GDP Cycles Determine the Intracellular Location of the Late Endocytic Compartments and Contribute to Myogenic Differentiation

Experimental Cell Research 246, 56 – 68 (1999) Article ID excr.1998.4284, available online at http://www.idealibrary.com on

RAP1A GTP/GDP Cycles Determine the Intracellular Location of the Late Endocytic Compartments and Contribute to Myogenic Differentiation Ve´ronique Pizon,* ,1 Francisca Me´chali,† and Giuseppe Baldacci* *Laboratoire de Ge´ne´tique et Biologie Mole´culaire de la Re´plication, CNRS–UPR 9044, 7 rue Guy Moquet, BP 8, 94801 Villejuif, France; and †CRBM–CNRS, 1919, Route de Mende, BP 5051, 34283 Montpellier, Cedex 5, France

tion, myoblasts irreversibly withdraw from the cell cycle and fuse to form multinucleated myotubes [1–3]. Concomitant with cell differentiation, successive synthesis of a complex variety of proteins occurs. Expression of different proteins is relevant of the cell-differentiating stage. For instance, myogenin is expressed when myoblasts are triggered to differentiate [4] whereas troponin T is expressed in myotubes when differentiation is completed [5]. Myogenic differentiation is also accompanied by drastic morphological cell modifications. These changes are connected in part with the assembly of new membranes and in part with the relocation of subcellular organelles such as the nucleus, the centrosome, the Golgi complex, and the late endocytic compartments [6, 7]. Current research concerning myogenic differentiation suggests that lysosomal proteolytic enzymes could play an important role during differentiation [8 –11]. Indeed, these enzymes, located in the degradative organelles late endosome and lysosomes [12, 13], could support the intracellular reorganization processes needed for the conversion of myoblasts into myotubes. RAP1A protein is a small GTP-binding protein of the RAS superfamily [14]. Like RAS protein, RAP1A cycles between a GTP and a GDP-bound state. By analogy with RAS mutants, substitution of Gly with Val at position 12 of RAP1A (RAP1AV12) reduces RAP1A GTPase activity and consequently gives rise to a constitutively GTP-bound protein. Conversely, substitution of Ser by Asn at position 17 of RAP1A (RAP1AN17) confers a higher affinity for GDP [15]. GTP/GDP cycles are regulated by RAP1A interactions with proteins displaying GTPase-activating activity [16 –18] or exchange factor functions [19, 20]. Various proteins interacting with RAP1A also interact with Ras or Ras-related proteins. Interactions with Raf-1 [21], RasGAP [22], RalGDS [23], RGL [24], Rlf proteins [25], Krit1 [26], and Nore1 [27] could account for the various functions described for RAP1A. RAP1A protein was initially hypothesized to antagonize oncogenic RAS function [28]. The physiological significance of

RAP1A protein is a small Ras-like GTPase that accumulates during muscle differentiation. In this study, we observed variable intracellular location of the endogenous RAP1A protein and concomitant relocation of the late endocytic compartments in differentiating myogenic cells. By monitoring the nucleotide-bound form of RAP1A protein, we established that the various protein localizations were related to the GTP/ GDP-bound state. To carry on our study, we raised stable myogenic cell lines overexpressing wild-type or mutated forms of RAP1A. Myoblasts overexpressing the GTP-bound mutant did not display specific changes of RAP1A and of late endocytic compartments locations. In contrast, the GDP-bound mutant clustered with acidic structures in the perinuclear region of myoblasts. In addition, we observed that overexpression of GDP-bound RAP1A protein induces disturbances in the maturation process of the lysosomal enzyme cathepsin D. Whereas ectopic expression of wildtype or GTP-bound RAP1A proteins inhibited myogenic differentiation, the GDP-bound mutant favors myotubes formation. From our results, we propose that RAP1A protein may regulate the morphological organization of the late endocytic compartments and therefore affect the intracellular degradations occurring during myogenic differentiation. © 1999 Academic Press

Key Words: RAP1A; Ras-like protein; late endosome; lysosomes; myogenic differentiation.

INTRODUCTION

Differentiation of skeletal muscle cells involves three main stages characterized by specific gene expression and cellular morphological features. In culture, myoblasts grown in the presence of growth factors remain in a proliferative state. Following growth factor deple1

