ProsomeCytodistribution Relative to Desmin and Actin Filaments in Dividing C2.7 Myoblasts and during Myotube Formationin Vitro

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

233, 99 –117 (1997)

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Prosome1 Cytodistribution Relative to Desmin and Actin Filaments in Dividing C2.7 Myoblasts and during Myotube Formation in Vitro F. De Conto,*,† S. Missorini,*,† C. Arcangeletti,* ,† F. Pinardi,*,† D. Montarras,‡ C. Pinset,‡ J. Vassy,§ G. Ge´raud,* C. Chezzi,† and K. Scherrer*,2 *Institut Jacques Monod du CNRS, Universite´ Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France; †Istituto di Microbiologia, Universita` degli Studi, Parma, Italy; ‡Institut Pasteur, 25 rue du Dr.Roux, 75014 Paris, France; and §A.I.P.C., Universite´ Paris 7, Hoˆpital Saint Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France

muscle satellite cells, which made it possible to monitor the complete buildup of the sarcomeric structure.

Prosomes constitute the multicatalytic proteinase (MCP) core of the 26S proteasomes, but were first observed as subcomplexes of untranslated mRNP; this suggests that they play a putative role in the control of protein biosynthesis in addition to their catabolic enzymatic function. In previous investigations it was shown that some prosomes colocalize with the intermediate filaments (IF) of the cytoskeleton, of the cytokeratin type in epithelial cells, and of the vimentin type in fibroblasts. Studies on adult rat muscle carried out with prosome-specific monoclonal antibodies (pmAbs) have shown, surprisingly, that specific types of prosomes predominantly occupy a particular zone in between the M and the Z lines of the sarcomeric structure. The data presented here show that the subunit composition of prosomes changes when the dividing C2.7 myoblasts fuse into myotubes. We show furthermore that, in dividing C2.7 myoblasts, prosomes colocalize with the desmin network as well as with that of actin, in a distribution that changes with the subunit pattern of the prosomes investigated by individual pmAbs. Surprisingly, when myogenic fusion is induced, specific types of prosomes move first to the nuclei; later on, they reappear in the cytoplasm. There, superimposing initially onto the reorganizing desmin filaments that run from one pole of the prefusion myoblast to the other, prosomes gradually colocalize with the actin fibers in the fusing myotubes, finally forming a ‘‘pearl on a string’’ pattern. These results are discussed in relation to parallel observations of prosome distribution between the actin and IF networks not only in epithelial cells but also in fusing

q 1997 Academic Press

INTRODUCTION

Many functions have been attributed to the cytoskeleton. In particular, a transport function was assigned to the tubulin system [6–9], which also plays a mechanical role in, for example, the flagella [7, 10]. The actin system is supposed to have an ‘‘architectural’’ role [11 – 16], and in particular the ‘‘stress’’ fibers, observed mainly in tissue culture cells [17]. Within the framework of the present investigation it is of importance to recall that the actin-based microfilaments (MFs)3 have been shown to be the physical support of the polyribosomes and thus of the translation machinery [18 –21], in addition to their more classical functions. The notion that the nuclear matrix and cytoskeletal systems participate in gene expression has been reinforced by recent data showing that specific types of mRNAs are transported to specific cell compartments, and that de novo synthesized proteins are deposited and used in a cotranslational manner [18, 21, 22]. No clear-cut function had been thus far assigned to the third cytoskeletal system, the intermediate filaments (IFs), beyond a putative role in cell architecture [23 –28]. In this context, the observation that the prosome particles colocate with the IFs in epithelial and fibroblastic cells [29–31] may be of interest, since they are putative markers of mRNA in the nontranslated state. Prosomes are small, ‘‘facultative’’ ribonucleoprotein particles (Mr 720,000, 12 1 17 nm), which were first observed on mRNPs, as trans-acting factors specifically associated with mRNAs in the nontranslated state ([1,

1 We use here the term ‘‘prosome’’ introduced by our laboratory [1] for the then unknown mRNA-associated 19 –20S particle and, when speaking of its proteinase activity, the term ‘‘multicatalytic proteinase or MCP,’’ as recommended by the group of enzymologists concerned [2, 3], in preference to the term ‘‘proteasome’’ suggested by Arrigo et al. [4]. In general, the term prosome relates to the biosynthetic, mRNA- and cytoskeleton-related, and proteasome to the catabolic pathway of protein homeostasis controlled, in part, by this system. 2 To whom correspondence and reprint requests should be addressed. Fax: 33 1 44 27 76 47. E-mail: [email protected].

3

Abbreviations used: CSK, cytoskeleton; EM, electron microscopy; IIF, indirect immunofluorescence; IFs, intermediate filaments; MCP, multicatalytic proteinase; MFs, microfilaments; MTs, microtubules; p-mAb, anti-prosome monoclonal antibody; p23K, p25K, p27K, p31K, prosomal subunit proteins of Mr 23,000, 25,000, 27,000, 31,000, respectively. 99

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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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32], for review see [33]), but constitute also the proteolytic core of the 26S proteasomes (reviews in [33 –35]). Cross-linking experiments demonstrated early on that they are specifically present at the level of the ribosome-free mRNP and absent in polyribosomes [32]. In view of their multicatalytical proteinase (MCP) activity, prosomes are also called ‘‘MCP complexes’’ or ‘‘20S proteasomes.’’ In the 26S proteasome complex, the basic prosome particle is associated with an extensive and variable combinatory set of ATPases, proteases and activators and inhibitors, which confer on the structure a very selective and variable substrate specificity [36]. In the archeobacterium Thermoplasma acidophilum pro(tea)somes are present in a primitive form, composed of four superposed rings of seven subunits each, in a a14 b14 stoechiometry [37, 38]. Indistinguishable at first glance from the archeobacterial particles under the electron microscope, prosomes in higher eukaryotes show characteristic differences in their internal structure [39]. This is probably due to the fact that they are, in contrast with the unique bacterial proteasomes, a population of ‘‘mosaic-like’’ particles built also of 28 subunits (Mr 20,000 to 36,000) but in a variable combinatory set of specific proteins. This ‘‘mosaic’’ varies according to cell type and physiological or pathological state [40–44]. Immunoprecipitation with p-mAbs has directly demonstrated that particles of different subunit mosaics coexist in a given cell [45]. Monoclonal antibodies created and extensively characterized in our laboratory [29–31, 42, 43, 46, 47] have made it possible to trace prosomes of specific subunit composition in a wide variety of cells and species ([31, 48], discussion in [33]). Since its original observation [29], the prosome –IF colocalization has been widely investigated in various types of epithelial cells and fibroblasts [30, 31, 46] but, thus far, only preliminary data have been published on muscle cells containing the desmin system, representing the muscle cells differentiation-specific variant of the IF networks [31]. The muscle system possesses, however, a particular advantage for the investigation of cytoskeletal networks, as well as for analysis of the role of prosomes because of their putative function as trans-acting factors operating upstream of the translation machinery; as pointed out, this is itself linked to the MF system [18 –21]. This advantage is based on several facts: (i) undifferentiated muscle cells, in particular the satellite cells of skeletal muscle and early embryonic presumptive myoblasts gradually extend the organ-specific desmin network during terminal differentiation and muscle maturation, in parallel to the preexisting vimentin IF system; (ii) a profound reorganization of the cytoskeleton takes place when muscle cells fuse into myotubes which will form, eventually, the sarcomeric structure; (iii) the reorganization of the cells’ morphology and cytoskeletal systems is conditioned by an extensive reprogramming of gene expres-

