Assembly and dynamics of of the longitudinal and junctional sarcoplasmic reticulum in skeletal muscle cells

Vincenza Cusimano, Francesca Pampinella, Emiliana Giacomello, and Vincenzo Sorrentino1

Molecular Medicine Section, Department of Neuroscience, and Interuniversity Institute of Myology, University of Siena, 53100 Siena, Italy

Edited by Clara Franzini-Armstrong, University of Pennsylvania Medical Center, Philadelphia, PA, and approved January 12, 2009 (received for review October 11, 2008) The sarcoplasmic reticulum (SR) of skeletal muscle cells is a complex tially localized near the M band and, to a lesser extent, to the network of tubules and cisternae that share a common lumen Z disk (11–16). delimited by a single continuous membrane. The SR contains The longitudinal SR tubules regularly merge into large structures longitudinal and junctional domains characterized by distinctive named terminal cisternae. The region of the terminal membrane patterns of localization, but how SR proteins reach and/or cisternae that faces the T tubule/plasma membrane represents the are retained at these sites is not known. Here, we report that the junctional SR domain, where the Ca2ϩ-release channels of skeletal organization of longitudinal SR proteins is a slow process charac- muscle cells (ryanodine receptor type 1; RyR1) and other junctional terized by temporally distinct patterns of protein localization. In SR proteins localize (17–19). The voltage-activated Ca2ϩ channels, contrast, junctional SR proteins rapidly and synchronously assem- dihydropyridine receptors (DHPRs), are organized in tetrads on bled into clusters which, however, merged into mature triadic the plasma membrane/T tubule system that faces the junctional SR junctions only after completion of longitudinal SR protein organi- (2, 20, 21). The juxtaposition of junctional SR and T tubule/plasma zation. Fluorescence recovery after photobleaching experiments membranes is essential for a functional coupling between the RyR1 indicated that SR organization was accompanied by significant and the ␣1S subunit of the DHPR (22, 23). In contrast to longitu- changes in the dynamic properties of longitudinal and junctional dinal SR proteins, junctional proteins are initially imaged as clusters proteins. The decrease in mobility that accompanied organization with no apparent relationship to the contractile apparatus. At a of the longitudinal SR proteins ank1.5-GFP and GFP-InsP3R1 was later stage, these clusters converge into triadic structures at the I-A abrogated by deletion of specific binding sites for myofibrillar or band borders (2, 20). cytoskeletal proteins, respectively. Assembly of junctional SR do- It appears, therefore, that although the SR is a unique mains was accompanied by a strong decrease in mobility of organelle delimited by a single continuous membrane, the junctional proteins that in triadin appeared to be mediated by its longitudinal and junctional SR domains are characterized by intraluminal region. Together, the data suggest that the organi- specific patterns of protein localization (2). However, little is zation of specific SR domains results from a process of membrane known about the mechanisms that mediate sorting or retention reorganization accompanied by the establishment of multiple of SR proteins at these specific sites (5). To address these points, protein–protein interactions with intrinsic and extrinsic cues. the temporal and spatial organization of longitudinal and junc- tional SR membrane proteins was followed in differentiating calcium store ͉ excitation–contraction coupling ͉ muscle differentiation ͉ primary myotubes. Additionally, dynamics of GFP-tagged SR protein dynamics proteins was analyzed by using fluorescence recovery after photobleaching (FRAP) and inverse FRAP (iFRAP) techniques. he sarcoplasmic reticulum (SR) of muscle cells is a special- Tized form of the endoplasmic reticulum (ER) dedicated to Results ϩ storage and release of Ca2 . Assembly of the SR starts in Localization Kinetics of Longitudinal and Junctional SR Proteins in embryonic muscle cells, where an apparently disorganized ac- Differentiating Myotubes. Labeling of 2-day differentiated myo- cumulation of membranes is observed (1, 2). Following a series tubes with a polyclonal antibody against ank1.5 revealed, in most of modifications occurring throughout embryonic life and the cells, a diffused signal throughout the entire SR (Fig. 1A). first postnatal weeks, the SR develops in a highly structured 3D However, in a small fraction of cells, ank1.5 immunostaining was network of tubules and cisternae arranged in close association concentrated in a region of the SR near the M band (Fig. 1D). with the contractile apparatus (2–5). From a structural and All cells labeled with an antibody against SERCA presented a functional point of view, 2 regions are identified in the SR: the diffused staining (Fig. 1B). Staining with antibodies against RyR longitudinal and the junctional SR. The longitudinal SR is and triadin yielded a partially superimposable punctate pattern morphologically characterized by a series of parallel tubules and (Fig. 1 G and H), in agreement with previous data (4, 21). In is specialized in Ca2ϩ uptake, a process operated by the SR/ER ϩ 4-day differentiated myotubes, ank1.5 was organized in bands in Ca2 -ATPase (SERCA) pumps (6, 7). The longitudinal SR Ϸ55% of the myotubes. In approximately half of these cells, corresponds to the larger part of the SR and covers most of the ank1.5 was only found at the level of M bands (Fig. 1J), whereas sarcomere. However, a major density of membrane is observed in the remaining cells, a second band was detected at the level in correspondence with the Z disk, where some ER domains may of the Z disk (Fig. S1 A–C). In approximately 1/3 of 4-day also be localized (8) and, to a minor extent, near the M band (2, 9). Accordingly, immunofluorescence staining of adult skeletal muscle sections has shown that most longitudinal SR proteins, Author contributions: V.C., F.P., E.G., and V.S. designed research; V.C., F.P., and E.G. including SERCA pumps and the inositol-trisphosphate recep- performed research; V.C. and V.S. analyzed data; and V.C. and V.S. wrote the paper. tors (InsP3R), are detected predominantly in correspondence The authors declare no conflict of interest. with the Z disk and with a weaker signal over the M band (10). This article is a PNAS Direct Submission. In contrast, the small muscle-specific isoform of 1 1To whom correspondence should be addressed. E-mail: [email protected]. CELL BIOLOGY (ank1.5), a transmembrane protein of the longitudinal SR known This article contains supporting information online at www.pnas.org/cgi/content/full/ to interact with the myofibrillar protein , is preferen- 0810243106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810243106 PNAS ͉ March 24, 2009 ͉ vol. 106 ͉ no. 12 ͉ 4695–4700 Downloaded by guest on October 1, 2021 Table 2. Mobility of longitudinal SR and junctional GFP-protein ABC constructs expressed in NIH3T3 cells

