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Developmental Biology 262 (2003) 324Ð334 www.elsevier.com/locate/ydbio

Membrane segregation and downregulation of raft markers during sarcolemmal differentiation in cells

A. Draeger,a,* K. Monastyrskaya,a F.C. Burkhard,b A.M. Wobus,c S.E. Moss,d and E.B. Babiychuka,e

a Department of Cell Biology, Institute of Anatomy, University of Bern, Switzerland b Department of Urology, University Hospital of Bern, Switzerland c In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany d Department of Cell Biology, Institute of Ophthalmology, University College, London e The Institute of Physiology, Kiev Taras Schevchenko University, Ukraine Received for publication 24 April 2003, accepted 16 June 2003

Abstract implies flexibility in combination with force resistance and requires a high degree of sarcolemmal organization. Smooth muscle cells differentiate largely from mesenchymal precursor cells and gradually assume a highly periodic sarcolemmal organization. Skeletal muscle undergoes an even more striking differentiation programme, leading to cell fusion and alignment into myofibrils. The lipid bilayer of each cell type is further segregated into raft and non-raft microdomains of distinct lipid composition. Considering the extent of developmental rearrangement in skeletal muscle, we investigated sarcolemmal microdomain organization in skeletal and smooth muscle cells. The rafts in both muscle types are characterized by marker proteins belonging to the family which localize to the inner membrane leaflet, as well as glycosyl-phosphatidyl-inositol (GPI)-anchored enzymes attached to the outer leaflet. We demonstrate that the profound structural rearrangements that occur during skeletal muscle maturation coincide with a striking decrease in membrane lipid segregation, downregulation of 2 and 6, and a significant decrease in raft-associated 5Ј-nucleotidase activity. The relative paucity of lipid rafts in mature skeletal in contrast to smooth muscle suggests that the organization of sarcolemmal microdomains contributes to the muscle-specific differences in stimulatory responses and contractile properties. © 2003 Elsevier Inc. All rights reserved.

Keywords: Calcium; Transverse tubular system; Membrane microdomains; Annexins; Caveolin

Introduction muscle sarcolemma have greatly benefited from the cell’s well-structured appearance (Ockleford et al., 2002). Their Skeletal muscle contraction is an immediate explosive protein arrangement is governed by costameres, which par- action, triggered by neuronal stimulation, whereas speed is allel the regular sarcomeric arrangement of the underlying rarely of the essence during smooth muscle shortening. contractile apparatus (Bloch et al., 2002). In smooth muscle, Smooth muscle contractile responses can be elicited by a the plasma membrane is organized into alternating mac- variety of signals (mechanical, chemical, neuronal) which rodomains of firm, actin-attachment sites, and flexible ve- are additionally subject to extensive modulation. For either sicular domains (Draeger et al., 1989; North et al., 1993). cell type, there exists a wealth of data relating to the orga- Yet, the lipid moiety has only recently emerged as being nization of protein constituents within the sarcolemma (for more than a mere anchorage site of cytoskeletal and extra- review, see Small et al., 1992). Investigations of skeletal cellular matrix components, passively responding to the constraints imposed on it by its proteinaceous partners. It is now well established that specific lipids associate to form * Corresponding author. Fax: ϩ41-31-631-38-07. functional units (rafts), creating substructures which are E-mail address: [email protected] (A. Draeger). instrumental for the deployment of proteins, activation of

0012-1606/$ Ð see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0012-1606(03)00398-1 A. Draeger et al. / Developmental Biology 262 (2003) 324–334 325 receptors, and triggering of signalling cascades (Brown and London, 1998, 2000; Simons and Ikonen, 1997; Simons and Toomre, 2000). These lipid microdomains are further sub- divided into “caveolar” and “non-caveolar rafts” (Abrami et al., 2001). In the present study, we show that the plasma membrane of skeletal muscle undergoes a striking reorganization of its lipid and protein components during differentiation in the early perinatal period. Undifferentiated skeletal myoblasts and myotubes closely resemble smooth muscle cells in their expression of raft-associated lipids and proteins. Moreover, the enzymatic activity of GPI-anchored 5Ј-nucleotidase in membrane rafts purified from skeletal muscle of neonatal mice correlates well with the levels measured in smooth muscle cells. 5Ј-Nucleotidase activity markedly decreases in the early perinatal period, concomitantly with transverse tubular (T-tubular) invagination and terminal maturation of the skeletal muscle sarcolemma. Simultaneously, membrane microdomain segregation becomes less apparent and raft protein markers (annexins 2 and 6) are downregulated. An- nexins are diffusely distributed within myotubes, transiently colocalize with caveolin 3 during embryogenesis, and are expressed in the T-tubules within mature myocytes. In contrast to smooth muscle cells, the caveolar and noncaveolar raft domains of fully differentiated skeletal muscle largely occupy separate locations. Given their dis- tinct tasks, it is not surprising that sarcolemmal protein distribution differs between smooth and skeletal muscle. The observed lipid segregation and Ca2ϩ-dependent redis- tribution of membrane components may account for the higher degree of adaptability and contractile modulation of smooth muscle cells (with abundant lipid rafts), in contrast to the all-or-nothing contractile response observed in skel- etal muscle cells.

