Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, Schematic of βII and βIII spectrins expression in the somatodendritic compartment, axon initial segment (AIS) and distal unmyelinated axon. b, Comparison of βII and βIII spectrins sequences and binding domains. c, d, e, Representative confocal images for βII-spectrin (orange), βIII-spectrin (magenta), CAAX-GFP (grey) and Homer1C (cyan) in a filopodium (a), a mature spine (b) or a dendrite (c) at 21 DIV. Scale bars, 1 µm for filopodium and spine, 2 µm for dendrite. Note that the display contrast was purposely kept constant. f, g, Fluorescence intensity of βII-spectrin and βIII-spectrin in spine necks and dendrites at 10 DIV and 21 DIV normalized to the average intensity of filopodia at the same time-point. Each box shows the median ± percentile. n between 98 and 160 spines or 84 and 97 dendrites from 20 to 30 neurons examined over 3 independent experiments for each time point. Mann-Whitney test, *: p < 0.05, ***: p < 0.001, ****: p < 0.0001. h, i, Correlation scatter plots between βII-spectrin and βIII-spectrin normalized fluorescence intensities in spine neck or dendrite at 10 DIV and 21 DIV. n between 98 and 160 spines or 84 and 97 dendrites from 20 to 30 neurons examined over 3 independent experiments for each time point. r , Pearson’s r coefficient; P, P value.

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques: Expressing, Comparison, Binding Assay, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, b, c, Representative d STORM images of βII-spectrin and its 1D autocorrelation amplitude in axons (a), dendrites (b), and spine necks with PSD95 labelling observed in epifluorescence (c). The non-periodic control was obtained by measuring the 1D autocorrelation amplitude of a cytosolic mCherry labelling in axons, dendrites or spine necks. n between 45 and 80 regions of interest examined over at least 3 independent experiments. d, Spacing between βII-spectrin epitopes. e, f, Representative d STORM images of βIII-spectrin and its 1D autocorrelation amplitude in dendrites (e) and spine necks (f). n between 45 and 80 regions of interest examined over at least 3 independent experiments. g, Spacing between βIII-spectrin epitopes. h, i, Representative two-color STORM images of βII-spectrin and βIII-spectrin and autocorrelation (AC) or cross-correlation amplitudes in dendrites (h) and spine necks with Homer1C labelling (i). n = 76 regions of interest examined over 3 independent experiments. j, Spacing between β-spectrin epitopes after merging the two channels. Each box shows the median ± percentile. Scale bars, 1 µm. Mann-Whitney test, ns: non-significant, ****: p < 0.0001.

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques: Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, Schematic of βII-, βIII- and ɑII-spectrin in the dendrite. Arrowheads correspond to the same area. b , Quantification of the distances between clusters of localizations on XZ projections. c-f , Two-color 3D MINFLUX of βII- and βIII-spectrins in dendrite (c and d) and dendritic spine neck (e and f). Scale bars, 500 nm in c and e, 100 nm in d and f. Arrowheads highlight clusters of localizations that appear in pairs, independently of the β-spectrin isoform, mostly visible in dendrites. (d) and (f) XZ projections of regions in (c) and (e) respectively, show discrete clusters regularly spaced independently of the β-spectrin paralogue, suggesting that βII- and βIII-spectrins are radially periodic.

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques:

Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, b, c, Representative d STORM images of βII-spectrin in a βIII-spectrin KO axon (a), dendrite (b) and spine (c), followed by βII-spectrin localization intensities, 1D autocorrelation amplitudes and spacing measured in the associated neurites. The non-periodic controls are the same as in . PSD95 was imaged using epifluorescence microscopy. n between 30 and 88 regions of interest per neurite examined over 5 independent experiments. d, e, Representative d STORM images of βIII-spectrin in a βII-spectrin KO dendrite (d) and spine (e), followed by βIII-spectrin localizations intensities, 1D autocorrelation amplitudes and spacing measured in the associated neurites. n between 30 and 88 regions of interest per neurite examined over at least 3 independent experiments. Each box represents the median with percentile. Scale bars, 1 µm. Mann-Whitney tests, ns: non-significant, *: p < 0.05, ****: p < 0.0001.