2 D (␮m /s) Mf (%) n D EF GFP-SERCA2a 0.17 Ϯ 0.07 92.2 Ϯ 9.2 13 GFP-InsP3R1 0.12 Ϯ 0.10 72.2 Ϯ 16.0 18 ank1.5-GFP 0.37 Ϯ 0.16 87.1 Ϯ 9.7 20 G H I GFP-RyR1 0.20 Ϯ 0.18 75.3 Ϯ 11.5 17 GFP-␣1S 0.17 Ϯ 0.10 67.1 Ϯ 15.7 12 GFP-JP1 reticular 0.06 Ϯ 0.02 72.3 Ϯ 19.4 14 GFP-JP1 clusters 0.06 Ϯ 0.05 51.8 Ϯ 11.0 19 JKL Triadin-GFP 0.22 Ϯ 0.15 87.1 Ϯ 8.9 15 Junctin-GFP 0.39 Ϯ 0.29 92.8 Ϯ 7.1 18

Diffusion constants (D) and mobile fractions (Mf) of GFP fusion constructs M N O are shown; n indicates the number of experiments; all means are reported with SDs.

myotubes where ank1.5 was organized at least at the M band. P Q R RyR staining was still found in distinct clusters in the majority of 6-day differentiated myotubes (Fig. 1T), although in Ϸ10% of these myotubes, it was detected at the border between A and I S T U bands (Fig. 1W). Interestingly, this triadic pattern of RyR1 was observed only in those myotubes were SERCA was organized at the Z disk (Fig. S1 G–I) and ank1.5 at both the M band and the VWX Z disk regions (Fig. 1 V–X). The relative percentages of myo- tubes based on the localization pattern of SR proteins at 2, 4, and 6 days of differentiation are reported in Table 1.

