Dynamic early clusters of nodal proteins contribute to node of Ranvier assembly during myelination of peripheral neurons
- Centre for Discovery Brain Sciences, University of Edinburgh, United Kingdom
- Biodiscovery Institute, University of Nottingham, United Kingdom
- Centre for Microsystems & Photonics, Dept. Electronic and Electrical Engineering, University of Strathclyde, United Kingdom
Figures
Figure 1 with 2 supplements
SEP-Nfasc186 and β1Nav-EGFP are targeted to the axonal plasma membrane during myelination and assemble into early clusters.
(A, B) Live imaging and immunostaining of SEP-Nfasc186+ (A) or β1Nav-EGFP+ (B) axons in dorsal root ganglia (DRG) neuron-Schwann cell co-cultures. Both proteins are targeted to early clusters (solid arrowheads) flanked by diffuse signal at the axolemma on unmyelinated axons. SEP-Nfasc186 and β1Nav-EGFP are targeted to heminodes (open arrowheads) and nodes (white arrows) and cleared from the internodal axonal membrane. Immunostaining for GFP identifies SEP-Nfasc186 or β1Nav-EGFP; myelin-associated glycoprotein (MAG) is a component of ensheathing Schwann cells and AnkyrinG (AnkG) and βIV-spectrin are nodal proteins. Scale bars, 3 µm.
Figure 1—figure supplement 1
SEP-Nfasc186 (A) and β1Nav-EGFP (B) in early clusters (arrowheads) colocalise with nodal proteins.
(A, B) Live imaging shows that fluorescent fusion proteins in early clusters in myelinating dorsal root ganglia (DRG) neuron-Schwann cell co-cultures 10 days after the induction of myelination have not been cleared from the axonal membrane. Immunostaining reveals their colocalisation with nodal markers. Scale bars, 3 µm.
Figure 1—figure supplement 2
Early clusters assemble in WT axons in vitro and in WT, β1Nav-EGFP, and SEP-Nfasc186 mice in vivo.
(A) Immunostaining of wild-type (WT) myelinating dorsal root ganglia (DRG) neuron-Schwann cell co-cultures 10 days after the induction of myelination shows that neurofascin (Nfasc) clusters with AnkyrinG (AnkG) in unmyelinated axons (solid arrowheads). Immunostaining for the myelin marker myelin-associated glycoprotein (MAG) showed myelinated segments at heminodes (open arrowheads) and nodes (arrows) in the same cultures. Scale bars, 3 µm. (B) Immunostaining of WT sciatic nerve fibres from neonatal WT mice (P1) shows an early cluster immunostained for Nfasc and Nav in the absence of the myelin marker P0. In contrast, heminodes are shown adjacent to myelinating Schwann cells. Scale bars, 3 µm. (C) Immunostaining of sections of sciatic nerves from WT, β1Nav-EGFP, and SEP-Nfasc186 mice at P1 for βIV-spectrin (nodal clusters - green), Caspr (paranodes - magenta), and P0 (myelin - blue). Arrowheads point to nodal clusters with paranodal Caspr. Scale bar, 5 µm.
Figure 2 with 2 supplements
Early cluster assembly requires myelinating conditions and occurs close to Schwann cells independently of clear axo-glial membrane specialisations.
