Nodal β spectrins are required to maintain Na+ channel clustering and axon integrity
- Department of Neuroscience, Baylor College of Medicine, United States
- Program in Developmental Biology, Baylor College of Medicine, United States
- Department of Pathology, Yale University, United States
- Department of Physiology and Cell Biology, The Ohio State University, United States
- Biomedical Engineering, The Ohio State University, United States
Figures
Figure 1
Mice lacking β4 spectrin in PNS sensory neurons have normal behaviors, action potential conduction, and Nav channel clustering at nodes or Ranvier.
(A) Immunoblotting of brain homogenates from 3 month-old Sptbn4F/F and Nes-cre;Sptbn4F/F mice using antibodies against the C-terminal SD domain of β4 spectrin and actin. (B) Immunostaining of …
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Figure 1—source data 1
Original immunoblotting image for Figure 1A and statistical summary for Figure 1C,D,F,H.
- https://cdn.elifesciences.org/articles/52378/elife-52378-fig1-data1-v1.xlsx
Figure 2
β1 spectrin and AnkR rescue Nav channel clustering at nodes in β4 spectrin deficient dorsal roots.
(A) Immunostaining of dorsal roots from 1.5 month-old Sptbn4F/F and Avil-cre;Sptbn4F/F mice using antibodies against pan-neurofascin (blue), β4 spectrin SD-domain (green), and β1 spectrin (red). …
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Figure 2—source data 1
Statistical summary for Figure 2C.
- https://cdn.elifesciences.org/articles/52378/elife-52378-fig2-data1-v1.xlsx
Figure 3
Mice lacking β1 spectrin in PNS sensory neurons have normal behaviors, action potential transmission, and Nav channel clustering at nodes.
(A) Immunoblotting of brain homogenates from 3 month-old SptbF/F and Nes-cre;SptbF/F mice using antibodies against β1 spectrin and actin. (B) Immunostaining of cortex, hippocampal CA1 region and …
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Figure 3—source data 1
Original immunoblotting image for Figure 3A and statistical summary for Figure 3C,D,F,H.
- https://cdn.elifesciences.org/articles/52378/elife-52378-fig3-data1-v1.xlsx
Figure 4
Mice lacking both β1 and β4 spectrin in PNS sensory neurons have motor dysfunction and impaired action potential conduction.
(A) Avil-cre;SptbF/F;Sptbn4F/F mice have a hindlimb clasping reflex. (B) Footprint assay in 6 week-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice. (C) Accelerating rotarod test performed …
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Figure 4—source data 1
Statistical summary for Figure 4C,D,F.
- https://cdn.elifesciences.org/articles/52378/elife-52378-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
Mice lacking both β1 and β4 spectrin in PNS sensory neurons progressively lose their nodal Nav channels.
(A) Immunostaining of dorsal roots from P8, 1 month-old and 6 month-old SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice using antibodies against pan-neurofascin (blue) and panNav channels …
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Figure 5—source data 1
Statistical summary for Figure 5B,C.
- https://cdn.elifesciences.org/articles/52378/elife-52378-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Quantification of nodes per field of view (FOV) by paranodal neurofascin immunostaining in SptbF/F;Sptbn4F/F and Avil-cre;SptbF/F;Sptbn4F/F mice dorsal root at the indicated ages.
N = 3 animals per genotype per time point, except N = 4 in 6 month-old Avil-cre; SptbF/F;Sptbn4F/F. Data are represented as mean ± SEM. 1M: p=0.897; 6M: p=0.7976.
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Figure 5—figure supplement 1—source data 1
Statistical summary for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/52378/elife-52378-fig5-figsupp1-data1-v1.xlsx
Figure 6
PNS sensory axons lacking nodal spectrins have an axon injury response, deformation, and hypermyelination.
(A) Immunostaining of DRG from 1 and 9 month-old Avil-cre;SptbF/F;Sptbn4F/F mice using antibodies against ATF3 (green) and the axonal marker Tuj1 (red). Scale bar, 200 μm. (B) Quantification of the …
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Figure 6—source data 1
Statistical summary and individual data for Figure 6B,C,E,F,G,H,I.
- https://cdn.elifesciences.org/articles/52378/elife-52378-fig6-data1-v1.xlsx
Figure 7
Cartoon illustrating the phenotypes of the mice analyzed here.
Mice lacking β1 spectrin have normal nodes of Ranvier. Mice lacking β4 spectrin have normal nodes of Ranvier due to compensation by a β1 spectrin/AnkR protein complex. Mice lacking all nodal β …
Videos
Video 1
9 month-old WT and Avil-cre;SptbF/F;Sptbn4F/F mice.