Materials and methods

Fig. 1. Annexins are symmetrically aligned with mitochondria in skeletal Tissue preparation muscle. Fluorescent micrographs of ultrathin longitudinal cryosections of human skeletal muscle (M pyramidalis), dual labelled with a polyclonal Since several of the antibodies employed in this study antibody against cytochrome C (green: aÐc), which delineates the mito- have a restricted cross-reactivity, it was sometimes neces- chondria, and a monoclonal antibody against annexin 1 (red: a), 4 (red: b), sary to use human material for immunolabelling. Consent or 6 (red: c). The mitochondria are characteristically situated at the junction between A- and I-bands, and the annexins are located on either side of for working with this tissue was obtained from the Medical these. Bar, 5 ␮m. Ethical Commission of the University of Bern. Thin strips of human M. pyramidalis (striated) and M. detrusor vesicae (smooth) were obtained during routine (140 mM KCl, 5 mM NaCl, 1 mM MgCl2, 10 mM glucose, urinary bladder surgery. Samples of murine m. pectoralis and 10 mM Hepes, pH 7.4) containing 2 mM CaCl2. The tissue (striated) were obtained from decapitated animals (1 and 4 was then fixed in Naϩ-orKϩ-Tyrode’s solution containing 4% days after birth). The processing of tissue for contraction/ paraformaldehyde and further processed for immunohisto- relaxation experiments and ultrathin cryosectioning were chemistry as described previously (Babiychuk et al., 1999). performed as described previously (Babiychuk et al., 1999). For immunoelectron microscopy, the muscle strips were fixed Some muscle strips were incubated for 2Ð8 h either in overnight in Hepes-buffered saline containing 4% paraformal- ice-cold, Ca2ϩ-free Naϩ-Tyrode’s solution (140 mM NaCl, 5 dehyde and 0.2% picric acid, dehydrated, embedded in LR- mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM Hepes, White resin, and sectioned for viewing in an electron micro- pH 7.4) containing 2 mM EGTA or in Kϩ -Tyrode’s solution scope (Zaugg et al., 1999). 326 A. Draeger et al. / Developmental Biology 262 (2003) 324–334

tions were performed in compliance with the protocol estab- lished by Harlow and Lane (1988). The antiserum was specific for annexin 6, cross-reactivity with other mammalian proteins having been undetected by Western blotting. Fluorescent labelling was performed by using Cy3-(Jack- son, Baltimore, USA) or Alexa-conjugated (Molecular Probes, Eugene, USA) secondary antibodies. A gold-conjugated sec- ondary antibody (Sigma, Buchs, Switzerland) was used for immunoelectron microscopy. Negative controls were gener- ated by absorbing the antibody with purified antigen (for an- nexin 6) or by applying a nonbinding primary antibody. Immunostained ultrathin cryosections were viewed in a Zeiss Axiophot fluorescent microscope, and images were collected with a digital CCD camera (Ultrapix, Astrocam). Immunogold stained ultrathin LRWhite sections were ex- amined in a ZEISS 400 electron microscope.

