Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination
- Jena University Hospital – Friedrich Schiller University Jena, Germany
Figures
Figure 1 with 2 supplements
Generation of syndapin III KO mice.
(a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 …
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Figure 1—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 1 (i.e. of Figure 1l and q).
- https://doi.org/10.7554/eLife.29854.005
Figure 1—figure supplement 1
The syndapin III F-BAR domain localizes to the plasma membrane and decorates distinct membrane domains (enlarged images Figure 1c–d).
(c–e) Merges of MIPs of NIH3T3 cells transfected with GFP, GFP-syndapin III1-70 (putatively remaining upon KO; composed of aa1-70 peptide of syndapin III and five unrelated aa resulting from the …
Figure 1—figure supplement 2
A putative syndapin III1-70 peptide does not interfere with syndapin III F-BAR domain functions (enlargement of images shown in Figure 1i).
MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization and membrane tubulation abilities of GFP-syndapin III …
Figure 2
Syndapin III KO leads to a loss of plasma membrane invaginations with caveolar morphology.
(a–b’) TEM of 50 nm sections of chemically fixed primary cardiomyocytes isolated from WT (a,a’) and syndapin III KO mice (b,b’), respectively. Marked are deep membrane invaginations hit by the …
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Figure 2—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 2 (i.e. of Figure 2c,f and h).
- https://doi.org/10.7554/eLife.29854.007
Figure 3 with 3 supplements
Syndapin III KO leads to impairments in the formation of cav3-coated caveolae.
(a,b) Details of wide-field TEM images of anti-syndapin III immunogold-labeled P-faces of freeze-fractured plasma membranes of cardiomyocytes (CDMC) from WT (a) and syndapin III KO mice (b). (c) …
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Figure 3—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 3 (i.e. of Figure 3f,g,j–p).
- https://doi.org/10.7554/eLife.29854.012
Figure 3—figure supplement 1
Syndapin III is present at cav3-labeled caveolae, shallow membrane indentations and flat membrane areas (picture from which Figure 3h' and h'' were taken).
Syndapin III (15 nm gold) is present at caveolae highlighted by anti-cav3 labeling (10 nm gold) in primary mouse cardiomyocytes. Labelings at caveolae are marked by black arrows, at shallow …
Figure 3—figure supplement 2
Syndapin III is present at cav3-labeled caveolae, shallow membrane indentations and flat membrane areas and syndapin III KO impairs the formation of caveolae (non-colored data corresponding to images shown in Figure 3h–h’’ and and i,i’).
Non-color-marked electron micrographs shown in Figure 3h–h’’ and in Figure 3i,i’ showing that syndapin III (15 nm gold) is present at caveolae highlighted by anti-cav3 labeling (10 nm gold) in …
Figure 3—figure supplement 3
Cav1 and cav3/syndapin III show distinct patterns of localization in primary cardiomyocytes, hearts and skeletal muscles with cav3 and syndapin III being restricted to myocytes.
(a) Sections of skeletal muscles of WT mice and of syndapin III KO mice (specificity control for anti-syndapin III staining) subjected to immunofluorescence analyses showed no spatial overlap of …
Figure 4 with 1 supplement
Impairment of caveolar invagination by syndapin III KO does not lead to dissociation of CAVIN-1 from the plasma membrane.
(a,b) Quantitative western blot analyses of homogenates of hearts and skeletal muscles from WT and syndapin III KO mice showing that the levels of the important cav3 coat component CAVIN-1 are …
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Figure 4—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 4 (i.e. of Figure 4a,b,d,f,i,j,k and l).
- https://doi.org/10.7554/eLife.29854.015
Figure 4—figure supplement 1
Impairment of caveolar invagination by syndapin III KO does not lead to dissociation of CAVIN-1 from the plasma membrane (non-colored images corresponding to Figure 4g and h and further example images).
Upper panels, non-color marked electron micrographs of anti-CAVIN-1 immunogold labeling of freeze-fractured cardiomyocytes isolated from WT (left) and syndapin III KO mice (right) (in combination …
Figure 5
Syndapin III shapes liposomes into tubules with caveolar diameters and localizes to the rim of cav3 coats.
