During 2011-2012, we have gained a good understanding of the organization of microtubules in skeletal muscle. Our first goal was to determine whether muscle microtubules are dynamic, i.e. constantly grow, move, and shrink. To reach this goal, we have injected cDNA encoding microtubule markers into the mouse footpad. These injections, followed by electroporation, introduce the cDNA into the Flexor Digitorum Brevis (FDB) muscle. Six days after the cDNA injection, 30 to 80% of FDB fibers (i.e. the large multinucleated cells that make skeletal muscle) express the cDNA and are thus fluorescent. We can then prepare single muscle fibers by enzymatic digestion of the muscle and plate them on a support suitable for confocal microscopy. Our efficiency of plating has improved considerably and we now routinely obtain coverslips with more than 50 fibers which can be observed live, possibly after treatment with various drugs such as the microtubule-depolymerizing drug nocodazole. We have used three cDNA constructs which we previously verified to serve as faithful markers of the endogenous proteins. Each one encodes a protein tagged by green fluorescent protein (GFP). One construct, tubulin-GFP, encodes the protein that forms the core of microtubules. The second construct, EB3-GFP, encodes a protein of the microtubule tip that leads the way in motile microtubules. The third construct, Gal-TFase-GFP, encodes the Golgi complex protein Galactosyl transferase. EB3-GFP is found all over the fibers and is very motile. It travels mostly in 2 orthogonal directions, along the fiber axis and along the costameres. Nucleation points can be observed constantly, even at steady state. In contrast, much of GFP-tubulin is immobile and appears to form a stable network. In double transfections, with EB3 labeled with GFP and tubulin with a red protein (mCherry), each keeps the same behavior and EB3 travels along tubulin tracks. We believe this dichotomy to reveal the double character of the microtubule network in muscle: there is a stable network which may contribute to reinforce the fiber against mechanical stress, and a motile component, responsible for the transport and organelle functions. What if the isolation procedure of the fibers affected microtubule dynamics? To rule this out, we have examined microtubule dynamics in the whole muscle, in the live anesthetized animal, by intravital microscopy. We were able to visualize microtubules at high magnification and record images from several muslce fibers within one frame. These experiments produced similar results, therefore validating the use of the single fibers. EB3 shows a speed of 8 micrometers/ min in fibers and 5 micrometers/ min intravitally, with no significant difference. This is important since dissociated fibers are denervated and we know that denervation causes profound changes in microtubule organization within a few days. This and other results suggest that the microtubules form tracks or bundles of a few microtubules. We have used super-resolution microscopy to demonstrate indeed that single but thick microtubules in "diffraction- limited" microscopy are resolved in a few microtubules in super-resolution, confirming our predictions. By expressing and following Golgi complex markers, we have shown that the Golgi elements of muscle are immobile, in agreement with our hypothesis that they are maintained near special domains of the endoplasmic/ sarcoplasmic reticulum (ER/ SR) by their association with ER exit sites. The association of Golgi elements with microtubules is therefore not due to the transport of Golgi elements along microtubules. Since results obtained with fixed fibers indicate that microtubules in muscle fibers originate from the Golgi elements , this explains the association of dynamic microtubules with immoblie Golgi elements. Is this a phenomenon similar to the nucleation of microtubules from the Golgi complex of some non-muscle cells, or muscle-specific? To answer this question we have knocked down the Golgi complex protein GM130. This protein is an attractive target because 1) it has been proposed to play a role in nucleation of microtubules from the Golgi of non-muscle cells;2) it is rather abundant in muscle and we can detect it by immunofluorescence or immunoblotting. Muscle fibers depleted of GM130 by shRNA injection show differences in the organization of other Golgi complex proteins but microtubule nucleation and steady-state microtubule organization appear normal, suggesting