The βII-spectrin membrane periodic skeleton (MPS) assembles progressively.

(A) Representative micrographs of single optical sections from STED nanoscopy of DIV1, DIV2, DIV4, and DIV6 DRG axons stained with βII-spectrin antibody. Scale bar: 1 µm. (B) Average autocorrelation curves across multiple axonal regions. (C) The normalised autocorrelation amplitudes of the βII-spectrin rings. Amplitudes were quantified as the difference between the first peak and the average of the first two valleys of the autocorrelation curve. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games-Howell’s test. (D) Periodicity across DIVs demonstrates a reduction in the spacing between the βII-spectrin rings. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games-Howell’s test. The numbers below each data set indicate the coefficient of variation of the distribution. (E) Plot of the normalised autocorrelation amplitudes against periodicity for all DIVs, indicating the degree of autocorrelation and the relative distribution of MPS periodicity. (F) βII-spectrin ring diameter across DIVs. The data were analysed using the Kruskal-Wallis test with multiple comparisons corrected by Dunn’s test. For A-F, 80-100 axonal segments from 3 biological replicates for each DIV were analysed. (G) The evolving gap length between cut ends following ablation is represented as a one-phase fit of the means (± SEM). Curve fits were compared using the extra sum-of-squares F-test. The effect sizes and significance, represented by Cohen’s d and two-sided t-test, obtained by comparing the gap length in the last 10 frames (till 0.74 sec) using Estimation statistics (Fig S1C) are indicated. The data are derived from n=19 (DIV1), n=15 (DIV2) and n=16 (DIV4) axons from at least three biological replicates. (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001).

Destabilisation of F-actin and microtubules affects MPS development and stability.

(A,D,G) Average autocorrelation curves of axonal segments across DIVs treated with DMSO (control), Jasplakinolide (Jasp), Latrunculin-A (Lat-A), Paclitaxel (Taxol), and Nocodazole (Noco). (B,E,H) The normalised autocorrelation amplitudes of the βII-spectrin rings across treatments and DIVs. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games-Howell’s test (ns, p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001). (C, F, I) Periodicity (spacing) across DIVs treated with indicated drugs. The numbers below each data set indicate the coefficient of variation of the distribution. The numbers above each data set indicate the coefficient of variation of the distribution. For A-I, 60-95 axonal segments from 3 biological replicates for each DIV were analysed.

Microtubule dynamics are crucial for MPS formation.

(A) Timeline of the experiment. 1 nM Taxol or 10 nM Nocodazole was added 16 hr after seeding the neurons and fixed either after another 32 hrs (DIV2) or after 80 hrs (DIV4). (B,C) Average autocorrelation curves drop for βII-spectrin stained axons from cultures treated with 10 nM Nocodazole (green dotted curve) and 1 nM Taxol (pink dotted curve) compared to DMSO (black curve). (D)The normalised autocorrelation amplitudes of the βII-spectrin rings. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games-Howell’s test (***, p ≤ 0.001; ****, p ≤ 0.0001). (E) Periodicity across DIVs demonstrates an increase in variability between the βII-spectrin rings. The numbers below each data set indicate the coefficient of variation of the distribution. For B-E, 82-100 axonal segments from 3 biological replicates for each DIV were analysed.

Actin stabilisation or myosin contractility does not affect MPS formation.

(A) Timeline of the experiment. 10 nM Jasplakinolide or 10 μM Blebbistatin was added 16 hr after seeding the neurons and fixed either after another 32 hrs (DIV2) or after 80 hrs (DIV4). (B, C) Average autocorrelation curves show no difference for βII-spectrin stained axons from cultures treated with 10 μM Blebbistatin (green dotted curve) and 10 nM Jasplakinolide (pink dotted curve) compared to DMSO (black curve). (D)The normalised autocorrelation amplitudes of the βII-spectrin rings. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games-Howell’s test (ns, p > 0.05). (E) Periodicity across DIVs demonstrates variability between the βII-spectrin rings. The numbers below each data set indicate the coefficient of variation of the distribution. For B-E, 79-98 axonal segments from 3 biological replicates for each DIV were analysed.

Formins and Arp2/3 affect MPS formation and maintenance.