(A) SEP-Nfasc186+ dorsal root ganglia (DRG) neurons were grown in matrigel- or collagen-coated microfluidic chambers for 10 days with or without rat Schwann cells, and with myelinating medium or C-medium for further 10 days. Early cluster quantitation: n = 3 independent cultures, unpaired two-tailed Student’s t-test, **p<0.01, ****p<0.0001. (B) Schwann cells (immunostained for periaxin) surround an axon with early clusters (solid arrowheads) in myelinating co-cultures. Myelin at heminodes (open arrowhead) was immunostained with periaxin antibodies. Scale bar, 5 µm. (C) Early clusters are in close proximity to Schwann cells. (a) Early clusters in a SEP-Nfasc186+ co-culture imaged 10 days after myelination induction (scale bar, 50 µm). (b) Magnification of the box in (a) (scale bar, 5 µm). (c) 3D model of the early clusters shown in (b). Arrowhead shows the cluster viewed by electron microscopy (EM) (scale bar, 5 µm). (d) The early cluster model shown in (c) is superimposed on an EM slice of an axon and (e) on a 3D reconstruction obtained from serial EM sections of the same axon. (f) EM section through the early cluster indicated by the arrow in (c). The dotted green line denotes the position of the early cluster. (g) 3D model of the early cluster (dark green) is superimposed on the same EM section shown in (f). A Schwann cell (magenta) and an axon (pale green) are pseudocoloured to show their close proximity. Scale bar, 2 µm.
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Early clusters are not contacted by Schwann cell microvilli.
(A) Live imaging and immunostaining of SEP-Nfasc186+ dorsal root ganglia (DRG) neuron-Schwann cell co-cultures show that the Schwann cell microvillar marker pERM is not present at early clusters (solid arrowheads) but is detectable at heminodes (open arrowhead) and nodes (arrow). (B) The Schwann cell microvillar proteins dystrophin (Dp116) and radixin are also absent in early clusters (solid arrowheads) but are detectable at heminodes (open arrowhead) and nodes (arrow). Dotted magenta lines in the bright-field images show the location of myelin. Scale bars, 5 µm.
Figure 2—figure supplement 2
Imaging of early clusters by CLEM.
(A) Correlation between LM and electron microscopy (EM) images of early clusters. Diagram (a) and tiled bright-field image of the somal compartment (b) of a microfluidic device. The red box in (b) corresponds to the area shown in (c–f). (c) Early clusters in a SEP-Nfasc186+ co-culture imaged 10 days after the induction of myelination. (d) Fluorescence image shown in (c) overlaid to the corresponding bright-field image. The same area was re-identified on the resin (e) and EM slice (f) using dorsal root ganglia (DRG) somas (arrows) as biological fiducials. Scale bars, 50 µm. (B, C) Serial EM slices of the early clusters indicated by the red arrowheads in (c). The axon in each EM slice is pseudocoloured in yellow, and the position of the early cluster is shown by a green dotted line. A 3D model of the early cluster is superimposed on the EM image corresponding to the centre of the stack. Scale bar, 2 µm.
Figure 3 with 3 supplements
Early clusters are dynamic and exchange SEP-Nfasc186 with the surface pool by lateral diffusion in DRG neuron-Schwann cell co-cultures.
(A) Still video images of fluorescence recovery after photobleaching (FRAP) of a naked axon, an early cluster (solid arrowhead), a heminode (open arrowhead), and a node (arrow). FRAP regions of interest (ROIs) are in white boxes. Magenta lines outline myelin in bright-field images. (B, C) The fluorescent signal at unmyelinated axons, early clusters, and heminodes in vitro recovers after photobleaching. n≥3 independent cultures, ≥10 axons per culture, one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test; ***p<0.001. (D) Still video images of FRAP with bilateral fluorescence loss in photobleaching (FLIP) at an SEP-Nfasc186+ early cluster (solid arrowhead) and heminode (open arrowhead) and asymmetric FLIP at an SEP-Nfasc186+ heminode (FRAP ROIs in white boxes and FLIP ROIs in green boxes). (E–G) Signal recovery at early clusters and at heminodes is by lateral diffusion. n≥3 independent cultures, ≥10 axons per culture; early clusters: unpaired two-tailed Student’s t-test. Heminodes: one-way ANOVA followed by Sidak’s multiple comparisons test; **p<0.01; ***p<0.001; ****p<0.0001; ns = not significant. Scale bars, 5 µm.
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
SEP-Nfasc186 in early clusters is dynamic in the intercostal nerves of triangularis sterni explants.
