Author Correction Na,K-ATPase in skeletal muscle: two populations of - control localization in the but not partitioning between the sarcolemma and the transverse tubules McRae W. Williams, Wendy G. Resneck, Tamma Kaysser, Jeanine A. Ursitti, Connie S. Birkenmeier, Jane E. Barker and Robert J. Bloch

Journal of Cell Science 123, 1970 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.074260

There was an error published in J. Cell Sci. 114, 751-762.

In Fig. 1, the authors mistakenly included several panels taken from quadriceps muscle that had been used in a previous publication, rather than original images of the extensor digitorum longus. The corrected figure is shown below.

The authors apologize for this error. RESEARCH ARTICLE 751 Na,K-ATPase in skeletal muscle: two populations of β-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules

McRae W. Williams1, Wendy G. Resneck1, Tamma Kaysser2,*, Jeanine A. Ursitti 1, Connie S. Birkenmeier2, Jane E. Barker2 and Robert J. Bloch1,‡ 1Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA 2The Jackson Laboratory, Bar Harbor, ME 04609, USA *Present address: Motorola BioChip Systems, 4088 Commercial Ave., Northbrook, IL 60062, USA ‡Author for correspondence (e-mail: [email protected])

Accepted 28 November 2000 Journal of Cell Science 114, 751-762 © The Company of Biologists Ltd

SUMMARY

We used immunological approaches to study the factors that this isoform of is not necessary to link the controlling the distribution of the Na,K-ATPase in fast Na,K-ATPase to the spectrin-based membrane skeleton. twitch skeletal muscle of the rat. Both α subunits of the In immunofluorescence and subcellular fractionation Na,K-ATPase colocalize with β-spectrin and ankyrin 3 experiments, the α2 but not the α1 subunit of the Na,K- in , structures at the sarcolemma that lie ATPase is present in transverse (t-) tubules. The α1 subunit over Z and M-lines and in longitudinal strands. In of the pump is not detected in increased amounts in the t- immunoprecipitates, the α1 and α2 subunits of the Na,K- tubules of muscle from the ja/ja mouse, however. Our ATPase as well as ankyrin 3 associate with β-spectrin/α- results suggest that the spectrin-based membrane skeleton, fodrin heteromers and with a pool of β-spectrin at the including ankyrin 3, concentrates both isoforms of the sarcolemma that does not contain α-fodrin. Myofibers of Na,K-ATPase in costameres, but that it does not play a mutant mice lacking β-spectrin (ja/ja) have a more uniform significant role in restricting the entry of the α1 subunit distribution of both the α1 and α2 subunits of the Na,K- into the t-tubules. ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K- ATPase in wild-type muscle requires β-spectrin. The Na,K- Key words: Na,K-ATPase, Spectrin, Fodrin, Ankyrin, Sarcolemma, ATPase and β-spectrin are distributed normally in muscle Skeletal muscle, , , Spectrin-based membrane fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting skeleton, Sarcolemma, Transverse (t-) tubule