To whom correspondence and reprint requests should be addressed at IFRC1, CNRS–UPR 9044, 7 rue Guy Moquet, BP8, 94801 Villejuif, France. Fax: 133146789980. E-mail:[email protected]. 0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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this inhibition remains uncertain and little data indicate that RAP1A could play such a role in nontransformed cells. Although the precise function of RAP1A remains unresolved, its presence in the late endocytic compartments of various cell types suggests that it might be involved in degradative processes [29]. Analyses of RAP1A expression and location during myogenic differentiation also support this speculation. Indeed, RAP1A protein builds up when drastic cell architectural modifications occur during myogenic differentiation. Furthermore, during skeletal myogenesis, RAP1A accumulates in muscle regions subject to significant morphological reorganization [30]. To study whether RAP1A protein is required during myogenic differentiation and whether it is involved in intracellular degradative processes, stable myogenic cell lines expressing wild-type or mutated proteins were derived. After evaluating the effect of the various ectopic RAP1A proteins on cell growth and differentiation, we determined their intracellular location. Visualization of the late endocytic compartments showed that location of these compartments was related to the nucleotide bound form of RAP1A protein. Furthermore, analysis of cathepsin D indicated that relocation of the late endosomal compartments had physiological effects on this lysosomal enzyme maturation. Since late endocytic compartment relocation is a normal process of myogenic differentiation, we suggest that RAP1A protein may regulate the structural organization of late endosome and lysosomes and therefore influence intracellular degradative pathways. MATERIALS AND METHODS Chemicals. DOTAP, hygromycin B, and restriction enzymes were from Boehringer. Glutathione–Sepharose 4B was from Pharmacia Biotech; acridine orange, cytosine b-D-arabinofuranoside, Giemsa stain, and tetracycline hydrochloride (Tc) were from Sigma; May– Grunwald stain was from RAL Inc. Taq polymerase was from Epicentre technologies. Geneticin, gentamicin, Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were from Gibco BRL. Fetal calf serum was from D. Dutsher Inc. Plasmids constructs. Inducible RAP1A vectors: human RAP1A cDNA [14] was cloned in the pGEM3 vector. Mutants were constructed by site directed mutagenesis with the oligonucleotides: 59CTTGGTTCAGTAGGCGTT-39, for RAP1AV12; 59-CGTTGGGAAGAATGCTCTGACA-39, for RAP1AN17. From these constructs a Sty1 fragment encompassing the mutated nucleotides was inserted into the corresponding sites of the pGEM3 RAP1A cDNA. The various pGEM3 RAP1A constructs were then introduced into the EcoRI– BamHI sites of the pUDH10-3 [31]. The N-terminal GFP-tagged constructs were generated by polymerase chain reaction by introducing an EcoRI site before the initiation codon of the pGEM3 RAP1A constructs with the primer 59-GTGAATTCTATGCGTGAG-39. This EcoRI site was used to ligate the RAP1A cDNAs to the pEGFP-C1 vector (Clontech Laboratories). All the constructs were sequenced completely. Establishment of the RAP1A cell lines. For constructing stable transfected cell lines overexpressing RAP1A proteins in an inducible manner, mouse myogenic C2 cells [32] grown as described previously [30] were cotransfected with the pUHD15-1 plasmid that provides

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the coding sequence of the tetracycline-controlled transactivator [31] and with the pSV2-neo plasmid conferring resistance to geneticin. After selection, using a luciferase activity assay, several cell lines were screened for their tetracycline-regulatable expression. The C2RT20 clone displaying a barely detectable luciferase activity in the presence of Tc was subsequently cotransfected with the RAP1A plasmids and with the pREP4 vector (Invitrogen) conferring resistance to hygromycin B. Several stable cell lines were isolated and analyzed for each RAP1A construct. The control cell line was established by transfecting C2RT20 cells with the empty pUDH10-3 vector. To generate cell lines expressing the GFP-tagged RAP1A proteins, C2 cells were transfected with the various GFP constructs. Single clones selected by growing cells in the presence of geneticin were isolated and recombinant proteins expression was verified by immunofluorescence experiments. All transfections were performed using DOTAP as described by the manufacturer. Cell culture and vital staining. RAP1A transfectants were grown in DMEM supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, 0.25 mg/ml streptomycin, 250 mg/ml hygromycin B, and 800 mg/ml geneticin. Cells were induced to differentiate by growth in 1% FCS. A total of 1.5 mg/ml of Tc was added daily in the medium when cells were grown in presence of the drug. For nucleotide-bound RAP1A determination experiments, 24 h prior protein being extracted, cells were exposed to 10 mM cytosine b-D-arabinofuranoside to eliminate undifferentiated myoblasts. For cytological analysis cells were fixed in methyl blue– eosine methanol medium for 30 min before Giemsa staining for 15 min (May–Grunwald Giemsa staining). For acridine orange vital staining, cells plated on glass coverslips were incubated in DMEM phenol red-free medium for 5 min at 37°C in 10% CO 2 atmosphere with 2.5 mg/ml acridine orange prepared in PBS. After three washes with warm medium (37°C), cells were mounted and viewed immediately with a Leica DMRD microscope using a rhodamine filter. Antibodies, immunofluorescence microscopy, and confocal laser scanning microscopy. Affinity-purified RAP1 antibody was prepared as described previously [29]. Anti-troponin T antibody was from Amersham. Anti-myogenin antibody F5D was kindly provided by Dr. S. Leibovitch. Monoclonal M3A5 (Golgi apparatus) and polyclonal anti-rab7 antibodies were respectively provided by Dr T. Kreis and Dr M. Zerial. Anti-cathepsin D antibody was from Transduction Laboratories. Horseradish peroxidase and TRITC- and FITC-conjugated antibodies against rabbit, mouse, and rat IgG were from Pierce. Cell lines overexpressing RAP1A proteins by the Tet expression system were plated on glass coverslips prior fixation in methanol at 220°C for 3 min. Indirect immunofluorescence experiments were then performed as described [30]. Immunofluorescence of the various GFP-tagged RAP1A proteins was observed directly in living cells grown on coverslips. All immunofluorescence observations were performed with a Leica DMRD microscope using suitable filters. Confocal laser scanning microscopy was performed using a Leica confocal imaging system (TCS4D). Micrographs were processed in a Macintosh computer with Adobe Photoshop 4.0. Images were assembled and printed directly on dye sublimation printer (Colorease Kodak). Protein extracts and Western blots. Cells were washed twice and then scraped in PBS. Samples were centrifuged at 3000 rpm at 4°C in a table-top centrifuge. Pellets were homogenized in buffer containing 50 mM Tris, pH 8, 20 mM EDTA, 2% SDS, 1 mM PMSF (phenylmethylsulfonyl fluoride), and a mixture of protease inhibitors (10 mg/ml leupeptin and aprotinin, 1 mg/ml of pepstatin). Protein concentration was determined by the Pierce Micro BCA protein assay system according to the supplier’s protocol. Ten to 30 mg protein was loaded on a 13% SDS–polyacrylamide gel and transferred onto 0.2 mm Trans-Blot membrane (Bio-Rad). Blocking, antibodies incubations, washing, and chemiluminescence detection were performed as previously described [29]. Western blot photographs were digitized