(a–c) Analyses of tubules induced by incubating liposomes with syndapin III (a,b) and GST (c), respectively, by freeze-fracturing/TEM (a) as well as by cryo-TEM (b,c). (d,e) Quantitative analyses of …
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Figure 5—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 5 (i.e. of Figure 5d and e).
- https://doi.org/10.7554/eLife.29854.017
Figure 6 with 1 supplement
Syndapin III is involved in the organization of cav3-containing membrane domains.
(a) Coprecipitation of endogenous cav3 from heart lysates with the indicated immobilized GST-fusion proteins of syndapin III. (b,c) GFP-syndapin III F-BAR domain but not GFP clusters with cav3 at …
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Figure 6—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 6 and of the corresponding Figure 6—figure supplement 1 (i.e. of Figure 6h and i and Figure 6—figure supplement 1a–d).
- https://doi.org/10.7554/eLife.29854.022
Figure 6—figure supplement 1
Syndapin III plays an important role in organizing cav3-containing membrane domains.
(a–d) Quantitative comparisons of density gradient fractions of DRMs showing an altered cav3 distribution in syndapin III KO hearts (a,b) and skeletal muscles (c,d) compared to WT. Upon syndapin III …
Figure 7
Not all described cav3 loss-of-function phenotypes are found upon syndapin III KO and may thus not reflect impairments of caveolar invagination.
(a,b) Quantitative immunoblot analyses of ERK1/2 and phosphoERK1/2 in heart homogenates of WT and syndapin III KO mice (normalized to WT) show no alteration of MAPK signaling (pERK1/2/ERK1/2 …
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Figure 7—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 7 (i.e. of Figure 7b,h and j).
- https://doi.org/10.7554/eLife.29854.024
Figure 8 with 1 supplement
Syndapin III-mediated caveolar invagination counterpoises membrane tensions and thereby ensures cell integrity.
(a,b) Anti-cav3-labeled P-faces of freeze-fractured plasma membranes of cardiomyocytes from WT mice incubated for 5 min in isoosmotic (iso) (a) and hypoosmotic conditions (hypo; hypo15 buffer) (b). …
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Figure 8—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 8 and of the corresponding Figure 8—figure supplement 1 (i.e. of Figure 8c–h and of the Figure 8—figure supplement 1c–h).
- https://doi.org/10.7554/eLife.29854.027
Figure 8—figure supplement 1
Caveolar flattening upon induction of membrane tensions in NIH3T3 cells.
(a,b) Electron micrographs of P-faces of freeze-fractured plasma membranes of NIH3T3 cells incubated for 5 min in isoosmotic (iso) (a) and hypoosmotic (hypo) conditions (b) and labeled with …
Figure 9
Impairment of caveolar invagination upon syndapin III KO in heart tissue has no consequences on cardiac integrity.
(a–c) Wheat germ agglutinin stainings of 8 µm cryosections of hearts of 20 weeks old syndapin III KO mice (b) show no signs of cellular damage or alteration of cross-sectional areas of cardiac …
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Figure 9—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 9 (i.e. of Figure 9c–e,h and o–t).
- https://doi.org/10.7554/eLife.29854.029
Figure 10
Syndapin III KO causes impairments in caveolar invagination in skeletal muscle.
(a,b) Double-immunogold labeled, freeze-fractured skeletal muscles of WT and syndapin III KO mice (syndapin III, 15 nm gold; cav3, 10 nm gold) show that caveolar invagination is impaired in tissues …
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Figure 10—source data 1
This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 10 (i.e. of Figure 10c,d,g-l and n-t).
- https://doi.org/10.7554/eLife.29854.031
Figure 11
Syndapin III KO leads to skeletal muscle phenotypes reminiscent of clinical symptoms found in patients with myopathies associated with CAV3 mutation.
(a–f) Histological examinations of cryosections of musculus gastrocnemius from >64 weeks old WT and syndapin III KO mice (three animals each). Whereas no clear signs of cellular damage or …
Videos
Video 1
Electron tomogram data set of the caveolar invagination shown in Figure 5f,g.
