Journal: Molecular Biology of the Cell

Article Title: Dynamics of triadin, a muscle-specific triad protein, within sarcoplasmic reticulum subdomains

doi: 10.1091/mbc.E19-07-0399

Figure Lengend Snippet: T95 diffuses in SR membranes of cultured myotubes. (A) Schematic representation of an iteration experiment: a myotube expressing a PAGFP-tagged protein of the triads is repeatedly photoactivated in a specific ROI with a 405-nm laser, revealing more fluorescence of the proteins inside that region. The dynamic behavior of the activated proteins can be followed and recorded. One frame recorded every 2 s and one activation every four frames. Frames preactivation and after 1, 3, 6, and 10 activations (pink lines) are shown on B and C. (B) Iterative photoactivation of DIF3 (top panels) and DIF9 (bottom panels) myotubes coexpressing Sec61β-PAGFP (green) and DsRedKDEL(magenta). Shown are one image preactivation and four images postactivation (2, 20, 46, and 87 s). SUM images are sum intensity projections of all postactivation frames (87 s). Scale bars = 5 μm. (C) Iterative photoactivation of DIF3 (top panels) and DIF9 (bottom panels) myotubes coexpressing T95-PAGFP (green) and DsRedKDEL (magenta). Shown are one image preactivation and four images postactivation (2, 20, 46, and 87 s). SUM images are sum intensity projections of all postactivation frames (87 s). T95 clusters appear at distant sites from the activation ROI (white arrowheads) and organize on each side of the DsRedKDEL striations at DIF9. Scale bars = 5 μm. (D) Speed of the fluorescence wave of Sec61β-PAGFP and T95-PAGFP at DIF3 and DIF9. Values are represented as means ± c.i. obtained from linear regressions as indicated on Supplemental Figures S4 and S5; * p = 0.01232 using model comparison of linear regressions, as performed by GraphPad Prism 6.0 (see Materials and Methods ); n = 12 and 11 cells for Sec61β at DIF3 and DIF9, respectively, from three independent experiments; n = 10 and 7 cells for T95 at DIF3 and DIF9, respectively, from three independent experiments. (E) Fluorescence decay rates for Sec61β-PAGFP and T95-PAGFP at DIF3 and DIF9. Values are represented as means ± c.i. obtained from exponential decay modelization as indicated on Supplemental Figures S4 and S5; **** p < 0.0001 using model comparison of exponential decays, as performed by GraphPad Prism 6.0 (see Materials and Methods ); n = 12 and 11 cells for Sec61β at DIF3 and DIF9, respectively, from three independent experiments; n = 10 and 7 cells for T95 at DIF3 and DIF9, respectively, from three independent experiments.

Article Snippet: A nonlinear fit with an unconstrained model “One site binding–Specific binding” from GraphPad Prism 6.0 was applied and indicated Bmax values for each experiment.

Techniques: Cell Culture, Expressing, Fluorescence, Activation Assay