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sion, when muscle-specific mRNAs coding for myosin, titin, actinin, sarcomeric actin, tropomyosin, etc. are expressed under the influence of myogenic differentiation factors such as Myo D and myogenin [49]. It is particularly interesting within the perspective of our work that the cytoplasmic types of b and g actin, which are at the basis of the MF and stress fiber systems, are gradually replaced by sarcomeric actin; the latter brings about muscle contraction by exerting its effect on myosin within the sarcomeric structure. In view of the profound reprogramming of the translation machinery that accompanies the reorganization of the cytoskeletal systems during myotube formation, we investigated the relationship between prosomes and the cytoskeleton, and in particular the IF and actin systems in muscle cells, our aim being to better understand the function in myogenesis of both the prosomes and the specific types of cytoskeletal systems. In a parallel investigation on adult rat skeletal muscle we found, most surprisingly, that specific types of mosaic-patterned prosomes, including the p27K and p31K prosome subunits, are located mainly at the level of the I band, in between the M and Z lines of the sarcomeric structure [5]. This prosome-based ‘‘pseudosarcomeric’’ structure is gradually generated during the fusion of muscle precursor cells into myotubes [50], as detailed in a related paper describing the fusion of rat muscle satellite cells into myotubes in vitro (Foucrier et al., in preparation). Here we present a study of the C2.7 myoblasts, established myogenic precursor cells that can be kept indefinitely in tissue culture and, furthermore, can be induced to form myotubes without, however, reaching the stage of final sarcomeric organization [51]. For this investigation we applied a new type of fixation technique (‘‘instantaneous fixation’’), which gives a better analysis of the physiological steady state of cytoskeleton dynamics and protein biosynthesis [52, 53]. Interestingly, we found that prosomes in muscle cells colocate not only with the desmin-type IF but also, in part, with MFs and stress fibers. This raises the question of a possible functional interrelation between these two types of cytoskeletal systems, involving prosomes and the protein-synthesizing machinery. We show, using a series of prosome-specific monoclonal antibodies, that prosomes distribute themselves in between the desmin filaments and the microfilaments and stress fibers, in a pattern characteristic of the specific antigen tested. Most interestingly, biochemical analysis shows that the subunit pattern of prosomes in myoblasts changes when myotubes are formed. When fusion is induced, specific types of prosomes move to the nuclei, prior to their reappearance in the cytoplasm. When the desmin filaments reorient along the axis of the future myotubes, prosomes colocate increasingly with the actin system.

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MATERIALS AND METHODS Cell Culture Monolayer cultures of C2.7 myoblasts [54] derived from the C2 mouse myogenic cell line of Yaffe´ and Saxel [51] proliferated in DMEM (Gibco: Life technologie s.a.r.l., 95610 Eragny, France) supplemented with 20% fetal calf serum (FCS) (proliferation medium) (Bayer Diagnostic, 92807 Puteaux Cedex, France) and antibiotics (Gibco).

Two-Dimensional Gel Electrophoresis The prosome-containing fractions purified from the free cytoplasmic mRNP complexes were pooled, proteins were precipitated by TCA and analyzed by two-dimensional gel elctrophoresis as described earlier [40]. For isoelectric focusing (4 h, 200– 750 V), the first dimension gels contained 8 M urea and 2% ampholines (pH 3.5 to 10) (Pharmacia Biotech S.A., Saint Quentin, Yvelines, France). Second-dimension electrophoresis was for 90 min at 100 V on SDS– polyacrylamide (13%) gels. Western Blotting

Differentiation Protocol Monolayer cultures of C2.7 myoblasts (20,000 cells/dish) were seeded in 35-mm plastic petri dishes (Costar, Cambridge, MA) and allowed to grow in the proliferation medium for 3 days. Then the proliferation medium was replaced with the DME medium supplemented with a lower FCS concentration (1%) (differentiation medium). Prosome-Specific Monoclonal Antibodies Monoclonal antibodies directed against the prosomal proteins p23K (35A33), p25K (7A11), p27K (IB5), and p31K (AA4) were mouse ascitic fluids (Organon Teknika, Turnhout, Belgium). Purified prosomes from duck erythroblasts or HeLa cells were used as antigens for mouse immunization, according to the procedure described previously [48]. These antibodies were tested for the absence of crossreactivity with any other components in cell lysates and were successfully used for IIF and EM in a series of cytological investigations bearing on various types of cells [29– 31, 42, 43, 46, 47]. Cell Fractionation of C2.7 Myoblasts Exponentially cultured C2.7 cells were trypsinized (trypsin/EDTA in PBS, Gibco), washed twice with isotonic buffer (140 mM NaCl, 10 mM Hepes, pH 7.4, 5 mM KCl, 1.5 mM MgCl2) and pelleted again by low speed centrifugation (1500 rpm) for 10 min. All the following steps were performed at 47C. Cells were gently resuspended in 8 vol of hypotonic buffer (10 mM triethanolamine, pH 7.4, 10 mM KCl, 1 mM MgCl2 , 1 mM MnCl2 , 5 mM 2-mercaptoethanol) and homogenized in a tight Dounce homogenizer. Lysis was monitored by phasecontrast microscopy and isotonicity was restored after 3– 4 min by addition of 2 M sucrose (0.25 M final). Differential centrifugation was performed to obtain the fractions corresponding to nuclei, mitochondria, polyribosomes, and free mRNP as described previously [29, 55]. The nuclei were purified according to Ref. [56]. The mitochondria were washed twice in isotonic buffer (hypotonic buffer containing 0.25 M sucrose), centrifuged at 9000 rpm for 20 min, resuspended in TEK buffer (10 mM triethanolamine (pH 7.4), 50 mM KCl, 5 mM 2-mercaptoethanol), and sonicated twice for 5 s on ice. All final pellets were resuspended in TEK buffer and equal amounts of each fraction were analyzed by SDS–PAGE [57] after protein quantification [58]. Prosome Purification Prosomes were purified from the free cytoplasmic mRNP complexes by sedimentation, after resuspension of the mRNP pellets in TEK buffer. About 1 to 5 A260 units were loaded on a 5 –21% (w/w) isokinetic sucrose gradient containing 0.5 M KCl, and centrifuged on top of a 85% sucrose cushion (38,000 rpm, 11 h, 47C, Beckman rotor SW41). Peak fractions in the sedimentation zone of 19–20S were pooled and concentrated in TEK buffer by ultrafiltration (Amicon Centricon 30, Beverly, MA). The filtrate was then loaded on a 5 to 21% (w/w) isokinetic sucrose gradient containing 0.1% Sarkosyl and centrifuged on top of a 85% sucrose cushion (38,000 rpm, 13 h, 47C, Beckman rotor SW41).

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After electrophoresis in polyacrylamide gels, proteins were electrophoretically transferred onto nitrocellulose membrane (0.45 mm) according to Towbin et al. [59]. The incubation of the nitrocellulose membranes with the antibodies was as described previously [48]. Indirect Immunofluorescence C2.7 myoblasts (20,000 cells/dish) were seeded in 35-mm plastic petri dishes (Costar), pretreated with 100 mg/cm2 of a 0.1% collagen type I solution (Sigma Chemical Co., St. Louis, MO) for 6 h at 377C, and allowed to grow for 48 h. Triton X-100 extraction and fixation protocol (delayed fixation protocol). Exponential cultures of C2.7 myoblasts were briefly rinsed with CSK buffer (10 mM Pipes, pH 6.9, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 1 mM EGTA, 1.2 mM PMSF) at 377C and extracted with CSK buffer containing 0.2% Triton X-100 for 3 min. Extracted cells were carefully washed with CSK buffer, fixed with 4% paraformaldehyde in CSK buffer for 20 min at 377C, washed three times for 15 min in PBS (7 mM Na2HPO 4 , 1.5 mM KH2PO4 (pH 7,4), 137 mM NaCl, 2.7 mM KCl), and then processed for immunofluorescence as described below. Instantaneous fixation protocol. C2.7 myoblasts were simultaneously Triton-extracted and fixed with paraformaldehyde for 20 min (10% Triton X-100 and 1% paraformaldehyde in CSK buffer) at 377C, then washed three times in PBS for 5 min, and finally processed for immunofluorescence as described below. Staining protocol. The fixed cells were preincubated for 15 min with 1% BSA in PBS (to reduce background staining) and then incubated with the prosomal p-mAbs (anti-p23K, anti-p25K, and antip31K diluted 1:10) in PBS containing 0.2% BSA (incubation buffer) for 30 –60 min at 377C in a humid chamber. The cells were washed three times for 5 min with PBS and the fixed antibody was revealed by FITC- or RITC-conjugated anti-mouse IgG (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) diluted 1:30 in the incubation buffer for 40 min at 377C. The cells were washed abundantly with PBS and mounted in Mowiol (Calbiochem AG, Lucerne, Switzerland). In the case of double immunofluorescence, the p-mAbs were used in combination with either rabbit polyclonal anti-desmin antibodies (Unipath- Eurodiagnostic BV, Dardilly, France; diluted 1:40), or rabbit polyclonal anti-cytoplasmic actin (a kind gift of G.Gabbiani) diluted 1:80. The anti-cytoskeleton polyclonal antibodies used were tested in C2.7 myoblast and myotube lysates for their cross-reactivity with prosomal antigens. A slight reactivity was also found with vimentin in the case of the anti-desmin polyclonal antibody only (data not shown). The p-mAbs were secondarily detected with FITC (or RITC)-conjugated goat anti-mouse IgG, and the anti-cytoskeleton antibodies were revealed by Texas red-labeled (Amersham International plc, Little Chalfont, Buckinghamshire, England; diluted 1:30) or FITC-labeled (Kirkegaard & Perry Laboratories; diluted 1:30) goat anti-rabbit IgG. Negative controls were carried out by an identical procedure, except that the first antibody was replaced by the incubation buffer alone. Alternatively, the first antibodies were replaced, one by one, with irrelevant antibodies of identical isotype (IgG1; specifically, anti-human thyroglobulin monoclonal antibody or anti-gastrin poly-

