Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation
- Institut de Myologie, Sorbonne Université, INSERM, France
- Department of Physiology, Faculty of Medicine Pitié-Salpêtrière, Sorbonne Université, France
- Neuromuscular Morphology Unit, Institut de Myologie, Pitié-Salpêtrière Hospital, Sorbonne Université, France
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine (IBPS), Service de Microscopie Électronique (IBPS-SME), France
- Université Grenoble Alpes, INSERM, U1216, Grenoble Institut des Neurosciences, France
- Department of Neurology, National Reference Center for 'Rare Peripheral Neuropathies', University Hospital, France
- Institut de Recherche en Infectiologie de Montpellier, CNRS UMR 9004, Université de Montpellier, France
Figures
Figure 1 with 3 supplements
Cav3-positive caveolae form nanoscale rings in differentiated myotubes.
(A) Super-resolution images of a human myotube expressing Cav3GFP and labelled with antibodies against RyR1 (red). (B) Quantification of ring diameter from super-resolution images of differentiated …
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Figure 1—source data 1
Quantification of ring diameter in Cav3GFP and immunolabeled myotubes.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig1-data1-v2.zip
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Figure 1—source data 2
Quantification of ring diameter from human and mouse myotubes on PREM.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig1-data2-v2.zip
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Figure 1—source data 3
Quantification of the number of caveolae per ring.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig1-data3-v2.zip
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Figure 1—source data 4
Quantification of the average caveolae diameter.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig1-data4-v2.zip
Figure 1—figure supplement 1
Triad markers are organized longitudinally and then transversally during in vitro differentiation of myotubes into myofibers.
(A) Immunofluorescent DHPR (green) and Ryanodine receptor (RyR1) (red) labeling in primary mouse myotubes differentiated for 5 days. Labeling shows clusters of RyR1 and DHPR that appear …
Figure 1—figure supplement 2
Nascent T-tubules form ring-like structures in myotubes.
(A) Bin1 antibody labels a tubular network extending from the plasma membrane forming contacts with RyR1-positive fluorescent dots. Blue arrowheads and white arrows denote tubular and circular Bin1 …
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Figure 1—figure supplement 2—source data 1
Quantification of ring diameter labeled by MemBright.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig1-figsupp2-data1-v2.zip
Figure 1—video 1
T-tubule staining on live myotubes corresponding to Figure 1—figure supplement 2.
T-tubules from live primary mouse myotubes were stained with the MemBright lipid probe. Tubules emanate transversely from the sarcolemma and are mostly oriented longitudinally.
Figure 2 with 2 supplements
DHPR is enriched in Cav3 structures in contact with RyR1-positive SR cisternae.
(A–C) EM images showing caveolae rings in contact with cortical endo/sarcoplasmic reticulum cisternae (pseudo-colored purple) on the cytosolic part of the adherent sarcolemma. Yellow arrows indicate …
Figure 2—figure supplement 1
Additional CLEM of DHPR in caveolin-positive ring-like structures.
(A–C) Two-color CLEM analysis in unroofed primary mouse myotubes transduced with GFP-tagged α1s-subunit of the DHPR (green) and labeled with antibodies against Cav3 (red). (C) Higher magnification …
Figure 2—video 1
Correlative super-resolution/PREM for Cav3 and RyR1 corresponding to Figure 2D.
The unroofed myotube is shown with successive fluorescent microscopy images (Cav3 in green, RyR1 in red), low-magnification PREM image (grid appears white), super-resolution image and …
Figure 3 with 3 supplements
Caveolae rings extend into Bin1-positive tubules.
(A–C) High magnification PREM views of caveolae rings on unroofed myotubes from primary mouse cultures. In (B) caveolae are pseudo-colored in light purple. White arrows indicate beaded tubes …
Figure 3—figure supplement 1
Thin-section EM analysis of extensively differentiated myotubes.
(A–D) Gallery of thin-section EM from the adherent surface of extensively differentiated mouse primary myotubes as in Figure 3D. The tubular structures emanating from the caveolae ring-like …
Figure 3—video 1
Correlative super-resolution/PREM of Cav3 and Bin1 corresponding to Figure 3E–L.
The unroofed myotube is shown with successive fluorescent microscopy images (Cav3 in green, Bin1 in red), low-magnification PREM image (grid appears white), super-resolution image and …
Figure 3—video 2
Correlative super-resolution/PREM of Cav3 and Bin1 corresponding to Figure 3E-L.