Cell culture

Primary cultures of human skeletal muscle myoblasts were obtained from Promocell (Heidelberg, Germany). They were cultivated on glass coverslips in skeletal muscle- adapted medium for 48 h and then in differentiation medium (Promocell). Myotube formation was observed 4Ð5 days later. For biochemical experiments, the C2C12 murine skel- etal myoblast cell line was used. It was cultivated as de- scribed by Kuch et al. (1997). Human myometrial smooth muscle cells were cultivated and used as described in Babiy- chuk et al. (2002). The cells were fixed with 4% parafor- maldehyde in Naϩ-Tyrode’s solution, and the coverslips Fig. 2. Annexin 6 associates with the transverse tubular system. Fluores- cent micrographs of thin longitudinal cryosections of human skeletal mus- were inversed over drops of antibody solution for immuno- cle (M. pyramidalis), labelled in (a) with a polyclonal antibody against cytochemical staining. Optical sections were obtained with calsequestrin (green) and a monoclonal antibody against annexin 6 (red), a Biorad MicroRadiance confocal microscope, and col- and in (b) with a polyclonal antibody against the dihydropyridine receptor lected images were processed with a Silicone Graphics (green) and a monoclonal antibody against annexin 6 (red). In (a), annexin workstation, using IMARIS multi-channel-image process- 6 is associated exclusively with the transverse tubular system (t-tub), without overlap with the calsequestrin-marked (sr). ing software (Bitplane AG, Zurich, Switzerland). In (b), annexin 6 colocalizes extensively with the dihydropyridine receptor- marked transverse tubular system. Bar, 10 ␮m. Isolation of lipid rafts and 5Ј-nucleotidase assay

Immunohistochemistry and immunoelectron microscopy For the 5Ј-nucleotidase assays, abdominal skeletal muscle was dissected from newborn and adult mice, and smooth mus- Immunolabelling was performed as described by Jostarndt- cle (uterus) was taken from adult animals only. Pieces of Fo¬gen et al. (1998). Monoclonal antibodies against annexins 1, frozen tissue (approx. 100 mg each) were finely sliced and 2, 4, and 6 and against caveolin 3 and polyclonal antibodies homogenised on ice in (0.5 ml) of BW buffer (20 mM Imida- ϩ against caveolin 1 3 and caveolin 3 were purchased from zole, 40 mM KCl, 2 mM MgCl2, pH 7.0) using Polytron Transduction Laboratories (Lexington, USA). Polyclonal anti- homogeniser. Following a 30-min incubation of the homoge- bodies against calsequestrin and the dihydrophyridine receptor nate on ice in presence of 0.4% NP40 and 0.2 mM CaCl2, the were from Upstate Technology (NY, USA), and a polyclonal sucrose content of the homogenate was then adjusted to 40% antibody against cytochrome C was purchased from Santa by the addition of 80% sucrose in BW buffer. A 30-5% Cruz (USA). For dual-labelling purposes, a polyclonal anti- discontinuous six-step (5%/step) sucrose gradient was layered body against annexin 6 was generated in a rabbit. The cDNA over the homogenate. The gradients were centrifuged for 16 h of human annexin 6 was cloned into pET-28a(ϩ) vector (No- at 33,000 rpm in a SW50.1 rotor at 4¡C. Then, 8 ϫ 600 ␮l vagen). Recombinant annexin 6 was expressed in Escherichia fractions were collected from the top of the gradient, and Ј coli (BL21 strain) as a fusion product with the N-terminal His6 Western blotting analysis as well as 5 nucleotidase activity tag; it was purified on Ni-agarose (Qiagen) according to the assays were performed. Protein concentration in fractions was manufacturer’s recommendations. Subcutaneous immuniza- determined by using BCA assay (Sigma) as described by the A. Draeger et al. / Developmental Biology 262 (2003) 324–334 327

2ϩ Fig. 3. Annexins redistribute with changes in [Ca ]free.(aÐc) Fluorescent micrographs of thin cryosectioned human skeletal muscle (M. pyramidalis), 2ϩ labelled with a monoclonal antibody against annexin 6. In myofibres contracted with potassium chloride (high [Ca ]free), annexin 6 is associated with both 2ϩ the sarcolemma and the transverse tubular system. In muscle relaxed by incubation in an EGTA-containing buffer [low [Ca ]free) (b)], annexin 6 is diffusely distributed throughout the cytoplasm. Between individual fibres, annexin 6-positive pericytes, belonging to the vascular system, can be discerned. In transversely sectioned skeletal muscle (c), contracted fibres are distinguished by their sarcolemmal labelling with annexin 6. (d, e) Electron micrographs of longitudinally sectioned human skeletal muscle (M. pyramidalis), immunostained with a monoclonal antibody against annexin 4, and tagged with gold particles. In contracted fibres (d), clusters of gold particles are associated with the transverse tubular system (encircled). In relaxed fibres (e), gold particles (arrows) are diffusely distributed. Bars, 50 ␮m (a, b); 500 ␮m (c); 0.5 ␮m (d, e). manufacturer. 5Ј-Nucleotidase activity in each fraction was mon- formed as described previously (Babiychuk and Draeger, itored as Pi release, which was measured spectrophometrically at 2000; Babiychuk et al., 2002). Protein content of the indi- a wavelength of 820 nm (A820) according to a modification of the vidual bands following SDS-PAGE and Western blotting method described by Fiske and Subarrow (1925), using 5Ј-AMP was estimated by using ONE-Dscan 1.0 software. as a substrate (Babiychuk et al, 1999). Activity was expressed as an optical density at 820 nm per ␮g protein. Data represent the Results average of three independent experiments. Skeletal muscle annexins translocate from the cytoplasm to the Miscellaneous sarcolemma and transverse tubular system during contraction