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clonal antibody was used, diluted 1:100 and kindly provided by Dr. A. Natali) (data not shown). Observations were made under a fluorescence microscope (Leitz Laborlux S, Wild-Leitz, Wetzlar, RFA) and photographs were made using HP5 film (400 ASA; Ilford Ltd., Basilden, Essex, England). Confocal Laser Scanning Microscopy Two different confocal microscopes and imaging systems were used. Analysis of prosome–IF cytolocalization in C2.7 myoblasts (Figs. 4 and 5), prosomes (Fig. 6), and prosome–actin cytolocalization (Fig. 7) during the differentiation process was carried out using an MRC-600 system (Bio-Rad, UK) mounted on a Optiphot II Nikon microscope equipped with a 601 objective (Plan Apo; NA 1.4). For FITC and Texas red excitation, a multiple-line argon laser beam (25 mW) ajusted to 514 nm was used. For each optical section, double fluorescence images were acquired simultaneously (dual mode acquisition). Power line excitation, photomultiplier gain, and pinholes were ajusted to avoid potential red channel contamination by leaking FITC-specific emission (‘‘cross-talk’’); Figs. 4C and 4C* (upper right corner) indicate absence of cross-talk. Weak signals were digitalized in photon counting mode (Bio-Rad system) in order to increase the signal versus background ratio; each section was scanned 20 times. Z series were carried out using 0.5-mm steps between each focal plane. The two images were merged to check the relative position of the two fluorochromes. Analysis of prosome –actin filament interactions in C2.7 myoblasts during myotube formation (Figs. 7 and 8) was performed using the TCS40 (Leica, Germany) confocal imaging system, equipped with a 631 objective (plan apo; NA 1.4). For FITC and Texas red excitation, an argon –kripton ion laser adjusted at 488 and 568 nm was used. For each optical section, double fluorescence images were acquired in sequential mode (i.e., FITC first and Texas red second). The signal was treated using line averaging to integrate the signal collected over 8 lines in order to reduce noise. For high resolution, we defined a set of acquisition parameters, which took into account Nyquist’s principle. The confocal pinhole was closed to yield a minimum field depth (about 0.6 mm). A focal series was collected for each specimen. The focus step between these sections was generally 0.3 mm and the XY pixelation was set to 100 nm. Each selected section level was then processed to produce a single high spatial resolution red/green composite image. Photographs were printed on a sublimation laser printer (Colorease Kodak) with Photoshop software. Image Analysis Double staining microfluorometry analysis (Fig. 8) was performed using the Multicolor analysis software (Leica, France) running on the TCS (Leica) confocal microscope. In the cytofluorogram, the pixel to pixel correlation between two channels (red and green) can be outlined; colocalized or closely related areas can be selected (yellow cloud) and quantified. The selected cytofluorogram is surperimposed in white color onto the merged images of this same area (Fig. 8).

RESULTS

The main aim of the present investigation was to characterize the cytodistribution of prosomes in C2.7 myoblasts in relation to the cytoskeleton, and to study the reorganization of prosome and cytoskeletal networks during induced myotube formation which results in major morphological and biochemical remodeling of these cells. We have applied here a new methodology [53] which allows observation of the cytoskeletal networks after ‘‘instantaneous’’ fixation of cells at physiological temperature (377C). The dynamic steady state

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of both the cytoskeleton and the machinery of protein biosynthesis is thus preserved. Furthermore, the new method discriminates between proteins truly integrated into the cytoskeletal system and their nonpolymerized ‘‘free’’ building blocks [52, 53]. The main tools for this study were prosome-specific monoclonal antibodies (p-mAbs) created by our laboratory [29], which have been extensively characterized and successfully used in a series of investigations [29 – 31, 42, 43, 46, 47]; they were found to be free of any cross-reactivity with other antigens in lysates of various cell types. Biochemical Characterization of Muscle Prosomes Prior to exploring the cytolocation of prosomes by indirect immunofluorescence (IIF), we undertook the study, by biochemical and immunological methods, of the cytodistribution of muscle prosomes present in the C2.7 myoblasts, and the characterization of their subunit protein composition. As found previously (review in [33]), prosomes are complexes of variable mosaic subunit composition, and every type of cell thus far investigated has enabled us to observe populations of particles possessing distinct variations in their composition [40, 41, 45, 47]. Furthermore, it was particularly important within the IIF studies reported below, to investigate biochemically the possible presence of free prosome protein antigens outside the 20S particles. Interestingly, in none of the cells thus far investigated has a pool of free prosomal proteins outside the 20S particles been observed, except in heat shock conditions [60]. Indeed de novo synthesized prosomal proteins integrate immediately into preprosomes [61]. C2.7 myoblasts kept in tissue culture were fractionated by a standard protocol (see Materials and Methods) that permitted the evaluation of their presence in the nucleus and the various subcytoplasmic fractions, by one-dimensional gel electrophoresis and Western blotting, using the p-mAbs. The results (data not shown) were identical to those reported previously for various types of epithelial cells and fibroblasts [30, 31], indicating the presence of relatively few particles in the nucleus, in particular of p27K-specific prosomes, the majority of prosomes being found in the pellet containing the free mRNP. The finding that, as in all cells tested thus far [33], prosome antigens are absent in the cytosol fraction of C2.7 myoblasts and myotubes, was particularly important (data not shown; cf. [31]). Indeed, in this fraction of the postmitochondrial supernatant, the free (‘‘soluble’’) proteins which should include prosomal proteins outside the 20S particles, if present, sediment at 2–4S. Their absence in the cytosol fraction indicates that the IIF data shown below represent patterns of 20S prosome particles exclusively, and not free subunit antigens.

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FIG. 1. Purification of prosomes from C2.7 myoblasts by sucrose gradient centrifugation and detection of prosomal proteins by Western blotting, using the anti-p25K, anti-p27K, and anti-p31K p-mAbs. (A) About 1 A260 unit of ribosome-free mRNPs of C2.7 myoblasts was dissociated by 0.2% Sarkosyl and loaded on a 5 to 21% (w/w) isokinetic sucrose gradient containing 0.1% Sarkosyl (Beckman rotor SW41, 38,000 rpm, 13 h, 47C). (B) Each two subsequent fractions of the gradient were pooled and proteins were precipitated by TCA, dissolved in 2% SDS, and electrophoretically separated on a 13% polyacrylamide gel and silver-stained. (C) Western blot analysis of the proteins separated as in B; proteins were electrophoretically transferred onto a nitrocellulose membrane, and immunoreacted successively with the anti-p25K, anti-p27K, and anti-p31K mAbs. Bars to the right indicate the position of the individual prosomal antigens. Marker proteins are indicated on the left side.

Figure 1 reports the isolation of muscle prosomes and their biochemical characterization in the presence of the strong nonionic detergent Sarkosyl, which strips

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off any loosely associated protein contaminating the prosome particles. Fractionation on sucrose gradients of the pellet containing the free mRNP and prosomes from C2.7 myoblasts showed that prosome antigens are present exclusively in the 19–20S position and are absent from the pool of free proteins sedimenting in the 2–4S range at the top of the gradient. Whereas free cytosolic proteins and other dissociated mRNA-associated trans-acting factors sediment in between 2S and about 10S, the prosomes identified by the monoclonal antibodies p25K, p27K, and p31K sediment, largely free of contaminants, in the 19–20S position. Nevertheless, an unusual protein of Mr 56,000 absent from other recently investigated types of cells, such as epithelial cells or fibroblasts [31], was observed on this gradient fraction at the prosomes position. A 56 K protein was observed by two-dimensional electrophoresis in prosomes of avian erythroblasts also (see [60]) and may represent a genuine prosome protein, or might possibly be contained within another stable complex sedimenting to the same position. The subunit composition of the muscle prosomes present in C2.7 myoblasts (Fig.2A) resembles that of many other cells investigated, with subunits within the usual Mr 20,000 –35,000 range. Nevertheless, comparing the bidimensional electrophoretic pattern of these Sarkosyl-purified mouse myoblast prosomes (Fig. 2A) with those of rat erythroblasts (Fig.2C), and with, as a reference, the classic pattern of HeLa cells (Fig.2D), several spots can be observed that are qualitatively or quantitatively different. Interestingly, the analysis of the subunit pattern comparing the prosomes of muscle cells prior to and after differentiation into myotubes (Figs. 2A and 2B) showed quantitative (open arrowheads) as well as qualitative changes in protein composition (solid arrowheads). Even correcting for the different staining intensities of the two pictures shown, the myotube pattern seems more dense, showing several new spots (upper right). Of particular interest is a new row of spots (arrow), aligning with a major protein, seen in both patterns to the left. This possibly indicates the chemical modification of a protein upon differentiation, whereas the spots shown by solid arrowheads seem to correspond to newly induced protein subunits. On the basis of this biochemical investigation we may conclude that prosomes in C2.7 myoblasts possess characteristics very similar to those isolated from other types of cells, although they present, as generally observed in different types of cells [41], particularities in the composition of their subunit. The most significant new observation is the modification of the myoblast pattern upon differentiation into myotubes. This indicates once more the variable, mosaic-like subunit composition of this type of particles. It is of prime importance for the IIF studies reported below that, as already mentioned, no free prosome antigens seem to exist in C2.7 myoblasts outside the 20S particles.