The unroofed myotube is shown with successive fluorescent microscopy images (Cav3 in green, Bin1 in red), low-magnification PREM image (grid appears white), super-resolution image and …
Figure 4 with 4 supplements
Time-lapse imaging of Cav3/Bin1 tubules extending from rings.
(A) Gallery of consecutive frames from two time-lapse sequences of Cav3GFP expressing human myotubes (green). The gallery shows four consecutive frames every 40 s followed by four consecutive frames …
Figure 4—video 1
Live imaging of Cav3GFP structures on the surface of human myotubes corresponding to Figure 4A and B.
Time-lapse shows a tubule emanating from a Cav3-positive ring acquired at 1 frame every 10 s using a spinning-disk microscope equipped with a SR module.
Figure 4—video 2
Live imaging of Cav3GFP structures on the surface of human myotubes corresponding to Figure 4A and B.
Time-lapse shows a tubule emanating from a Cav3-positive ring acquired at 1 frame every 10 seconds using a spinning-disk microscope equipped with a SR module.
Figure 4—video 3
Live imaging of Bin1GFP structures on the surface of human myotubes corresponding to Figure 4C and D.
Time-lapse shows show a tubule emanating from a Bin1-positive ring acquired at 1 frame every 10 s using a spinning-disk microscope equipped with a SR module.
Figure 4—video 4
Live imaging of Bin1GFP structures on the surface of human myotubes corresponding to Figure 4C and D.
Time-lapse shows a tubule emanating from a Bin1-positive ring acquired at 1 frame every 10 seconds using a spinning-disk microscope equipped with a SR module.
Figure 5 with 2 supplements
Bin1 forms rings and tubes in vitro and in cellulo.
(A) Super-resolution fluorescence microscopy image of an unroofed human myotube transduced with Bin1GFP (green). Insets numbered 1–3 show the circular organization (white arrowheads) of the …
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Figure 5—source data 1
Measurements of ring diameters and tube length in vitro.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig5-data1-v2.zip
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Figure 5—source data 2
Measurements of ring diameters and tube length in cellulo.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig5-data2-v2.zip
Figure 5—figure supplement 1
Bin1 tubulation assay in mouse myotubes.
(A) Immunofluorescence images of murine primary myotubes transduced with adenoviruses expressing either Bin1GFP or Bin1-ex11GFP and labeled with antibodies against Cav3 (red). Note the presence of …
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Figure 5—figure supplement 1—source data 1
Quantification of tubule density in Bin1-exon11 vs Bin1 +exon 11.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
Quantification of caveolae ring density in Bin1-exon11 vs Bin1 +exon 11.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig5-figsupp1-data2-v2.zip
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Figure 5—figure supplement 1—source data 3
Quantification of caveolae ring diameter in Bin1-exon11 vs Bin1 +exon 11.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig5-figsupp1-data3-v2.zip
Figure 5—figure supplement 2
Bin1 forms persistent rings and tubules in vitro.
(A) Confocal time-lapse images showing the dynamics of 1 µM Bin1-Alexa647 (magenta) organization in rings and tubes on 5% mol PI4,5P2-containing membranes. Airyscan image acquired at the same region …
Figure 6 with 1 supplement
CLEM evidence of Bin1 tubules and rings in human and murine myotubes.
(A) High magnification PREM image of an unroofed human myotube transduced with an adenovirus expressing Bin1GFP forming a characteristic pearled tubule with caveolar material. A 3D anaglyph of the …
Figure 6—video 1
Correlative super-resolution/PREM for Bin1GFP corresponding to Figure 6I-K.
The unroofed myotube is shown with successive fluorescent microscopy images (Cav3 in red, Bin1GFP in green), low-magnification PREM image (grid appears white), super-resolution image and …
Figure 7
Bin1 tubules are in contact with the extracellular medium and depletion of Cav3 decreases Bin1-induced tubulation.
(A–B) PREM images of intact myotubes transduced with Bin1GFP. Yellow arrows indicate necks of caveolae (25–40 nm) seen from the extracellular side of the plasma membrane organized in a circular (A) …
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Figure 7—source data 1
Quantification of Cav3 protein levels in myotubes treated with siRNA against Cav3.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig7-data1-v2.zip
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Figure 7—source data 2
Quantification of Bin1GFP fluorescence intensity in control and siCav3 myotubes.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig7-data2-v2.zip
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Figure 7—source data 3
Western blot uncropped membranes.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig7-data3-v2.zip
Figure 8 with 2 supplements
Cav3 mutations disorganize caveolae rings in patient-derived cells and T-tubules in patient muscle biopsies.
