Restricted Distribution of mRNAs Encoding a Sarcoplasmic Reticulum or Transverse Tubule Protein in Skeletal Myofibers
Department of Anatomy and Cell Biology (MN,TK,PS,JP,KM), and Department of Dermatology (JP), University of Oulu, Oulu, Finland
Correspondence to: Kalervo Metsikkö, Department of Anatomy and Cell Biology, P.O. Box 5000 (Aapistie 7), FIN-90014, University of Oulu, Oulu, Finland. E-mail: kalervo.metsikko{at}oulu.fi
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: in situ hybridization skeletal muscle sarcoplasmic reticulum
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have analyzed the localization of mRNA species encoding proteins that are translocated to the ER and then sorted into organelles located throughout the fibers. For this purpose, we selected transcripts that encode calsequestrin (CSQ), a peripheral protein of the SR lumen, and dihydropyridine receptor (DHPR) 1 subunit that is transported from the ER to the transverse tubule membrane. CSQ is a high-capacity calcium-binding protein that provides a reserve for calcium within the SR. DHPR is a dihydropyridine-sensitive L-type calcium channel that functions as a voltage sensor of the transverse tubule triad region (MacLennan 2000
). In situ hybridization studies showed that in the myotube stage, there was no compartmentalization of the transcripts, and their distribution mimicked that of the conventional ER markers. In the adult myofibers, the distribution surprisingly did not match the distribution of the ER, the SR, or the transverse tubule markers, but the mRNAs were restricted beneath the sarcolemma. Interestingly, all the nuclei were active. Because the inner regions of the fibers seemed to be lacking transcripts, it is reasonable to conclude that the respective proteins were synthesized beneath the sarcolemma.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Probes
Approximately 0.2 kb-cDNA fragments of the CSQ and dihydropyridine-sensitive L-type calcium channel (DHPR) 1 were cloned by RTPCR using total RNA from rat skeletal muscle. Primers CSQ-forward (GAA GGA TCC TCA AAG ACC CAC CCT AC) and CSQ-reverse (AGT CGT CTG GGT GAA TTC ACA AGA TG) were chosen for CSQ2 cDNA amplification from gene bank sequence U33287 (bases 8551064) and EcoRI and BamHI cloning sites were added to the primers. Similarly, primers DHPR
1-forward (GCA ATG AAT TCT CCA AGA TGA CTG) and DHPR-reverse (TCC TAG GCC TTG TAC AGT AGC TGT) were designed from the gene bank sequence L04684 to clone DHPR
1 cDNA fragment (bases 730928). The restriction sites for EcoRI and NcoI were added to the DHPR
1 primer sequences. Both PCR products were cloned into pGEM-T vector (Promega Corp.; Madison, WI) according to standard procedure. The plasmid DNAs were isolated and sequenced to certify correct insert sequences and orientations. The digoxigenin (DIG)-labeled RNA antisense and sense probes were synthesized from the linearized CSQ and DHPR
1 cDNA clones according to the instructions of the DIG-RNA labeling kit (Roche Diagnostics GmbH; Penzberg, Germany) using SP6- or T7-polymerase transcription. The concentrations of the DIG-labeled probes were estimated by the spot test according to the protocol from Roche Diagnostics.
RNA In Situ Hybridization
In situ hybridizations were performed based on protocol described by Ylä-Outinen et al. (2002) with slight modifications. Briefly, PFA-fixed samples were washed in 0.1 M phosphate buffer, permeabilized by treatment with 0.5% Triton X-100 in PBS, treated with proteinase K (10 µg/ml), washed with cold water, incubated in 0.2 M HCl containing 0.15 M NaCl, and washed in saline sodium citrate buffer (SSC). The myotube samples were not treated with proteinase K. The samples were acetylated with 0.25% acetic anhydride and incubated overnight at 45C in the hybridization solution containing DIG-labeled RNA probe (300 ng/ml), 50% formamide, 0.6 M NaCl, 2 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1.0 mg/ml BSA, 0.02% polyvinylpyrrolidine, 0.02% Ficoll, 10 mM dithiothreitol, 0.2 mg/ml ssDNA, and 10% dextran sulfate. Posthybridization treatment included stringent SSC washes at 50C and a wash with RNase A for 30 min at 37C.
