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In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans.

Zhan H, Stanciauskas R, Stigloher C, Dizon KK, Jospin M, Bessereau JL, Pinaud F - Nat Commun (2014)

Bottom Line: Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans).In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms.Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles.

View Article: PubMed Central - PubMed

Affiliation: University Claude Bernard Lyon 1, CGphiMC UMR CNRS 5534, Villeurbanne 69622, France.

ABSTRACT
Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals.

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Diffusion analysis of individual VDCC at the sarcolemma of normal C. elegans worms expressing UNC-36-split-GFP.(a) Distribution of diffusion coefficients for 10,481 VDCC trajectories (39 muscle cells, six worms) determined from individual MSD analysis and showing that two populations of slow and fast diffusing channels co-exist at the sarcolemma of resting muscles. (b) Example of individual VDCC trajectories and the evolution of their MSD over time. VDCC undergo either confined or short-scale free diffusion as determined using 2D diffusion models that best fit the MSD (red). Scale bars: 200 nm. (c) Ensemble probability distribution of the squared displacement (PDSD) analysis of VDCC diffusive behaviours (10,481 VDCC, 39 muscle cells, six worms) in resting muscles. (d) Ensemble PDSD analysis of VDCC diffusive behaviours (36,325 VDCC, 62 muscle cells, 18 worms) in muscles under sustained contraction with levamisole. (e) Diffusion coefficients (±s.d.) for both fast and slow VDCC populations in resting and contracted muscles (F-test, *P<0.05, ***P<0.001). (f) VDCC nanodomain nearest-neighbour distances at the sarcolemma of resting and contracted muscles. The central squares and bars represent the mean of the distribution and its median, respectively. The box length represents the interquartile range and the error bars the s.d. of the mean (Wilcoxon sign-rank test, **P<0.01 compared with resting muscles).
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f5: Diffusion analysis of individual VDCC at the sarcolemma of normal C. elegans worms expressing UNC-36-split-GFP.(a) Distribution of diffusion coefficients for 10,481 VDCC trajectories (39 muscle cells, six worms) determined from individual MSD analysis and showing that two populations of slow and fast diffusing channels co-exist at the sarcolemma of resting muscles. (b) Example of individual VDCC trajectories and the evolution of their MSD over time. VDCC undergo either confined or short-scale free diffusion as determined using 2D diffusion models that best fit the MSD (red). Scale bars: 200 nm. (c) Ensemble probability distribution of the squared displacement (PDSD) analysis of VDCC diffusive behaviours (10,481 VDCC, 39 muscle cells, six worms) in resting muscles. (d) Ensemble PDSD analysis of VDCC diffusive behaviours (36,325 VDCC, 62 muscle cells, 18 worms) in muscles under sustained contraction with levamisole. (e) Diffusion coefficients (±s.d.) for both fast and slow VDCC populations in resting and contracted muscles (F-test, *P<0.05, ***P<0.001). (f) VDCC nanodomain nearest-neighbour distances at the sarcolemma of resting and contracted muscles. The central squares and bars represent the mean of the distribution and its median, respectively. The box length represents the interquartile range and the error bars the s.d. of the mean (Wilcoxon sign-rank test, **P<0.01 compared with resting muscles).

Mentions: In resting muscles, two populations of slow and fast diffusing VDCC undergoing confined or short-scale free diffusion were identified at the sarcolemma (10,481 trajectories, 39 muscle cells, six worms, Fig. 5a,b). Ensemble PDSD analyses revealed that most channels (81%) diffused slowly (Dvdcc−slow=1.22 × 10−3±1.2 × 10−4 μm2 s−1) in membrane nanodomains 82±2 nm in diameter (Fig. 5c,e and Supplementary Table 1). The second VDCC population (19%) diffused 20-fold faster (Dvdcc−fast=2.22 × 10−2±1 × 10−3 μm2 s−1) and underwent free diffusion for a length scale of 230±8 nm (Fig. 5c,e and Supplementary Table 1), after which membrane obstacles induced a subdiffusive behaviour. Both populations were independently distributed at the sarcolemma (Supplementary Fig. 5), did not change diffusion and diffused more slowly than CD4-split-GFP, indicating that we imaged two behaviours of stably assembled VDCC rather than a dynamic association of the glycosylphosphatidylinositol-anchored UNC-36 (ref. 23) with the channels’ α1 subunit.


In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans.