Cav3 and syndapin III are indicated by the electron-dense gold particles (anti-syndapin III, 15 nm; anti-cav3, 10 nm). Reconstruction and tomographic sectioning carried out with IMOD software. Bar, …
Video 2
Rotation of the 3D segmentation of a syndapin III and cav3-coated caveolar invagination shown in Figure 5h.
Syndapin III is indicated by the red spheres and cav3 is indicated by the yellow spheres. The Pt/C layer of freeze-fractured cardiomyocyte plasma membrane is indicated (green). Segmentation carried …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (mouse and rat) | PACSIN3/syndapin III | this paper | ||
| Strain, strain background (Mus musculus) | C57BL/6J | Jackson Labs (Bar Harbor, Maine) | IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | 129/SvJ mouse genomic λ library | Stratagene (San Diego, California) | ||
| Cell line (Mus musculus) | NIH3T3 | Cell Lines Services GmbH (Eppelheim, Germany) | CVCL_0594 | |
| Cell line (human) | HEK293 | Cell Lines Services GmbH | CVCL0045 | |
| Transfected construct (rat) | GFP-syndapin III F-BAR (aa 1–336; extended F-BAR) | this paper | ||
| Transfected construct (mouse) | Syndapin III aa1-70 peptide (plus five unrelated aa encoded by exon5/6 deletion-induced frame shift and multiple stop codons) | this paper | ||
| antibody | guinea pig anti-syndapin I, rabbit anti-syndapin II, rabbit anti-syndapin III, anti-GST | Koch et al. (2011), EMBO J 30:4955–4969., Qualmann et al. (1999), Mol Biol Cell 10:501–513. Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 | anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) | |
| Antibody | goat anti-GAPDH (polyclonal) | Santa Cruz (Dallas, Texas) | sc48167 AB_1563046 | 1:1000 |
| Antibody | mouse anti-cav3 (monoclonal ) | Santa Cruz | sc-5310, AB_626814 | 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) |
| Antibody | goat anti-cav3 (polyclonal) | Santa Cruz | sc-7665 AB_637945 | 1:500 (western blot), 1:50 (IHC) |
| Antibody | mouse anti-cav1 (monoclonal) | Santa Cruz | sc-53564, AB_628859 | 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) |
| Antibody | rabbit anti-cav1 (polyclonal) | Santa Cruz | sc-894 AB_2072042 | 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) |
| Antibody | rabbit anti-IRTK (polyclonal) | Santa Cruz | sc-710, AB_631106 | 1:1000 (western blot) |
| Antibody | mouse anti-CAVIN-1 (monoclonal) | BD Bioscience (Franklin Lakes, New Jersey) | 611258, AB_398788 | 1:50 (FRIL) |
| Antibody | mouse anti-PDI (monoclonal) | Stressgene (Farmingdale, New York) | ADI-SPA-891, AB_10615355 | 1:1000 (western blot) |
| Antibody | mouse anti-β-actin (monoclonal) | Sigma | A5441, AB_476744 | 1:5000 (western blot) |
| Antibody | mouse anti-β-tubulin (monoclonal) | Sigma | T4026, AB_477577 | 1:1500 (western blot) |
| Antibody | rabbit anti-CAVIN-1 (polyclonal) | Proteintech (Rosemont, Illinois) | 18892–1-AP,AB_10596795 | 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) |
| Antibody | rabbit anti-CAVIN-3 (polyclonal) | Proteintech | 16250–1-AP AB_2171894 | 1:1000 (western blot) |
| Antibody | goat anti-CAVIN-2 (polyclonal) | R & D Systems (Minneapolis, Minnesota) | AF5759AB_2269901 | 1:200 (western blot) |
| Antibody | rabbit anti-CAVIN-4 (polyclonal) | Sigma | HPA020987AB_1853080 | 1:400 (western blot) |