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with an AGFA Studio Scan IIsi scanner and proteins quantified using NIH Image software package. GTP/GDP bound RAP1A determination. The pGEX-RGF97 vector was a generous gift of Dr. A. Wittinghofer (Max-Planck-Institut, Dortmund, Germany). The GST-tagged Rap binding domain of the Ral-guanine nucleotide exchange factor (RalGDS-RBD) was expressed in Escherichia coli and purified as described [33]. Cells were washed three times in ice-cold PBS, scraped, pelleted for 5 min at 3000 rpm at 4°C, and then overlaid with lysis buffer (50 mM Tris, pH 7.4, 200 mM NaCl, 1% NP-40, 1 mM MgCl 2, 1 mM PMSF, and protease inhibitors) for 20 min on ice. Lysates were centrifuged at 15,000 rpm for 10 min at 4°C and 30 mg of RalGDS-RBD coupled to glutathione–Sepharose beads were added to 600 mg of supernatant proteins. After 1 h at 4°C with slight agitation, beads were centrifuged, and washed two times in lysis buffer before Laemmli sample buffer was added. Precipitated and supernatant proteins were further analyzed by Western blot experiments.

RESULTS

Concomitant Intracellular Relocation of RAP1A Protein and of Late Endocytic Compartments during C2 Cells Differentiation The intracellular distribution of the endogenous RAP1 protein was studied by indirect immunofluorescence experiments during C2 cells differentiation. Using purified anti-RAP1 antibody undifferentiated myoblasts (Fig. 1A) and differentiated myotubes (Fig. 1C) displayed the typical RAP1A labeling (Figs. 1A9 and 1C9) observed previously [30]. Surprisingly, after 54 h of differentiation when fusion of adjacent C2 cells occurred (Fig. 1B), most of the RAP1A protein accumulates in the perinuclear region of the fused cells (Fig. 1B9). Given the association of RAP1A protein with late endosome and lysosomes, we investigated the intracellular distribution of these organelles during myogenic differentiation. Because of their internal content in acid hydrolases, these degradative compartments can be visualized within living cells with acridine orange, a metachromatic vital dye used as a fluorescent marker [34]. Whereas fluorescence was dispersed throughout all the cytoplasm of the C2 myoblasts (Fig. 2A) and myotubes (Figs. 2D and 2E), neighboring cells that just achieved fusion (Fig. 2B) displayed a perinuclear labeling (Fig. 2C). Altogether these data show the intracellular relocation of the late endocytic compartments during myogenic differentiation. They also point out that clustering of the acidic organelles parallels the perinuclear relocation of the RAP1A protein at the time of cell fusion. Analysis of the Nucleotide Bound RAP1A during C2 Cell Differentiation Studies based on the overexpression of mutated Ras-related proteins established that depending on their nucleotide linkage, these proteins can display different intracellular locations [35–37]. To investigate the nucleotide bound state of the RAP1A protein