INTRODUCTION Spectrin is one of the major components of the membrane cytoskeleton responsible for maintaining the biconcave shape The organization of the plasma membrane into distinct of the mammalian erythrocyte (reviewed by Bennett, 1990; structural domains underlies the ability of cells to perform Gallagher and Forget, 1993; Hassoun and Palek, 1996). In the many specific functions, from maintaining ionic homeostasis erythrocyte, spectrin is composed of heterodimeric complexes to synaptic transmission. Understanding how such domains of αI and βI subunits (Winkelmann and Forget, 1993), products form has been difficult, even in bipolar cells like epithelia. It of the human erythroid spectrin , SPTA1 and SPTB, is especially challenging in excitable cells that, in addition to respectively. αβ-Spectrin heterodimers can associate head-to- the usual set of intracellular membrane compartments, also head to form tetramers and higher oligomers that can contain many local plasmalemmal domains devoted to synaptic polymerize further by virtue of their ability to bind transmission, ion transport, and metabolic trafficking. We are (Bennett, 1990; Pumplin and Bloch, 1993; Hartwig, 1995; Viel studying skeletal muscle as a model excitable cell, as its plasma and Branton, 1996). This network is anchored to the membrane, or sarcolemma, is accessible in biochemical erythrocyte membrane through its interaction with erythroid amounts, repetitive in organization, and composed of fewer ankyrin, ankyrin 1, which links the β subunit of spectrin to the types of domains than the plasma membranes of , with bicarbonate/chloride exchanger, an integral . which it otherwise shares many similarities. Our goal is to Mutation or loss of either spectrin or ankyrin results in understand the unusual structure and function of the hemolytic anemia, consistent with the idea that both sarcolemma at the molecular level. Here we focus on the role are vital to the proper support of the red cell membrane of the spectrin-based membrane skeleton in organizing the (Gallagher and Forget, 1993; Hassoun and Palek, 1996). Na,K-ATPase in the sarcolemma and in the transverse tubule Spectrin is also a component of the membrane-associated system, to which the sarcolemma is connected. cytoskeleton in skeletal muscle (Nelson and Lazarides, 1983; 752 JOURNAL OF CELL SCIENCE 114 (4) Craig and Pardo, 1983; Porter et al., 1992; Vybiral et al., 1992; We have used a combination of immunofluorescence and Porter et al., 1997; Zhou et al., 1998), the importance of which immunoprecipitation approaches, as well as mouse mutants has been underscored because it contains , the that are missing βI-spectrin or the erythroid form of ankyrin protein missing in patients with Duchennes Muscular (ankyrin 1 or Ank1; AnkR), to determine the nature of the Dystrophy (Hoffman et al., 1987). Spectrin is present at the sarcolemmal complex containing β-spectrin. Our results sarcolemma in two distinct populations, both of which contain indicate that both populations of β-spectrin are anchored to the the alternatively spliced product of βI-spectrin expressed by Na,K-ATPase in the sarcolemma via ankyrin 3 (Ank3 or striated muscle (βIΣ2: Winkelmann et al., 1990). One AnkG). This suggests that the distribution of the two population, selectively enriched at sites overlying Z-lines, populations of β-spectrin into distinct sarcolemmal domains contains the muscle form of β-spectrin associated with α- cannot readily be explained by an association with different fodrin (αII-spectrin), expressed by the SPTAN1 . The sets of and integral membrane proteins. other population, selectively enriched at sites overlying M- We further report on the subcellular distribution of the two lines and in longitudinally oriented strands, contains muscle β- α subunits of the Na,K-ATPase present in normal and mutant spectrin without significant amounts of an identifiable α skeletal muscle fibers. Although both the α1 and α2 forms of subunit (Porter et al., 1997; Zhou et al., 1998). Viewed in the Na,K-ATPase are present at the sarcolemma, where they longitudinal sections, the network of β-spectrin at the associate with muscle β-spectrin, only α2 is present in sarcolemma appears as a rectilinear grid or lattice (Porter et al., significant amounts in the transverse tubules (t-tubules; Lavoie 1992; Porter et al., 1997; Williams and Bloch, 1999a,b), the et al., 1997). We show that this differential distribution is not elements of which have been termed ‘costameres’ (Pardo et al., altered in mutant muscle lacking β-spectrin. Thus, their 1983; Porter et al., 1992). Although the molecular cloning and association with the spectrin-based membrane skeleton at the sequencing of β-spectrin from muscle is not yet complete, the sarcolemma does not influence the relative abilities of the α1 available information (Winkelmann et al., 1990; Weed, 1996; and α2 subunits of the Na,K-ATPase to partition into the t- Zhou et al., 1998) suggests that the same isoform of βI-spectrin tubules. is present in the two populations of β-spectrin at the sarcolemma. The population of muscle β-spectrin that is associated with MATERIALS AND METHODS α-fodrin forms heterodimers that resemble the αβ-spectrin heterodimers of the erythrocyte (Porter et al., 1997). This Materials similarity suggests that the association of β-spectrin/α-fodrin Unless otherwise specified, all materials were purchased from Sigma heteromers with the sarcolemma is likely to be mediated by Chemical Co. (St Louis, MO) and were the highest grade available. ankyrin, several isoforms of which are present in skeletal muscle (Nelson and Lazarides, 1984; Flucher and Daniels, Antibodies The rabbit antibody, 9050, was made against purified human 1989; Birkenmeier et al., 1993; Birkenmeier et al., 1998; β β Σ Devarajan et al., 1996; Zhou et al., 1997; Kordeli et al., 1998; erythrocyte -spectrin ( I 1) and was affinity-purified over a column of erythrocyte β-spectrin and cross-adsorbed against β-fodrin (βII) Tuvia et al., 1999; Wood and Slater, 1998). Possible candidates and α-fodrin (αII), purified from bovine brain (Porter et al., 1997; for the integral sarcolemmal protein(s) to which ankyrin in turn Zhou et al., 1998). Additional antibodies to erythroid β-spectrin were binds include the chloride-bicarbonate exchanger, the Na,Ca- generated in laying hens and purified with the EggStract kit (Promega, exchanger (Li et al., 1993), the voltage-gated Na channel Madison, WI) and affinity purification, as described for 9050. (Srinivasan et al., 1988; Flucher and Daniels, 1989; Wood and Specificity was demonstrated by immunoblotting (J. A. Ursitti et al., Slater, 1998), cell adhesion proteins (Davis and Bennett, 1993), unpublished). The generation and purification of rabbit anti-α-fodrin, and the Na,K-ATPase (Nelson and Veshnock, 1987; 9053, has been described (Porter et al., 1997). Madreperla et al., 1989; Morrow et al., 1989; Davis and Monoclonal antibodies to the α1 and α2 subunits of the Na,K- Bennett, 1990; Devarajan et al., 1994; Jordan et al., 1995; ATPase, McK1 and McB2 (Felsenfeld and Sweadner, 1988; Urayama Thevananther et al., 1998; Zhang et al., 1998), all of which bind β to ankyrin in other excitable cells. As -spectrin alone is β α α capable of binding ankyrin (Kennedy et al., 1991), it seems Fig. 1. Colocalization of -spectrin, ankyrin 3 and the 1 and 2 subunits of the Na,K-ATPase in costameres. Perfusion-fixed EDL likely that the population of muscle β-spectrin that lacks an µ α muscle was snap frozen and 20 m thick longitudinal sections were identifiable subunit associates with the membrane similarly. cut on a cryostat. Sections were labeled with affinity-purified chicken How the two populations of β-spectrin form in skeletal (B,E,K) or rabbit (H) anti-β-spectrin, followed by fluoresceinated muscle, and how they generate distinctive domains at the secondary antibodies. Individual samples were double labeled with sarcolemma, are still poorly understood. One possibility is that polyclonal rabbit antibodies to the α1 (A), and α2 (D) subunits of the different structures, approaching the sarcolemma from the Z Na,K-ATPase, or with monoclonal antibodies to ankyrin 3 (G), and M-lines of the nearby contractile apparatus, interact followed by tetramethylrhodaminylated secondary antibodies. Non- selectively with different components of the membrane immune rabbit serum was used as a control (J). Similar controls with non-immune chicken and mouse antibodies gave similar results (not skeleton, helping to stabilize and localize the two spectrin β populations. A second possibility is that the organization is shown). Composite images were constructed with -spectrin shown in green, the other antigens in red, and areas with both proteins in imposed by domains of the muscle’s basal lamina (e.g. yellow (C,F,I,L). The results show that all four proteins codistribute Colognato et al., 1999). Alternatively, the two populations in the rectilinear, costameric lattice. Bars, 5 µm. Insets in A-C: two- of β-spectrin may bind to different ankyrins that in turn are fold magnifications of the boxed regions indicate the domains of linked to different integral proteins concentrated in discrete costameres: longitudinal strands (arrowhead), Z line domains (large sarcolemmal domains. arrows) and M line domains (small arrow). Na,K-ATPase and spectrin skeleton in myofibers 753