| Antibody | mouse anti-β-dystroglycan (monoclonal) | Leica Biosystems (Wetzlar, Germany) | NCL-b-DG,AB_442043 | 1:1000 (western blot); 1:200 (IHC) |
| Antibody | mouse anti-dystrophin (monoclonal) | Leica Biosystems | NCL-DYS1,AB_442080 | 1:200 (western blot); 1:50 (IHC) |
| Antibody | mouse anti-GM130 (monoclonal) | BD Biosciences | 610822AB_398141 | 1:1000 (western blot) |
| Antibody | mouse anti-β-catenin (monoclonal) | BD Biosciences | 610153, AB_397554 | 1:1000 (western blot) |
| Antibody | mouse anti-ERK1/2 (monoclonal) | Cell Signalling (Danvers, Massachusetts) | #9107AB_2235073 | 1:5000 (western blot) |
| Antibody | mouse anti-pERK1/2 (monoclonal) | Cell Signalling | #9106, AB_331768 | 1:1000 (western blot) |
| Antibody | mouse anti-GFP (monoclonal) | Clontech (Mountain View, California) | 632380,AB_10013427 | 1:8000 (western blot) |
| Antibody | rabbit anti-Cherry(polyclonal) | Abcam (Cambridge, UK) | ab167453AB_2571870 | 1:1000 (western blot) |
| Antibody | Alexa Fluor555-labeled wheat germ agglutinin (WGA) | Molecular Probes (Eugene, Oregon) | W32464 | 1:2000 (IF) |
| Antibody | Alexa Fluor488-labeled wheat germ agglutinin (WGA) | Molecular Probes | W11261 | 1:2000 (IF) |
| Antibody | Alexa Fluor488-labeled goat anti-guinea pig | Molecular Probes | AB_142018 | 1:1000 (IF) |
| Antibody | Alexa Fluor568-labeled goat anti-guinea pig | Molecular Probes | AB_141954 | 1:1000 (IF) |
| Antibody | Alexa Fluor488-labeled donkey anti-mouse | Molecular Probes | AB_141607 | 1:1000 (IF) |
| Antibody | Alexa Fluor568-labeled donkey anti-mouse | Molecular Probes | AB_2534013 | 1:1000 (IF) |
| Antibody | Alexa Fluor647-labeled goat anti-mouse | Molecular Probes | AB_141725 | 1:1000 (IF) |
| Antibody | Alexa Fluor488-labeled donkey anti-rabbit | Molecular Probes | AB_141708 | 1:1000 (IF) |
| Antibody | Alexa Fluor568-labeled goat anti-rabbit antibodies | Molecular Probes | AB_143011 | 1:1000 (IF) |
| Antibody | Alexa Fluor647-labeled goat anti-rabbit antibodies | Molecular Probes | AB_141775 | 1:1000 (IF) |
| Antibody | Alexa Fluor680-labeled donkey-anti-goat | Molecular Probes | AB_141494 | 1:10000 (western blot) |
| Antibody | Alexa Fluor680-labeled goat-anti-rabbit | Molecular Probes | AB_2535758 | 1:10000 (western blot) |
| Antibody | Alexa Fluor680-labeled goat-anti-mouse | Molecular Probes | AB_1965956 | 1:10000 (western blot) |
| Antibody | goat anti-rabbit-peroxidase | Dianova (Hamburg, Germany) | AB_2337945 | 1:10000 (western blot) |
| Antibody | goat anti-guinea pig-peroxidase | Dianova | AB_2337405 | 1:10000 (western blot) |
| Antibody | goat anti-mouse-peroxidase | Dianova | AB_2338523 | 1:10000 (western blot) |
| Antibody | DyLight800-conjugated goat anti-rabbit | Pierce/Thermo (Waltham, Massachusetts) | AB_2556775 | 1:10000 (western blot) |
| Antibody | DyLight800-conjugated goat anti-mouse | Pierce | AB_2556774 | 1:10000 (western blot) |
| Antibody | IRDy 800CW-conjugated donkey anti-goat | BioTrend (Köln, Germany) | AB_220102 | 1:10000 (western blot) |
| Antibody | anti-guinea pig antibodies coupled to IRDye680 | LI-COR Bioscience (Lincoln, Nebraska) | AB_10956079 | 1:10000 (western blot) |
| Antibody | anti-guinea pig antibodies coupled to IRDye800 | LI-COR Bioscience | AB_1850024 | 1:10000 (western blot) |
