1:1000 (WB) | BD Biosciences (cat. #612563) |
| α-II-spectrin | Mouse | 1:400 (ICC), 1:1000 (WB) | BioLegend (cat. #803201) |
| βIII-tubulin | Rabbit | 1:1000 (ICC) | BioLegend (cat. #PRB-435P) |
| βIII-tubulin | Chicken | 1:1000 (ICC) | Abcam (cat. #1107216) |
| αII-tubulin | Rat | 1:1000 (ICC) | Sigma-Aldrich (clone α-3A1, cat. #T5168) |
| Caspase-3 | Rabbit | 1:1000 (WB) | Cell Signaling (cat. #9662) |
| Cleaved-Caspase-3 | Rabbit | 1:500 (ICC/WB) | Cell Signaling (cat. #9661) |
| Protein 4.1N | Rabbit | 1:400 (ICC) | Abcam (cat. #ab244499) |
| SNTF | Rabbit | 1:500 (ICC), 1:5000 (WB) | Millipore (cat. #ABN2264) |
| NF-H | Chicken | 1:1000 (ICC) | Abcam (cat. #ab4680) |
| NF-M | Rabbit | 1:1000 (ICC) | Millipore (cat. #AB1987) |
| Secondary antibodies (ICC) | |||
| Goat anti-mouse IgG-STAR ORANGE | 1:250 | Abberior (cat. #STORAGE-1001) | |
| Goat anti-rabbit IgG-STAR ORANGE | 1:250 | Abberior (cat. #STORAGE-1002) | |
| Goat anti-rabbit IgG-STAR RED | 1:250 | Abberior (cat. #STRED-1002) | |
| Goat anti-mouse IgG-Atto 647N | 1:250 | Millipore (cat. #50185) | |
| Donkey anti-chicken IgY-Alexa488 | 1:500 | Jackson Immunoresearch (cat. #703-545-155) | |
| Goat anti-rat IgG-Dylight488 | 1:500 | Abcam (cat. #ab96887) | |
| Phalloidin-Atto 647N | 1:200 | Sigma-Aldrich (cat. #65906) | |
Appendix 1—table 3
List of secondary antibodies for western blot.
| HRP-conjugated antibodies (WB) | ||
|---|---|---|
| Goat anti-mouse IgG (H+L) | 1:10,000 | Jackson Immunoresearch (cat. #115-035-003) |
| Goat anti-rabbit IgG (H+L) | 1:10,000 | Jackson Immunoresearch (cat.111-035-144) |
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BetaII-spectrin gaps and patches emerge from the patterned assembly of the actin/spectrin membrane skeleton in human motor neuron axons
eLife 14:RP108021.
https://doi.org/10.7554/eLife.108021.3
