Rearrangement of mRNAs for costamere proteins during costamere development in cultured skeletal muscle from chicken.
Mature skeletal myofibrils are surrounded by costameres, ribs of metavinculin, vinculin, intermediate filaments, and other proteins that connect the myofibril to the extracellular matrix. Costameres have recently been shown to be the sites at which the forces generated by the myofibril are transduced laterally into the extracellular matrix. We observed costameres developing in cultured skeletal muscles, grown in micromass culture from cells taken from embryonic chicken leg. We detected proteins by immunofluorescence and mRNA by in situ hybridization. Antibody and probe signals were imaged by laser scanning confocal microscopy. Antibody to vimentin protein is first detected in stripes in register with the Z line of the myofibril, at approximately day 12 after fusion; soon thereafter probe to vimentin mRNA is also detected in the same stripes. Optical sections indicate that vimentin mRNA and protein are very close, no more than 0.1 mm apart and possibly in immediate contact. Antibody to vimentin is detected in stripes only in cells that twitch spontaneously. Antibodies and probes to desmin and vinculin protein and mRNA are next detected in stripes of the same periodicity, at approximately day 17 after fusion. Vinculin protein (but not mRNA) is detected at focal contacts much earlier in development. Controls for bleed through of fluorescence, RNase H sensitivity, hybridization without probe, and binding to the myofibril all gave appropriate results. Probes to glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme, stained diffusely and did not associate with the myofibril. These results show that components of the costamere arrive at the structure in a defined sequence, and that mRNA organization is a conspicuous, precise and temporally controlled aspect of costamere development. These results may have wider implications. In these cells, some mRNAs are positioned with submicrometer precision in space and differentially over time. Particular mRNAs differ in the time and place of such positioning. This implies both that cellular structures provide physical cues for such positioning and that mRNA contains information that interacts with such cues in a message-specific manner. If such precision in mRNA location is found in other somatic cells, it could have significant implications for the ways in which cells generate and maintain cellular structures.
Morris
E J
EJ
Department of Biochemistry, University of Iowa, Iowa City 52242.
Fulton
A B
AB
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002645:Chickens; D004247:DNA; D003893:Desmin / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D024510:Muscle Development; D009124:Muscle Proteins / Q000235:genetics* / Q000648:ultrastructure; D009132:Muscles / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism*; D014746:Vimentin / Q000648:ultrastructure; D016596:Vinculin / Q000648:ultrastructure
D003893:Desmin; D009124:Muscle Proteins; D012333:RNA, Messenger; D014746:Vimentin; D016596:Vinculin; D004247:DNA