Fig. 1 754 JOURNAL OF CELL SCIENCE 114 (4) et al., 1989), were from Dr K. J. Sweadner (Massachusettes General quadriceps muscles were removed and incubated for an additional 5 Hospital, Boston, MA). Rabbit antibodies to the α1 and α2 subunits, minutes in 2% paraformaldehyde in PBS. Tissue was blotted dry, snap and monoclonal mouse antibody to α1, were purchased from Upstate frozen and cryosectioned (20 µm thickness). Samples were collected Biotechnologies (Lake Placid, NY). Antibodies to ankyrins 2 and 3 on slides coated with a solution of 0.5% gelatin, 0.05% chromium were generously provided by Dr V. Bennett (Howard Hughes Medical potassium sulfate, and stored at −70°C. Tissue for cross sections (20 Institute, Duke University, Durham NC) and to ankyrin 3 by Dr J. µm) was obtained without perfusion or fixation, but was otherwise Morrow (Department of Pathology, Yale University School of handled as above. The preparation of unfixed longitudinal sections has Medicine, New Haven CT). Rabbit antibodies to the spectrin-binding been reported (Williams and Bloch, 1999a). domain of ankyrin 1, p65, have been described (Zhou et al., 1997). Non-immune mouse monoclonal antibodies, MOPC21, were obtained Fluorescent immunolabeling and imaging from Sigma Chemical Co. (St Louis, MO). Sections were incubated in PBS/BSA (PBS containing 1 mg/ml BSA, Secondary antibodies included goat anti-rabbit, goat anti-mouse, 10 mM sodium azide) for 15 minutes and then in primary antibody in donkey anti-mouse, and goat anti-sheep IgGs, and goat anti-chicken PBS/BSA for 2 hours at room temperature, or overnight at 4°C. When IgY, conjugated to fluorescein or tetramethylrhodamine for use in sheep antibodies to the dihydropyridine receptor (DHPR) were used, immunofluorescence experiments, or to alkaline phosphatase for use in immunoblotting. These antibodies, as well as non-immune sheep and rabbit sera, were from Jackson Immunoresearch Laboratories (West Grove, PA). All secondary antibodies were species-specific with minimal cross reactivity. Animals Female Sprague-Dawley rats, aged 6 months to 1 yr (Zivic-Miller, Zelienople, PA) were used. Ankyrin 1-deficient mice (nb/nb: White et al., 1990), β-spectrin-deficient mice (ja/ja: Bodine et al., 1984; Bloom et al., 1994), and age- matched controls were bred and raised in the Barker laboratory. As soon as they suckled, newborn ja/ja mice were transfused through the superficial temporal vein with 0.1 ml of concentrated erythrocytes, obtained from blood Fig. 2. The α1 and α2 subunits of the Na,K-ATPase, ankyrin 3 and β-spectrin drawn from the retroorbital sinus of a C57BL/6J coprecipitate. Homogenates of rat hindlimb muscle were prepared, precleared twice, and (B6)-+/+ mouse. Transfusion allowed the ja/ja then incubated either with Sepharose-Protein A bound to normal rabbit IgG (‘NRS pellet’) mice, which normally die within 7 days after or to affinity-purified rabbit antibodies to β-spectrin, 9050 (‘β-sp pellet’). Pellets were birth, to survive for an average of 3.7 months analyzed by SDS-PAGE and immunoblotting with 9050 anti-β-spectrin (A), McK1 (Kaysser et al., 1997). antibodies to the α1 subunit of the Na,K-ATPase (B), McB2 antibodies to the α2 subunit of the Na,K-ATPase (C), monoclonal antibodies to ankyrin 3 (D), and MopC control mouse Cryosectioning IgG (E). Samples for B, C and E were dissolved in SDS-PAGE buffer at 37°C; samples for Animals were anesthetized and sacrificed by A and D were boiled. The results show that ankyrin 3 and both the α1 and α2 subunits of perfusion fixation, as described (Williams and the Na,K-ATPase are present in the precipitates generated with anti-β-spectrin (lanes 2), Bloch, 1999a). The tibialis anterior (TA), but not with non-immune serum (lanes 1). All immunolabeling of the anti-β-spectrin extensor digitorum longus (EDL) and pellets is specific.

