Visualizing sarcomere and cellular dynamics in skeletal muscle to improve cell therapies
- Translational Cardiology and Functional Genomics, Max Delbrück Center for Molecular Medicine, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site, Germany
- Charité Universitätsmedizin, Germany
- Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Germany
- Computational Biology, Max Delbrück Center for Molecular Medicine, Germany
- Heart Research Center Göttingen, Cellular Biophysics and Translational Cardiology Section, University Medical Center Göttingen, Germany
Figures
Figure 1 with 1 supplement
Generation and validation of a titin Z-disk knock-in with enhanced fluorescence.
(a) Targeting strategy to insert mCherry into titin’s Exon 28 (Z-disk). (b) mCherry is integrated outside the Z9 domain (red arrow) and GFP at the c-terminus (green arrow). (c) Sarcomeric structure …
-
Figure 1—source data 1
Measured signal intensity of mCherry and dsRed from 0 to 14 hr depicted in panel f.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Knock-in of mCherry at titin’s Z-disk.
(a) Representative images of extensor digitorum longus (EDL) sections of homozygous, heterozygous, and WT Ttn(Z)-mCherry mice with significantly different fluorescence intensities (b); one-way ANOVA …
-
Figure 1—figure supplement 1—source data 1
File with values for measured signal intensity at the Z-disk of mCherry in EDL muscle of homozygote (tg/tg), heterozygote (tg/wt), and wildtype (wt/wt) animals, corresponding to panel b; bodyweight (g), corresponding to panel c; heart weight and HW/BW ratio corresponding to panel d.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig1-figsupp1-data1-v1.tif
-
Figure 1—figure supplement 1—source data 2
Original titin gel for panel e indicating the relevant bands of titin (N2A, T2) and MHC of wildtype (+/+), heterozygote (+/m), and homozygote (m/m) mCherry skeletal muscle.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig1-figsupp1-data2-v1.tif
-
Figure 1—figure supplement 1—source data 3
Original file for titin gel displayed in panel e.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig1-figsupp1-data3-v1.xlsx
Figure 2 with 1 supplement
Titin mobility in myotubes at Z-disk and M-band.
(a) Representative recovery of the sarcomeric titin signal within 14 hr. Intensity profiles of the bleached regions (white rectangle). Scale bar 10 µm. (b) The mCherry-labeled Z-disk titin (mCh-Z) …
-
Figure 2—source data 1
Measured intensity of mCherry-Z and GFP-M intensity at different timepoints from prebleached to 8 hr post bleaching, corresponding to panel a.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig2-data1-v1.xlsx
-
Figure 2—source data 2
Values of normalized intensity of mCherry-Z and GFP-M signal from 0 to 840 min of recovery after bleaching, corresponding to panel b.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig2-data2-v1.xlsx
-
Figure 2—source data 3
Values of GFP-M and mCh-Z of mobile fraction and mobile fraction (in %) corresponding to panel c, exchange half-life (min) corresponding to panel d, and fast phase (in %) corresponding to panel e.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig2-data3-v1.xlsx
-
Figure 2—source data 4
Values of normalized fluorescence intensity of integrated (GFP-M and mCh-Z) and between (GFP-Z and mCh-M) from 0 to 840 min of recovery after bleaching, corresponding to panel f.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig2-data4-v1.xlsx
Figure 2—figure supplement 1
Titin kinetics in double-heterozygous myotubes.
(a) Representative images of FRAP in fixed cells (n = 4) reveal no recovery of titin signal over 14 hr at Z-disk and M-band. Scale bar 10 µm. (b) No difference in the reactivation of the fluorophore …
-
Figure 2—figure supplement 1—source data 1
File with values of normalized intensities from 0 to 8 hr of GFP-M and mCherry-Z signal in fixed cells, corresponding to panel b; normalized intensity of mCherry-Z signal in live and fixed cells, corresponding to panel c; normalized intensity of GFP-M signal in live and fixed cells, corresponding to panel d; normalized intensity of mCherry-M and GFP-Z signal, corresponding to panel e; values of mobile fraction (in %) corresponding to panel f; exchange half-life (min) corresponding to panel g; two-phase recovery (in%) corresponding to panel h.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
After cell fusion, titin is distributed throughout the myotube.
Satellite cells were isolated from homozygous titin(Z)-mCherry and titin(M)-eGFP mice. After co-cultivation and differentiation to myotubes, cells were fixed at different stages of cell fusion, from …
Figure 3—figure supplement 1
Titin distribution after cell fusion.
Satellite cells isolated from Ttn(Z)-mCherry and Ttn(M)-eGFP animals were co-cultured, fixed at different fusion states (a) early fusion, (b) early distribution, and (c) late distribution and …
Figure 4 with 1 supplement
Cell fusion and redistribution of red and green titin in skeletal muscle cells.
(a) Live imaging of cell fusion of homozygous Ttn(Z)-mCherry and Ttn(M)-eGFP myogenic cells (two frames per hour). Arrows indicate the initiation of cell fusion between a small Ttn-eGFP myocyte and …
-
Figure 4—source data 1
Area occupied by fluorophore (red and green) from 0 up to 4 or 8 h of myotube maturation, corresponding to panel b and immature myotubes corresponding to panel c.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig4-data1-v1.xlsx
-
Figure 4—source data 2
Values of mature and immature cells from 0 to 8 hr of relative area (%), corresponding to panel d, and absolute mixed area, corresponding to panel e.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig4-data2-v1.xlsx
Figure 4—video 1
Fusion movie: time lapse of cell fusion.
Cell fusion and redistribution of red (mCherry) and green (eGFP) titin in skeletal muscle cells from 0 to 8 hr. The same scale applies as in the originals in Figure 4 with the appropriate scale-bar.
Figure 5 with 1 supplement
Distribution of titin mRNA in skeletal muscle cells undergoing cell fusion determined by smFISH detecting mCherry and GFP coding region.
(a) Model of titin mRNA and protein synthesis and localization. (b) Representative image of a fusing myotube, where the distribution of titin mRNA has just started. While we find both titin-mCh and …
-
Figure 5—source data 1
File with values for mRNA and protein of cell fusion events, corresponding to panel b.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Titin mRNA localization after cell fusion.
Satellite cells were isolated from homozygous Ttn(Z)-mCherry and Ttn(M)-eGFP mice, co-cultured, differentiated to myotubes and fixed after fusion. The cartoon indicates the position of nuclei …
-
Figure 5—figure supplement 1—source data 1
File with values for RNA and protein overlap correlation of fusion events, corresponding to panel b, and signal localization/overlap of RNA protein of mCherry and GFP in fusion events corresponding to panel c.
- https://cdn.elifesciences.org/articles/95597/elife-95597-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
Titin distribution during regeneration.
(a) Tibialis anterior (TA) muscle of Ttn(Z)-mCherry mice was injured by injection of cardiotoxin (CTX) followed by transplantation of Ttn(M)-eGFP myoblasts on the following day (n = 3). Controls …
Figure 6—figure supplement 1
Titin mobility and integration upon in vivo regeneration and cell transplantation.
(a) Transversal sections of tibialis anterior (TA) muscles of the control group (cardiotoxin [CTX] only, n = 1) and the noninjected contralateral muscle (b). (c) Longitudinal sections of TA muscles …
