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Published June 17, 2015 | Supplemental Material + Published
Journal Article Open

Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line

Abstract

Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine) ^(ct90a)). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle.

Additional Information

© 2015 Ruf-Zamojski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: October 10, 2014; Accepted: May 1, 2015; Published: June 17, 2015. This work was supported by a National Human Genome Research Institute (NHGRI) Center of Excellence in Genomic Science grant P50HG004071 (SEF) and by a Muscular Dystrophy Association Development grant (FRZ). We would like to thank members of the Fraser laboratory for technical help and discussions. We would like to thanks all members of the Bernheim laboratory, specifically Dr. Stéphane König and Prof. Laurent Bernheim, as well as Dr. Richard Fish at the University of Geneva, for their help and helpful discussions. The Dmd MANDRA-1, S58, F59 and CH1 antibodies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. Author Contributions: Conceived and designed the experiments: FRZ VT LAT SEF. Performed the experiments: FRZ VT LAT. Analyzed the data: FRZ VT LAT SEF. Wrote the paper: FRZ VT LAT SEF. Competing interests: The authors have declared that no competing interests exist.

Attached Files

Published - journal.pone.0128944.pdf

Supplemental Material - journal.pone.0128944.s001.EPS

Supplemental Material - journal.pone.0128944.s002.EPS

Supplemental Material - journal.pone.0128944.s003.EPS

Supplemental Material - journal.pone.0128944.s004.MOV

Supplemental Material - journal.pone.0128944.s005.DOCX

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