Fig. 3. The α1 and α2 subunits of the Na,K-ATPase and ankyrin 3 co-immunoprecipitate with β- spectrin that is not associated with α-fodrin, as well as with β- spectrin/α-fodrin heteromers. Homogenates, prepared and precleared as in Fig. 2, were subjected to immunoprecipitation twice with anti-α-fodrin (9053; ‘α-fo pellet I’ and ‘α-fo pellet II’), and once with anti-β-spectrin (9050; ‘β-sp pellet’). Controls were prepared with normal rabbit serum (‘NRS pellet’). Pellets were collected, and processed for SDS-PAGE. Immunoblotting was with 9050 antibodies to β-spectrin (A), 9053 antibodies to α-fodrin (B), McK1 (C) and McB2 (D) antibodies to the α1 and α2 subunits of the Na,K-ATPase, and with monoclonal antibodies to ankyrin 3 (E). The results show that 9053 anti-α-fodrin immunoprecipitates the α1 and α2 subunits of the Na,K-ATPase, together with β-spectrin and ankyrin 3 (A-E, lanes 2 and 3). The same proteins are immunoprecipitated by 9050 anti-β-spectrin (lanes 4 in A, C-E), without α-fodrin (B, lane 4). None are precipitated by NRS (lanes 1). This suggests that ankyrin 3 and the α1 and α2 subunits of the Na,K-ATPase associate both with β-spectrin/α-fodrin heteromers, and with the pool that contains β-spectrin with no known α-spectrin-like subunit. Na,K-ATPase and spectrin skeleton in myofibers 755 solutions contained 0.01% Triton X-100. After washing, samples were protease inhibitors (Porter et al., 1992), homogenized in a Polytron incubated for 1 hour in secondary antibodies in PBS/BSA, washed PT 10/35 Brinkmann homogenizer at 4°C for 1 minute (4× 15 again and mounted (Williams and Bloch, 1999a). Samples were seconds), and incubated for 1 hour at 4°C. Insoluble material was observed with a Zeiss 410 confocal laser scanning microscope removed by centrifugation at 16,000 RPM for 1 hour (4°C, SS-34 equipped with a ×63, NA 1.4 plan-apochromatic objective. The rotor, Sorvall Instruments, Newton, CT), and the supernatant was pinholes for fluorescein and tetramethylrhodamine were 18. All stored at −70°C. labeling was shown to be specific through the use of the appropriate non-immune controls (e.g. Fig. 1). Subcellular fractionation To generate figures, images were arranged and labeled with Corel T-tubule membranes (fraction F3) and sarcolemmal fractions (fraction Draw 6 (Corel Corporation Ltd, Ottawa, Ontario). Insets were PF6) were isolated from rabbit skeletal muscle, as described prepared with Metamorph (Universal Imaging, West Chester, PA) and (Dombrowksi et al., 1996). magnified 2-fold with Corel Draw 6. SDS-PAGE and immunoblotting Muscle homogenates Proteins were separated by SDS-PAGE on 5-15% acrylamide minigels Sprague-Dawley rats, anesthetized as above, were perfused (Porter et (Hoefer, San Francisco, CA) as described (Laemmli, 1970), except al., 1997). The major muscle groups from the hindlimb were dissected that samples to be tested for the Na,K-ATPase were incubated in and frozen in liquid nitrogen. While submerged in liquid nitrogen, the sample buffer at 37°C for 15 minutes instead of boiling. Molecular sample was ground to a fine powder with a mortar and pestle. This mass standards were acquired from Bethesda Research Laboratories powder was suspended in a solution (modified from Hoffman et al., (Bethesda, MD). Some gels were visualized with Coomassie Brilliant 1989) containing 1% deoxycholate, 1% NP-40, 10 mM sodium Blue or silver staining. For immunoblotting, proteins were transferred phosphate, 0.14 M NaCl, 2 mM EDTA, pH 6.8, supplemented with to nitrocellulose (Burnette, 1981). Blots were incubated briefly in

Fig. 4. Loss of the Na,K-ATPase from costameres in β-spectrin-deficient but not ankyrin 1-deficient mice. Perfusion-fixed tibialis anterior muscle of wild-type (A,B,E,F), ankyrin-1 deficient, nb/nb (C,D,G,H), and β-spectrin-deficient, ja/ja (I-L) mice were cryosectioned and labeled as in Fig. 1. β-Spectrin was labeled with affinity-purified chicken antibodies (A,C,E,G,K) or non-immune chicken IgY (L), followed by fluorescein-conjugated goat anti-chicken IgY. The α1 (B,D,I) and the α2 (F,H,J) subunits of the Na,K-ATPase were labeled by rabbit antibodies followed by tetramethylrhodamine-conjugated goat anti-rabbit IgG. Labeling of ja/ja muscle shows no β-spectrin at the sarcolemma (K); low levels of cytoplasmic labeling detected in this sample are also seen with non-immune antibody (L, control) and hence are non-specific. The results show costameric localization of the α1 and the α2 subunits of the Na,K-ATPase in wild-type and nb/nb muscle, but more uniform distributions in the ja/ja sample. Bars, 5 µm. 756 JOURNAL OF CELL SCIENCE 114 (4)

Fig. 5. Differential fractionation of the α1 and the α2 subunits of the Na,K-ATPase. Fractions enriched in transverse tubules (lanes 1) and the sarcolemma (lanes 2) were isolated from rabbit hindlimb muscle and analyzed by SDS-PAGE and immunoblotting. Blots were probed with McK1, McB2, and monoclonal antibodies to the DHPR, a t- tubule marker. Both the α1 and the α2 subunits of the Na,K-ATPase are present in the sarcolemmal fraction, but only the α2 subunit is present in the t-tubule fraction.

milk-PTA (3% milk solids 10 mM NaN3, 0.5% Tween-20 in PBS) and then overnight at 4°C or for 2 hours at room temperature in primary antibody in milk-PTA. After washing, samples were incubated with secondary antibodies in milk-PTA for 1 hour at room temperature. Bound antibody was visualized chromogenically (Kirkegaard and Perry, Gaithersburg, MD) or by chemiluminescence (‘Western Light Detection’ kit, Tropix Laboratories, Bedford, MA). Immunoprecipitation Aliquots of rat muscle homogenate containing 1 mg protein were incubated overnight with mouse IgG bound to Sepharose beads, to