| Antibody | gold-labeled goat anti-rabbit (10 nm) | British Biocell International (Cardiff, UK) | AB_1769130 | 1:50 (FRIL) |
| Antibody | gold-labeled goat anti-rabbit (15 nm) | British Biocell International | AB_1769134 | 1:50 (FRIL) |
| Antibody | gold-labeled goat anti-mouse (10 nm) | British Biocell International | AB_1769156 | 1:50 (FRIL) |
| Antibody | gold-labeled goat anti-mouse (15 nm) | British Biocell International | AB_2715551 | 1:50 (FRIL) |
| Recombinant DNA reagent | rat syndapin III full length (aa 1–424) in pGEX-5X1 (plasmid) | Braun et al. (2005) Mol Biol Cell, 16:3642–3658. | ||
| Recombinant DNA reagent | rat syndapin III F-BAR (aa 1–336; extended F-BAR) in pGEX-5X1 (plasmid) | this paper | ||
| Recombinant DNA reagent | rat syndapin III ∆F-BAR (aa 336–424) in pGEX-5X1 (plasmid) | this paper | ||
| Recombinant DNA reagent | rat syndpin III SH3 (aa 366–424) in pGEX-5X1 (plasmid) | this paper | ||
| Recombinant DNA reagent | rat syndapin III ∆SH3 (aa 1–365) in pGEX-5X1 (plasmid) | this paper | ||
| Recombinant DNA reagent | GFP-rat syndapin III F-BAR (aa 1–336; extended F-BAR) in pEGFP-C2 (plasmid) | this paper | ||
| Recombinant DNA reagent | mouse syndapin III aa1-70 peptide (plus five unrelated aa encoded by exon5/6 deletion-induced frame shift and multiple stop codons) plasmids including pGEM-T, mCherry-pCMV-Tag2b, pEGFP-C2 and pGEX-5X-1 | this paper | ||
| Peptide, recombinant protein | rat GST-syndapin III full length (aa 1–424) and rat syndapin III full-length (untagged) | Braun et al. (2005) Mol Biol Cell 16:3642–3658 (for plasmid and (uncut) GST-fusion protein) | Untagged syndapin III was obtained from GST-syndapin III upon cleavage of the GST tag with 6 U precission protease/mg protein and overnight dialysis against 20 mM HEPES, 150 mM NaCl, 2 mM EDTA und 2.5 mM DTT pH 7.4 at 4°C. | |
| Peptide, recombinant protein | rat GST-syndapin III F-BAR (GST+Sdp III aa 1–336; extended F-BAR) | this paper | ||
| Peptide, recombinant protein | rat GST-syndapin III ∆F-BAR (GST+Sdp III aa 336–424) | this paper | ||
| Peptide, recombinant protein | rat GST-syndapin III SH3 (GST+SdpIII aa 366–424) | this paper | ||
| Peptide, recombinant protein | rat GST-syndapin III ∆SH3 (GST+Sdp III aa 1–365) | this paper | ||
| Commercial assay or kit | NucleoSpin Plasmid | Macherey-Nagel (Düren, Germany) | 740.588.250 | |
| Commercial assay or kit | NucleoBond Xtra Midi | Macherey-Nagel | 740.410.100 | |
| Commercial assay or kit | Rediprime II Random Prime Labelling System | GE Healthcare (Chicago, Illinois) | #RPN1633 | |
| Chemical compound, drug | α P32 dCTP | GE Healthcare (Chicago, Illinois) | ||
| Software, algorithm | AxioVision 4.8.2 | Zeiss (Oberkochen, Germany) | SCR_002677 | |
| Software, algorithm | ZEN 2012 | Zeiss | SCR_013672 | |
| Software, algorithm | Prism6 | GraphPad (La Jolla, California) | SCR_002798 | |
| Software, algorithm | imageJ | other | SCR_003070 | open source software |
| Software, algorithm | IMOD package | Kremer et al. (1996) Journal of Structural Biology, 116, 71–76 | SCR_003297 | open source software |
| Software, algorithm | IMARIS 8.4 | Bitplane (Zürich, Switzerland) | SCR_007370 | |
| Other | DAPI stain | Molecular Probes | (1:10000) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29854.033