SDS PAGE, Western blotting, thin-layer chromatogra- During the development of skeletal muscle, the synthesis phy (TLC), and sucrose gradient centrifugations were per- of myofibrillar proteins and their regular arrangement into 328 A. Draeger et al. / Developmental Biology 262 (2003) 324–334

Fig. 4. Membrane rafts in skeletal muscle sarcolemma. (a) Thin-layer chromatograms (TLC) of smooth (porcine stomach, Sm.m) and skeletal muscle (porcine leg muscles, Sk.m) microsomes extracted in the presence of the nonionic detergent NP-40. Detergent-resistant (pel) and soluble (sup) fractions were separated by low-speed centrifugation. In contrast to skeletal muscle, extraction of smooth muscle microsomes resulted in preferential solubilization of phosphati- dylethanolamine (PE) and phosphatidylcholine (PC) and the formation of detergent-resistant pellets enriched in cholesterol (Ch) and sphingomyelin (SM). 2ϩ (b) Skeletal muscle microsomes were subjected to sucrose-gradient centrifugation in the presence of 2% NP-40 and either 0.2 mM CaCl2 (Ca )or2mM EGTA. Fractions (1 ml) of either 40% sucrose (fractions 1Ð2) or 30Ð5% sucrose gradient (fractions 3Ð7) were analyzed by TLC for lipid—or by Western Blotting for protein—composition. Irrespective of their sterol or glycerophospholipid nature, most of the lipids are present in their soluble form in fractions 1Ð3, where they colocalize with the nonraft protein markers annexin 1 (anx 1) and annexin 4 (anx 4). However, the raft markers caveolin and, in the presence of Ca2ϩ, annexins 2 and 6, are preferentially found within the buoyant fractions (4 and 5), demonstrating that, despite their very low abundance, the rafts are still present within skeletal muscle sarcolemma.

sarcomeres follows a well-documented course (Fu¬rst tions, reveals an overlapping but not identical pattern of et al., 1989; Young et al., 2001). Adult human skeletal staining along the transverse tubular system and sarco- muscle fibres display a pattern of alternating A-(myosin) lemma. But presumably due to steric hindrance, immu- and I-bands (actin), with the mitochondria characteristi- nolabelling varied in intensity according to the order of cally localized at their junction or at the I-band (Ogata incubation with the various types of antibodies (not and Yamasaki, 1997; Fig. 1a). In contracted fibres, anti- shown). bodies against annexins 1, 4, 6 (Fig. 1aÐ c), and 2 (not The membranous distribution of Ca2ϩ-sensitive an- shown) colocalize lateral to this region and also along nexins in contracted skeletal muscle fibres (Fig. 3a and d) 2ϩ the sarcolemma (Fig. 2). Dual labelling with a polyclonal is lost as the elevated [Ca ]i drops towards resting levels antibody against calsequestrin (Fig. 2a) excludes annexin and the cells relax. In relaxed fibres, the annexins detach 6 from the sarcoplasmic reticulum, but colocalization from all membrane systems and are diffusely distributed with the dihydropyridine receptor confirms its distri- within the cytoplasm (Fig. 3b and e). In transversely bution along the transverse tubular system (Fig. 2b). sectioned skeletal muscle, the contractile state of indi- Dual labelling with polyclonal and monoclonal anti- vidual fibres can be ascertained by their labelling pattern bodies against different annexins in various combina- with antibodies against annexins (Fig. 3c). A. Draeger et al. / Developmental Biology 262 (2003) 324–334 329