Fig. 6. Subcellular localization of the α1 and the α2 subunits of the Na,K-ATPase in ja/ja muscle. Frozen cross-sections of unfixed rat or mouse muscle were extracted briefly with detergent and labeled by immunofluorescence. (A-F) Sections of rat EDL were double labeled with McK1 antibody to the α1 (A) or McB2 antibody to the α2 (D) subunit of the Na,K-ATPase, together with polyclonal sheep antibodies to the DHPR (B,E). Secondary antibodies were followed by tetramethylrhodamine-conjugated goat anti-mouse IgG and fluorescein-conjugated donkey anti- sheep IgG. The color composite images (C,F) show the Na,K-ATPase subunit in red, the DHPR in green, and structures containing both labels in yellow. The results show that the α2 but not the α1 subunit of the Na,K-ATPase codistributes with the DHPR in the t-tubules, but that both subunits are present at the sarcolemma. (G-J) Sections of the tibialis anterior muscle of wild-type (G,I) and β-spectrin-deficient, ja/ja mice (H,J) were immunolabeled with McK1 (I,J) and McB2 (G,H). The subcellular localization of both α subunits is identical in both samples. Bars, 5 µm. Na,K-ATPase and spectrin skeleton in myofibers 757 remove nonspecifically bound protein. (Early experiments had shown structures containing the two populations of muscle β-spectrin a nonspecific reaction of mouse secondary antibodies with a ~100 kDa (Williams and Bloch, 1999a,b), one over Z-lines that is protein in immunoblots of unboiled samples that was eliminated by composed of β-spectrin and α-fodrin, the other over M-lines this step.) Beads were pelleted by centrifugation (14,000 rpm, 5 and in longitudinal strands that contains β-spectrin without seconds, Eppendorf 5415 centrifuge). The pellet was mixed with a any identifiable α subunit (Porter et al., 1997). We use volume of sample buffer (Laemmli, 1970) equal to 1/3 of the original immunofluorescence and immunoprecipitation protocols, as homogenate volume. The remaining supernatant was then precipitated well as mouse mutants that lack key components of the overnight with Protein A-Sepharose beads (Pharmacia, LKB, α α Uppsala, Sweden), to remove more non-specific binding components. membrane skeleton, to study the ability of the 1 and 2 After centrifugation, the remaining supernatant was incubated with subunits of the Na,K-ATPase to associate with ankyrin 3 and Protein A-Sepharose beads bound overnight at 4°C to the appropriate spectrin at the sarcolemma, and their ability to partition antibodies. Aliquots containing 10 µg of rabbit antibody or the between the sarcolemma and the transverse tubules of equivalent in normal rabbit serum were used for each mg protein in myofibers. the original homogenate. Beads were mixed overnight at 4°C with supernatant. After centrifugation, the pellet was washed and mixed Immunofluorescent localization in costameres with SDS-PAGE sample buffer, as above, and divided into two equal Double immunofluorescence labeling experiments were used fractions. One fraction was incubated at 37°C for 15 minutes; the to examine the distribution of the α1 and α2 subunits of the other was boiled for 5 minutes. Beads and sample buffer were β separated by low speed centrifugation through a Centricon filter (pore Na,K-ATPase and -spectrin in paraformaldehyde-fixed, size, 0.45 µm: Amicon, Danvers, MA). longitudinal cryosections of rat extensor digitorum longus We used the affinity-purified antibody, 9050, for (EDL) muscle. Primary antibodies were visualized with immunoprecipitation of β-spectrin. For sequential species-specific secondary antibodies coupled to fluorescein immunoprecipitations, homogenates were precipitated first with and tetramethylrhodamine. As reported previously (see 9053 anti-α-fodrin, then with 9053 again, to assure that all the α- Introduction for references), muscle β-spectrin is concentrated fodrin was removed, and finally with 9050, to precipitate the in costameres, present at the sarcolemma over Z and M-lines remaining β-spectrin (Porter et al., 1997). and in longitudinal strands (Fig. 1A and inset). The distribution of the α1 subunit of the Na,K-ATPase was nearly identical to that of β-spectrin, with high concentrations at the costameres RESULTS and no significant staining in the intercostameric regions (Fig. 1B; see Williams and Bloch, 1999b). This was confirmed by Our experiments were designed to elucidate the relationship computer-generated composite pictures (Fig. 1C). We obtained between the two isoforms of the Na,K-ATPase expressed in similar results when we compared the distribution of the α2 skeletal muscle, and the two distinct populations of β-spectrin subunit of the Na,K-ATPase with β-spectrin (Fig. 1D-F). We present at the sarcolemma. We chose to study the Na,K-ATPase never observed regions of the sarcolemma that contained because it has been associated with the spectrin-based significant amounts of β-spectrin without also containing the membrane skeleton in other cells, and isoform-specific α1 or α2 subunits of the Na,K-ATPase, or that contained antibodies are available to the two forms, α1 and α2, expressed significant amounts of one of the subunits of the Na,K-ATPase in skeletal muscle (Orlowski and Lingrel, 1988). We focus without also containing β-spectrin. Controls omitting one of on fast twitch muscle, because it displays clearly defined the primary antibodies showed that this colocalization was not