The DIG-labeled RNARNA hybrids were detected by immunofluorescence. Briefly, samples were blocked by incubation with 6% BSA-PBS solution and then incubated with mouse anti-DIG antibody (1:100 dilution in 6% BSA-PBS; Roche). After washes in PBS, the AlexaFluor 488-conjugated IgG (Molecular Probes; Leiden, the Netherlands) was used for detection. The samples were counterstained with the nuclear Hoechst Dye 33,258 according to the standard protocol. The mounted samples were examined with a Zeiss LSM 510 (Carl Zeiss Inc.; Jena, Germany) confocal laser scanning microscope. Confocal section thickness was 0.5 µm. Intensity profiles across the scanned images were displayed by the LSM 510 examiner program. In situ hybridizations with the sense probes were used as negative controls in all experiments.
RNA Isolations and Northern Analysis
Total RNAs from adult rat muscle tissues were isolated with Trizol reagent (Gibco Life Technologies; Karlsruhe, Germany) according to the manufacturer's instructions. RNA samples (10 µg) were loaded on the denaturating agarose gel, electrophoresed, and transferred to a nylon membrane (Roche Diagnostics) by capillary transfer and fixed with UV-crosslinker (Stratagene GmbH; Heidelberg, Germany). DHPR1 antisense probe hybridization and washes were performed at 68C. CSQ antisense probe was hybridized at non-stringent temperatures (48C) and washed at 52C to detect the transcripts of the CSQ1 and CSQ2 isoforms. The CSQ and DHPR
1 DIG-labeled antisense riboprobes (100 ng/ml) were used for hybridization with DIG EASY hyb hybridization solution (Roche Diagnostics). After washes, alkaline phosphataseconjugated sheep anti-DIG antibody and CSPD chemiluminescent reagent (Roche Diagnostics) were used for detection of the hybrids according to the manufacturer's instructions.
Immunolabeling
Monoclonal antibodies against CSQ, DHPR1 subunit, and protein disulfide isomerase (PDI) were from Affinity BioReagents, Inc. (Golden, CO). Anti-tubulin antibody (clone DM1A) and polyclonal anti-desmin antibodies were obtained from Sigma (St Louis, MO). Routine immunohistochemical protocols were used. Briefly, PFA-fixed samples were permeabilized with 0.5% Triton X-100, and the nonspecific binding was blocked with 1% BSA-PBS solution. The primary antibodies were applied on the samples for 1 hr at 37C. AlexaFluor-488 or -568-conjugated secondary antibodies were used for detection. The samples were mounted and examined with a Zeiss LSM510 confocal laser-scanning microscope.
Drug Treatment, Metabolic Labeling, and SDS-PAGE
Myofibers were cultured overnight before incubation for 1 or 2 hr in the presence of nocodazole (10 µM), cytochalasin D (20 µM), latrunculin A (5 µM), puromycin (200800 µg/ml), or cycloheximide (40 µg/ml). After treatment with the drugs, the myofibers were subjected to in situ hybridization. Metabolic labeling for the puromycin- or cycloheximide-treated samples was performed by incubation for 1 hr in the presence of [35S]methionine (5 µCi/ml). SDS-PAGE was performed on 10% polyacrylamide gels as described by Laemmli (1970) using the Bio-Rad Mini Protean 3 cell (Bio-Rad Laboratories; Hercules, CA). The electrophoresis was performed at 50 mA current for 4 hr. The gel was dried, and radioactivity incorporated into proteins was detected with Fuji Bioimager (BAS 1800 II, Fuji Fujifilm; Sendai, Japan). Images were processed with Adobe Photoshop6.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
To obtain an overall view of the distribution of the CSQ and DHPR1 mRNA in the myofibers, we performed in situ hybridization on isolated myofibers. The procedure was done either immediately after the isolation process or after cultivation on Matrigel substratum. Confocal sectioning indicated a strictly subsarcolemmal distribution in both cases and with both probes. Remarkably, the labeling pattern comprised cross-striations that often appeared as double strands (Figure 4)
. A prominent perinuclear staining was present around every nucleus. The cross-striated staining distributed uniformly over the entire myofiber length with the exception that the intensity often decreased with the distance from the nuclei (not shown). High-stringency hybridization conditions essentially weakened the labeling in all fibers, but the staining patterns remained unaltered. Double staining with antibodies against desmin indicated that the cross-striated labeling was over the I-band areas (Figure 4, right panels).