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FIG. 2. Two-dimensional electrophoretic pattern of prosomal proteins in C2.7 myoblasts and myotubes in comparison with those of rat erythroblasts or HeLa cells. Prosomes were purified from the free cytoplasmic mRNP complexes by sedimentation of the resuspended mRNP pellets in TEK buffer. About 1 to 5 A 260 units was loaded on a 5 to 21% (w/w) isokinetic sucrose gradient containing 0.5 M KCl on top of an 85% sucrose cushion bottom and centrifuged (38,000 rpm, 11 h, 47C, Beckman rotor SW41). Peak fractions in the sedimentation zone of 19– 20S were pooled and concentrated in TEK buffer by ultrafiltration. The filtrate was then loaded on a 5 to 21% (w/w) isokinetic sucrose gradient containing 0.1% Sarkosyl on top of an 85% sucrose cushion bottom and centrifuged (38,000 rpm, for 13 h, 47C, Beckman rotor SW41). Then the prosomal fractions were pooled, proteins were precipitated by TCA and analyzed by twodimensional gel elctrophoresis as described earlier [40]. Isoelectric focusing (4 h, 200–750 V) was on gels containing 8 M urea and 2% ampholines (pH 3.5 to 10). Second-dimension (13% gels) electrophoresis was for 90 min at 100 V. Silver stains of (A) prosome proteins from C2.7 myoblasts, (B) prosome proteins from C2.7 myotubes (3rd day of differentiation), (C) prosome proteins from mouse erythroblasts, (D) prosome proteins from HeLa cells (for C, D cf. [40]) are shown. Marker proteins are indicated on the left side.

Prosome Cytodistribution in Exponentially Growing Myoblasts In contrast to previously reported studies on prosome cytodistribution, analyzed after extraction of cells by Triton X-100 prior to fixation with paraformal-

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dehyde [31], we applied in the present study the ‘‘instantaneous’’ fixation technique reported in detail under Materials and Methods. In short, cells are fixed by 1% paraformaldehyde and extracted simultaneously by 10% Triton X-100 at 377C, resulting in instantaneous arrest of the dynamic physiological steady state. High-resolution microscopic observation is thus possible of filaments that are otherwise obscured by soluble proteins, fixed but not really integrated into the cytoskeletal filaments. Optimized by a systematic technical study to be reported elsewhere ([53], submitted), this permits observation of the cellular filamentous networks in their physiological steady state. In PtK1 epithelial cells, this new technique shows a variable distribution of specific prosomes in between the IFs and the actin-based microfilament (MF)/stress fiber system [52, 53]. As seen in Fig. 3, an analogous observation can be made by IIF with C2.7 myoblasts using instantaneous fixation (A and B). If the pattern produced by (Fig. 3A) p25K and (Fig. 3B) p31K p-mAbs is compared with staining (Figs. 3A* and 3B*) by a polyclonal anti-desmin antibody, the prosomes are distributed between two types of networks which seem to coincide (i) with the desmin filaments (and, possibly, to a minor degree with vimentin; see Materials and Methods), and (ii) the MFs and stress fibers. By double-label IIF using anti-cytoplasmic actin antibodies, the large and straight fibers observed in the prosome pattern can be identified by confocal microscopy as representing the actin-containing MFs and stress fibers (see below). This stressfiber-like pattern is not seen in Figs. 3C and 3D (delayed fixation) showing C2.7 myoblasts, double-labeled with (Fig. 3C) the p25K and (Fig. 3D) p31K p-mAbs and with (Figs. 3C* and 3D*) a desmin-specific antibody, which were fixed after prior extraction by Triton X-100 at 377C. In these pictures the prosomes seem to coincide largely with the desmin network. This indicates that the prosomes remain firmly attached to the IFs in the presence of Triton X-100, whereas their interactions with MFs and stress fibers are sensitive to this detergent. Nevertheless, there are areas in these micrographs where the IF and prosome systems do not coincide and where, therefore, prosomes might still be present on MFs. What seems totally absent after Triton extraction, however, is the stress-fiber type of pattern, clearly seen in Figs. 3A and 3B. The distribution of prosomes in between the desmintype IFs and the actin networks of the cytoskeleton observed here seems in apparent contradiction with previously reported data concerning other types of cell. In the latter, after Triton extraction, an almost exclusive presence of prosomes on the IF was observed [31]. The new results seem to be due to two parameters of the analytical procedure: (1) ‘‘instantaneous fixation’’ in contrast to prior Triton extraction adopted in the

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FIG. 3. Colocalization of prosomes with intermediate filaments in C2.7 myoblasts observed after ‘‘instantaneous’’ and ‘‘delayed’’ fixation. C2.7 myoblasts kept at 377C were (A, A*, B, B*) instantaneously fixed with 1% paraformaldehyde in the presence of 10% Triton X-100 for 20 min or (C, C*, D, D*) Triton-extracted (0.2%, for 3 min) and then fixed with 4% paraformaldehyde for 20 min. The cells were stained with (A, C) the prosomal anti-p25K p-mAb or (B, D) the anti-p31K p-mAb, in combination with (A*, B*, C*, D*) rabbit polyclonal anti-desmin antibodies. Immunoreaction was revealed with RITC-labeled goat anti-mouse IgG for prosome-specific mAbs, and FITC-conjugated goat anti-rabbit IgG for cytoskeletal antibodies. Black insets in B, B*, D, and D* show negative controls, using identical procedures, except that the first antibody was replaced by 0.2% BSA in PBS (bars, 20 mm).

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previous experimentation, and (2) the temperature of the cells at the time of fixation. The Three-Dimensional Prosome Network in C2.7 Myoblasts in Continuous Culture Observed by Confocal Microscopy In order to characterize the new type of prosome filaments observed and to analyze the distribution of prosomes between these newly observed actin-like and the desmin-like patterns, we resorted to confocal microscopy thus allowing analysis of these filaments in the three-dimensional space. Serial focal sections of C2.7 myoblast monolayers (Z series of 4 to 6 focal sections, step 0.5 mm) were analyzed and the top and bottom sections selected and shown in Figs. 4 and 5; in both the top and bottom sections are separated by 1.5 mm. Figure 4 reports the data we obtained using the same prosomal antibodies (Figs. 4A and 4B, p25K; Figs. 4C and 4D, p31K) as those used for Fig.3, relative to staining by actin (Figs. 4A*–4D*). For these experiments, an anti-actin antibody was used and detected by Texas red-labeled goat anti-rabbit IgG, in preference to phalloidin. This antibody (kindly provided by G. Gabbiani) is specific to cytoplasmic actin (and does not cross-react with prosomal antigens; not shown). The following observations can be made: (1) There is a clear coincidence, in the micrographs shown, of the (Figs. 4A and 4B) p25K and (Figs. 4C and 4D) p31K-specific prosome networks with (Figs. 4A*–4D*) the actin pattern, although to a variable extent, in particular at the periphery of the cells and when comparing the top and bottom sections. This coincidence is more obvious than that observed with desmin, shown in Fig.5. Nevertheless, it is very clear that there are areas in which the p25K prosome, as well as the p31K prosome, and the actin pattern do not coincide, in particular in the perinuclear zone where, as observed in Fig.5, desmin is most abundant. (2) A differential cytodistribution of the two prosome antigens is again clearly evident; the p31K antigen is relatively abundant in both the nucleus (excluding dark patches corresponding, probably, to the nucleoli) and the cytoplasm, whereas the p25K antigen is more

FIG. 4. Serial focal sections in the confocal microscope of prosome and actin networks in C2.7 myoblasts. C2.7 myoblasts were instantaneously fixed with 1% paraformaldehyde in the presence of 10% Triton X-100 at 377C for 20 min. The cells were stained with (A, B) antip25K or (C, D) anti-p31K p-mAbs, in combination with (A*, B*, C*, D*) polyclonal anti-cytoplasmic actin antibodies. The fixed prosomal antibodies were secondarily detected by FITC-conjugated goat antimouse IgG. The anti-actin antibodies were revealed with Texas redlabeled goat anti-rabbit IgG. (A, A*) and (C, C*) show the bottom sections close to the solid support; (B, B*) and (D, D*) are the top sections. The bottom and top sections shown are separated by 1.5 mm (bars, 25 mm).