(A) Still video images of fluorescence recovery after photobleaching (FRAP) in an unmyelinated axon, an early cluster (solid arrowhead), a heminode (open arrowhead), and a node (arrow). FRAP regions of interest (ROIs) are shown as white boxes. The image of the node is superimposed on the corresponding bright-field image in the inset, confirming the presence of flanking myelin (black). Scale bar, 5 µm. (B, C) FRAP curves show that the fluorescent signal in nodal complexes in unmyelinated axons, early clusters, and heminodes in triangularis sterni explants recovers after photobleaching but does not recover at nodes. n≥5 animals, total ≥ 24 axons; one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test; **p<0.01; ****p<0.0001.
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Vesicular transport in DRG axons is unaffected by FRAP/FLIP.
(A–C) Movement of axonal β1Nav-EGFP transport vesicles in a myelinating dorsal root ganglia (DRG) neuron-Schwann cell co-culture was recorded for 1 min immediately before and after performing fluorescence recovery after photobleaching combined with fluorescence loss in photobleaching (FRAP/FLIP) for 15 min (FRAP at magenta region of interest (ROI) and FLIP at green ROI). Analysis of kymographs of vesicular movement showed that FRAP/FLIP had no effect on vesicle motility or speed. n = 5 axons; paired two-tailed Student’s t-test; ns = not significant.
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Figure 3—figure supplement 2—source data 1
Source data for Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 3
Delivery of β1Nav-EGFP to early clusters is dynamic and independent of microtubule-based vesicular transport.
(A, B) Still video images of fluorescence recovery after photobleaching (FRAP) at β1Nav-EGFP+ early clusters (solid arrowheads) in dorsal root ganglia (DRG) neuron-Schwann cell co-cultures. FRAP and control regions of interest (ROIs) are shown as white and green boxes, respectively. 4 hr after photobleaching, the fluorescence intensity at β1Nav-EGFP+ early clusters was significantly higher than the fluorescence intensity recorded in the adjacent control ROI. n = 3 independent cultures, ≥10 axons per culture, one-sample t-test, null hypothesis: FRAP/control ROI intensity 4 hr after photobleaching = 1; **p<0.01. Scale bar, 5 µm. (C, D) Movement of β1Nav-EGFP+ vesicles in DRG axons was recorded for 1 min immediately before, and 1 hr and 4.5 hr after adding either dimethyl sulfoxide (DMSO) or nocodazole (30 µM). Analysis of kymographs of vesicular movement showed that nocodazole treatment for 1 hr or 4.5 hr significantly reduced the proportion of motile vesicles. Unpaired two-tailed Student’s t-test, *p<0.05, n = 3 independent cultures, ≥10 axons per culture. (E) Still video images of FRAP at β1Nav-EGFP+ early clusters (solid arrowheads) in co-cultures treated with DMSO or nocodazole (30 µM) throughout imaging. DMSO or nocodazole was added to the cultures 30 min before photobleaching. FRAP and control ROIs are shown as white and green boxes, respectively. Bright-field images confirm the absence of myelin. The same axons are shown after immunostaining for GFP, βIV-spectrin, and β3-tubulin. Open arrowheads point to tubulin blebs. Scale bar, 5 µm. (F, G) Nocodazole treatment does not influence the extent of fluorescent signal recovery at β1Nav-EGFP+ early clusters nor the fluorescence intensity in the control region 4 hr after photobleaching. n = 4 independent cultures, ≥10 axons per culture, unpaired two-tailed Student’s t-test, ns = not significant.
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Figure 3—figure supplement 3—source data 1
Source data for Figure 3—figure supplement 3.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig3-figsupp3-data1-v2.xlsx
Figure 4 with 2 supplements
Axo-glial junctions restrict the dynamics of nodal complexes.
