Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte

Figures

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  Fig. 1. Dynamic microtubules are stabilized at the Z-disk and preferentially interact with desmin intermediate filaments. a Schematic of the transition states of microtubule dynamics. b Representative kymograph from control cardiomyocytes transduced with AdV-EB3-GFP; black arrows denote Z-disk and colored arrows denote transition events. c Quantification of initiation, rescue, pause, and catastrophe events On and Off the Z-disk in control cardiomyocytes (N = 19 cells, n = 228 events). The bar represents mean ± 1SEM; statistical significance determined with Two Sample Kolmogorov–Smirnov Test. d Representative EM images from transverse sections of isolated cardiomyocytes. Microtubules are denoted by white arrows. In the right-hand panel, the area between the myofibrils is filled by membranous and filamentous structures consistent with intermediate filaments, which are bisected by microtubules. e Representative immunofluorescent images and f quantification of a-actinin-EB1 or desmin-EB1 PLA interactions in control cardiomyocytes (N = 3 rats, n = 10 cells per rat). The box represents the 25th and 75th percentiles ± 1SD, bolded line represents the mean; statistical significance was determined…

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  Fig. 2. Desmin stabilizes dynamic microtubules at the Z-disk. a Overview of the cell fractionation assay adapted from Fassett et al. [15] that allows for the separation of free tubulin and polymerized microtubules within the dynamic tubulin pool. b Representative western blot and c quantification of α-tubulin in free and dynamic microtubule fractions (top) or of total dTyr-tubulin, α-tubulin, and acetylated tubulin in the whole-cell lysate (bottom) from control (Scram) or Desmin knock-down (Des KD) cardiomyocytes (N = 3 rats, n = 5 WB technical lanes for dtyr and 6 for acetyl and tubulin fractions). d Representative EB3-GFP kymograph from Scram (top) or Des KD (bottom) cardiomyocytes. e Quantification of catastrophe, pause, and rescue event frequencies and f event locations in Scram or Des KD cardiomyocytes (N = cells, n = events). The bar represents mean ± 1SEM; statistical significance for C was determined with two-sample Student’s T test, and for E and F was determined with two-sample Kolmogorov–Smirnov test

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  Fig. 3. TTL reduces microtubule stability through its tyrosinase activity. a Representative western blot (top) and quantification (bottom) of α-tubulin and detyrosinated (dTyr) tubulin in free and cold-sensitive dynamic microtubule fractions from adult rat cardiomyocytes treated with null, TTL, or TTL-E331Q adenoviruses; detyrosinated tubulin values are normalized to α-tubulin in cold-sensitive fraction (N = 4 rats, n = 8 WB technical lanes). b Representative western blot (top) and quantification (bottom) of α-tubulin and acetylated tubulin in whole-cell lysate from null, TTL, or E331Q expressing cardiomyocytes (N = 3 rats, n = 6 WB technical lanes). c Validation of HDAC6 and αTAT1 constructs and Tubastatin A (TubA) treatment. Representative western blot (top) and quantification (bottom) of a-tubulin and acetylated tubulin in whole-cell lysate from adult rat cardiomyocytes treated with null, HDAC6, or αTAT1 adenoviruses, or DMSO or 1 mM TubA treatment overnight (N = 3 rats, n = 6 WB technical lanes). d Representative western blot (top) and quantification (bottom) of α-tubulin and acetylated tubulin, in free and polymerized dynamic fractions. Lysates from cardiomyocytes were infected with…

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  Fig. 4. Tyrosinated microtubules are more dynamic. a Representative kymographs from cardiomyocytes treated with EB3-GFP plus null, TTL, or E331Q adenoviruses. b Quantification of catastrophe and pause event frequencies and c event locations in cardiomyocytes treated with EB3-GFP plus null, TTL, or E331Q adenoviruses (N = cells, n = events). d Gross measurements of microtubule dynamics. (Left) Tortuosity, the distance a microtubule grows divided by its displacement, & (right) number of catastrophes in relation to the number of successful Z-disk crossing in cardiomyocytes treated with EB3-GFP plus null, TTL, or E331Q adenoviruses. e Z-disc bias score (log2 transformation of the ratio of events that occurred On vs. Off the Z-disk) for all experimental conditions. The bar represents mean ± 1SEM; statistical significance was determined with Kruskal–Wallis ANOVA with post hoc test

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  Fig. 5. Tyrosination promotes EB1 and CLIP170 interactions on cardiomyocyte microtubules. a Representative AiryScan Joint Deconvoluted immunofluorescent images of EB1-CLIP170 PLA interactions in adult rat cardiomyocytes treated with null, TTL, or TTL-E331Q adenoviruses. b Representative western blot (top) and quantification (bottom) of EB1 in whole-cell lysate from adult rat cardiomyocytes treated with null, TTL, or E331Q adenoviruses for 48 h (N = 3 rat, n = 3 WB technical lanes). c Representative immunofluorescent images (left) and quantification (right) of CLIP170 in adult rat cardiomyocytes treated with null, TTL, or E331Q adenoviruses for 48 h (N = 3 rats, n = 10 cells per rat). d Quantification of EB1-CLIP170 PLA interactions in adult rat cardiomyocytes treated with null, TTL, or TTL-E331Q adenoviruses (N = 3 rats, n = 10 cells per rat). The bar represents mean ± 1SEM, and the middle line in the box graph represents mean ± 1SEM; statistical significance for (b) was determined with one-way ANOVA with post hoc test, and for (c) and (d) was determined with Kruskal–Wallis ANOVA with post hoc test