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Rearrangement of Mrnas for Costamere Proteins During Costamere Development in Cultured Skeletal Muscle from Chicken

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Rearrangement of Mrnas for Costamere Proteins During Costamere Development in Cultured Skeletal Muscle from Chicken Journal of Cell Science 107, 377-386 (1994) 377 Printed in Great Britain © The Company of Biologists Limited 1994 Rearrangement of mRNAs for costamere proteins during costamere development in cultured skeletal muscle from chicken E. J. Morris and A. B. Fulton* Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA *Author for correspondence SUMMARY Mature skeletal myofibrils are surrounded by costameres, earlier in development. Controls for bleed through of fluo- ribs of metavinculin, vinculin, intermediate filaments, and rescence, RNase H sensitivity, hybridization without probe, other proteins that connect the myofibril to the extracellu- and binding to the myofibril all gave appropriate results. lar matrix. Costameres have recently been shown to be the Probes to glyceraldehyde-3-phosphate dehydrogenase, a sites at which the forces generated by the myofibril are glycolytic enzyme, stained diffusely and did not associate transduced laterally into the extracellular matrix. We with the myofibril. These results show that components of observed costameres developing in cultured skeletal the costamere arrive at the structure in a defined sequence, muscles, grown in micromass culture from cells taken from and that mRNA organization is a conspicuous, precise and embryonic chicken leg. We detected proteins by immuno- temporally controlled aspect of costamere development. fluorescence and mRNA by in situ hybridization. Antibody These results may have wider implications. In these cells, and probe signals were imaged by laser scanning confocal some mRNAs are positioned with submicrometer precision microscopy. Antibody to vimentin protein is first detected in space and differentially over time. Particular mRNAs in stripes in register with the Z line of the myofibril, at differ in the time and place of such positioning. This implies ~day 12 after fusion; soon thereafter probe to vimentin both that cellular structures provide physical cues for such mRNA is also detected in the same stripes. Optical sections positioning and that mRNA contains information that indicate that vimentin mRNA and protein are very close, no interacts with such cues in a message-specific manner. If more than 0.1 mm apart and possibly in immediate contact. such precision in mRNA location is found in other somatic Antibody to vimentin is detected in stripes only in cells that cells, it could have significant implications for the ways in twitch spontaneously. Antibodies and probes to desmin and which cells generate and maintain cellular structures. vinculin protein and mRNA are next detected in stripes of the same periodicity, at ~day 17 after fusion. Vinculin Key words: in situ hybridization, costamere, myofibrillogenesis, protein (but not mRNA) is detected at focal contacts much intermediate filaments, mRNA INTRODUCTION of proteins and occurring in highly restricted locations, costameres are one of the cellular structures whose assembly Costameres are ribs of protein that contain vinculin and lie must be explained in order to understand the mature phenotype between the cell membrane and the Z line in some skeletal of a muscle cell. muscles and in cardiac muscle (Craig and Pardo, 1983; Pardo We reported the organization of vimentin protein into bands et al., 1983). Costameres encircle the myofibril and offer a with the spacing of the Z line in muscles grown in micromass contact point to couple contractions of the myofibril to the cultures (Cripe et al., 1993). In these cells, vimentin mRNA extracellular matrix along the length of muscle cells (Shear and was also organized into bands with the same spacing. We were Bloch, 1985). A recent report established that costameres are interested to discover whether these bands of vimentin were sites of force transduction in cardiomyocytes (Danowski et al., costameres developing in cultured muscle cells and whether 1992). Costameres include various proteins known elsewhere message organization might play a role in the development of to be associated with cell matrix adhesion sites. These proteins these structures. For these reasons, we have examined the include talin (Beckerle and Yeh, 1990), spectrin and ankyrin organization of vimentin, vinculin and desmin protein and (Nelson and Lazarides, 1984), intermediate filament proteins, mRNAs during the later stages of muscle development. and dystrophin (Porter et al., 1992). Danowski et al. (1992) presented a detailed review of recent work on costameres. MATERIALS AND METHODS Costameres are one structure among several that permit muscle cells to function as contractile machinery and transduce Muscle culture contraction to the rest of the organism. Composed of a variety Cultures were prepared weekly from thigh muscle of 12-day-old 378 E. J. Morris and A. B. Fulton chicken embryos (Denning et al., 1988). Synchronized muscle cells showed