(A) Still video images of fluorescence recovery after photobleaching (FRAP) at SEP-Nfasc186+ early clusters (solid arrowheads), heminodes (open arrowheads), and nodes (white arrows) in Cntnap1+/+ or Cntnap1-/- dorsal root ganglia (DRG) neurons. FRAP regions of interest (ROIs) are shown as white boxes. (B) The same axons are shown after immunostaining for GFP, AnkyrinG (AnkG), and myelin-associated glycoprotein (MAG) or GFP, Caspr, and MBP (MAG and MBP are myelin markers). Scale bar, 3 µm. (C) Analysis of FRAP curves shows that the fluorescent signal at early clusters and heminodes recovered to the same extent in Cntnap1+/+ and Cntnap1-/- (Caspr-null) axons 15 min after photobleaching. In contrast, signal recovery after photobleaching was significantly higher at nodes from Cntnap1-/- mice. n≥3 independent cultures, ≥10 axons per culture, unpaired two-tailed Student’s t-test, *p<0.05.
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Axo-glial junctions restrict the dynamics of nodal complexes ex vivo.
Still video images of fluorescence recovery after photobleaching (FRAP) at SEP-Nfasc186+ heminodes (A) and nodes (B) in wild-type (WT), Caspr/-, or Nfasc-/- nerve-muscle explants. FRAP regions of interest (ROIs) are shown by white boxes. Scale bars, 5 µm. (B) Analysis of FRAP curves shows that 15 min after photobleaching, the fluorescent signal at Cntnap1-/- and Nfasc-/- heminodes recovered to the same extent as WT heminodes. By contrast, fluorescence recovery was significantly higher at Cntnap1-/- and Nfasc-/- nodes compared to WT nodes. n≥5 animals, total ≥23 axons, one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test, ****p<0.0001, ns = not significant.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Paranodal axo-glial junctions prevent the lateral diffusion of nodal SEP-Nfasc186.
(A) Still video images of fluorescence recovery after photobleaching (FRAP) and FRAP combined with fluorescence loss in photobleaching (FRAP/FLIP) (FRAP regions of interest (ROIs) shown at white boxes and FLIP ROIs shown at green boxes) at SEP-Nfasc186+ nodes in wild-type (WT) and Cntnap1-/- dorsal root ganglia (DRG) neurons. Dotted magenta lines indicate the location of myelin. Scale bar, 5 µm. (B, C) FRAP curves show that recovery of the fluorescent signal at nodes after photobleaching is completely prevented by FLIP. n = 3 independent cultures, ≥10 axons per culture, unpaired two-tailed Student’s t-test, **p<0.01. (D) Still video images of FLIP (green boxes) at SEP-Nfasc186+ nodes in WT and Cntnap1-/- DRG neurons. Dotted magenta lines denote the location of myelin. Scale bar, 5 µm. (E, F) FLIP results in significant loss of the fluorescent signal from Cntnap1-/- nodes, but not from WT nodes. n = 3 independent cultures, ≥10 axons per culture, unpaired two-tailed Student’s t-test, ****p<0.0001.
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig4-figsupp2-data1-v2.xlsx
Figure 5 with 2 supplements
Early clusters are mobile and plastic in DRG neuron-Schwann cell co-cultures.
(A–D) Still video images of SEP-Nfasc186+ early clusters at 10 days after the initiation of myelination. Dashed lines outline the imaged axons. Arrowheads of different colours are used to indicate multiple early clusters on the same axon. (A) An individual early cluster migrating along an axon. (B) Two early clusters can be seen moving in the same direction along an axon. One early cluster crosses an axonal branching point (white arrow), while the other moves past a crossing point between axons (green arrow). (C) An early cluster fragmenting and disappearing. (D) A group of early clusters moving synchronously along an axon. Scale bars, 5 µm.
Figure 5—figure supplement 1
Protracted imaging does not affect myelination.