Fig. 1. Sequential organization of longitudinal and junctional SR proteins Dynamics Studies of Longitudinal and Junctional SR Proteins in NIH during skeletal muscle cell differentiation. Immunolabeling of primary rat skeletal muscle cells at 2 (A–I),4(J–O), and 6 (P–X) days after induction of 3T3 Cells. The specific localization of SR proteins observed in differentiation. The majority of 2-day differentiated myotubes showed a mature myotubes may result from the reorganization of the SR diffused staining for ank1.5 (A) and SERCA (B). Only in a minority of myotubes membrane around the developing (2, 8, 24) and ank1.5 was observed at the level of the M band (D), in an alternating pattern from the establishment of interactions with intracellular struc- with ␣-. (E) In all 2-day differentiated myotubes, RyR (G) and triadin (H) tures, like the , the myofilaments, and the plasma formed clusters. Improved organization of ank1.5 (J) and SERCA (K) was membrane (25). To address this question, a set of transmem- observed in 4-day differentiated myotubes, whereas RyR (M) and triadin (N) were still organized in clusters. In 6-day differentiated myotubes, a further brane proteins representative of longitudinal and junctional SRs improvement in ank1.5 (P, S, and V) and SERCA (Q) localization was observed. were cloned in frame with the GFP (Table 2). The dynamic RyR staining was still observed in clusters in the majority of the myotubes (T), properties of these GFP-fusion proteins were analyzed by FRAP although in a fraction of cells triadic structures were formed (W). (Scale bar: experiments to calculate the diffusion constant (D), which 5 ␮m.) measures the rate of protein movement, and the mobile fraction (Mf), which measures the fraction of fluorescent proteins that are able to move. At first, experiments were performed in NIH differentiated myotubes, SERCA staining formed a striated pattern close to the Z disk (Fig. 1K). It is noteworthy that 3T3 cells to analyze the properties of the GFP proteins in the ER SERCA started to be organized at the Z disk only in those of nonmuscle cells. In NIH 3T3 cells, all GFP-tagged proteins myotubes where ank1.5 was organized in a banded pattern. After studied showed a reticular pattern, except Junctophilin 1 (JP1) 4 days of differentiation, RyR and triadin colocalized in clusters GFP-fusion protein (GFP-JP1) which, in addition to a reticular in Ͼ90% of the myotubes (Fig. 1 M and N). After 6 days of organization, also formed discrete clusters (Fig. S2). Fluores- differentiation, immunostaining for ank1.5 revealed a localized cence recovery measurements revealed that all GFP-tagged signal in Ϸ70% of the myotubes. In approximately half of these proteins with a reticular pattern had diffusion constant values in myotubes, ank1.5 was organized at the level of the M band (Fig. the range of those reported for membrane proteins (25) and were S1 D–F), whereas in the remaining cells, it was localized both at highly mobile (Table 2). FRAP experiments on GFP-JP1 orga- M bands and Z disks (Fig. 1 P, S, and V). At this stage, SERCA nized in clusters revealed a significantly lower mobility compared staining was organized in bands at the level of the Z disk in with that observed for GFP-JP1 organized in a reticular network, approximately half of the myotubes (Fig. 1Q), and again, orga- although no difference was observed in the diffusion constants nization of SERCA at the Z disks was detected only in those (Table 2).