cells in their lipid microdomain organization (compare Fig. 5a, right, and Fig. 1 in Babiychuk et al., 1999), as well as in their annexin expression profiles (Fig. 5c). In parallel with the decrease in lipid compartmentaliza- tion and downregulation of annexins 2 and 6, we observed a marked decrease of raft-associated 5Ј-nucleotidase activ- ity in differentiated skeletal muscle tissue (Fig. 6; and Babiychuk and Draeger, 2000). Sucrose gradient centrifu- gation of adult smooth, as well as neonatal and adult skeletal Fig. 5. Downregulation of membrane rafts during skeletal muscle differ- muscle lysates prepared in presence of 0.4% NP40 and 0.2 2ϩ entiation. (a) Equal amounts (100 mg) of skeletal muscle tissue (diff.: mM Ca displays a codistribution of the raft-markers porcine leg) and undifferentiated cultured cells (undiff.: C2C12 murine caveolin and annexin 2 (Fig. 6a, c and e). A parallel deter- ϩ skeletal muscle cell line) were extracted in Na -Tyrode’s buffer containing mination of 5Ј-nucleotidase activity in the gradient demon- 0.2 mM CaCl2 and 0.8% NP-40. The supernatant obtained after low-speed centrifugation (6000g, 30 min) was subjected to high-speed centrifugation strates that the peak of enzymatic activity coincides with the (50,000g, 90 min) and the resulting pellets and supernatants were analyzed raft-containing fractions (Fig. 6b, d, and f). However, the by TLC. In differentiated skeletal muscle, the lipid composition of the raft-associated 5Ј-nucleotidase activity differs significantly pellets and supernatants is the same. But in undifferentiated cells, the pellet between the tissue samples (Fig. 6g). Its value in neonatal is enriched in, while the supernatant is depleted of the raft-markers cho- skeletal muscle (Fig. 6b) approximates to the value in lesterol and sphingomyelin. (b) Samples derived from equal amounts (100 mg) of smooth (diff.: sm., porcine stomach) and skeletal (diff.: sk. porcine smooth muscle (Fig. 6f), and its decline in adult skeletal leg) muscle tissue and from undifferentiated cultured cells (undiff., sm.: muscle (Fig. 6d) correlates well with the decrease in an- human myometrium; undiff.: sk.-C2C12 cells) were analyzed by Western nexin 2 expression in that tissue (Fig. 6a and c). In contrast blotting. Note the significant downregulation of annexins 1, 2, and 6 in to annexin 2 and 5Ј-nucleotidase, overall caveolin expres- differentiated skeletal muscle. sion (isoforms 1 and 3) is not significantly diminished during skeletal muscle maturation, indicating that the gross sarcolemmal structure, which forms after myoblast fusion, Annexins discriminate between raft- and non-raft regions is well developed before birth. in skeletal muscle sarcolemma