Still video images of SEP-Nfasc186+ heminodes (solid arrowheads) merging to form a node (open arrowheads). Note that the location of the newly assembled node becomes fully stabilised 24 hr after the start of imaging, and the node is maintained for at least 28 hr after that. Dotted magenta lines denote the location of myelin. Scale bar, 5 µm.
Figure 5—figure supplement 2
Movement of early clusters is impaired by disruption of the actin cytoskeleton.
(A) Still video images of SEP-Nfasc186+ early clusters (solid arrowheads) in co-cultures treated with dimethyl sulfoxide (DMSO) or the actin-depolymerising agent latrunculin A (50 nM) for 16 hr. Scale bar, 5 µm. (B) Immunostaining of the same axons shown in (A) immediately after live imaging shows that the actin cytoskeleton in axons and adjacent Schwann cells (detected by staining with phalloidin) was effectively disrupted by treatment with latrunculin A. Confocal images of DMSO- and latrunculin A-treated axons were acquired using identical settings. Scale bar, 5 µm. (C, D) Quantification of early cluster movement in SEP-Nfasc186+ DRG neuron-Schwann cell co-cultures revealed that both the percentage and the speed of early clusters that are mobile are significantly reduced by latrunculin A treatment. n = 3 independent cultures, ≥10 axons per culture, unpaired two-tailed Student’s t-test, **p<0.01.
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Figure 5—figure supplement 2—source data 1
Source data for Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig5-figsupp2-data1-v2.xlsx
Figure 6
Assembly of early clusters precedes heminode formation in co-cultures and in vivo.
(A) SEP-Nfasc186+ early clusters, heminodes, and nodes in dorsal root ganglia (DRG) neuron-Schwann cell co-cultures were quantitated at the indicated time points in the somal (proximal) and axonal (distal) compartments of the microfluidic device. n = 3 independent cultures. (B, C) Immunostained sections of sciatic nerves from wild-type (WT) mice at P3 for βIV-spectrin (nodal clusters - green), Caspr (paranodes - red), and P0 (myelin - blue). Early clusters (closed arrowhead), heminodes (open arrowhead), and nodes (arrow) were quantitated at P1, P3, and P5, showing the sequential appearance of early clusters, heminodes, and nodes; n≥2 mice. Scale bar, 5 µm.
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/68089/elife-68089-fig6-data1-v2.xlsx
Figure 7
Early clusters merge with heminodes.
(A, B) Still video images of SEP-Nfasc186+ early clusters 10 days after myelination was initiated. Dashed lines outline the imaged axons. Dotted magenta line indicates the location of myelin. Arrowheads of different colours are used to indicate multiple early clusters on the same axon. Three early clusters can be seen merging in (A). A single early cluster (solid arrowhead) is shown merging with a heminode (open arrowhead) in (B). (C) Model depicting early clusters migrating, disaggregating, merging, or fusing with a heminode. Key to the colours: dark grey, myelinating Schwann cell with myelin; pale grey, pre-myelinating Schwann cell in contact with the axon; magenta, axo-glial junction; green, nodal complex. Asterisks identify groups of nodal complexes and their fate.
Videos
Video 1
FRAP (white box) of SEP-Nfasc186 in an axon with no Schwann cells (A), early cluster (B), heminode (C), and node (D).
Real interframe interval: 4 s.
Video 2
Bilateral FRAP/FLIP of SEP-Nfasc186 at an early cluster (top panel) and a heminode (middle panels) and unilateral FRAP/FLIP at a heminode (bottom panel).
Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) regions of interest (ROIs) are delimited by white and green boxes, respectively. Real interframe interval: 4 s.
Video 3
An SEP-NFASC186+ early cluster moving along an axon.
The video was acquired 10 days after the induction of myelination. Real interframe interval: 2 hr.
Video 4
An SEP-NFASC186+ early cluster breaking up into three smaller early clusters and eventually disappearing.
The video was acquired 10 days after the induction of myelination. Real interframe interval: 2 hr.