Table 1. Percentages of myotubes presenting SR proteins organized at specific sites Days of ank1.5 at M ank1.5 at M SERCA at Z RyR and triadin RyR and triadin differentiation band band and Z disk disk in clusters in triads

2 7.4 Ϯ 3.3 0 0 100 0 4 31.8 Ϯ 12.4 24.0 Ϯ 15.2 32.0 Ϯ 30.7 93.7 Ϯ 12.5 6.3 Ϯ 12.5 6 32.2 Ϯ 22.7 39.2 Ϯ 14.0 49.5 Ϯ 15.7 82.2 Ϯ 16.5 17.8 Ϯ 16.5

Values represent means Ϯ SDs from at least 4 independent experiments, with more than 100 cells scored per experiment.

4696 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810243106 Cusimano et al. Downloaded by guest on October 1, 2021 2 C 0.20 Ϯ 0.1 ␮m /s, respectively), as shown in Fig. 2C. The diffusion A B constant of GFP-InsP3R1 was similar in undifferentiated and differentiated myotubes (D of 0.13 Ϯ 0.07 ␮m2/s and 0.07 Ϯ 0.05 2 ␮m /s, respectively). However, the Mf of GFP-InsP3R1 was reduced from 79.1% Ϯ 11.2% (n ϭ 20) to 62.8% Ϯ 11.3% (P Յ 0.01, n ϭ 14) when the protein was organized at the Z disk level (Fig. 2F). InsP3R1 has been described to associate with the D E F cytoskeleton through its interaction with protein 4.1N (26, 27). Accordingly, a GFP-InsP3R1 construct missing the C-terminal 14 aa that are responsible for binding to protein 4.1N (28) was prepared (GFP-InsP3R1⌬14). In differentiated myotubes, the GFP-InsP3R1⌬14 was still observed in bands near the Z-disk ␣ I region (Fig. 2H; Inset shows overlay with -actinin staining). GH However, the mobile fraction and the diffusion constant of GFP-InsP3R1⌬14, either distributed all over the SR (Fig. 2G)or localized in bands at the level of the Z disk (Fig. 2H), were not significantly different (D of 0.1 Ϯ 0.1 ␮m2/s and 0.16 Ϯ 0.2 ␮m2/s and Mf of 75.7% Ϯ 15.3% (n ϭ 19) and 76.2% Ϯ 8.9% (n ϭ 18), J K L respectively; Fig. 2I). Thus, deletion of the binding site for protein 4.1N abolished the reduction in mobility but not Z band localization of InsP3R1 in differentiated myotubes. The last longitudinal SR protein analyzed was ank1.5. The ank1.5-GFP fusion protein recapitulated the localization pattern observed for the endogenous protein, although localization at the Z band was M N O observed less frequently. Photobleaching of selected areas of undifferentiated myotubes expressing ank1.5-GFP where the protein was homogeneously distributed in the SR (Fig. 2 J) revealed a high diffusion coefficient (D of 0.20 Ϯ 0.12 ␮m2/s), with a mobile fraction of 88.0% Ϯ 6.6% (n ϭ 21), as shown in Fig. 2L. In differentiated myotubes, where ank1.5-GFP was mainly localized at the M band (Fig. 2K), a significant decrease Fig. 2. Dynamics of longitudinal SR proteins in differentiating myotubes. Localization of GFP-SERCA2a (A and B), GFP-InsP3R1 (D and E), GFP- in the diffusion constant and mobile fraction was observed (D of Ϯ ␮ 2 Յ Ϯ ϭ InsP3R1⌬14 (G and H), ank1.5-GFP (J and K), and ank1.5⌬-GFP (M and N)in 0.07 0.06 m /s, P 0.01 and Mf of 55.9% 13.8%, n 21, undifferentiated (A, D, G, J, and M) and differentiated (B, E, H, K, and N) P Յ 0.01), indicating that the mobility of ank1.5-GFP became myotubes. Insets in B, E, H, K, and N show the relative position of the bands reduced when the protein was organized at the M band. Local- observed for the GFP-proteins (in green) in differentiated myotubes, with ization of ank1.5 has been shown to depend on its interaction respect to the staining of ␣-actinin (in red) that labels the Z disks. Graphic with obscurin (12–16). Therefore, to further understand the representations of the mobile fraction (Mf in %) of GFP-SERCA2a (C), GFP- molecular basis of