Zhan H, Stanciauskas R, Stigloher C, Dizon KK, Jospin M, Bessereau JL, Pinaud F - Nat Commun (2014)

Diffusion analysis of individual VDCC at the sarcolemma of normal C. elegans worms expressing UNC-36-split-GFP.(a) Distribution of diffusion coefficients for 10,481 VDCC trajectories (39 muscle cells, six worms) determined from individual MSD analysis and showing that two populations of slow and fast diffusing channels co-exist at the sarcolemma of resting muscles. (b) Example of individual VDCC trajectories and the evolution of their MSD over time. VDCC undergo either confined or short-scale free diffusion as determined using 2D diffusion models that best fit the MSD (red). Scale bars: 200 nm. (c) Ensemble probability distribution of the squared displacement (PDSD) analysis of VDCC diffusive behaviours (10,481 VDCC, 39 muscle cells, six worms) in resting muscles. (d) Ensemble PDSD analysis of VDCC diffusive behaviours (36,325 VDCC, 62 muscle cells, 18 worms) in muscles under sustained contraction with levamisole. (e) Diffusion coefficients (±s.d.) for both fast and slow VDCC populations in resting and contracted muscles (F-test, *P<0.05, ***P<0.001). (f) VDCC nanodomain nearest-neighbour distances at the sarcolemma of resting and contracted muscles. The central squares and bars represent the mean of the distribution and its median, respectively. The box length represents the interquartile range and the error bars the s.d. of the mean (Wilcoxon sign-rank test, **P<0.01 compared with resting muscles).
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Related In: Results  -  Collection

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Show All Figures
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f5: Diffusion analysis of individual VDCC at the sarcolemma of normal C. elegans worms expressing UNC-36-split-GFP.(a) Distribution of diffusion coefficients for 10,481 VDCC trajectories (39 muscle cells, six worms) determined from individual MSD analysis and showing that two populations of slow and fast diffusing channels co-exist at the sarcolemma of resting muscles. (b) Example of individual VDCC trajectories and the evolution of their MSD over time. VDCC undergo either confined or short-scale free diffusion as determined using 2D diffusion models that best fit the MSD (red). Scale bars: 200 nm. (c) Ensemble probability distribution of the squared displacement (PDSD) analysis of VDCC diffusive behaviours (10,481 VDCC, 39 muscle cells, six worms) in resting muscles. (d) Ensemble PDSD analysis of VDCC diffusive behaviours (36,325 VDCC, 62 muscle cells, 18 worms) in muscles under sustained contraction with levamisole. (e) Diffusion coefficients (±s.d.) for both fast and slow VDCC populations in resting and contracted muscles (F-test, *P<0.05, ***P<0.001). (f) VDCC nanodomain nearest-neighbour distances at the sarcolemma of resting and contracted muscles. The central squares and bars represent the mean of the distribution and its median, respectively. The box length represents the interquartile range and the error bars the s.d. of the mean (Wilcoxon sign-rank test, **P<0.01 compared with resting muscles).
Mentions: In resting muscles, two populations of slow and fast diffusing VDCC undergoing confined or short-scale free diffusion were identified at the sarcolemma (10,481 trajectories, 39 muscle cells, six worms, Fig. 5a,b). Ensemble PDSD analyses revealed that most channels (81%) diffused slowly (Dvdcc−slow=1.22 × 10−3±1.2 × 10−4 μm2 s−1) in membrane nanodomains 82±2 nm in diameter (Fig. 5c,e and Supplementary Table 1). The second VDCC population (19%) diffused 20-fold faster (Dvdcc−fast=2.22 × 10−2±1 × 10−3 μm2 s−1) and underwent free diffusion for a length scale of 230±8 nm (Fig. 5c,e and Supplementary Table 1), after which membrane obstacles induced a subdiffusive behaviour. Both populations were independently distributed at the sarcolemma (Supplementary Fig. 5), did not change diffusion and diffused more slowly than CD4-split-GFP, indicating that we imaged two behaviours of stably assembled VDCC rather than a dynamic association of the glycosylphosphatidylinositol-anchored UNC-36 (ref. 23) with the channels’ α1 subunit.

Bottom Line: Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans).In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms.Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles.

View Article: PubMed Central - PubMed

Affiliation: University Claude Bernard Lyon 1, CGphiMC UMR CNRS 5534, Villeurbanne 69622, France.

ABSTRACT
Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals.

Show MeSH
Related in: MedlinePlus