To advance towards objective 1, we have been working with the flexor digitorum brevis muscle of the mouse, from which collagenase-dissociated fibers can be reliably and reproducibly prepared, treated with drugs, stained with antibodies and observed in immunofluorescence. In experimental conditions of microtubule growth, we observe typical """"""""asters"""""""" of short microtubules. In most cell types such asters originate from the centrosome, although a population of specialized microtubules appears to be centered on the Golgi complex. During muscle development the centrosomes are disassembled. In muscle fibers, the asters we observe are centered on Golgi complex elements. We have shown that the Golgi complex elements are part of the nucleating mechanism by demonstrating that microtubule asters are severely reduced in number and in size in fibers treated with Brefeldin A, a drug that collapses the Golgi complex into the endoplasmic reticulum. We are now investigating the role of specific Golgi complex proteins in this nucleation. The first candidate is the Golgi complex protein GM130, a central Golgi matrix protein. In preliminary experiments we have tested shRNAs and succeded in knocking GM130 down in C2 muscle cell cultures. We have also optimized techniques for injecting cDNAs into mouse FDB muscle. We should therefore be able to knock down GM130 in FDB muscle fibers and assess the effects on microtubule nucleation and Golgi distribution in vivo. In order to observe microtubule dynamics in muscle in vivo (objective 2), we have constructed and tested DNA plasmids encoding fragments or the whole of two microtubule-associated proteins: MAP4, and the ensconsin microtubule-binding domain (EMTB),linked to fluorescent proteins. This preliminary work is necessary because the most obvious microtubule marker, tubulin-GFP, does not fully incorporate in existing microtubules, and does not track microtubule dynamics faithfully in C2 cultures. At low degrees of overexpression, MAP4 and EMTB show dynamic behavior in myoblasts and myotubes and, satisfy all our criteria for usefulness for in vivo experiments. The next stage will be to introduce them into muscle fibers in vivo. To evaluate the mechanism and consequences of the microtubule defects in disease models (objective 3) we have observed microtubule nucleation in mdx and Pompe disease mice and have found profound defects which we are currently analyzing. In the past year we have finalized a study showing that the initial reorganization of centrosomal proteins during myogenesis in muscle cell cultures drives all the other steps and takes place independently of microtubule reorganization. We have also continued our collaboration with Drs. Plotz and Raben on the study of Pompe Disease. Our results on microtubule nucleation in muscle in vivo are turning around the way we understand microtubule-organelle relationships. They will help us understand muscle phenotype in several diseases as well as the difficulties encountered by enzyme replacement therapy in Pompe disease.

Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2010
Total Cost
$393,487
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
City
State
Country
Zip Code
Iglesias-Bartolome, Ramiro; Uchiyama, Akihiko; Molinolo, Alfredo A et al. (2018) Transcriptional signature primes human oral mucosa for rapid wound healing. Sci Transl Med 10:
Duverger, Olivier; Carlson, Jenna C; Karacz, Chelsea M et al. (2018) Genetic variants in pachyonychia congenita-associated keratins increase susceptibility to tooth decay. PLoS Genet 14:e1007168
Tsai, Pei-Fang; Dell'Orso, Stefania; Rodriguez, Joseph et al. (2018) A Muscle-Specific Enhancer RNA Mediates Cohesin Recruitment and Regulates Transcription In trans. Mol Cell 71:129-141.e8
Lim, Jeong-A; Sun, Baodong; Puertollano, Rosa et al. (2018) Therapeutic Benefit of Autophagy Modulation in Pompe Disease. Mol Ther 26:1783-1796
Madsen, Agnete B; Knudsen, Jonas R; Henriquez-Olguin, Carlos et al. (2018) ?-Actin shows limited mobility and is required only for supraphysiological insulin-stimulated glucose transport in young adult soleus muscle. Am J Physiol Endocrinol Metab 315:E110-E125
Ferdinand, John R; Richard, Arianne C; Meylan, Françoise et al. (2018) Cleavage of TL1A Differentially Regulates Its Effects on Innate and Adaptive Immune Cells. J Immunol 200:1360-1369
Duverger, Olivier; Cross, Michael A; Smith, Frances J D et al. (2018) Enamel anomalies in a pachyonychia congenita patient with a mutation in KRT16. J Invest Dermatol :
Milgroom, Andrew; Ralston, Evelyn (2016) Clearing skeletal muscle with CLARITY for light microscopy imaging. Cell Biol Int 40:478-83
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
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

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