Fig. 7. Molecular model of the Na,K-ATPase, ankyrin 3 and two populations of spectrin at costameres: this cartoon shows the region of the muscle fiber that includes the sarcolemma and nearby structures. From the extracellular surface to the myoplasm, it depicts the Na,K-ATPase α (including α1 and α2 isoforms) and β subunits (α,β); the membrane skeletal proteins, ankyrin 3 (Ank), α-fodrin, and muscle β-spectrin; and the contractile proteins, actin , and α-. We propose that the population of β-spectrin associated with α-fodrin is concentrated over Z- lines by virtue of interactions between the membrane skeleton and structures, probably -based intermediate filaments, that emerge from the contractile apparatus at Z-lines. We further propose that the population of β-spectrin without α-fodrin is concentrated over M-lines and in longitudinal strands (not shown) by virtue of interactions with other structures, probably also intermediate filaments, that emerge from the contractile apparatus at M-lines. Not drawn to scale. 758 JOURNAL OF CELL SCIENCE 114 (4) due to fluorescence ‘bleed-through’ or species cross-reactivity precipitation with anti-α-fodrin again concentrated β-spectrin by secondary antibodies (e.g. Fig. 1J-L). As the primary and α-fodrin in the pellet, although in apparently smaller antibodies react specifically with their respective antigens (see amounts than in the previous precipitation (Fig. 3A,B, lanes 3), below), all the labeling was therefore specific. These results consistent with the nearly complete removal of the α-fodrin suggest that both the α1 and α2 subunits of the Na,K-ATPase from the supernatant (not shown). The final precipitation with concentrate with β-spectrin in costameres of fast twitch anti-β-spectrin concentrated most of the remaining β-spectrin myofibers of the rat. in the pellet (Fig. 3A, lane 4). Neither α-fodrin nor β-spectrin We next examined ankyrin 3, which has been associated with was precipitated non-specifically, as pellets generated with the Na,K-ATPase in other tissues. As reported above for the α non-immune rabbit serum did not contain either protein (Fig. subunits of the Na,K-ATPase, ankyrin 3 colocalized with β- 3A,B, lanes 1). Thus, sequential immunoprecipitation spectrin (Fig. 1G-I), suggesting that both ankyrin 3 and the two separates a population of β-spectrin that is associated with α- α subunits of the Na,K-ATPase are concentrated in all regions fodrin from one that is not (Porter et al., 1997). of costameres, together with muscle β-spectrin. Ankyrin 3 and We probed these samples with antibodies to the α1 and α2 the Na,K-ATPase are therefore likely to associate with both subunits of the Na,K-ATPase (Fig. 3C,D) and to ankyrin 3 (Fig. populations of β-spectrin (see Introduction), the population 3E). Both subunits of the Na,K-ATPase were present in the over Z-lines bound to α-fodrin, and the population over M- precipitates generated by anti-α-fodrin (Fig. 3C,D lanes 2,3). lines and in longitudinal domains that does not contain The final immunoprecipitation with anti-β-spectrin also significant amounts of α-fodrin. concentrated these proteins in the pellet (Fig. 3C,D, lanes 4). Ankyrin 3 was also present in the two immunoprecipitates Co-immunoprecipitation of the Na,K-ATPase, generated by anti-α-fodrin (Fig. 3E, lanes 2,3) and in the ankyrin 3 and β-spectrin immunoprecipitate generated by anti-β-spectrin (Fig. 3E, lane We used immunoprecipitation to study the association of the 4). Neither of the α subunits of the Na,K-ATPase nor ankyrin Na,K-ATPase and ankyrin 3 with muscle β-spectrin. 3 were concentrated in the non-immune precipitate (Fig. 3C- Immunoprecipitates generated from muscle homogenates with E, lanes 1). These results show that ankyrin 3 and both the α1 affinity-purified antibodies to β-spectrin (see Materials and and α2 subunits of the Na,K-ATPase associate specifically with Methods) were collected and examined by immunoblotting for both populations of β-spectrin in skeletal muscle. To our ankyrin 3 and for each of the α subunits of the Na,K-ATPase. knowledge, this is the first time that the biochemical Precipitation with anti-β-spectrin concentrated β-spectrin in association of ankyrin 3 with βI-spectrin has been the pellet (Fig. 2A, lane 2; band at ~265 kDa). The pellet also demonstrated to occur in situ. contained significant amounts of ankyrin 3 (Fig. 2D, lane 2; band at ~190 kDa; see also Thevananther et al., 1998) and the Immunofluorescence studies in mutant muscle α1 and α2 subunits of the Na,K-ATPase (Fig. 2B,C, lanes 2; We further tested the idea that the α1 and α2 subunits of the bands at ~100 kDa). We could not detect ankyrin 1 or ankyrin Na,K-ATPase associate with both populations of muscle β- 2 in the immunoprecipitate, however (not shown). The spectrin by examining the sarcolemma of two mouse mutants, presence of β-spectrin, ankyrin 3 and the two isoforms of the ja/ja and nb/nb, which lack βI-spectrin and ankyrin 1, Na,K-ATPase in the immunoprecipitate was specific, as they respectively (Bodine et al., 1984; White et al., 1990; Bloom et could not be detected in control immunoprecipitates generated al., 1994). Based on our results with rat muscle, we predicted with normal rabbit serum (Fig. 2A-E, lanes 1), nor were they that both forms of the Na,K-ATPase would be nearly uniformly labeled in the immunoblots by a non-immune mouse (Fig. 2E, distributed at the sarcolemma of ja/ja mice but would be found lane 2) or rabbit IgG (not shown). These results suggest that normally in costameres of nb/nb mice. muscle β-spectrin, ankyrin 3 and both the α1 and α2 subunits Double immunofluorescence labeling of the sarcolemma of of the Na,K-ATPase are associated in a complex in vivo. fast twitch, EDL muscle of wild-type mice showed the The presence of the Na,K-ATPase and ankyrin 3 at all costameric pattern described above. Muscle β-spectrin, costameric regions suggests that these proteins interact not recognized with affinity-purified chicken antibodies, was only with the population of β-spectrin at Z-lines, associated concentrated in a rectilinear array composed of longitudinal with α-fodrin, but also with the population of β-spectrin at M- strands and elements overlying Z-lines and M-lines (Fig. lines and in longitudinal domains, which do not contain 4A,E). Both the α1 and α2 subunits of the Na,K-ATPase were significant amounts of α-fodrin or any other identifiable α- concentrated in the same structures (Fig. 4B,F). We observed spectrin-like subunit (Porter et al., 1997; Zhou et al., 1998). We similar patterns in EDL fibers from the nb/nb mouse (Fig. employed sequential immunoprecipitation to examine this 4C,D,G,H), suggesting that ankyrin 1 is not necessary for the further. We used antibodies to α-fodrin in two rounds of organization of β-spectrin or either isoform of the Na,K- immunoprecipitation to obtain the β-spectrin/α-fodrin ATPase at the sarcolemma. heteromers, and then immunoprecipitated with antibodies to β- The organization of the