A new directionality tool for assessing microtubule pattern alterations

Abstract

The cytoskeleton (microtubules, actin and intermediate filaments) has a cell type-specific spatial organization that is essential and reflects cell health. We are interested in understanding how changes in the organization of microtubules contribute to muscle diseases such as Duchenne muscular dystrophy (DMD). The grid-like immunofluorescence microtubule pattern of fast-twitch muscle fibers lends itself well to visual assessment. The more complicated pattern of other fibers does not. Furthermore, visual assessment is not quantitative. Therefore we have developed a robust software program for detecting and quantitating microtubule directionality. Such a tool was necessary because existing methods focus mainly on local image features and are not well suited for microtubules. Our tool, TeDT, is based on the Haralick texture method and takes into account both local and global features with more weight on the latter. The results are expressed in a graphic form responsive to subtle variations in microtubule distribution, while a numerical score allows quantitation of directionality. Furthermore, the results are not affected by imaging conditions or post-imaging procedures. TeDT successfully assesses test images and microtubules in fast-twitch fibers of wild-type and mdx mice (a model for DMD); TeDT also identifies and quantitates microtubule directionality in slow-twitch fibers, in the fibers of young animals, and in other mouse models which could not be assessed visually. TeDT might also contribute to directionality assessments of other cytoskeletal components.

Microtubule binding distinguishes dystrophin from utrophin

“…Moreover, we quantitatively analyzed microtubule lattice images using a recently developed program (44) for analyzing microtubule directionality (TeDT). In agreement with previously published data (19,20,35,44,45), the directionality histograms ( Fig. S2B) resulting from TeDT analysis revealed a significant decrease in the presence of transversely oriented microtubules (centered around 90°) in mdx and Fiona-mdx mice that were present in WT, Dys-TG-mdx, and mini-Dys-TG-mdx mice (P < 0.05).…”

“…In the absence of dystrophin, the subsarcolemmal microtubule lattice becomes disorganized (19,20,35,44), and this disorganization contributes to the dystrophic phenotype of the mdx mouse (43,58). Like dystrophin, utrophin couples membranebound dystrophin-associated proteins with costameric actin filaments, suggesting that up-regulation of utrophin could compensate for the loss of dystrophin (28)(29)(30)(31).…”

Dystrobrevin increases dystrophin's binding to the dystrophin–glycoprotein complex and provides protection during cardiac stress

“…Microtubule organization was analyzed using texture detection technique (TeDT) [32]. Confocal images of cardiomyocyte α-tubulin immunofluorescence were analyzed.…”

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms

“…Microtubule directionality measurements were made using texture detection (TedT) software (44). Confocal images representing either a single optical section or a maximum projection of 1-3 sections containing the subsarcolemmal microtubule cytoskeleton were captured.…”

“…To quantitate the impact of nNOS deficiency on microtubules, subsarcolemmal microtubule lattice directionality was analyzed using custom-built texture detection (TedT) software ( Fig. 5C-E) (44). Loss of nNOS quantitatively disrupted subsarcolemmal microtubule organization (Fig.…”