during C2 cells differentiation, we used an assay previously developed to measure RAP1 activation in platelets [38]. This in vitro assay, based on the high affinity of the RAP-binding domain of Ral exchange factor (RBD) for the GTP-bound form of RAP1A protein, provides semiquantitative information since we cannot ascertain that all GTP-bound RAP1A is precipitated. As a matter of fact, we observed some differences in the amount of precipitated GTP-bound protein at the same time of differentiation in three independent experiments. Nevertheless, reported to the maximum of GDP- and of GTP-bound protein, we observed in each independent experiment reproducible variations of the relative amounts of each nucleotide-bound protein during the time course of the differentiation. Figure 3 shows the results obtained in one of these experiments. C2 myoblasts induced to differentiate for various time periods were lysed and equal amounts of protein were used to reveal total RAP1A protein and myogenic markers expressed during differentiation (Fig. 3D). GTP-bound RAP1A was identified by precipitation with equal amounts of purified GST-tagged RBD bound to glutathione beads (GST–RBD) as verified by red Ponceau staining of the membrane (Fig. 3C). GDP-bound RAP1A was estimated as the RAP1A protein remaining in the same amount of cell lysate after GST–RBD precipitation (Fig. 3B). The various proteins were further identified by Western blot analysis. After 96 h of differentiation, expression of the troponin T isoforms [39] was maximum (Fig. 3A, lane D96), indicating that myoblasts achieved their differentiation in myotubes. At that time maximum GTP-bound RAP1A protein was also detected. Compared to myoblasts (Fig. 3A, lane G), GTP-bound RAP1A was fivefold more abundant in myotubes than in myoblasts, as indicated by Western blot quantification (Fig. 3F). As expected [30], we detected threefold more total RAP1A protein in myotubes than in myoblasts (Fig. 3A). Therefore, the increase of GTP-bound RAP1A observed in myotubes could not solely result from protein accumulation inherent to myogenic differentiation. Myogenin is an early marker of differentiation expressed when myoblasts withdraw from the cell cycle and start to differentiate [40]. Since C2 cells cannot be synchronized, myogenin was mostly detected after 24 and 48 h of differentiation. By monitoring the GDP-bound RAP1A (Fig. 3A), we observed a transient increase of the protein at 48 and 72 h of differentiation when cells already stopped growing (Fig. 3E). Taken together, these data show that during myogenic differentiation maximum amounts of GDP- and GTP-bound RAP1A protein are detected at the time of cell fusion and in myotubes, respectively.

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FIG. 1. Immunofluorescence localization of endogenous RAP1A. Undifferentiated C2 cells (A, A9) and C2 cells induced to differentiate for 54 h (B, B9) or 72 h (C, C9) in 1% FCS. Indirect immunofluorescence experiments (A9, B9, C9) were performed with affinity purified anti-RAP1 antibody on cells displaying the phenotype observed by phase contrast optics (A, B, C). Bar, 30 mm.

Regulatable Expression of Wild-Type and Mutated RAP1A Proteins in Myogenic Cells To highlight biological effects induced by the nucleotide bound state of RAP1A protein during myogenic differentiation, stable myogenic cell lines overexpressing wild-type or mutated RAP1A proteins were established. We used an inducible system in which the ectopic expression can be repressed by Tc in the cell

culture medium (Tet expression system, [31]). Western blot analysis performed with total cellular proteins and purified anti-RAP1 antibody shows the extent of RAP1A GTPases synthesis in the different cell lines overexpressing the wild-type (wt), the GTP-bound (RAP1AV12) or the GDP-bound (RAP1AN17) RAP1A proteins (Fig. 4). Under proliferation conditions, Tc had no effects on the endogenous RAP1 protein expres-

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ever, when maximal activation of ectopic gene expression was achieved, 3 days after removal of Tc, about two-thirds of the clones displayed an additional threefold increase in RAP1A protein content. These data show the establishment of stable myogenic cell lines overexpressing, in a constitutive or regulatable manner, the wild-type or the mutated forms of RAP1A protein. Phenotypic Analysis of Stable Myogenic Cell Lines Expressing Various RAP1A Proteins In order to discriminate possible effects due to C2 cell subcloning, we analyzed the cell morphology of several comparable transfected clones. After May– Grunwald Giemsa staining, we observed that all the cell lines expressing the same RAP1A protein form display identical phenotypes. Representative cell morphology of each type of the transfected cells is shown in Fig. 5. Under growing conditions, C2RT20 cells (Fig. 5A) and cells overexpressing recombinant RAP1A proteins (Figs. 5B–5D) displayed an identical phenotype. Induced to differentiate for 3 days without Tc, C2RT20 cells (Fig. 5A9) and cells expressing the RAP1AN17 protein (Fig. 5D9) ceased proliferating and fused to form typical elongated polynucleated myotubes. In contrast, very few myotubes could be observed in cell lines overexpressing either wild-type (Fig. 5B9) or RAP1AV12 proteins (Fig. 5C9). Since growth arrest is an obligatory event in the normal program of myogenic differentiation [3], we verified that the inability of transfected cells to differentiate was not related to impaired growth arrest (data not shown). Altogether, these results indicate that forced expression of either wild-type or GTP-bound RAP1A proteins did not affect the myoblastic phenotype but blocked myotubes formation with a cell-cycle-independent mechanism. Biochemical Differentiation in Myoblasts Overexpressing Wild-Type or Mutated RAP1A Proteins