sarcolemma in the ja/ja mouse was spectrin to isolate β-spectrin that is free of α-fodrin (Porter et significantly altered, however. As expected from the nature of al., 1997). The pellets were analyzed, as above, for the the ja/ja mutation (Bloom et al., 1994), we detected no β- presence of β-spectrin, ankyrin 3, and the α1 and α2 subunits spectrin at the sarcolemma (Fig. 4K), which did, however, label of the Na,K-ATPase, as well as for α-fodrin. As controls we with antibodies to the α subunits of the Na,K-ATPase (Fig. used precipitates generated with non-immune sera. 4I,J). Unlike the wild type, both the α1 and the α2 subunits The first immunoprecipitation with anti-α-fodrin of the Na,K-ATPase were more broadly distributed in the concentrated both β-spectrin and α-fodrin in the pellet (Fig. sarcolemma of ja/ja muscle (Fig. 4I,J). Indeed, the α1 subunit 3A,B, lanes 2), as reported (Porter et al., 1997). The second appeared nearly uniformly distributed (Fig. 4I). The α2 subunit Na,K-ATPase and spectrin skeleton in myofibers 759 also redistributed, as it was clearly not confined to the DISCUSSION rectilinear lattice of costameres in ja/ja muscle, although it could still be detected in irregular longitudinal strands and in The formation of distinctive membrane domains requires that Z-lines (Fig. 4J). The α2 subunit is also present intracellularly particular membrane proteins be delivered to and retained in (Fig. 6D), so its apparent presence at Z-lines may be due to the the plasma membrane or in appropriate subcellular membrane inability of the confocal microscope to distinguish between fractions and, often, that these proteins be concentrated structures in the membrane and in nearby myofibrils. The together into characteristic regions or structures. The irregularity of the longitudinal strands containing α2 suggests cytoskeleton has been proposed to play a role in both the presence of folds, but the possibility remains that some of processes, first by helping to deliver proteins to particular the longitudinal domains of costameres are stable in ja/ja membrane systems, and then by interacting with proteins to muscle. Nevertheless, our results show that the sarcolemmal concentrate them into distinctive structures or domains. These organization of both the α1 and the α2 subunits of the Na,K- processes are not understood in excitable cells, nor have they ATPase is altered when β-spectrin is absent. We obtained been studied with proteins, like the Na,K-ATPase, that are similar results on muscle fibers that had been fixed in situ and targeted to more than one membrane system and that can on fibers that were cryosectioned from unfixed tissue, accumulate in distinctive structures in those membranes. Here suggesting that the altered distribution of the Na,K-ATPase was we study the role in these processes of one part of the not caused during the collecting or processing of tissue cytoskeleton, the spectrin-based membrane skeleton. We show samples. These results suggest that β-spectrin is required for that β-spectrin is required for the accumulation of both forms the localization of both the α1 and the α2 forms of the Na,K- of the Na,K-ATPase into costameres at the sarcolemma, but not ATPase at costameres. for the differential partitioning of the α1 and α2 forms of the Na,K-ATPase between the sarcolemmal and the t-tubular Subcellular distribution of the α1 and α2 subunits of membranes. the Na,K-ATPase in wild-type and ja/ja mice In addition to labeling at the sarcolemma, antibodies to the α2 Complex containing both forms of the Na,K-ATPase, subunit of the Na,K-ATPase, but not the α1 subunit, labeled ankyrin 3 and muscle β-spectrin structures in the myoplasm. The α2 subunit has been found in Association of , including the general tissue homolog, subcellular fractions enriched in t-tubules, while both the α1 fodrin, with the Na,K-ATPase has been studied extensively in and the α2 subunits are present in sarcolemmal fractions renal epithelium (Nelson and Veshnock, 1987; Morrow et al., (Lavoie et al., 1997). We confirmed this observation in rabbit 1989; Nelson and Hammerton, 1989; Davis and Bennett, 1990; calf muscle by immunoblotting fractions isolated from see also Madreperla et al., 1989; Smith et al., 1993). The sarcolemma and t-tubular membrane (Fig. 5). The t-tubule predominant epithelial spectrins are heterotetramers of α- and fraction, identified by the presence of high concentrations of β-fodrin ([αIIβII]2) that associate with the Na,K-ATPase the dihydropyridine receptor (DHPR: Fig. 5C) contained only through ankyrin 3 (AnkG: Nelson and Hammerton, 1989; Koob the α2 subunit and had no detectable α1 subunit (Fig. 5A,B, et al., 1990; Hu et al., 1995; Peters et al., 1995; Thevananther lanes 1). The sarcolemmal fraction, which lacked detectable et al., 1998). By contrast, mature, fast twitch skeletal muscle DHPR, contained both α subunits of the Na,K-ATPase (Fig. fibers express several ankyrins (see Introduction for 5A,B, lanes 2). references), as well as α-fodrin and an excess of β-spectrin We also used double immunofluorescence labeling of unfixed (Porter et al., 1997; Zhou et al., 1998), but no β-fodrin (Weed, cross sections of rat EDL muscle to examine the subcellular 1996; Zhou et al., 1998; Williams et al., 2000). Despite these distribution of the α1 and α2 subunits of the Na,K-ATPase. differences, our results suggest that the Na,K-ATPase at the Antibodies to the α2 (Fig. 6D) but not the α1 (Fig. 6A) subunit sarcolemma associates quite efficiently with ankyrin 3 and revealed a distinctive reticular pattern in the sarcoplasm that co- muscle β-spectrin. labeled with antibodies to the DHPR (Fig. 6D-F), consistent with Indeed, our immunofluorescence studies indicate that both the presence of the α2 subunit of the Na,K-ATPase in t-tubules. the α1 and α2 forms of the Na,K-ATPase are restricted to Antibodies to the α1 subunit of the Na,K-ATPase did not show costameres, and that this restriction requires β-spectrin, significant intracellular labeling and so gave no overlap with whether or not it is associated with α-fodrin. In wild-type DHPR (Fig. 6A-C). Thus, morphological studies confirm the muscle little or no labeling of the Na,K-ATPase could be results of subcellular fractionation. detected in sarcolemmal regions outside of costameres, We examined muscle from the ja/ja mouse to learn if whereas in ja/ja samples the Na,K-ATPase (especially the interactions with the spectrin-based membrane skeleton at the α1 subunit) seemed nearly uniformly distributed in the sarcolemma plays a role in excluding the α1 subunit of the sarcolemma. Our immunoprecipitation protocols confirmed the Na,K-ATPase from the t-tubules. Frozen cryosections of EDL association of both subunits of the Na,K-ATPase with β- muscle from ja/ja mice were double labeled with antibodies to spectrin. Surprisingly, considering the number of studies of the the α1 or α2 subunits of the Na,K-ATPase, together with Na,K-ATPase and its association with the membrane