Video 5
A group of SEP-NFASC186+ early clusters synchronously moving along an axon.
The video was acquired 10 days after the induction of myelination. Real interframe interval: 2 hr.
Video 6
Three SEP-NFASC186+ early clusters merging into one.
The video was acquired 10 days after the induction of myelination. Real interframe interval: 2 hr.
Video 7
An SEP-NFASC186+ early cluster migrating towards and eventually merging with a heminode (solid arrowhead).
The video was acquired 10 days after the induction of myelination. Real interframe interval: 2 hr.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Mus musculus), C57BL/6JOla, male and female | Nfasc-/- mice | Sherman et al., 2005 | Peter Brophy, University of Edinburgh | |
| Strain (Mus musculus), C57BL/6JOla, male and female | Cntnap1-/- mice | Gollan et al., 2003 | E Peles, Weitzmann Institute | |
| Strain (Mus musculus), C57BL/6JOla, male and female | Thy1-SEP-Nfasc186 | This paper | Peter Brophy, University of Edinburgh | |
| Strain (Mus musculus), C57BL/6JOla, male and female | ß1Nav-EGFP | Booker et al., 2020 | Peter Brophy, University of Edinburgh | |
| Antibody | P0 (chicken polyclonal) | Aves Labs | RRID:AB-2313561 | 1:100 |
| Antibody | Sodium channel (Pan) (mouse monoclonal) | Sigma-Aldrich | Cat# S8809 | 1:200 |
| Antibody | MAG (mouse monoclonal) | M Filbin | IgG1 | 1:50 |
| Antibody | ßIV-spectrin (rabbit polyclonal) | Desmazieres et al., 2014 | PJ Brophy | 1:200 |
| Antibody | ßIV-spectrin (rabbit polyclonal) | MN Rasband, Baylor College of Medicine | 1:400 | |
| Antibody | AnkyrinG (mouse monoclonal) | UC Davis/NIH NeuroMab | Cat# 75–147 | 1:50 |
| Antibody | GFP (chicken polyclonal) | Abcam | Cat# ab13970 | 1:1000 |
| Antibody | β3-tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T8660 | 1:100 |
| Antibody | Gliomedin (rabbit polyclonal) | E Peles | 1:100 | |
| Antibody | Caspr (rabbit polyclonal) | DR Colman | 1:5000 | |
| Antibody | Caspr (guinea-pig polyclonal) | M Bhat, University of Texas, San Antonio | 1:400 | |
| Antibody | MBP (rabbit polyclonal) | Vouyioukiis and Brophy, 1993 | PJ Brophy, pep7 | 1:1000 |
| Antibody | Periaxin (rabbit polyclonal) | Gillespie et al., 1994 | PJ Brophy, Repeat region | 1:5000 |
| Antibody | pERM (rabbit polyclonal) | Cell Signalling Technologies | Cat# 3141 | 1:200 |
| Antibody | Dystrophin (mouse monoclonal) | Sigma-Aldrich (MANDRA1) | Cat# D8043 | 1:200 |
| Antibody | Radixin (rabbit polyclonal) | Sherman et al., 2012 | P Brophy, RAD4 | 1:500 |
| Antibody | Neurofascin-Pan (rabbit polyclonal) | Tait et al., 2000 | PJ Brophy, NFC | 1:2000 |
| Antibody | Goat Anti-mouse IgG1 Alexa Fluor 488 | Invitrogen | Cat# A-21121 | 1:1000 |
| Antibody | Goat Anti-rabbit Alexa Fluor 647 | Invitrogen | Cat# A-32733 | 1:1000 |
| Antibody | Donkey Anti-rabbit Alexa Fluor 594 | Jackson ImmunoResearch | Cat# 111-585-14 | 1:1000 |
| Antibody | Donkey Anti-chicken Alexa Fluor 488 | Jackson ImmunoResearch | Cat# 703-545-155 | 1:1000 |
| Antibody | Goat Anti-mouse IgG2a Alexa Fluor 568 | Invitrogen | Cat# A-21134 | 1:1000 |