FIG. 2. Labeling of the intracellular acidic compartments during myogenic differentiation. Vital staining was performed on undifferentiated C2 cells (A) and on C2 cells induced to differentiate for 54 h (B, C) or 96 h (D, E) in 1% FCS. Cells were incubated with acridine orange in order to visualize the late endosome and the lysosomes compartments. The phenotypes of the differentiating cells are shown by phase contrast optics (B, E). Bar, 10 mm.

sion as observed in the parental C2RT20 and control cells. Despite the presence of the drug, leakage of the expression system causes a threefold increase of RAP1A protein in most of the transfected cells. How-

Myoblast fusion is not required for the induction of differentiation-specific gene expression [41]. Therefore, we tested whether the fusion-defective cells could express a biochemical marker of terminal differentiation. To limit or increase the amount of RAP1A protein before or at the time of differentiation, myoblasts were grown in 10% FCS in the presence (1) or absence (2) of Tc. Subsequently, after 3 days of differentiation in 1% FCS with or without Tc, expression of troponin T was followed by Western blot analyses. Independently of the presence of Tc, troponin T was detected in C2RT20 cells (Fig. 6A). In contrast, cell lines overexpressing wild-type RAP1A protein displayed a drastic inhibition of troponin T expression. Furthermore, accumulation of wt RAP1A protein in myoblasts inhibited troponin T

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FIG. 3. Analysis of the nucleotide bound form of RAP1 during differentiation. C2 cells induced to differentiate for the indicated time periods (hours) were lysed. 10 mg lysate was subjected to SDS–PAGE and Western blotting. The same red Ponceau stained membrane (D) was used to identify total RAP1A, myogenin, and troponin T proteins by immunoblots experiments (A). 30 mg of purified GST-tagged ral RBD bound to gluthatione–agarose beads was used to precipitate the GTP-bound RAP1A present in 600 mg of lysates. 30 mg depleted lysate was used to determine GDP-bound RAP1A. B and C, red Ponceau staining of membranes used to reveal RAP–GDP and RAP–GTP by immunoblotting (A), respectively. Quantification of immunoblot experiments (A) is shown in E and F.

expression even more. Identical results were obtained when the RAP1AV12 mutant was expressed (data not shown). When expressed at the time of differentiation, ectopic wt or GTP-bound RAP1A proteins also prevent troponin T synthesis as exemplified by cells expressing the RAP1AV12 mutant. As expected from phenotypic analysis, troponin T was expressed in the RAP1AN17 cell lines (Fig. 6B). By monitoring the appearance of the differentiation marker in the regulatable clones (17/1 and 17/3), we detected a 25% increase in the amount of troponin T when the RAP1AN17 protein was accumulated prior differentiation. This accumulation is not a side effect of the drug since troponin T expression was not influenced by Tc in control and in nonregulatable cells (17/8). To rule out the possibility that the nondifferentiating cells were arrested at an early stage of differentiation, we looked for the expression of myogenin. Since myogenin was barely detectable in

cells overexpressing wild-type and RAP1AV12 protein, we concluded that these proteins affect a very early stage of the differentiation program (data not shown). Collectively, these results indicate that forced expression of wild-type or GTP-bound RAP1A protein inhibits myogenic differentiation, whereas overexpression of the GDP-bound RAP1A protein seems to stimulate the differentiation program. Intracellular Distributions of the Mutated RAP1A Proteins The intracellular distribution of the different ectopic RAP1A proteins overexpressed with the Tet system were studied by indirect immunofluorescence confocal microscopy in nondifferentiated cells. Consistent with our previous observations, the endogenous RAP1A protein was associated with vesicular structures distrib-

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could be correlated with an increase of the GDP-bound RAP1A protein, location of the acidic organelles was investigated in cells expressing GFP-tagged RAP1A. Compared to control cells (Fig. 8E) or to cells expressing the GFP-RAP1AV12 protein (Fig. 8H), cells expressing GFP-RAP1AN17 (Figs. 8F and 8G) displayed acridine orange staining clustered nearby the cell nuclei. To verify that overexpression of GDP-bound RAP1A protein was indeed responsible for reclustering acidic organelles, these intracellular compartments were analyzed in Tet regulatable RAP1AN17 cell lines FIG. 4. Inducible RAP1A protein expression in stable cell lines. C2RT20 cells or selected clones transfected with the empty pUDH10-3 vector (Ct), wild-type RAP1A (wt18, wt28, wt19), RAP1AV12 (12/9, 12/12) ,or RAP1AN17 (17/1, 17/3, 17/6, 17/8, 17/9) cDNAs were grown in 10% FCS for 72 h in the presence (1) or absence (2) of 1.5 mg/ml of Tc. Total proteins (15 mg) were separated by 13% SDS–PAGE and analyzed by Western blot using affinity purified anti-RAP1 antibody.