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abundant in the cytoplasm, particularly in the perinuclear area. For instance, in Fig.4A (p25K, bottom section) a variable perinuclear stain can be observed for the p25K antigen, which is highly concentrated around the nucleus of some cells and more poorly represented in others. The same, although less evident, observation can be made for the p31K antigen (Fig.4C). (3) Taking advantage of confocal microscopy, we used serial focal sections across the cell to observe the cytoskeleton at different levels. The comparison of the top and bottom (close to the support) focal planes shown here indicates clearly the particular abundance of prosome networks close to the base, where the cells attach to the supporting material. It is noteworthy that the cytoplasmic pattern of the p25K antigen is limited to the sections at and below the nuclei, whereas the p31K pattern can be observed throughout the different focal planes, although in different amounts (compare in Fig.4 (p25K), A and B with (p31K) C and D). Interestingly, in an identical manner, the nuclei are stained apparently at the top focal plane by both antibodies. This type of experiment thus highlights once more the heterogeneity of the prosome subunit distribution throughout the cells relative, in this case, to the microfilament and stress fiber networks. Figure 5 shows the bottom and top sections of a series in which the cells were double labeled with the (A, B) p25K and (C, D) p31K prosome antibodies, and with (A*–D*), a polyclonal antibody specific for desmin. After instantaneous fixation, as in the micrographs based on conventional immunofluorescence microscopy (Fig. 3), one can observe in the same picture the two types of networks stained by the prosome antibodies, i.e. the more tangled filaments which coincide with desmin and the straight running filaments resembling the actin patterns seen in Figs. 4A and 4C. Whereas in some areas the coincidence of desmin and prosome filaments is clearly evident, in other areas it is not. If one compares the two prosome antigens, and also the bottom and top focal sections, it is interesting to note that the patterns shown in Fig. 5 are not identical. As seen previously, the p31K antigen is more abundant in the nuclear area and does not stain the nucleoli, whereas the p25K antigen is scarce and shows a more

FIG. 5. Serial focal sections in the confocal microscope of prosome and intermediate filament networks in C2.7 myoblasts. C2.7 myoblasts were instantaneously fixed with 1% paraformaldehyde in the presence of 10% Triton X-100 at 377C for 20 min. The cells were stained with (A, B) anti-p25K or (C, D) anti-p31K p-mAbs, in combination with (A*, B*, C*, D*) polyclonal anti-desmin antibodies. The fixed prosomal antibodies were secondarily detected by FITC-conjugated goat anti-mouse IgG. The anti-desmin antibodies were revealed with Texas red-labeled goat anti-rabbit IgG. (A, A*) and (C, C*) are the bottom sections; (B, B*) and (D, D*) are the top sections. The bottom and top sections shown are separated by 1.5 mm (bars, 25 mm).

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diffuse pattern in the nucleus, as well as in the cytoplasm. Furthermore, the p25K antigen seems to follow desmin filaments more closely than the p31K. The difference in the cytodistribution of the two antigens with regard to the desmin network is particularly evident in the nuclear area which is exempt of desmin and contains prosome antigens in different amounts. Interestingly, both antigens tested have a tendency to concentrate in the perinuclear area, where the desmin network is most dense. Altogether, the experiments shown in Figs. 4 and 5 indicate a heterogeneity in the distribution of the prosomes between the actin and desmin filaments throughout the C2.7 cells, and partial coincidence of the prosomes with the actin and desmin patterns. The Reorganization of the Prosome Networks Relative to the Cytoskeleton upon Induced Fusion into Myotubes of C2.7 Myoblasts C2.7 myoblasts are a myogenic cell line, originally developed by D. Yaffe´ [51], and which can be induced to form myotubes by drastically lowering the serum concentration in the differentiation medium (1% fetal calf serum), in comparison with the proliferation medium (20% fetal calf serum). The change of medium brings about myotube formation in a gradual manner; in our hands it was maximal after about 3 days of culture in differentiation medium. Prior to the demonstration and discussion of the IIF staining analyzed by confocal microscopy, several observations must be stressed a priori: (1) in early stages the fusion of myoblasts into myotubes is never synchronous; in other terms, at any given point in time, prefusion myoblasts and myotubes coexist. (2) In C2.7 cell fusion, myotube formation is never complete; at a given stage of myogenesis the rearrangement of the cytoskeleton and the buildup of the muscle system is abortive and no clear-cut sarcomeric organization is observed. This is in contrast with, for example, the Sol-8 myogenic cell lines which allow observation of fully differentiated sarcomeric structures and contracting myotubes in vitro [62]. (3) The arrest of normal myogenesis relates most obviously to the cytoskeletal organization of the syncitium formed by fusing myoblasts, since at late stages nuclei stick together like grapes, rather than aligning themselves in a well-separated longitudinal fashion (see e.g., Fig. 6). Figures 6 to 8 show the behavior of prosomes and the cytoskeleton of C2.7 myoblasts during the differentiation process, as observed by confocal microscopy and image analysis. In this series of experiments, we analyzed hundreds of focal sections and combinations of anti-prosome mAbs with anti-desmin or anti-actin polyclonal antibodies, observing the various prosomal antigens. Obviously, only a small selection of the data is presented. Note that in these experiments the p25K

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p-mAb was replaced by the p23K subunit-specific pmAb; furthermore, to reduce the number of pictures, micrographs showing various stages of differentiation were selected in preference to more homogeneous representation of given stages of fusion. Figure 6 shows the analysis by confocal microscopy of the dynamics of (a, b, c,) the p31K-specific prosomes and (d, e, f) the p23K-specific prosomes during the differentiation process ((a, d) Day 1; (b, e) Day 2; (c, f) Day 5 after induction of differentiation). Each image represents a projection in extended focus equivalent to three focal sections (section step 0.5 mm). Compared with growing myoblasts, there is an apparent immediate increase in the quantities of prosome antigens in postmitotic myoblasts and fusing myotubes. Most interestingly, prior to the fusion of the postmitotic myoblasts (Fig. 6a, Day 1), a particular abundance (if not exclusive presence) of some types of prosomes in the nucleus can be observed. This is the case for the p31K prosomes in Fig.6a (see cells at top right and bottom), where several myoblasts are visible, exclusively stained in the nucleus. (The observation reported here was also seen in fusing myogenic satellite cells [5]; the exclusive presence of prosome antigens in the nuclei of cells had been observed previously during early embryonic development, see [42, 43]; see Discussion.) It is interesting to note that the nuclear pattern observed here looks quite different from that obtained using the same antibody in growing myoblasts (Figs. 4C and 4D). In fact, in the case of dividing myoblasts, a fairly homogeneous staining pattern can be observed, except for the nucleoli, which are not labeled. On the other hand, in Fig. 6a, a speckled nuclear pattern can be seen for p31K-specific prosomes, in particular in the nuclei of postmitotic myoblasts prior to fusion. In the already fused or later phase postmitotic prefusion myoblasts (see cell at bottom right in Fig. 6a), increased quantities of these p31K-containing prosomes are visible in the cytoplasm of the syncitium, in particular in the perinuclear area. It is noteworthy that, although fusing C2.7 myoblasts never reach the stage of contracting myotubes, the beginning of a seemingly pseudo-sarcomeric organization can be observed in the p31K–prosome pattern (Fig. 6c, zone outlined by arrows). It seems that, in places, p31K-specific prosomes occupy specific regions in a striated pattern, rather than in isolated ‘‘pearl on a string’’ alignments. The p23K-specific prosomes follow a different pattern. Absent from the nuclei in dividing myoblasts (data not shown) and during the early stages of fusion (Fig.6d), p23K-stained prosomes appear transiently at Day 2 of differentiation in the nuclei of the syncitium (Fig.6e) and disappear at later stages (Fig.6f). Having reported this subtle but significant movement of prosome antigens to the nuclei at the onset of myogenesis, we may comment on more general obser-

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FIG. 6. Reorganization of the cytolocation of p31K- and p23K-specific prosomes observed by confocal microscopy in C2.7 myoblasts during myotube formation. C2.7 myoblasts were instantaneously fixed for 20 min with 1% paraformaldehyde in the presence of 20% Triton X-100 at 377C at different times after the induction of the differentiation process. The cells were stained with (a, b, c) the antip31K p-mAb or with (d, e, f) anti-p23K p-mAb. The fixed prosome antibodies were secondarily detected by FITC-labeled goat anti-mouse IgG. Each micrograph represents the projection in extended focus of the equivalent of three focal sections, obtained in a step of 0.5 mm. (a, d) Day 1, (b, e) Day 2, (c, f) Day 5 after differentiation. The arrows outline an area of c where a faint cross-striation of the prosome pattern may be noticed (bars, 10 mm).