Thin-layer chromatography of skeletal and smooth mus- Noncaveolar rafts spatially segregate from caveolae in the 2ϩ cle microsomes prepared at high [Ca ]free, revealed an sarcolemma of fully differentiated skeletal muscle cells abundance of detergent insoluble sphingomyelin-and cho- lesterol-enriched rafts in smooth compared to skeletal mus- During the differentiation of skeletal muscle cells, the cle (Fig. 4a) (Babiychuk et al., 1999). Despite the paucity of pattern of localization of membrane-associated proteins un- raft lipids within this tissue, the annexins still fall into two dergoes striking changes. We monitored the sarcolemmal categories determined by their association with raft or non- localization of caveolin 3 and annexin 6 during myoblast raft domains. As in smooth muscle (Babiychuk et al., 2000, fusion and development of the transverse tubular system 2002), annexins 1 and 4 partition to the soluble (nonraft) (Fig. 7). Within multinucleated myotubes, caveolin 3 is fraction of sucrose gradients performed in the presence of oriented parallel to the long axis (Fig. 7a and c), whereas the nonionic detergents, whereas annexins 2 and 6 codistribute annexins initially display a diffuse localization pattern (Fig. with detergent insoluble buoyant raft membranes, colocal- 7b and c). Invagination of the transverse tubular system izing with the raft-marker caveolin (Fig. 4b). At low begins during late embryogenesis and is completed during 2ϩ [Ca ]free, the annexins remain in the soluble fraction and the early perinatal period (Schiaffino and Margreth, 1969; do not display membrane attachment (Fig. 4b). Franzini-Armstrong, 1991). At day 1 postpartum, caveolin 3 is associated with the sarcolemma and the transverse tubular The segregation between raft and nonraft regions decreases system (Fig. 7d and f). At this stage, annexin 6 colocalizes during the sarcolemmal differentiation of skeletal muscle transiently with caveolin 3 (Fig. 7e and f). In the pectoral muscle of 4-day-old mice, caveolin 3 is distributed accord- In nondifferentiated skeletal muscle, the sarcolemma is ing to the adult pattern, i.e., it associates exclusively with compartmentalized into raft- and nonraft lipid-microdo- the sarcolemma (Fig. 7g and i). But the annexins [(annexin mains (Fig. 5a, right). But during maturation, this segrega- 6 in Fig. 7h and i) annexins 1, 2, 4 not shown] partially tion is lost and the membrane becomes more uniform in colocalize with caveolin 3 along the sarcolemma, as well lipid composition (Fig. 5a, left). These changes are closely as remaining attached to the transverse tubular compart- linked with the expression levels of annexins 2 and 6, which ment. decrease significantly during the course of differentiation In smooth muscle cells, which lack complex membrane (Fig. 5b). It is interesting to note that undifferentiated skel- infoldings, annexin 6 is arranged in periodic arrayment etal muscle myoblasts/myotubes resemble smooth muscle around the circumference (Fig. 7k; Babiychuk and Draeger, 330 A. Draeger et al. / Developmental Biology 262 (2003) 324–334 A. Draeger et al. / Developmental Biology 262 (2003) 324–334 331

2000), delineating the raft regions of the sarcolemma and al., 2000). Although invaginations of the transverse tubular colocalizing largely with caveolin (Fig. 7l and m). system are continuous with the sarcolemma, they do not directly participate in its load-bearing function. Instead, they are specialized sites for Ca2ϩ entry and excitationÐ Discussion contraction coupling. Protein segregation in connection with the anchorage of Precise coordination and feedback between the regula- cytoskeletal and components is a well- tory, signal-generating units of the plasma membrane and known concept (for recent reviews, see D’Atri and Citi, their executive, force-producing contractile apparatus is of 2002; Geiger and Bershadsky, 2002). But that specific lipids paramount importance