ank1.5 localization and dynamic properties, an InsP3R1 (F), GFP-InsP3R1⌬14 (I), ank1.5-GFP (L), and ank1.5⌬-GFP (O). (Scale ank1.5-GFP construct mutated in the binding site for obscurin ␮ Յ bar: 5 m.) **, Statistical significance with P 0.01; all means in the graphs are (ank1.5⌬-GFP) was characterized. In differentiated myotubes, indicated with SDs. ank1.5⌬-GFP did not localize at the M band, but in a region of the SR close to the Z disk region (Fig. 2N Inset shows overlay ␣ ⌬ Dynamics of Longitudinal SR Proteins in Primary Skeletal Muscle Cells. with -actinin staining). Furthermore, the mobility of ank1.5 - The dynamics of 3 GFP-fusion proteins representative of the GFP localized at the Z disk was similar to that measured for the Ϯ ␮ 2 Ϯ ϭ longitudinal SR, SERCA2a, InsP3R1 Ca2ϩ-release channel, and diffused protein (D of 0.4 0.2 m /s, Mf of 92.1% 7.2%, n Ϯ ␮ 2 Ϯ ϭ ank1.5 were then analyzed in muscle cells. In undifferentiated 21; D of 0.4 0.3 m /s and Mf of 92.2% 7.8%, n 22, myotubes, GFP-SERCA2a, GFP-InsP3R1, and ank1.5-GFP respectively), as shown in Fig. 2 M–O. Thus, deletion in ank1.5 were diffused throughout the SR (Fig. 2 A, D, and J), whereas of the binding site for obscurin resulted in mislocalization of the mutant protein near the Z disk and in the absence of localization- in differentiated myotubes they organized predominantly in dependent changes in protein dynamics. transverse bands (Fig. 2 B, E, and K). Counterstaining of these differentiated cells with an antibody against ␣-actinin showed Dynamics of Junctional Proteins. The key players in the process of that the fluorescent signals of GFP-SERCA2a and GFP- 2ϩ ␣ Ca release in skeletal muscle cells are RyR1 on the junctional InsP3R1 colocalized with -actinin at the Z disk (Fig. 2 B Inset SR and DHPR on the T tubule/plasma membrane. A protein and E Inset), whereas ank1.5-GFP signal was alternating with ␣ important for the docking of the SR to the T tubule/plasma that of -actinin in a pattern compatible with localization at the membrane is JP1, a of the junctional SR able M band (Fig. 2K Inset). Therefore, the transfected GFP-fusion to bind lipid components of the plasma membrane (19). In proteins distributed in a manner similar to that of endogenous differentiating myotubes, GFP-RyR1 was initially diffused in the proteins. It has to be noted that in differentiated myotubes, a SR but rapidly formed clusters that later assembled at the border small fraction of GFP-SERCA2a and GFP-InsP3R1, but not between the A and I bands (Fig. 3A). Fluorescence recovery ank1.5-GFP, remained diffused over the SR or formed brief after photobleaching in selected areas of the SR of undifferen- longitudinal striations (Fig. 2 B, E, and K), which are likely to tiated myotubes, where GFP-RyR1 was homogeneously distrib- reflect saturation of the membrane system by transfected proteins. uted, revealed a rapid recovery of fluorescence (D of 0.06 Ϯ 0.05 2 FRAP experiments revealed that the dynamics of GFP- ␮m /s) and an Mf of 72.0% Ϯ 12.8% (n ϭ 13), as shown in Fig. SERCA2a, either when diffused throughout the SR or when 3B. When GFP-RyR1 was organized in clusters and in triadic localized near the Z disk, did not show any difference in the structures, the mobile fraction decreased significantly (Mf ϭ mobile fraction and the diffusion constant (Mf of 88.8% Ϯ 9.1%, 43.9% Ϯ 12.1%, n ϭ 17, and 41.9% Ϯ 13.2%, n ϭ 16, respec- CELL BIOLOGY n ϭ 16, and 93.2% Ϯ 4.0%, n ϭ 23; D of 0.22 Ϯ 0.1 ␮m2/s and tively; P Յ 0.01), whereas the diffusion constant did not change