uted throughout the cytoplasm of C2RT20 myoblasts (Fig. 7A). A similar distribution was detected in myoblasts overexpressing either wild-type or RAP1AV12 proteins (cell line 12/12) (Figs. 7B and 7C). The staining pattern of the RAP1AN17 mutant (cell line 17/9) was strikingly different, most of the labeling being clustered in a perinuclear region (Fig. 7D). Since overexpression of the untagged RAP1A proteins was low using the Tet expression system, stable C2 cell lines expressing GFP-tagged mutated RAP1A proteins were established in order to better localize the ectopic proteins. Figure 8 shows the location of the GFPRAP1AN17 and the GFP-RAP1AV12 proteins observed by direct immunofluorescence on living cells. These pictures are representative of the location of the various mutated proteins observed in different cell lines expressing identical RAP1A mutant. As previously observed, RAP1AV12 protein displayed a vesicular staining distributed in the whole cell cytoplasm (Fig. 8D), quite different from the one revealed by control cell line expressing the GFP protein (Fig. 8A). In most of the GFP-RAP1AN17 expressing cell lines we analyzed, the mutated protein was restricted in a perinuclear region (Fig. 8B). However, in a few cells, the GFP-RAP1AN17 protein was localized at a single side of the nucleus (Fig. 8C). Thus, using two different expression systems, overexpressed GDP-bound RAP1A protein displays a specific intracellular location. Overexpression of the GDP-Bound RAP1A Protein Induces Clustering of the Late Endocytic Compartments in Myoblasts Since our data suggested that during C2 cell differentiation relocation of the late endocytic organelles

FIG. 5. Phenotypes of parental and RAP1A-transfected cells in growing and differentiating medium. C2RT20 (A), wtRAP1A clone 28 (B), RAP1AV12 clone 12/12 (C), and RAP1AN17 clone 17/8 (D) cells were cultivated in growing cell medium (10% FCS) for 72 h in the absence of Tc before May–Grunwald Giemsa staining. To analyze the phenotype of the same cell lines in differentiating medium, cells grown in 10% FCS without Tc were switched to serum-free medium (1% FCS) in the absence of Tc. Three days later they were subjected to staining: (A9) C2RT20, (B9) wtRAP1A, (C9) RAP1AV12, and (D9) RAP1AN17. Magnification, 2003.

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ments showed that the Golgi apparatus location remained unchanged in these cells (data not shown). Collectively these experiments indicate that overexpression of wild-type or GTP-bound RAP1A proteins has no effect on the late endocytic organelles distribution. In contrast, expression of the GDP-bound RAP1A protein induces a specific relocation of the late endosomal compartments nearby the nucleus of undifferentiated myoblasts. Expression of Cathepsin D in Myoblasts Overexpressing GDP- or GTP-Bound RAP1A Proteins FIG. 6. Troponin T expression in C2RT20 and in cells overexpressing RAP1A proteins. (A) Protein extracts were prepared from C2RT20 or fusion-defective cell lines expressing wild-type RAP1A (wt19, wt28) or RAP1AV12 (12/9, 12/12) mutant. Cells were grown for 72 h in 10% FCS in the presence (1) or absence (2) of 1.5 mg/ml of Tc prior being switched to differentiating medium (1% FCS) for 3 days in the presence (1) or absence (2) of the drug. (B) Protein extracts were prepared from C2RT20 and various RAP1AN17-expressing cells (17/1, 17/3, 17/8) grown for 72 h in 10% FCS in the presence (1) or absence (2) of 1.5 mg/ml of Tc prior differentiation in 1% FCS for 3 days in the presence (1) or absence (2) of Tc. Western blot analyses were performed with 30 mg of total proteins using anti-troponin T antibody.

(cell lines 17/1 and 17/3). Tc did not affect acridine orange labeling since independently of the presence of the drug, C2RT20 cells displayed prominent staining of numerous vesicles randomly dispersed throughout the cytoplasm (Figs. 9A and 9A9). In contrast, the vital dye pattern was strikingly different in cell line 17/1 cultivated in the presence or absence of Tc. When expression of the ectopic protein was inhibited (Fig. 9B), location of the acidic compartments was similar to that observed in C2RT20 cells. Upon expression of the RAP1AN17 protein, acridine orange labeling was almost completely clustered near the nucleus (Fig. 9B9). The same results were obtained with the cell line 17/3 (data not shown). Since cells overexpressing wild-type or RAP1AV12 proteins displayed the same acidic organelles aspect and distribution as those observed within C2RT20 cells (data not shown), we concluded that clustering of the endocytic compartments was specific for the expression of GDP-bound RAP1A. Relocation of these compartments was confirmed by control immunofluorescence experiments. Indeed, the small GTPase rab7, a marker of late endosomes [42] was present on expanded vesicular structures in C2RT20 cells and was mainly observed on large perinuclear vesicles in RAP1AN17-overexpressing cells (data not shown). Since relocation of late endosome and lysosomes could be a mere reflection of a general intracellular organelles reorganization, we also investigated the Golgi apparatus location in C2RT20, RAP1AV12, and RAP1AN17 cells. Immunofluorescence experi-