(A) High-magnification PREM images of caveolae rings on the cytosolic side of the plasma membrane of sonicated myotubes from patients with the Cav3 R26Q mutation. Caveolae are pseudocolored purple …
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Figure 8—source data 1
Quantification of caveolae ring density ctrl vs R26Q vs R26Q+Cav3-GFP.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig8-data1-v2.zip
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Figure 8—source data 2
Quantification of caveolae ring diameter in ctrl vs R26Q vs R26Q+Cav3-GFP.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig8-data2-v2.zip
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Figure 8—source data 3
Quantification of tubule density in control and patient myotubes transduced with Bin1 +ex11.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig8-data3-v2.zip
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Figure 8—source data 4
Quantification of tubule density in control and patient myotubes transduced with Bin1exon11.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig8-data4-v2.zip
Figure 8—figure supplement 1
Cav3 protein levels and characteristic ultrastructure of beaded caveolae tubes in caveolinopathy patient myotubes and characteristic histology of caveolinopathy patient muscle biopsies.
(A) Western-blot analysis of Cav3 protein levels in myotubes from patients with P28L and R26Q mutations (n=3 independent experiments). (B) Densitometric quantification of Cav3 protein levels …
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Figure 8—figure supplement 1—source data 1
Densitometric quantification of Cav3 protein levels in patient myotubes.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig8-figsupp1-data1-v2.zip
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Figure 8—figure supplement 1—source data 2
Western blot uncropped membranes.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig8-figsupp1-data2-v2.zip
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Figure 8—figure supplement 1—source data 3
Western blot uncropped membranes.
- https://cdn.elifesciences.org/articles/84139/elife-84139-fig8-figsupp1-data3-v2.zip
Figure 8—video 1
Tomogram from a semi-thin section of a caveolinopathy patient presenting a bifurcation and corresponding to Figure 8M.
High magnification EM images were acquired with electron tomography by collecting images at different tilt angles up to ±60° relative to the plane of the sample with a 2° increment between each frame.
Figure 9
Model of Bin1 and Cav3-mediated ring formation and tubulation.
(1) Bin1 molecules polymerize into ring-like structures and recruit Cav3-positive caveolae. (2) Caveolae assemble into circular structures while additional caveolae accumulate at specific spots on …
Author response image 1
Author response image 2
Time-lapse imaging of Cav3/Bin1 tubules extending from rings.
Gallery of consecutive frames from the time-lapse sequences of Cav3GFP expressing myotubes from figure 4 and movies 5 and 6. The gallery shows consecutive frames every 10s of a tubule emanating from …
Tables
Table 1
List of primary antibodies.
| Antibodies | Provider | Product ref |
|---|---|---|
| Cav3 (Mouse) monoclonal | BD Biosciences | 610421 |
| Cav3 (Rabbit) polyclonal | Abcam | ab2912 |
| Cav-1 (Rabbit) polyclonal | Santa Cruz | sc894 |
| Cavin 4 (MURC) (Rabbit) polyclonal | Merck | HPA020973 |
| DHPR (Mouse) monoclonal | Abcam | ab2862 |
| RyR1 (Rabbit) polyclonal | Custom | Marty et al., 1994 |
| Bin1 (Mouse) monoclonal | Merck | 05-449-C |
| Bin1 (Rabbit) polyclonal | Custom | Nicot et al., 2007 |
| GFP (Rabbit) polyclonal | Thermofischer | A11122 |
| -actinin 2(Mouse) monoclonal | Sigma Aldrich | A7811 |
| Junctophilin 2 (Rabbit) polyclonal | Thermofischer | PA5-20642 |
| GAPDH (Rabbit) polyclonal | Santa Cruz | sc25778 |
| -tubulin (Mouse) monoclonal | Thermofischer | 236-10501 |
Table 2
List of siRNA sequences.
| Target | Human siRNASequence |
|---|---|
| Cav3 (1) | 5’-CAGAUCUCGAGGCCCAGAUCG-3’ |
| Cav3 (2) | 5’-AAGCACAAUGGCCCUUCGCUC-3’ |
| Target | Murine siRNASequence |
| Cav3 | 5’-GGUUCCUCUCAAUUCCAC-3’ |