for muscle contraction. Since con- can by themselves form spatially and functionally separate tinuous recording and tight control of intracellular Ca2ϩ units is only now becoming apparent. These assemblies of 2ϩ concentration ([Ca ]i) are vital in order to trigger the cholesterol and glycosphingolipids form dynamic units corresponding contractile response, this information must (rafts), which are interspersed with glycerophospholipid- be constantly supplied to the signal-generating apparatus enriched nonraft regions. Individual membrane rafts con- responsible for Ca2ϩ-entry and removal. taining characteristic sets of proteins can self-associate to form higher-order structures which constitute hubs of sig- Annexins in Ca2ϩ-signalling nalling activity (Brown and London, 1998, 2000; Simons and Ikonen, 1997; Simons and Toomre, 2000). Here, we Members of the annexin protein family have been as- demonstrate that, although being less abundant in skeletal cribed a role in the Ca2ϩ homeostasis of cardiac (Hawkins than in smooth muscle, membrane rafts might play a role in et al., 1999) and smooth muscle (Babiychuk et al., 1999; the Ca2ϩ-dependent compartmentalization of the cell sarco- Babiychuk and Draeger, 2000). They have been localized lemma and be subject to annexin-dependent regulatory with the sarcolemma and intercalated disks in cardiomyo- pathways as proposed for smooth muscle (Babiychuk and cytes (Kaetzel et al., 1994; Luckcuck et al., 1997), or with Draeger, 2000; Babiychuk et al., 2002). transverse tubules in developing and mature skeletal muscle Membrane rafts can be grouped into at least two catego- (Arcuri et al., 2002; Barrientos and Hidalgo, 2002). In ries: large, stationary ones, which are associated with the cardiomyocytes derived from annexin 6Ϫ/Ϫ mice, the rate of caveolar regions, and smaller, presumably more flexible Ca2ϩ-clearance is accelerated (Song et al., 2002); in trans- ones (Abrami et al., 2001). The stable caveolar rafts, which genic animals overexpressing this protein within myocardial have been shown to harbour receptors for certain growth cells, Ca2ϩ homeostasis is disrupted (Gunteski-Hamblin et factors (Maatev and Smart, 2002), might be involved in al., 1996). signalling activities of long duration. But the highly dy- 2ϩ 2ϩ In the present study, we demonstrate that, when [Ca ]i namic, Ca -regulated association of annexin-dependent is elevated during the contraction of skeletal muscle cells, noncaveolar rafts suggests that they are involved in the members of the annexin-family are able to translocate from rapid fine-tuning of excitationÐcontraction coupling (Babiy- the cytoplasm to the sarcolemma and transverse tubular chuk and Draeger, 2000). Although caveolar and Ca2ϩ/ system. This translocation could conceivably furnish the annexin-regulated rafts occupy largely separate regions in muscle cell with a means of conveying information about its skeletal muscle, in smooth muscle cells they reside in the 2ϩ actual [Ca ]i to the membrane-localized machinery re- same (or largely overlapping) membrane domain. sponsible for Ca2ϩ entry and/or removal. Differentiation of the transverse tubular system Plasma membrane segregation During the differentiation of skeletal muscle cells, the The plasma membrane of skeletal muscle fibres displays contractile proteins rapidly assemble into sarcomeres. But an extraordinary degree of surface specialization. The sar- maturation of the plasma membrane is a much slower pro- colemma possesses the structural rigidity to withstand re- cess, which can be protracted well into the postnatal period peated contractionÐrelaxation events (Campbell, 1995), its in some mammals (Schiaffino and Margreth, 1969; Fran- costameres constituting a strong mechanical link between zini-Armstrong, 1991). In contrast to the well-investigated the membrane and the contractile apparatus (Rybakova et myofibrillogenesis, sarcolemmal maturation does not dis-