During successive stages of lysosomal enzyme maturation, various forms of cathepsin D can be assigned to specific intracellular organelles [43]. After its synthesis in the rough endoplasmic reticulum, cathepsin D is translocated into the Golgi apparatus as a proenzyme. Passage of the enzyme through late endosomes is marked by a proteolytic cleavage leading to intermediate forms (I). In lysosomes, cathepsin D is further processed into the mature form (M). Therefore, variations in the amount of the different forms of cathepsin D should reflect traffic disturbances between the various sites of the enzyme maturation. Cathepsin D was revealed by immunoblot experiments performed with lysates of C2RT20 cells and myoblasts overexpressing

FIG. 7. Confocal laser scanning microscopy of cells overexpressing RAP1A proteins. C2RT20 (A), wt RAP1A clone 28 (B), RAP1AV12 clone 12/12 (C), and RAP1AN17 clone 17/9 (D) cells were grown for 3 days on coverslips in 10% FCS without Tc. Cells were then subjected to indirect immunofluorescence analysis with the affinity purified RAP1 antibody. Bars: A–C, 10 mm; D, 5 mm.

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endocytic compartments but could also disturb the maturation of lysosomal enzymes. DISCUSSION

A previous study established that RAP1A is regulated developmentally during myogenic differentiation [30]. The present work stems from the observation that according to its nucleotide linkage the endogenous RAP1A protein displayed specific intracellular location during myogenic C2 cells differentiation. At the time of cell fusion, transient accumulation of the GDP-bound

FIG. 8. Immunofluorescence microscopy of stable cells lines overexpressing the GFP protein alone (A, E), the GFP-RAP1AN17 fusion protein (cell line GFP17/1) (B, F), cell line GFP17/2 (C, G), and the GFP–RAP1AV12 fusion protein (D, H). Green fluorescence was observed directly in living cells (A–D). Cells were incubated with acridine orange in order to visualize the late endosome and lysosomes compartments (E–H). Bar, 10 mm.

GDP- or GTP-bound RAP1A proteins. Figure 10 is representative of two independent experiments. Due to the very short half-life of the proenzyme, the anticathepsin D antibody only revealed the intermediate and mature forms of the enzyme in all the cells analyzed. Although the amount of the mature enzyme seems identical in all the cells, the intermediate forms of cathepsin D appeared much more abundant in cells overexpressing RAP1AN17. These results indicate that forced expression of the GDP-bound RAP1A protein not only induces morphological modifications of the late

FIG. 9. Effect of the overexpression of the GDP-bound RAP1A mutant on the late endocytic compartments. C2RT20 (A, A9) and RAP1AN17 clone 17/1 (B, B9) cells were grown for 3 days on coverslips in 10% FCS with (A, B) or without (A9, B9) Tc. Cells were then incubated with acridine orange in order to visualize the late endosome and lysosomes compartments. Bar, 10 mm.

RAP1A NUCLEOTIDE CYCLES AND MYOGENIC DIFFERENTIATION

FIG. 10. Expression of cathepsin D in C2RT20 and in RAP1Aoverexpressing cell lines. Total proteins were prepared from C2RT20, RAP1AN17 (clones 17/1, 17/8), and RAP1AV12 (clone 12/9) grown for 3 days in 10% FCS without Tc. 30 mg protein was used for Western blot analyses. Anti-cathepsin D antibody detects intermediate forms (I) and mature form (M).

RAP1A protein is accompanied by concomitant perinuclear relocation of RAP1A and of acidic organelles. Using two different expression systems, we confirmed the clustering of the GDP-bound RAP1A protein in a limited perinuclear region or at one side of the nucleus. These two locations could be related to the level of expression of the mutant protein within individual cell or to specific physiological events leading to different RAP1A location. Indeed, in BHK cells endogenous RAP1A protein has been localized predominantly in the late endocytic compartments although some protein also localized in the rough endoplasmic reticulum [29]. Further characterization is required to identify these intracellular regions. However, our results indicate clearly that overexpression of the GDP-bound RAP1A induces specific morphological changes of the late endocytic compartments in nondifferentiated myoblasts. Thus, RAP1A protein may regulate the intracellular location of late endosome and of lysosomes as do other Ras-related proteins [44, 45]. We also investigated whether RAP1A could affect myogenic differentiation. Induced to differentiate, myoblast overexpressing the wild-type or the GTP-bound RAP1A failed to differentiate. It appeared that a threefold increase of RAP1A protein was sufficient to inhibit differentiation. Thus, active RAP1A protein acts as a potent inhibitor of the differentiation program. Since we previously observed that RAP1A protein accumulates during myogenic differentiation [30], our present results could appear conflicting. However, this work establishes that the amounts of the two nucleotide-bound forms of the protein are tightly regulated during the different stages of differentiation. Eventually, the total amount of RAP1A protein is higher in myotubes than in myoblasts. Thus, forced expression of GTP-bound RAP1A in myoblasts could prevent myogenic differentiation by disturbing the normal regulation of the endogenous protein. The molecular mechanism involving RAP1A in myogenic differentiation remains unknown. RAP1A has been shown to antagonize Ras transformation, to interact with various Ras-interacting proteins and to