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FIG. 7. Relocalization of the p31K-containing prosomes observed by confocal microscope in C2.7 myoblasts during myotube formation. C2.7 myoblasts were instantaneously fixed with 1% paraformaldehyde in the presence of 10% Triton X-100 at 377C for 20 min, at different times after the induction of the differentiation process. The cells were stained with (a, b, c) anti-p31K p-mAb in combination with (a*, b*, c*) a polyclonal anti-cytoplasmic actin antibody. (a9, b9, c9) show colocalization (yellow), indicated by computer superposition of the two signals; not colocalizing areas maintain the original colors (green, prosomes; red, actin). The fixed prosome antibodies were secondarily detected by FITC-labeled goat anti-mouse IgG. The anti-actin antibodies were revealed with Texas red-labeled goat anti-rabbit IgG. Micrographs obtained from single focal planes are shown. (a) Day 2, (b) Day 4, (c) Day 5 after induction of differentiation (bars, 10 mm).

vations regarding the cytoskeleton and prosome patterns during myotube formation. The most striking feature in these pictures of the cytoskeleton during the differentiation process is the disappearance of the mul-

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tidirectional organization of the tangled desmin filaments and the straighter microfilaments, along with the gradual buildup of a longitudinal filamentous alignment, running from one pole to the other of fusi-

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form prefusion myoblasts as well as in the myotubes (Figs. 7a*, 7b*, and 7c*; and data not shown). The prosome pattern follows this cytoskeletal reorganization. At the same time there is a striking increase in concentration of prosomes in the nucleus and then in the cytoplasm of fused myoblasts. At early stages of fusion a partial colocalization of p23K-containing (or p25K-containing) and p31K-containing prosomes with desmin filaments can be observed. This desmin coincidence starts at and around the nuclei where prosomes assemble and form sort of ‘‘caps’’ which eventually fuse together (see in Figs. 6a– 6c single cells and growing myotubes stained by the p31K p-mAb). Desmin filaments coinciding with prosome antigens can be better observed on individual focal planes; at later stages, when nuclei start to glue together, the segregation of prosomes from the desmin filaments becomes more pronounced (data not shown). Figures 7 and 8 show the results concerning the prosomes in relation to the actin network. Prosome –actin colocalization seems to be significant throughout the differentiation process. When viewing these pictures stained for actin, one should remember that growing myoblasts, like all types of cells, contain the cytoplasmic types of actin, organized into microfilaments and stress fibers; this should be clearly distinguished from the sarcomeric actin in the adult myotubes, which is concentrated around the Z lines. Figure 7 shows corresponding single focal sections obtained by double-labeling immunofluorescence and observation by confocal microscopy using the anti-p31K prosome antibody in combination with anti-cytoplasmic actin polyclonal antibodies at different days after differentiation ((a, a*,a9) Day 2, (b, b*, b9) Day 4, (c, c*, c9) Day 5 after induction). As already mentioned, at early stages of fusion, the bundles of cytoplamic actin and stress fibers, which can be observed to run in all directions throughout the growing myoblasts, are very rapidly replaced by longitudinal actin filaments which run parallel to the desmin system. It is noteworthy that at these stages, when a considerable number of postmitotic myoblasts are present and an exclusive nuclear segregation of the p31K-specific prosomes is observed, there is no appreciable colocalization between

prosomes and actin filaments (Fig. 7a9). This is in contrast with what was observed in dividing myoblasts, where an extensive coincidence of prosome and actin networks is present, as visualized by IIF and confocal analysis (cf. Figs. 4C and 4C*). A partial colocalization can again be observed in fusing but still mononucleated cells (Fig. 7a9, upper right corner), where coincidence is quite evident, in particular on fibers lining the perinuclear area. As cells fuse, there are larger zones of prosome –actin coincidence in the cytoplasm, whereas in the nuclei 31 K prosomes do not seem to colocalize with actin (Figs. 7a9– 7c9). Only single focal sections are presented in Figs. 7 and 8, to ensure that one is observing real colocalization between prosomes and actin filaments, and not just an artifactual mixing of colors that can occur in multiple projections. Furthermore, as an additional control, we performed a quantitative analysis of pixel relation in small selected areas (Figs. 8: a, a*, b, b*). In these areas the percentage of the two signals for each spot was calculated at pixel level to test for real colocalization. Figure 8 shows a test of coincidence in which prosomes stained by (Figs. 8a, 8a*, 8a9) the p31K and (Figs. 8b, 8b*, 8b9) p23K pmAbs (red/gray) are double-labeled for actin (Figs. 8a, 8a*, 8a-, 8b, 8b*, 8b-, polyclonal anticytoplasmic actin Ab, green/gray). The cytofluorograms of the inserts indicate in the framed area real coincidence (yellow) by graphic overlay representation. The black and white magnifications of the ROI of Fig. 8 (a9, a-) and (b9, b-) indicate alignment of prosome staining in a pearl on a string (or rather ‘‘bird on the wire’’) fashion which is paralleled by the linear actin stain (the faint cross-striations in (a-, b-) are artefacts due to a 907 rotation of the pictures during image processing). Single pixel coincidence analysis (white spots in Figs. 8a* and 8b*) shows colocalization (1:1) on finer actin bundles of both types of prosomes which seems to exclude the fatter actin fibers. If one compares Fig. 8a* with 8b*, prosomes seem to occupy the fine bundles in a discontinuous spotted manner and in an amount that differs according to the p-mAb used. To summarize the overall observations (Figs. 6–8) concerning the dynamics of specific prosomes and the reorganization of the prosome network in relation to

FIG. 8. Test of coincidence by confocal microscope and image analysis of p31K- and p23K-containing prosomes and actin filaments in C2.7 myoblasts during myotube formation. C2.7 myoblasts were instantaneously fixed with 1% paraformaldehyde in the presence of 10% Triton X-100 at 377C for 20 min at Day 2 after the induction of the differentiation process. The cells were stained with (a, a*, a9) anti-p31K p-mAb and (b, b*, b9) anti-p23K p-mAb in combination with (a, a*, a-, b, b*, b-) a polyclonal anti-cytoplasmic actin antibody. The fixed prosome antibodies were secondarily detected by FITC-labeled goat anti-mouse IgG. The anti- actin antibodies were revealed with Texas red-labeled goat anti-rabbit IgG. (a, b) Composite overlay image of single optical sections of about 0.6 mm showing the (a) p31K-specific and (b) p23K-specific prosome stain in combination with actin (red, p23K and p31K prosomes, green, actin; note that colors are inverted compared to Fig. 7). The inset in the left bottom corner shows the cytofluorograms of the region of interest (ROI). The selected yellow area represent (a) p31K or (b) p23K colocalization with actin (red:green stain, 1:1). (a*, b*) Colocalizing spots are superimposed in white color onto the composite overlay image (a, b) defined by the selected yellow cytofluorogram area (see Discussion for the interaction between p31K and actin). (a9, b9, a-, b-) Magnifications of the ROI selected in a and b. (a9, b9) p31K and p23K stain shows punctuated linear patterns; (a-, b-) show the linear actin network (bundles exhibit a jagged aspect; this artefact is due to ROI image rotation: ‘‘aliasing’’ effect). The white dots correspond to signal superposition (1:1) analyzed at individual pixel level (bars, 10 mm).