(A) Representative micrographs of single optical sections from STED nanoscopy of DIV2, DIV4, and DIV6 DRG axons stained with βII-spectrin post-treatment with DMSO control, Formin inhibitor (SMIFH2; 20 µM, 30 min) or Arp2/3 inhibitor (CK666; 20 µM, 30 min). Scale bar: 1 µm (B) Average autocorrelation curves across multiple axonal regions for all treatments. (C) The normalised autocorrelation amplitudes of the βII-spectrin rings. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games -Howell’s test (ns, p > 0.05; ***, p ≤ 0.001, ****, p ≤ 0.0001) (D) Periodicity across DIVs demonstrates an increase in variability between the βII-spectrin rings at DIV2 and 4. The numbers below each data set indicate the coefficient of variation of the distribution. For B-D, 60-70 axonal segments from 3 biological replicates for each DIV were analysed.

Spectrin mobility is correlated with MPS maturation and dependent on F-actin-mediated cortical recruitment.

(A) βII-spectrin-GFP FRAP recovery curves for the DIV1, DIV2, and DIV4 axons. The recovery curves are represented as a one-phase fit of the means (± SEM). Plateau of curves were compared using the extra sum-of-squares F-test. (B) Quantification of the % mobile fraction. The data were analysed using the Mann-Whitney test. (C) t1/2 values of recovery curves obtained from one-phase fit of Latrunculin A and DMSO-treated DRG neurons. Data were analysed using the Kruskal-Wallis test. For A-C, 16-30 neurons for all DIVs from 3 biological replicates were analysed (D) βII-spectrin-GFP FRAP recovery curves for DRG axons treated with Lat-A across DIV1, DIV2 and DIV4. The green and black traces represent the average one-phase fit for Lat-A- and DMSO-treated neurons, respectively. The recovery curves are represented as a one-phase fit of the means (± SEM). Plateau of curves were compared using the extra sum-of-squares F-test. (E) Quantification of the % mobile fraction. The data were analysed using the Mann-Whitney test. In this experiment, 12-24 neurons for all DIVs from 3 biological replicates were analysed. (F) t1/2 values of recovery curves obtained from one-phase fit of Latrunculin A and DMSO-treated DRG neurons. Data were analysed using the Kruskal-Wallis test. 12-24 neurons for all DIVs from 3 biological replicates were analysed. (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01).

MPS in differentiated SH-SY5Y neurons. (A) Timeline indicating the differentiation protocol of SH-SY5Y cells.

(B) Representative micrographs of single optical sections from STED nanoscopy of βII-spectrin stained differentiating neurons (4, 6, and 8 days post-BDNF). Scale bar: 1 µm. (C, D) Average autocorrelation curves and quantification of normalised autocorrelation amplitudes across time points. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games-Howell’s test. (**, p ≤ 0.01). (E) Periodicity across days demonstrates a reduction in variability. The numbers below each data set indicate the coefficient of variation of the distribution. For B-E, 65 axonal segments from 3 biological replicates for each time-point were analysed. (F) Immunoblot validating the CRISPR-Cas9-based generation of βII-spectrin knockout line in SH-SY5Y cells (βII-Spectrin KO). KO-1 was used for all subsequent analyses.

Actin-binding and plasma membrane interactions of βII-spectrin are necessary for MPS development.

(A) Timeline indicating the differentiation protocol of βII-Spectrin KO cells and experimental procedures. βII-spectrin KO were transfected mid-differentiation with βII-spectrin variants indicated. The domains are denoted as follows: GFP-tag in green, actin-binding domain (ABD) in grey, ankyrin domain-binding domain in orange, and plasma membrane-binding domain in blue. (B) Representative micrographs of single optical sections from STED nanoscopy of anti-GFP stained differentiated neurites (day 6 post-BDNF) transfected with βII-spectrin variants. (C) Average autocorrelation curves and quantification of normalised autocorrelation amplitudes for all βII-spectrin variants. The data were analysed using the Brown-Forsythe and Welch ANOVA test with multiple comparisons corrected by Games-Howell’s test. (E) Periodicity analysis demonstrates high variation in the spacing between the βII-spectrin rings in K227Q and ΔABD variants. The numbers below each data set indicate the coefficient of variation of the distribution. In this experiment, 40–60 axonal regions were examined for each condition across 3 biological replicates. (ns, p > 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Actin-binding and membrane interactions drive βII-spectrin recruitment and mobility.

(A) FRAP recovery curves of mutant and βII-FL-spectrin expressing βII-Spectrin KO cells 6 days after transfection and BDNF treatment. The recovery curves are represented as a one-phase fit of the means (± SEM). Plateau of curves were compared using the extra sum-of-squares F-test. (B) Quantification of the % mobile fraction. The data were analysed using the Mann-Whitney test. (C) t1/2 values extracted from the fit were analysed using the one-way ANOVA test. In this experiment, 12-17 neurons for all DIVs from 3 biological replicates were analysed (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001).