a striated pattern of protein, mRNA, or both. The highest level were grown on collagen-coated glass coverslips employing a newly of organization in a given cell was the level at which the cell was developed micromass method (Cripe et al., 1993). scored. A minimum of 15 fields of view were scored for each coverslip. The percentage of cells displaying a given trait was calcu- Fixation lated and recorded. Cells were fixed in 1:9 (v/v), formaldehyde/methanol for 20 minutes These cultures were fairly synchronous throughout the first week. at −20°C. Solutions of 37% formaldehyde sealed under N2 were With longer times in culture, the muscles showed some variation in obtained from Electron Microscopy Sciences, and a new ampule was the times at which given patterns were seen, but did not vary in the used each time. This procedure is modified from that of Bresser and sequence in which they were seen. To compare samples cultured in Evinger-Hodges (1987). If fixed cells were stored in 70% ethanol at different weeks, samples were grouped into 3- or 4-day periods (7- −20°C, there was some loss of resolution. All the images shown here 10, 14-17, 20-24). The data from these periods were then averaged to were photographed on the same day on which the cells were fixed. account for variability within any one culture and between different Some samples were also fixed for protein staining only, in absolute cultures depending on the overall maturity. In addition, since these ethanol at −20°C. To optimize double staining for desmin and were micromass cultures, the cells in the centers of the micromasses vinculin, other samples were fixed in 0.1% glutaraldehyde in were at higher densities and showed more advanced development than methanol for 10 minutes at −20°C. All three fixation methods were cells at the edge. For these two reasons, reporting development as the compared for all three proteins. average percentage gives an impression of precision that could be misleading. For example, within a single culture, the cells in the center In situ hybridization of the micromass might have 70% of the cells with periodic vimentin The procedure of Lawrence and Singer (1985, 1986) was used with a mRNA and 35% periodic desmin mRNA; at the edge of such a mass, few modifications, described previously (Cripe et al., 1993). This the comparable numbers might be 40% and 20%. Since it was imposs- procedure was optimized by Lawrence and Singer for efficient hybrid- ible to collect data as a function of the distance from the micromass, ization and reduced background. It was shown to detect single copy the averaged data for development was expressed on a scale from 1 genes of DNA and cytoplasmic mRNAs of low abundance. We chose to 4, based on the percentage of cells that showed a striated pattern the present fixation because it improved cellular preservation and (0, did not stain for the protein (or mRNA); 1, stained diffusely; 2, retention of mRNA in these cells. between 0 and 30% were periodic; 3, between 30 and 70% were periodic; and 4, between 70 and 100% were periodic). Between 14 Probes and 21 samples were scored for each data point. A 425 base pair DNA probe to a vimentin-specific sequence, a 450 The variations in development after the first week make tests such base pair DNA probe to a vinculin-specific sequence, and a 389 base as Student’s t-test inappropriate for detecting differences within a pair DNA probe to a desmin-specific sequence were generated by the single culture. Therefore, the probability (P) that the fraction of cells PCR technique. The probes were constructed using digoxigenin-11- showing a pattern of vimentin protein in stripes was different from dUTP in the reaction mixture. A 640 base pair DNA probe to glyc- the fraction of cells showing vimentin mRNA in stripes was calcu- eraldehyde-3-phosphate dehydrogenase (GAPDH) was generated in a lated using the Wilcoxon two-sample test. Probabilities (P) for the similar fashion. Details of the PCR conditions and the probes to testing of the vimentin, desmin and vinculin protein or message were vimentin and GAPDH have been described (Cripe et al., 1993; Zehner calculated using the Mann-Whitney U-test. and Paterson, 1985). The 389 bp desmin mRNA probe was constructed using the same PCR technique. The plasmid pD8 was supplied by S. Capetanaki. The RESULTS 5′ 24-nucleotide primer was derived from the nucleotide sequence beginning at nucleotide 56 (5′-ATGTCAAGATGGCCTTG- GACGTGG-3′) and the 3′ 24-nucleotide primer ended with nucleotide We have previously shown that vimentin mRNA is localized 445 (5′-CTGGTCTTGCGCTAGCGCGAAGCC-3′). The 450 bp in cultured muscle cells (Cripe et al., 1993). The patterns seen vinculin mRNA was constructed using the same PCR technique. The for vimentin mRNA change during development. The most plasmid p1020 was supplied by S. Craig. The 5′ 30-nucleotide primer highly organized pattern detected is a series of stripes, coinci- was derived from the nucleotide sequence beginning at nucleotide dent with the vimentin protein, that appear after muscles begin 2704 (5′-ATCAGCCCATGATGATGGCTGCTAGGCAGT-3′) and to contract.
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