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Earlier studies have located mRNAs encoding sarcolemmal proteins such as dystroglycan (Mitsui et al. 1997) or sodium channel (Awad et al. 2001
) preferentially beneath the sarcolemma. An explanation for this could be that the large muscle cells contain ER subcompartments, and transcripts encoding exported proteins are sorted to ER domains beneath the sarcolemma. CSQ, on the contrary, is not exported from the ER to the sarcolemma but is targeted to the SR that occupies the inner regions of the fibers. Therefore, it is surprising that its transcripts were restricted beneath the sarcolemma as well. The transcripts of DHPR
1 were also restricted beneath the sarcolemma, although the protein product distributed throughout the fiber cross-section. These findings, together with previous in situ hybridization studies, suggest that in skeletal muscle fibers, transcripts encoding proteins to be translocated to the ER are restricted beneath the sarcolemma. If so, the rough ER would be restricted to subsarcolemmal regions as well. This is compatible with the notion that the SR membranes occupying the interfibrillar spaces are smooth-surfaced (Saito et al. 1984
), implying that they do not contain ribosomes and do not participate in protein synthesis. One explanation for why ER-specific proteins that are not directly linked to calcium storage are present in the entire SR is that they are needed for chaperoning processes in the muscle cells that are regularly subjected to stress conditions. It is notable that the smooth ER of hepatocytes also contains PDI (Akagi et al. 1988
) and the folding-defective tsO45 viral glycoprotein or BiP has free access to the smooth-ER compartment in UT-1 cells (Bergmann and Fusco 1990
).
We found that in the primary myotubes, the mRNAs encoding ER-translocated proteins were distributed throughout the multinucleated cells. This resembles the situation in C2 myotubes, where transferrin receptor mRNA was concentrated in the core region, although its distribution did not fully match the distribution of the ER (Ralston et al. 1997). In the primary myotubes, the mRNAs seem to also occupy the inner regions, in contrast to the situation in adult myofibers. Accordingly, mRNA compartmentalization mechanisms seem to appear during the terminal differentiation process. Studies supporting this include the finding that the transcripts of a cytoplasmic nitric oxide synthase shifted from their intracellular location to the subsarcolemmal regions upon terminal differentiation (Luck et al. 1998
). Similarly, vimentin and vinculin transcripts were diffuse during the first week of development but were detected in costameres as soon as costameres developed (Cripe et al. 1993
; Morris and Fulton 1994
). It should be noted that the inner regions of myofibers do not exclude all mRNA species, because myosin light-chain mRNA has been found evenly distributed within the cross-section of the fibers (Billeter et al. 1992
). Other studies have reported preferentially peripheral localization to myosin mRNA (Dix and Eisenberg 1991
; Jostarndt et al. 1994
), but this seems to depend on the type of chain the transcript encodes (Shoemaker et al. 1999
). Myoglobin transcripts have been located at the A bands of interfibrillar spaces (Mitsui et al. 1994
), in total contrast to the location of the CSQ or DHPR
1 transcripts.
Because the mRNA encoding CSQ was not detected in the inner regions of the myofibers, the protein product has to move relatively long distances from the subsarcolemmal areas to the core of the cell. Providing that the synthesis compartment has a common lumen with the SR, diffusion is one possibility, but experimental evidence favors the view that membrane vesicles carry CSQ from the ER to the terminal cisternae of the SR (Thomas et al. 1989; Nori et al. 2004
). In contrast, DHPR is transported through the Golgi elements to transverse tubules, and in myofibers a majority of the Golgi elements are located beneath the sarcolemma (Lu et al. 2001
). Furthermore, microtubules that act as tracks for vesicular trafficking in mononucleated cells are dense in the subsarcolemmal region of myofibers (Boudriau et al. 1993
). These findings, together with the idea that transcripts of ER-translocated proteins reside beneath the sarcolemma, suggest that membrane protein synthesis, as well as exocytic trafficking, is restricted beneath the sarcolemma in skeletal myofibers. We could not elucidate a crucial role for microtubules or actin filaments, or association with the ER in keeping the transcripts beneath the sarcolemma. Therefore, the positioning mechanisms of mRNAs in myofibers remain an open question.
Earlier work has shown that gene products in myotubes do not reach far from the nucleus of origin (Pavlath et al. 1989; Ralston and Hall 1989
). Regarding adult myofibers, it has been shown that gene products located at the neuromuscular junction are solely encoded by the nuclei in the junctional region (Merlie and Sanes 1985
). It has remained obscure, however, whether the extrajunctional nuclei are equal or if there are quiescent nuclei present. Our present results show that the genes encoding CSQ or DHPR
1 were active in all the nuclei, and it seems that there were no marked differences in activity between the nuclei.