skeleton antibodies to the DHPR, to mark t-tubules (not shown). As with in epithelia, ours appears to be the first definitive demonstration controls (Fig. 6G), the α2 subunit was present both at the that the Na,K-ATPase can associate with βI-spectrin in sarcolemma and in the t-tubules of ja/ja muscle (Fig. 6H), while mammalian cells (see also Vladimirova et al., 1998; Williams the α1 subunit remained restricted to the sarcolemma (Fig. 6I,J). and Bloch, 1999a). Thus, there is no significant change in the subcellular Our results further suggest that the linkage of the Na,K- distribution of either form of the Na,K-ATPase when β-spectrin ATPase to the spectrin-based membrane skeleton is mediated is absent from the sarcolemmal membrane skeleton. to a significant extent by ankyrin 3, which associates with 760 JOURNAL OF CELL SCIENCE 114 (4) muscle β-spectrin whether or not it is bound to α-fodrin. subunit. This complex is responsible for the localization of both Indeed, our results are the first to demonstrate biochemically the α1 and α2 isoforms of the Na,K-ATPase to costameres at the association in vivo of βI-spectrin (the product of the the sarcolemma of fast twitch myofibers, but it appears to play erythroid spectrin gene) with ankyrin 3. We have not yet no significant role in the partitioning of the α1 and α2 forms of detected either ankyrin 1 or ankyrin 2 in our the Na,K-ATPase between the sarcolemma and the t-tubules. immunoprecipitates (not shown), but this result is difficult to Our results do not readily explain why β-spectrin/α-fodrin interpret. Both ankyrin 1 and 2 are present at the sarcolemma heteromers concentrate over Z-lines, while β-spectrin without (Zhou et al., 1997; Tuvia et al., 1999) and should be able to a paired α subunit concentrates over M-lines and in bind simultaneously to integral membrane proteins and to β- longitudinal elements. Our findings suggest that any model spectrin. These ankyrins may dissociate more readily than proposing that the differential distribution of the two ankyrin 3 under the conditions we used, or they may be populations of spectrin is due to their binding to distinct selectively degraded in muscle homogenates. Although this complexes of ankyrins and integral membrane proteins is question warrants further study, our current evidence suggests highly unlikely. We now favor a model in which the differential that the linkage of the Na,K-ATPase to spectrin is mediated distribution of the two populations of spectrin at costameres is predominantly by ankyrin 3. determined by links between the sarcolemma and the contractile apparatus of nearby myofibrils (Fig. 7). Partitioning of Na,K-ATPase isoforms between the In our model, these links are made by intermediate filaments, sarcolemma and the t-tubules which have been shown to connect the sarcolemma to the Adult skeletal muscle expresses two of the three known α contractile apparatus at the level of Z- and M-lines (Pierobon- subunits of the Na,K-ATPase, α1 and α2 (Orlowski and Bormioli, 1981; Street, 1983; Shear and Bloch, 1985). Desmin Lingrel, 1988). It has been reported (Lavoie et al., 1997) that, concentrates selectively around Z disks (Lazarides, 1978; while both forms were present in the sarcolemma, only α2 was Granger and Lazarides, 1979; Richardson et al., 1981). It is present in significant amounts in the t-tubules of skeletal also present at the sarcolemma at Z line domains, but as it does muscle. We have confirmed this observation by not associate to a significant extent with longitudinal or M line immunofluorescence techniques, as well as by subcellular domains (A. O’Neill et al., unpublished), we have proposed an fractionation. The basis for the differential partitioning of the additional structure that performs this function (‘connectors’ in α1 and α2 subunits of the Na,K-ATPase between the Fig. 7). We postulate further that the desmin filaments promote sarcolemma and the t-tubules is still not understood, but our the accumulation of the β-spectrin/α-fodrin heteromers at Z results suggest that interactions with the spectrin-based line domains, while the ‘connectors’ promote the accumulation membrane skeleton are not involved. Both forms of the Na,K- of the population of β-spectrin that lacks an α subunit at ATPase associate with spectrin at the sarcolemma, and both longitudinal and M line domains. Intermediate filaments have become more uniformly distributed in the sarcolemma of ja/ja been shown to bind spectrin and ankyrin (Langley and Cohen, mice. Nevertheless, the α1 form of the Na,K-ATPase remains 1986; Langley and Cohen, 1987; Georgatos and Blobel, 1987), restricted to the sarcolemma in ja/ja muscle. This observation consistent with the possibility that these links are established can be explained in several ways. (i) The α1 form of the Na,K- by direct interactions of the filaments with the spectrin-based ATPase can indeed enter the t-tubule system in skeletal membrane skeleton. The concentration of spectrin and its myofibers of the ja/ja mouse, but once in the t-tubule, it is associated proteins into costameres should in turn concentrate rapidly removed or degraded. (ii) Although they are more the Na,K-ATPase. Rigorous testing of this model will help to uniformly distributed in the sarcolemma of the ja/ja mouse, reveal the identity and function of the proteins that organize both forms of the Na,K-ATPase remain bound to other integral the sarcolemma and how they interact simultaneously with the or peripheral sarcolemmal proteins that prevent their contractile apparatus and the membrane at costameres. As movement into the t-tubule system. (iii) A diffusion barrier, changes in the organization of costameres have been linked to located near the junction of the sarcolemma with the t-tubules, muscular dystrophy (Porter et al., 1992; Ehmer et al., 1997; restricts the movement of sarcolemmal proteins into the t- Williams and Bloch, 1999a), the factors that organize the tubular membrane. We cannot now distinguish among these sarcolemma are likely to serve an important function in the possibilities. Whichever explanation applies, our results clearly physiology of muscle. show that the spectrin-based membrane skeleton at the We are grateful to A. O’Neill for her assistance, to Drs K. sarcolemma does not play a significant role in determining the Sweadner, V. Bennett and J. S. Morrow for their generous gifts of distribution of the Na,K-ATPase between the sarcolemma and antibodies, and to Drs N. C. Porter, D. W. Pumplin, W. R. Randall, the t-tubules. It seems likely that other factors, perhaps M. P. Blaustein and M. F. Schneider for useful discussions. Our including targeting sequences in the α subunits of the Na,K- research has been supported by grants to R. J. Bloch from the National ATPase and their association with different β subunits, direct Institutes of Health (NS 17282, HL64304) and from the Muscular the α2 but not the α1 subunit to the t-tubules. 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