that the nucleation of microtubules from Golgi elements in muscle follows its own mechanism. Our observation of gamma-tubulin associated with Golgi elements suggests that small and incomplete centrosomes are nearby, possibly associated with the ER exit sites. These results are the object of a manuscript in preparation (Oddoux et al.). In summary, we have considerably advanced in understanding microtubule organization in sleletal muscle and believe that this work will allow to better understand the microtubule changes occurring in DMD. Our interest in applying microscopy techniques for the detection and analysis of muscle defects in myopathies, coupled with our collaborations with Dr. Raben on Pompe disease and with Dr. Ploug on metabolic defects, has led us into developing new software for the quantitation of muscle anomalies. Various forms of microscopy reveal muscle defects such as changes in the periodicity of the contractile proteins, inclusion of non-contractile bodies etc. It is however very difficult to quantitate these defects and therefore to compare the severity of disease between different biopsies or the evolution of muscle health following treatments such as enzyme replacement therapy in the case of Pompe disease. The new software based on the Matlab platform uses different filters and the concept of image "texture" to quantitate muscle defects without prior assumption of the type of defects. We have shown that it properly ranks muscle fibers from human and mouse (Liu, Raben &Ralston, submitted). The software, which we call MARS (muscle assessment and ranking scores), will be available for licensing through the NIH website. A new algorithm has been developed recently to evaluate and quantitate directionality of a line pattern such as an image of microtubules. Muscle microtubules form a complex network and visual assessment can be difficult. The new algorithm is much more sensitive than our eyes and will be helpful for unbiased observation of changes in microtubule patterns. For example, when we compared microtubules of the mdx mouse (which lacks Dystrophin and serves as a model for DMD) to those of control mice, it was easy to decide by visual examination that in fast fibers the microtubule pattern is different. But in slow muscles it was not possible to make such a call because slow fibers have a dense, intricate network of microtubules. Nevertheless, a re-examination of the images with the new software allowed us to see a significant difference in the orientation of the microtubules in mdx soleus compared to wild- type soleus. Thus we have developed image analysis tools that will allow us to determine microtubule and muscle damage in mouse and human biopsies in an unbiased way.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Milgroom, Andrew; Ralston, Evelyn (2016) Clearing skeletal muscle with CLARITY for light microscopy imaging. Cell Biol Int 40:478-83
Liu, Wenhua; Ralston, Evelyn (2014) A new directionality tool for assessing microtubule pattern alterations. Cytoskeleton (Hoboken) 71:230-40
Feeney, Erin J; Austin, Stephanie; Chien, Yin-Hsiu et al. (2014) The value of muscle biopsies in Pompe disease: identifying lipofuscin inclusions in juvenile- and adult-onset patients. Acta Neuropathol Commun 2:2
Suzuki, Ryo; Leach, Sarah; Liu, Wenhua et al. (2014) Molecular editing of cellular responses by the high-affinity receptor for IgE. Science 343:1021-5
Liu, Wenhua; Raben, Nina; Ralston, Evelyn (2013) Quantitative evaluation of skeletal muscle defects in second harmonic generation images. J Biomed Opt 18:26005
Oddoux, Sarah; Zaal, Kristien J; Tate, Victoria et al. (2013) Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements. J Cell Biol 203:205-13
Feng, Xin; Zhang, Tan; Ralston, Evelyn et al. (2012) Differences in neuromuscular junctions of laryngeal and limb muscles in rats. Laryngoscope 122:1093-8
Raben, Nina; Wong, Amanda; Ralston, Evelyn et al. (2012) Autophagy and mitochondria in Pompe disease: nothing is so new as what has long been forgotten. Am J Med Genet C Semin Med Genet 160C:13-21
Zaal, Kristien J M; Reid, Ericka; Mousavi, Kambiz et al. (2011) Who needs microtubules? Myogenic reorganization of MTOC, Golgi complex and ER exit sites persists despite lack of normal microtubule tracks. PLoS One 6:e29057
Raben, Nina; Schreiner, Cynthia; Baum, Rebecca et al. (2010) Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease. Autophagy 6:1078-89

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