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mediate sustained MAP kinase activation in PC12 cells [46]. Since overexpression of the oncogenic Ras proteins inhibits myogenic differentiation [3] and since conflicting data have involved or not the Ras/Raf/MAP kinase pathway in myogenic differentiation [47–52], it is possible that overexpressed active RAP1A protein may perturb differentiation directly or by interfering with the Ras signaling pathway. These hypotheses are under investigation. By analogy with the corresponding mutation in the Ras protein [53], RAP1AN17 should act as a dominant negative mutant. However, conflicting data have been reported concerning this RAP1A mutant. Whereas some reports proposed a dominant negative effect of RAP1AN17 [54 –56], others indicated that this mutant is inactive [15, 57]. In our study, considering myogenic differentiation, RAP1AN17 mutant does not behave as a dominant negative mutant since it does not inhibit differentiation. However, expression of the RAP1AN17 protein alters the endosomal compartments organization suggesting that it could interfere with the endogenous protein. Interestingly, recent data show that bound to GDP or GTP, Bud1, the yeast RAP1A homologue, can associate with different partners [58]. In myogenic cells, accordingly to the nucleotide bound form, RAP1A could also interact with different molecules leading to specific biological effects. Current research suggests that lysosomal proteolytic enzymes may be involved in the reorganizational changes associated with myogenesis. Support for this role comes from various observations: (i) myosin can be degraded by cathepsins B and D [59]; (ii) inactivation of one allele of cathepsin B induces deficiency in myoblast fusion [11]; (iii) activities of lysosomal proteinases increase during myogenic differentiation [8 –10]. Owing to gradual increase of acidity encountered between late endosome and lysosomes [12, 34], lysosomal enzyme activities must be regulated all along the late endocytic pathway. Since endocytosed substrate degradation has been shown to occur in lysosomes and in the late endosome [60], these endocytic organelles may display specific affinities toward different substrates. During differentiation, myoblasts first synthesize short half-life proteins like MyoD, a myogenic regulator [61]. After fusion, myotubes synthesize relatively stable proteins like the components of the contractile apparatus, actin, and myosin. The catabolic regulation of these proteins could take place in different stations of the degradative pathway, in agreement with the intracellular redistribution of the acidic organelles observed during myogenic differentiation [6]. In this work, we established that lysosomal enzyme maturation was affected concomitant with the perinuclear relocation of the late endosomal compartment. Indeed, accumulation of intermediate forms of cathepsin D was revealed in cells overexpressing the GDP-bound RAP1A mutant. By

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regulating the intracellular traffic between late endosome and lysosomes, RAP1A could indirectly regulate the activity of lysosomal enzymes and therefore interfere with the stability of specific myogenic proteins during differentiation. Since relocation of the late endosomal compartments reflects a normal physiological event occurring during myogenic differentiation, the differentiation advantage displayed by cells overexpressing GDP-bound RAP1A suggests that clustering of the late endocytic compartments may be an essential step required for myogenic differentiation. Consequently, by disturbing the physiological relocation of the degradative organelles, overexpression of activated RAP1A protein could inhibit myogenic differentiation. In conclusion, this work shows for the first time that relocation of the endogenous RAP1A protein is a physiological event occurring during C2 cells myogenic differentiation. It points out that, depending of its GTP/ GDP bound form, RAP1A protein displays different intracellular locations and different biological effects in mammalian cells. It also indicates a possible role of RAP1A in the organization of the late endosome and lysosomes compartments and suggests a strict regulation of this small GTP-binding protein during the orchestrated protein degradations necessary for cell differentiation. We are indebted to Drs. H. Bujard and M. Gossen for their generous gift of the tetracycline gene expression system. We are grateful to Drs. T. Kreis, S. Leibovitch, and M. Zerial for providing antibodies used in this study. We also thank members of the imagery department of the Jacques Monod institute in Paris. We thank Dr. D. Bouvier for assistance in protein quantification. We are grateful to Dr. P. Hughes and Dr. D. Bouvier for critically reading the manuscript. This work was supported in part by CNRS and INSERM and in part by a grant from the Association Franc¸aise de Lutte contre les Myopathies (AFM).

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