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques: Epifluorescence Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, b, c, Representative d STORM images of ɑII-spectrin in control or KOs axons (a), dendrites (b) and spines (c), followed by ɑII-spectrin mean intensities, 1D autocorrelation amplitudes and spacing measured in the corresponding compartments. Only conditions where ɑII-spectrin mean intensity median value was above one-quarter of the controls ɑ2-spectrin values were analysed for autocorrelation and spacing. The non-periodic controls are the same as . PSD95 was imaged using epifluorescence microscopy. n between 14 and 53 regions of interest per neurite examined over 3 independent experiments. Each box represents the median with percentile. Scale bars, 1 µm. Mann-Whitney tests, ns: non-significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques: Control, Epifluorescence Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, Scheme of βII- and βIII-spectrins highlighting the mutations disrupting interaction with phosphoinositides, ankyrins or actin. b, c, Representative spinning disk images of neurons re-expressing βII- (a) or βIII-spectrins (b) WT and mutant all tagged with eGFP, and expressing an intrabody labelling PSD95 . Scale bars in top images, 20 µm. Scale bars in bottom images, 1 µm. d, e, Ratios of the mean fluorescence intensity measured in axons, dendrites and spines in endogenous and re-expression conditions. n between 9 and 22 neurons obtained from 3 to 5 independent experiments for βII-spectrin, n between 7 and 16 neurons obtained from 3 to 4 independent experiments for βIII-spectrin. f, g, Mobile fractions of βII/III-spectrins measured 180 seconds after photobleaching. Each box represents the median with percentile. n between 12 and 24 regions of interest per neurite obtained from 3 to 5 independent experiments for βII-spectrin, n between 13 and 18 regions of interest per neurite obtained from 3 to 4 independent experiments for βIII-spectrin. Each box represents the median with percentile. Mann-Whitney tests, ns: non-significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques: Expressing, Mutagenesis, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, b Representative d STORM images of wild-type and mutant βII-spectrins (a) and wild-type and mutant βIII-spectrins (b) imaged in re-expression condition in the indicated neuronal compartments. The non-periodic controls are the same as in . PSD95 was imaged using epifluorescence microscopy. Scale bars, 1 µm. c, d βII-spectrin (a) and βIII-spectrins (b) 1D autocorrelation amplitudes measured in the indicated compartments. n between 31 and 162 regions of interest per compartment obtained from 3 to 5 independent experiments for βII-spectrin. n between 73 and 213 regions of interest per compartment obtained from 3 to 4 independent experiments βIII-spectrins. Each box represents the median with percentile. Mann-Whitney tests, ns: non-significant, *: p < 0.05, **: p < 0.01, ****: p < 0.0001. e, f, Spacing between βII-spectrin (e) or βIII-spectrin (f) epitopes obtained from the same datasets as in (c) and (d).

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques: Mutagenesis, Expressing, Epifluorescence Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

doi: 10.64898/2026.03.23.713115

Figure Lengend Snippet: a, Schematic of the periodic actin-spectrin cytoskeleton in axons (βII-spectrin mostly), dendrites and dendritic spines (random βII and βIII spectrins distribution with the presence of sparse βII/βIII heterotypic tetramers). Spectrin signals are excluded from post-synaptic densities. b, Schematic of βII- and βIII-spectrins sensitivity to their binding partners.

Article Snippet: The following primary antibodies were used: βII-spectrin (BD Biosciences, 612 563, mouse IgG1 monoclonal, 1/500), βIII-spectrin (Novus Biotechnologies, NB110 58346, rabbit polyclonal, 1/500 or Santa Cruz, sc515737, monoclonal, 1/100), αII-spectrin (Biolegend, 803206, mouse IgG2b monoclonal, 1/1000), α-Adducin (Abcam, 51130, rabbit IgG polyclonal, 1/200), GFP (Avès, GFP-1020, chicken monoclonal, 1/2000), DsRed (Takara, 632496, rabbit polyclonal, 1/2000), PSD-95 (ThermoFisher, MA1-046, mouse IgG1 monoclonal, 1/500), Homer 1C (Synaptic Systems, 160004, guinea pig polyclonal, 1/500).

Techniques: Binding Assay