Fig. 6. Decrease of noncaveolar raft markers in skeletal muscle sarcolemma after birth. Neonatal (a, b) and adult skeletal (c, d) and smooth muscle (e, f) lysates 2ϩ prepared in the presence of 0.4% NP-40 and 0.2 mM CaCl2 (Ca ) were adjusted to 40% sucrose, overlayed with a discontinuous 30Ð5% sucrose gradient and subjected to centrifugation (16 h at 33,000 rpm in SW50.1 rotor at 4¡C). Fractions (0.6 ml) were collected from the top of the gradient and analyzed by Western blotting with a monoclonal antibody against annexin 2 and a polyclonal one against caveolin (a, c, and e). Caveolin (isoforms 1 and 3) and annexin 2 are present within the buoyant fractions (3Ð6). In contrast to caveolin, annexin 2 expression declines after birth, correlating with the simultaneous decrease in enzymatic activity of 5Ј-nucleotidase (b, d, f, g) within the same fractions. The level of enzyme activity in differentiated smooth muscle (f, g) is similar to that in immature skeletal muscle before membrane invagination has taken place. 332 A. Draeger et al. / Developmental Biology 262 (2003) 324–334

Fig. 7. Relocation of annexin 6 and caveolin 3 during skeletal muscle differentiation. (aÐc) Myotubes derived from human primary cultures after the induction of differentiation, dual labelled with a polyclonal antibody against caveolin 3 (red: a, c) and a monoclonal antibody against annexin 6 (green: b, c). Caveolin 3 is longitudinally orientated, whereas annexin 6 is diffusely distributed. (dÐf) Thin longitudinal cryosection of murine pectoral muscle 1 day after birth, dual labelled with a monoclonal antibody against caveolin 3 (red: d, f) and a polyclonal antibody against annexin 6 (green: e, f). Caveolin 3 is associated predominantly with the sarcolemma, labelling of the developing transverse tubular system being much weaker. Annexin 6 is associated with both the sarcolemma and the developing transverse tubules. (gÐi) murine pectoral muscle 4 days after birth. At this stage, caveolin 3 associates exclusively with the sarcolemma. Annexin 6 associates with both the sarcolemma and the fully differentiated transverse tubules, (kÐm) Thin transverse sections of human urinary bladder smooth muscle, dual labelled with a polyclonal antibody against caveolin 1 (red: k, m) and a monoclonal antibody against annexin 6 (green: l, m). Both caveolin 1 and annexin 6 reside in largely overlapping sarcolemmal domains. Fluorescent micrographs. Bars, 3 ␮m(aÐc); 20 ␮m(dÐi); 5 ␮m(kÐm). close its secrets readily—terminal differentiation and T- replaces the caveolin 1 isoform present in myoblasts (Parton tubular formation cannot be observed in cell culture. Mor- et al., 1997). In terminally differentiated cells, caveolin 3 is phological studies have indicated a developmental no longer detectable within the mature, transversely orien- relationship between the sarcolemma and the transverse tated tubular system; it recedes to the plasma membrane, tubular system. The T-tubular system is enriched in choles- where it becomes exclusively associated with sarcolemmal terol and shaped by the membrane-deforming properties of caveolae (Parton et al., 1997). Caveolar and noncaveolar amphiphysin 2 (Lee et al., 2002). When it first appears, rafts most likely become separated during terminal mem- originating from caveolae at the cell surface (Parton et al., brane differentiation, some time after the completion of 1997), it displays an exclusively longitudinal orientation sarcomere organization. Commencing with myoblast fu- (Franzini-Armstrong, 1991). After fusion, the intercon- sion, the regular membrane attachment of nascent sarco- nected precursor tubules are lined with caveolin 3, which meres enables the immature cell to contract, while architec- A. Draeger et al. / Developmental Biology 262 (2003) 324–334 333 tural details, such as sarcolemmmal infoldings which Babiychuk, E.B., Monastyrskaya, K., Burkhard, F.C., Wray, S., Draeger, harbour ion channels, are still missing. A., 2002. Modulating signaling events in smooth muscle: cleavage of The downregulation of noncaveolar raft lipid and protein annexin 2 abolishes its binding to lipid rafts. FASEB J. 16, 1177Ð1184. Babiychuk, E.B., Palstra, R.J., Schaller, J., Ka¬mpfer, U., Draeger, A., 1999. components occurs during the perinatal period, concomitant Ј Annexin VI participates in the formation of a reversible, membrane- with the decline in 5 -nucleotidase activity. Previous studies cytoskeleton complex in smooth muscle cells. J. Biol. Chem. 274, have established an important role for protein C in 35191Ð35195. skeletal muscle differentiation (Rafuse and Landmesser, Barrientos, G., Hidalgo, C., 2002. Annexin VI is attached to transverse- 1996; Fedorov et al., 2002). It is interesting to note that this tubule membranes in isolated skeletal muscle triads. J. Membr. Biol. raft-associated protein (Babiychuk et al., 2002) has been 188, 163Ð173. reported to interact with annexin 6 in skeletal muscle Bloch, R.J., Capetanaki, Y., O’Neill, A., Reed, P., Williams, M.W., Res- neck, W.G., Porter, N.C., Ursitti, J.A., 2002. Costameres: repeating (Schmitz-Peiffer et al., 1998), and is also downregulated structures at the sarcolemma of skeletal muscle. Clin. Orthop. 403 during skeletal muscle myogenesis (Goel and Dey, 2002) (Suppl), S203ÐS210. similar to 5Ј-nucleotidase and raft-markers belonging to the Brown, D.A., London, E., 1998. Functions of lipid rafts in biological annexin-protein family. membranes. Annu. Rev. Cell Dev. Biol. 14, 111Ð36. Our results suggest that cellular maturation leads to pro- Brown, D.A., London, E., 2000. Structure and function of sphingolipid- found structural changes within the sarcolemma. 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Myogenesis in the mouse all-or-nothing response in skeletal muscle as opposed to a embryo: differential onset of expression of myogenic proteins and the multicomponent regulatory mechanism (involving neural, involvement of titin in myofibril assembly. J. Cell Biol. 109, 517Ð527. chemical, and mechanical stimuli) and a finely tuned con- Galbiati, F., Razani, B., Lisanti, M.P., 2001. Caveolae and caveolin-3 in tractile response in smooth muscle. muscular dystrophy. Trends Mol. Med. 7, 435Ð441. Geiger, B., Bershadsky, A., 2002. Exploring the neighborhood: adhesion- coupled cell mechanosensors. Cell 110, 139Ð142. Acknowledgments Gerke, V., Moss, S.E., 2002. Annexins: from structure to function. Physiol Rev. 82, 331Ð371. We thank Dr. Ceri England for critically reading the Goel, H.L., Dey, C.S., 2002. PKC-regulated myogenesis is associated with manuscript. The excellent technical assistance of C. 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