Mice lacking both β1 and β4 spectrin in sensory neurons are ataxic.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Sptb | https://www.ncbi.nlm.nih.gov/gene/20741 | Gene ID: 20741 | |
| Gene (M. musculus) | Sptbn4 | https://www.ncbi.nlm.nih.gov/gene/80297 | Gene ID: 80297 | |
| Genetic reagent (M. musculus) | Advillin-cre | F. Wang (Duke University, Durham, NC) | The Jackson Laboratory (Stock No:032536) | |
| Genetic reagent (M. musculus) | Sptbflox/flox | This paper | See Materials and methods, Section Animals | |
| Genetic reagent (M. musculus) | Sptbn4flox/flox | PMID: 30226828 | ||
| Antibody | Anti-Ankyrin G (Mouse monoclonal) | Neuromab | Clone: N106/36; RRID: AB_10673030 | IF (1:500) |
| Antibody | Anti-PanNav (Mouse monoclonal) | Neuromab | Clone: N419/78; RRID: AB_2493099 | IF (1:300) |
| Antibody | Anti-PanNav (Mouse monoclonal) | Sigma-Aldrich | Clone: K58/35; RRID: AB_477552 | IF (1:300) |
| Antibody | Anti-Kv1.2 (Mouse monoclonal) | Neuromab | Clone: K14_16; RRID: AB_2296313 | IF (1:500) |
| Antibody | Anti-β1 spectrin (Mouse monoclonal) | Neuromab | Clone: N385/21; RRID: AB_2315815 | IF (1:500) WB (1:1000) |
| Antibody | Anti-actin (Mouse monoclonal) | Millipore | Cat.#: MAB1501; RRID: AB_2223041 | WB (1:4000) |
| Antibody | Anti-β2 spectrin (Mouse monoclonal) | BD Biosciences | Cat.#: 612563; RRID: AB_399854 | IF (1:1000) |
| Antibody | Anti-Tuj1 (Mouse monoclonal) | BioLegend | Cat.#: 801201; RRID: AB_2313773 | IF (1:800) |
| Antibody | Anti-β3 spectrin (Rabbit polyclonal) | Novus | Cat.#: NB110-58346; RRID: AB_877723 | IF (1:1000) |
| Antibody | Anti-ATF3 (Rabbit polyclonal) | Santa Cruz | Cat.#: SC-188, RRID: AB_2258513 | IF (1:1000) |
| Antibody | Anti-β4 spectrin NT antibody (Rabbit polyclonal) | PMID: 28123356 | RRID: AB_2315634 | IF (1:50) |
| Antibody | Anti-β4 spectrin SD antibody (chicken polyclonal) | PMID: 28123356 | IF (1:200) | |
| Antibody | Anti-β4 spectrin SD antibody (Rabbit polyclonal) | PMID: 28123356 | IF (1:500) WB (1:1000) | |
| Antibody | Anti-Ankyrin R (Rabbit polyclonal) | PMID: 25362473 | IF (1:500) | |
| Antibody | Anti-Caspr (Rabbit polyclonal) | PMID: 10460258 | RRID: AB_2572297 | IF (1:500) |
| Antibody | Anti-Pan Neurofascin (Chicken polyclonal) | R and D Systems | Cat.#: AF3235; RRID: AB_10890736 | IF (1:500) |
| Sequence-based reagent | Genotyping primer for Sptbn4flox/flox mouse (sense) | PMID: 30226828 | 5’-GAGCTGCATAAGTTCTTCAGCGATGC-3’ | |
| Sequence-based reagent | Genotyping primer for Sptbn4flox/flox mouse (anti-sense) | PMID: 30226828 | 5’-ACCCCATCTCAACTGGCTTTCTTGG-3’ | |
| Sequence-based reagent | Genotyping primer for Sptbflox/flox mouse (sense) | This paper | 5’- ACAGAGACAGATGGCCGAAC-3‘ | |
| Sequence-based reagent | Genotyping primer for Sptbflox/flox mouse (anti-sense) | This paper | 5’-CTCTGGTTCCCAGGAGAGC-3’ | |
| Sequence-based reagent | Genotyping primer for Avil-cre mouse (primer 1) | PMID: 29038243 | 5’-CCCTGTTCACTGTGAGTAGG-3’ | |
| Sequence-based reagent | Genotyping primer for Avil-cre mouse (primer 2) | PMID: 29038243 | 5’- AGTATCTGGTAGGTGCTTCCAG-3’ | |
| Sequence-based reagent | Genotyping primer for Avil-cre mouse (primer 3) | PMID: 29038243 | 5’-GCGATCCCTGAACATGTCCATC-3’ | |
| Software, algorithm | Fiji | National Institutes of Health | RRID:SCR_002285 | |
| Software, algorithm | Qupath | PMID: 29203879 | ||
| Software, algorithm | Prism | Graph Pad | RRID:SCR_002798 | Version 6 |
| Software, algorithm | pClamp | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | MultiClamp | Molecular Devices | ||
| Software, algorithm | Clampfit | Molecular Devices |