Taken together, CSQ and DHPR1 transcripts neither followed the distribution of classical ER markers nor had the respective target organelles. Our findings here locate ER-translocated mRNAs to perinuclear regions and repeating rib-like structures that are in register with I bands beneath the sarcolemma. The results imply that the synthesis machinery of the respective proteins was similarly organized. Furthermore, there was no marked division of labor among the nuclei to transcribe the mRNAs of interest.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akagi S, Yamamoto A, Yoshimori T, Masaki R, Ogawa R, Tashiro Y (1988) Distribution of protein disulfide isomerase in rat hepatocytes. J Histochem Cytochem 36:15331542[Abstract]
Awad SS, Lightowlers RN, Young C, Chrzanowska-Lightowlers ZM, Lomo T, Slater CR (2001) Sodium channel mRNAs at the neuromuscular junction: distinct patterns of accumulation and effect of muscle activity. J Neurosci 21:84568463
Bergmann JE, Fusco PJ (1990) The G protein of vesicular stomatitis virus has free access into and egress from the smooth endoplasmic reticulum in UT-1 cells. J Cell Biol 110:625635[Abstract]
Billeter R, Messerli M, Wey E, Puntschart A, Jostarndt K, Eppenber HM, Parriard JC (1992) Fast myosin light chain expression in chicken muscle studied by in situ hybridization. J Histochem Cytochem 40:15471557
Boudriau S, Vincent M, Cote CH, Rogers PA (1993) Cytoskeletal structure of skeletal muscle: identification of an intricate exosarcomeric microtubule lattice in slow- and fast-twitch muscle fibers. J Histochem Cytochem 41:10131021
Cripe L, Mollis L, Fulton AB (1993) Vimentin mRNA location changes during muscle development. Proc Natl Acad Sci USA 90:27242728
Dix DJ, Eisenberg BR (1991) Distribution of myosin mRNA during development and regeneration of skeletal muscle fibers. Dev Biol 143:422426[CrossRef][Medline]
Fliegel L, Leberer E, Green NM, McLennan DH (1989) The fast-twitch muscle calsequestrin isoform predominates in rabbit slow-twitch soleus muscle. FEBS Lett 242:297300[CrossRef][Medline]
Fontaine B, Changeux J-P (1989) Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick. J Cell Biol 108:10251037[Abstract]
Jasmin BJ, Lee RK, Rotundo RL (1993) Compartmentalization of acetylcholinesterase mRNA and enzyme at the vertebrate neuromuscular junction. Neuron 11:467477[Medline]
Jolly C, Morimoto RI, Robert-Nicoud M, Vourc'h C (1997) HSF1 transcription factor concentrates in nuclear foci during heat shock: relationship with transcription sites. J Cell Sci 110:29352941
Jostarndt K, Puntschart A, Hoppeler H, Billeter R (1994) The use of 33P-labelled riboprobes for in situ hybridizations: localization of myosin alkali light-chain mRNAs in adult human skeletal muscle. Histochem J 26:3240[Medline]
Kaisto T, Metsikkö K (2003) Distribution of the endoplasmic reticulum and its relationship with the sarcoplasmic reticulum in skeletal myofibers. Exp Cell Res 289:4757[CrossRef][Medline]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680685[Medline]
Lodish HF, Froshauer S (1977) Binding of viral glycoprotein mRNA to endoplasmic reticulum membranes is disrupted by puromycin. J Cell Biol 74:358364[Abstract]
Lu Z, Joseph D, Bugnard E, Zaal KJ, Ralston E (2001) Golgi complex reorganization during muscle differentiation: visualization in living cells and mechanism. Mol Biol Cell 12:795808
Luck G, Oberbäumer I, Blottner D (1998) In situ identification of neuronal nitric oxide synthase (NOS-1) mRNA in mouse and rat skeletal muscle. Neurosci Lett 246:7780[CrossRef][Medline]
MacLennan DH (2000) Ca2+ signalling and muscle disease. Eur J Biochem 267:52915297
Merlie JP, Sanes JR (1985) Concentration of acetylcholine receptor mRNA at synaptic regions of adult muscle fibers. Nature 317:6668[Medline]
Mitsui T, Kawai H, Naruo T, Saito S (1994) Ultrastructural localization of myoglobin mRNA in human skeletal muscle. Histochemistry 101:99104[CrossRef][Medline]
Mitsui T, Kawai H, Shono M, Kawajiri M, Kunishige M, Saito S (1997) Preferential subsarcolemmal localization of dystrophin and beta-dystroglycan mRNA in human skeletal muscles. J Neuropathol Exp Neurol 56:94101[Medline]