| Antibody | Goat Anti-mouse IgG2a Alexa Fluor 647 | Invitrogen | Cat# A-21241 | 1:1000 |
| Antibody | Goat Anti-mouse IgG2b Alexa Fluor 488 | Invitrogen | Cat# A-21141 | 1:1000 |
| Antibody | Anti-mouse IgG1 Alexa Fluor 594 | Invitrogen | Cat# A-21125 | 1:1000 |
| Antibody | Goat Anti-mouse IgG1 Alexa Fluor 647 | Invitrogen | Cat# A-21240 | 1:1000 |
| Antibody | Goat Anti-mouse IgG2b DyLight 405 | Jackson ImmunoResearch | Cat# 115-475-207 | 1:100 |
| Antibody | Goat Anti-guinea pig Alexa Fluor 594 | Invitrogen | Cat# A-11076 | 1:8000 |
| Antibody | Goat Anti-rabbit Alexa Fluor 488 | Invitrogen | Cat# 710369 | 1:1000 |
| Antibody | Goat Anti-chicken Alexa-Fluor 647 | Invitrogen | Cat# A-21449 | 1:1000 |
| Chemical compound, drug | Phalloidin AlexaFluor 568 | Invitrogen | Cat# A-12380 | 1:50 |
| Chemical compound, drug | DAPI (4 ′, 6-diamidino-2-phenylindole) | Sigma-Aldrich | Cat# D9542 | 1 μg/ml |
| Chemical compound, drug | BrainStain fluorescent dye mix | ThermoFisher | Cat# B34650 | 1:300 |
| Chemical compound, drug | DMSO | Sigma-Aldrich | Cat# D2650 | |
| Chemical compound, drug | Poly-D-lysine | Sigma-Aldrich | Cat# P6407 | |
| Chemical compound, drug | B-27 | Thermo Fisher Scientific | Cat# 17504044 | |
| Chemical compound, drug | Matrigel | Corning | Cat# 356231 | |
| Chemical compound, drug | Fish skin gelatin | Sigma-Aldrich | Cat# G7765 | |
| Chemical compound, drug | Nocodazole | Sigma-Aldrich | Cat# SML1665 | |
| Chemical compound, drug | Latrunculin A | Merck | Cat# 428026 | |
| Chemical compound, drug | para-amino-Blebbistatin | Cayman Chemical | Cat# 22699 | |
| Software, algorithm | FIJI | Schindelin et al., 2012 | RRID:SCR_002285 | https://imagej.net/Fiji |
| Software, algorithm | Prism 8.0 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | KymoTool Box | Zala et al., 2013 | F Saudou, University Grenoble Alpes | |
| Software, algorithm | Amira | ThermoFisher | RRID:SCR_007353 |
Additional files
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Source data 1
Early clusters in vivo.
- https://cdn.elifesciences.org/articles/68089/elife-68089-data1-v2.xlsx
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Source data 2
Early clusters in Nfasc-/- neurons.
- https://cdn.elifesciences.org/articles/68089/elife-68089-data2-v2.xlsx
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Source data 3
Early clusters in Cntnap1-/- neurons.
- https://cdn.elifesciences.org/articles/68089/elife-68089-data3-v2.xlsx
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Source data 4
Blebbistatin treatment.
- https://cdn.elifesciences.org/articles/68089/elife-68089-data4-v2.xlsx
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Source data 5
Percentage and speed of mobile early clusters.
- https://cdn.elifesciences.org/articles/68089/elife-68089-data5-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68089/elife-68089-transrepform-v2.doc
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Dynamic early clusters of nodal proteins contribute to node of Ranvier assembly during myelination of peripheral neurons
eLife 10:e68089.
https://doi.org/10.7554/eLife.68089