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the cytoskeletal systems during myotube formation of C2.7 myoblasts, we would like to stress the following points: (1) One of the earliest signs of transformation of the exponentially growing myoblasts into still mononuclear prefusion myoblasts is the movement of some prosome antigens to the nuclei, exemplified here by the p31K antigen which, during a short period, can be seen in the nucleus exclusively. The other antigen analyzed, the p23K-type prosomes, behaves in a different fashion: scarcely present in the nuclei of prefusion myoblasts, they enter the nuclei for a short period, when syncitia are already formed and resort quite rapidly to the cytoplasm. (2) Localization of prosomes of a specific kind coincides, to some extent, with the cytoskeletal network of desmin type at an early stage of fusion, and more massively and progressively with the actin filaments, in both prefusion myoblasts and during fusion. (3) The main reorganization of the cytoskeletal network concerns the longitudinal alignment of both desmin and actin filaments running in parallel fibers from pole to pole in the cells and the syncitium. In the exponentially growing myoblasts, these run in all directions, being tangled in the case of IF and in parallel bundles, but aligned in different orientations, for actin. (4) The prosome–actin and desmin colocalization is transiently ruptured, when the prosomes tested move to the nuclei, and is progressively reestablished. This occurs already in the prefusion myoblasts, when the longitudinal alignment commences; this process follows a different time course depending on the different types of prosome tested. (5) When myotubes form, a striking increase in prosome concentration is observed, starting at the polar caps in the perinuclear area and progressively on filaments oriented toward the poles of the fusiform myoblasts, and later of the myotubes. (6) Somewhat diffuse desmin –prosome colocalization is observed early in fusion, when relatively few nuclei are present, and decreases later on, whereas the actin– prosome colocalization persists on clearly seen filaments in late stages of the fusion process. (7) Only part of the actin filaments seem to be occupied by prosomes of either kind, in subunit-specific intensity; finer bundles being preferred in relation to the fat ones (this is perhaps because the p-mAbs have difficulty in penetrating them). DISCUSSION

In this first systematic investigation of prosome distribution in myogenic cells and during fusion of myoblasts of the C2.7 cell line, we confirm a preliminary observation [31] that in muscle cells, as in the other types of cells studied, prosomes colocalize with the organ-specific IF network which, in this case, is desmin. But surprisingly, in contrast with previous investigations, we also observe colocalization of prosomes with the actin-based microfilaments and stress fibers. These

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new observations result from our use of a new technique of fixation/extraction [53], applied prior to IIF examination with p-mAbs. Furthermore, we report the reorganization during induced myotube formation of the prosome particles and their networks in relation to the cytoskeleton. Of prime importance in this context, which bears on a basic characteristic of the prosome system, is the biochemical observation of a change in the subunit composition of the particles upon differentiation of the myoblasts into myotubes. Prior to discussion and subsequent interpretation of our new observations, we must comment on some technical points. The prosome –IF colocalization reported previously [29 –31] was particularly evident in cells that were Triton-extracted prior to fixation (the method called here ‘‘delayed fixation’’) and IIF staining. The rationale for applying this technique which led to the extraction of 30–60% of prosomes (according to the antigen tested), leaving behind only the cytoskeletonbound particles, was dual: (i) Triton extraction removes all labile-bound proteins and permits observation of the cytoskeleton structure at high resolution, extracting soluble proteins in general, and, in particular, nonpolymerized monomers (prone to react with the Abs used); (ii) when cells are extracted by Triton in dynamic steady state (377C) or in ‘‘frozen’’ state (07C precisely), messenger RNA and polyribosomes remain bound to the cellular structures, and only free ‘‘runoff’’ ribosomes are extracted [63]. Since we were especially interested in the mRNA-bound prosomes [32, 40], this procedure was suitable. It was obvious, however, that cells treated in such a harsh manner could not possibly represent the in vivo situation. On the other hand, fixation at 377C by paraformaldehyde, followed by classic permeabilization by detergents for IIF, gave only diffuse patterns and did not permit detailed visualization of the various cytoskeletal networks. We therefore devised a technique (called here ‘‘instantaneous fixation’’) in which cells, maintained at physiological temperature, are simultaneously fixed and extracted. Accordingly, instantaneous fixation of proteins and crosslinking to the cytoskeleton by formaldehyde competes with simultaneous solubilization by the high detergent concentration (10% Triton X-100). This removes all proteins and protein complexes which, at the time of fixation, are not at a molecular distance that permits chemical reaction with the polymerized cytoskeletal filaments. This approach has proven to be successful, as shown here and in a parallel publication bearing on epithelial and fibroblastic cells [53], in that it allows clear observation of cytoskeletal networks, in spite of immediate fixation, and extraction of monomers, and of those pericytoskeletal structures that are not genuinely linked to their polymerized counterpart, e.g., a mutant type actin [53]. To understand the IIF patterns observed, it is important to recall the biochemical demonstration that

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in the myoblasts, as in all other types of cells tested previously, prosome antigens were found exclusively as part of the 20S particles and are not present as free subunit antigens (see Fig. 1 in this paper, [31], and discussion in Ref. [33]). The two-dimensional subunit pattern of prosomes from C2.7 myoblasts, shown here for the first time, falls within the framework of patterns observed previously in other types of cells, including those from embryonic chicken muscle [40, 64]. Nevertheless, viewing the four patterns shown, there are obvious differences with regard to the presence or absence, or the quantitative representation, of some of the prosome subunits. The difference between the prosome pattern of mouse (myoblasts) and its closest phylogenetic relation, the rat (erythroblasts), is particularly interesting. Prosomes are mosaic structures and this variation in subunit mosaic was found in general to depend on the type of cell and differentiation pattern (see discussion in [33]). The prosomes biochemically analyzed in C2.7 myoblasts seem to obey the same rule. Of prime importance in the prosome/proteasome story is, however, the observation reported here that in myotube formation the subunit pattern of the particles changes. Such changes were seen by IIF in several differentiating systems ([42, 43, 65], discussion in [33]) and were recently demonstrated biochemically by the variable subunit pattern of human blood cells [41, 47]. It has also been reported that in HeLa cells, treated with interferon, the change in physiological ‘‘status’’ brings about a variation in the prosome subunit pattern [61]. Such observations seem to indicate that the prosomes may modulate their functional specificity in protein homeostasis, controlled by protein synthesis and breakdown, to meet the particular needs of a given differentiated cell. Turning to the main point of the present investigation which concerns prosome cytodistribution in the cytoskeleton system, we first comment on the surprising presence of prosomes at the level of the actin system, which had not been observed previously. If one compares some of the micrographs reported in this investigation (Fig.3), it is evident, however, that the most stable relationship is that between prosomes and the IF system (here represented by desmin) which survives the rather harsh Triton extraction. Although supported by electron micrographs in immunohistochemical studies [29, 53], there is still no absolute proof that the colocalization observed amounts to a direct molecular interaction between prosomes and the intermediate filaments. As shown here, Triton extraction and, to a lesser extent lowering of the temperature, largely eliminates the pattern of prosome –actin colocalization; this interaction must therefore be of a dynamic nature. Its dissociation is reminiscent of the lability of microtubules at subphysiological temperatures, which, furthermore,

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partially disassemble when exposed to detergents unless stabilized by tubulin-stabilizing buffers [63, 66, 67]. It is surprising, in view of the apparent stability of the physiological IF–prosome interaction, and the lability of the prosome colocalization with the actin network reported here, that prosomes have been recently observed to interact directly with actin, at a molecular level, in an organized manner in vitro [53]. On the other hand, prosome – IF interaction in vitro has been observed but is not easily obtained (Bloemendal and colleagues, personal communication). What we observe in cells may therefore relate specifically to more complex biochemical in vivo interactions, rather than to a mere chemical tropism involving prosomes and IF molecules, as is the case for the prosome –actin interaction. The variations in the IIF patterns related to any given prosomal antigen studied are particularly interesting; they are evident throughout this study, apparently in a qualitative as well as a quantitative manner, and may relate to differential cytodistribution of specific prosome particles. One should point out in particular the staining by the p25K subunit-specific p-mAb (Fig.3A), and the absence in nucleoli of p31K-specific prosomes (Fig.3B). Another similar observation relates to the more evident desmin-like pattern of the p25Kcontaining prosomes (Figs.3A and 5) when compared to the preferential actin-like pattern of the p31K-specific prosomes (Figs.3B and 4). These data all confirm the variability of the prosomal composition observed biochemically; they seem to indicate that prosomes of a specific kind form subnetworks [46] and distribute in a particular manner among the cellular compartments, as well as among the intermediate filament and actin networks [5, 50, 65]. This is new evidence for the coexistence in one cell of prosomes possessing different subunit compositions observed first in Ref. [48]. Although in some cases the different pattern observed may occasionally be due to variations in exposure of specific antigenic epitopes of prosome complexes, in many cases this possibility seems excluded (see discussion in [33], and [53]). A further degree of heterogeneity in the prosome cytodistribution is introduced when the dividing myoblasts, stained for prosomes and the cytoskeleton, are observed in the three-dimensional space, which has become feasible with confocal microscopy. The data shown here indicate that both the density of the prosome networks and their distribution throughout the cell are heterogenous. Some types of prosomes are enriched in the perinuclear area, some follow the actin filaments at the bottom of the cell, close to the surface of adherence, whereas others seem to extend into the three-dimensional space, in some cases up to the top of the cell (see Fig.4D). It is impossible to draw any conclusions from the observations shown here apart from the general statement that, possibly, the distribu-