Morris EJ, Fulton AB (1994) Rearrangement of mRNAs for costamere proteins during costamere development in cultured skeletal muscle from chicken. J Cell Sci 107:377386
Nakata T, Nishita Y, Yorifuji H (2001) Cytoplasmic actin as a Z-disc protein. Biochem Biophys Res Commun 286:156163[CrossRef][Medline]
Neuhuber B, Gerster U, Döring F, Glossmann H, Tanabe T, Flucher BE (1998) Association of calcium channel 1S and ß1a subunits is required for the targeting of ß1a but not of
1S into skeletal muscle triads. Proc Natl Acad Sci USA 95:50155020
Nori A, Bortoloso E, Frasson F, Valle G, Volpe P (2004) Vesicle budding from endoplasmic reticulum is involved in calsequestrin routing to sarcoplasmic reticulum of skeletal muscle. Biochem J 379:502512
Park KW, Goo JH, Chung HS, Kim H, Kim DH, Park WJ (1998) Cloning the genes encoding mouse cardiac and skeletal calsequestrins: expression patterns during embryogenesis. Gene 217:2530[CrossRef][Medline]
Pavlath GK, Rich K, Webster SG, Blau HM (1989) Localization of muscle gene products in nuclear domains. Nature 337:570573[CrossRef][Medline]
Rahkila P, Alakangas A, Väänänen K, Metsikkö K (1996) Transport pathway, maturation, and targeting of the vesicular stomatitis virus glycoprotein in skeletal muscle fibers. J Cell Sci 109:15851596
Rahkila P, Luukela V, Väänänen K, Metsikkö K (1998) Differential targeting of vesicular stomatitis virus G protein and influenza virus hemagglutinin appears during myogenesis of L6 muscle cells. J Cell Biol 140:11011111
Rahkila P, Väänänen K, Saraste J, Metsikkö K (1997) Endoplasmic reticulum to Golgi trafficking in multinucleated skeletal muscle fibers. Exp Cell Res 234:452464[CrossRef][Medline]
Ralston E, Hall ZW (1989) Transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated myotubes. Science 244:10661069[Medline]
Ralston E, McLaren RS, Horowitz JA (1997) Nuclear domains in skeletal myotubes: the localization of tranferrin receptor mRNA is independent of its half-life and restricted by binding to ribosomes. Exp Cell Res 236:453462[CrossRef][Medline]
Ray A, Kyselovic J, Leddy JJ, Wigle JT, Jasmin BT, Tuana BS (1995) Regulation of dihydropyridine and ryanodine receptor gene expression in skeletal muscle. Role of nerve, protein kinase C, and cAMP pathways. J Biol Chem 270:2583725844
Reithmeier RA, de Leon S, MacLennan DH (1980) Assembly of the sarcoplasmic reticulum. Cell-free synthesis of the Ca2++ Mg2+-adenosine triphosphatase and calsequestrin. J Biol Chem 255:1183911846
Saito A, Seiler S, Chu A, Fleischer S (1984) Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J Cell Biol 99:875885[Abstract]
Scott BT, Simmerman HK, Collins JH, Nadal-Ginard B, Jones LR (1988) Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning. J Biol Chem 263:89588964
Shoemaker SD, Ryan AF, Lieber RL (1999) Transcript-specific mRNA trafficking based on the distribution of coexpressed myosin isoforms. Cells Tissues Organs 165:1015[CrossRef][Medline]
Taneja KL, Lifshitz LM, Fay FS, Singer RH (1992) Poly(A)mRNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J Cell Biol 119:12451260[Abstract]
Thomas K, Navarro J, Benson RJ, Campbell KP, Rotundo RL, Fine RE (1989) Newly synthesized calsequestrin, destined for the sarcoplasmic reticulum, is contained in early/intermediate Golgi-derived clathrin-coated vesicles. J Biol Chem 264:31403145
Volpe P, Martini A, Furlan S, Meldolesi J (1994) Calsequestrin is a component of smooth muscles: the skeletal- and cardiac-muscle isoforms are both present, although in highly variable amounts and ratios. Biochem J 301:465469[Medline]
Volpe P, Villa A, Podini P, Martini A, Nori A, Panzeri MC, Meldolesi J (1992) The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers. Proc Natl Acad Sci USA 89:61426146
Ylä-Outinen H, Koivunen J, Nissinen M, Bjorkstrand AS, Paloniemi M, Korkiamäki T, Peltonen S, et al. (2002) NF1 tumor suppressor mRNA is targeted to the cell-cell contact zone in Ca(2+)-induced keratinocyte differentiation. Lab Invest 82:353361[CrossRef][Medline]