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tion of specific types of prosomes in the three-dimensional space and among the cytoskeleton systems is related to particular functional sectors of the cell, such as the nucleoplasm, nuclear membrane, cytoplasm, plasma membrane. As reported previously, prosomes of a specific type were found at the level of the bile canaliculi in hepatocytes [65]. In the context of this paper, it is most interesting to recall their intercalation into the sarcomeric structure of skeletal muscle which was observed by our team ([5]; and Foucrier, Scherrer and colleagues, unpublished observations). To attempt to interpret this heterogeneity in relation to prosome function is, however, very difficult at the present stage of investigation. It may bear some relation to the mechanisms and dynamics of protein biosynthesis and turnover, as has been discussed elsewhere (review in [33]). Unfortunately, C2.7 myoblasts do not undergo a complete differentiation process, and never form a fully developed sarcomeric pattern, as can be observed upon fusion of satellite cells [50] or some types of myogenic cell lines such as the SOL-8 cells which reach the stage of in vitro contracting myotubes [62]. The small quantities of satellite cells that can be produced, and the necessity for growing SOL-8 cell lines on feeder layers of fibroblasts, excluded them from the present investigation. We chose C2.7 cells which can be investigated biochemically as well as cytologically. Furthermore C2.7 myoblasts represent a classical model for the study of myogenesis and the function of various myogenic factors by molecular genetics [68]; this approach is, henceforth, accessible on the basis of the present investigation for direct studies of the function of the prosome system. The process of differentiation of C2.7 myoblasts into myotubes was analyzed here by confocal microscopy and image processing, which enabled us to study prosomes in individual focal sections of the fusing myotubes. Close inspection of the superposition of confocal patterns obtained with two anti-prosome monoclonal antibodies compared with desmin (not shown) or, on single focal plans, with actin (Figs. 7 and 8) enabled us to make the following observations. (1) Once more, as in the dividing myoblasts discussed above, when one antigen is compared with the other in relation to the cellular compartments and the desmin and, in particular, the actin networks, the colocalizations differ quantitatively and qualitatively, in a time-related manner after induction of differentiation, indicating the existence of prosome subnetworks involving particles of particular subunit composition. (2) The most fundamental structural change in growing myoblasts is the rearrangement in prefusion myoblasts of the IF and actin filaments which begin to span from one pole of the fusiform cells to the other, gradually abandoning the tangled- or stress fiber-type patterns observed in the growing myoblasts. In the

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course of this realignment, prosomes seem to follow the desmin network and later, increasingly, the actin system. Taking into account the fact that both systems are known to carry mRNA [22] and the latter carry the polyribosomes [21, 63] this observation may possibly be related to a reprogramming of protein biosynthesis, given that prosomes are part of mRNP (review in [33]). (3) Also prior to fusion, there is a considerable increase in prosome concentration in the cytoplasm, and the sort of ‘‘polar cap’’ surrounding the nuclei in myoblasts (see Figs. 5A, 5B, and Figs. 6a–6c) is reinforced, before the prosomes align in relation to the poles of the fusiform prefusion myoblast and, later on, along the myotubes (Fig. 6). At this stage, the prosome –desmin colocalization is fairly strong in the perinuclear area but starts to extend into the filamentous system of the fusing myotubes. This observation may be of interest if one considers that in muscle cells specific mRNAs have been found by in situ hybridization in this perinuclear sector prior to and during myotube formation, whereas they are found at the periphery of the cells in myoblasts (review in [21]; [69 –71]; and De Conto, unpublished results). (4) The first important sign of modification of the prosomal cytolocalization is the movement of prosomes of a specific kind into the nuclei, and their almost complete disappearance from the cytoplasm. This may possibly indicate that the growing cells become prefusion myoblasts. This characteristic pattern changes more or less rapidly at later stages, when these prosomes partially (31K type) or completely (23K type) disappear from the nuclei; again this time-related phenomenon is related to the type of prosome analyzed, i.e., its subunit composition. This most interesting back and forth movement to and from the nucleus is reminiscent of that into the nuclei of all types of prosomes during blastulation in both amphibian and avian embryos [42, 43]. We may recall that in blastulation the zygotic genome is gradually activated, implying the differential modification of chromatin allowing for transcription. On this basis one might speculate that reprogramming at a genomic level may relate to this astonishing movement toward the nuclei of a small selected fraction of prosomes in prefusion myoblasts. Proteinase function of a particular kind, necessary for genomic reprogrammation, is one of the possibilities. On the other hand, it is known that prosomes (or 20S proteasomes) not only have been foud in mRNPs [32, 40] but also have been observed to associate with analogs of transcription factors within higher Mr complexes [72]. The presence and abundance of prosomes at the level of active chromatin reported previously [42] is not yet understood, but it is important to mention this point, since it may constitute the basis for further experimentation relating to reprogramming of gene expression. (5) This nuclear displacement of prosomes is related to the clear-cut rupture in their alignment with the

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cytoskeleton of both the IF and actin type; this occurs at a time when reprogramming of myogenesis takes place at the level of gene expression, i.e., during protein biosynthesis which is controlled by trans-acting factors at transcription level and beyond [68]. The reappearance of prosomes on the cytoskeleton occurs gradually and in a manner that depends on the type of cytoskeletal network and prosome specificity. Once more this phenomenon may be related to proteolysis as well as to mRNA processing and selective, cell-sector-specific transport and translation (discussion in [33]). (6) The superposition of the prosomes with actin in a pearl on a string manner is of great interest (Figs. 6 and 8). In some areas of growing myotubes a crossstriated pattern of prosome staining can be noted, formed by lateral alignment of such strings (see area outlined by arrows in Fig. 6c). This discontinous colocalization may coincide with the abortion of the buildup of the sarcomeric structure, which, in contrast, continues until completion in fusing satellite cells [50], and also in vivo during fetal myogenesis of the rat [5], when a pseudo-sarcomeric prosome pattern is fully formed. In both cases, the formation of a pseudo-sarcomeric structure by the prosomes is observed possibly concomitantly with or even prior to the integration of sarcomeric actin into the future sarcomeric structure (Foucrier and colleagues, unpublished observations). Indeed, it should be recalled once again that cytoplasmic ß- and g- actins exist in growing myoblasts, as in most types of cells, and are at the basis of the structure of microfilaments and stress fibers. The synthesis of sarcomeric actin starts prior to fusion of myoblasts into myotubes and the latter are thereafter built into the sarcomeric structure. One of the aims of our future work is to use antibodies specific to sarcomeric actin to distinguish at a fitting level of resolution for this type of investigation, the organization of the microfilament/ stress-fiber pattern relating possibly to protein biosynthesis, and thus compare it to the structural buildup of the sarcomeric actin pattern. These detailed highresolution investigations will be carried out on SOL-8 cells, which will allow us to obtain and analyze contracting myotubes. As a continuation of this investigation of C2.7 cells, it should be possible to address the question of the colocalization of prosomes with desmin and actin at a higher-resolution structural level, as well as biochemically, and to probe for their colocalization with mRNAs by in situ hybridization. Furthermore, we shall test the possibility that prosomes participate directly, in some way, in myogenesis. Indeed, the data reported here are encouraging this perspective, and provide the basis for an investigation of the function of prosomes in muscle differentiation by molecular genetics and biochemistry.

ies. We thank R. Schwartzman, F. Madrell, and A. Kropfinger for their help in the preparation of the manuscript. This research was supported by grants from the Centre National de la Recherche Franc¸aise (CNRS), the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), the Ligue Nationale Franc¸ aise contre le Cancer, and the Association pour la Recherche contre le Cancer (ARC). C. Chezzi was supported by Scientific Research grants from the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica, ‘‘Quota 60%,’’ U. Aebi and R. Sutterlin were supported by the Swiss National Science Foundation, and the M.E. Mu¨ller was supported by the Foundation of Switzerland.

We are grateful to J. Foucrier for many suggestions and to G. Gabbiani and A. Natali for helpful discussions and the gift of antibod-

27. Fuchs, E., and Weber, K. (1994) Annu. Rev. Biochem. 63, 345– 382.

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Received September 12, 1996 Revised version received January 21, 1997

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