Cusimano et al. PNAS ͉ March 24, 2009 ͉ vol. 106 ͉ no. 12 ͉ 4697 Downloaded by guest on October 1, 2021 Fig. 3. Dynamics of triadic proteins in differentiating myotubes. (A) Subcel- lular localization of GFP-RyR1 as a representative triadic protein. In poorly differentiated myotubes, GFP-RyR1 presented a diffused distribution (i), Fig. 4. The luminal region of triadin is responsible for its strong association whereas in more differentiated myotubes, GFP-RyR1 was organized in clusters to junctional domains. (A) Representative images of myotubes where triadin- (ii), and eventually in triadic structures (iii). Graphic representations of the GFP was observed in a reticular diffused state (i), in clusters (ii), and in triadic mobile fraction (M , in %) of GFP-RyR1 (B) and GFP-␣ (C) measured in a f 1S structures (iii). Graphic representations of the mobile fraction (M in %) of diffused distribution, organized in clusters and in triads (D). Graphic repre- f triadin-GFP (B), junctin-GFP (C), a triadin-junctin-GFP chimera (D), and a sentations of the mobile fraction of GFP-JP1 organized in clusters and in triadic junctin-triadin-GFP chimera (E). (Scale bar: 5 ␮m.) , Statistical significance structures. (Scale bar: 5 ␮m.) , Statistical significance with P Յ 0.01; all means * ** with P Յ 0.05; , statistical significance with P Յ 0.01; all means in the graphs in the graphs are indicated with SDs. ** are indicated with SDs.

ϭ Ϯ ␮ 2 Ϯ ␮ 2 (D 0.09 0.10 m /s and 0.07 0.08 m /s, respectively). complex with RyR1 and calsequestrin (18, 23, 29, 30). Triadin- These data indicate that the initially highly mobile pool of GFP presented an organization pattern similar to that of other GFP-RyR1 present in the undifferentiated SR becomes partially junctional proteins (Fig. 4A). Fluorescence recovery measure- immobilized once organized in clusters and triadic junctions. Given the functional association between RyR1 and the ments following photobleaching of areas of myotubes where triadin-GFP was diffused (Fig. 4Ai) indicated that the protein DHPR on the T tubule, we next extended our analysis to the 2 was highly mobile (D of 0.42 Ϯ 0.30 ␮m /s and Mf of 70.7% Ϯ dynamics of the DHPR ␣1S subunit. In undifferentiated myo- 29.0%, n ϭ 22). A major change in triadin-GFP mobility was tubes, homogeneously distributed GFP-␣1S moved rapidly (D of 0.40 Ϯ 0.24 ␮m2/s) and presented a relatively high mobility, with observed when this protein was organized in clusters, because Ϯ ϭ Յ an Mf of 74.8% Ϯ 13.7% (n ϭ 14), as shown in Fig. 3C. However, the Mf decreased to 20.9% 8.7% (n 23; P 0.01), as shown when GFP-␣1S was organized in distinct clusters and in triadic in Fig. 4B. The mobility of triadin-GFP was further reduced in junctions, the mobile fraction was reduced to 42.2% Ϯ 13.3% triadic junctions, because only in 50% of the myotubes was it (n ϭ 19) and 40.8% Ϯ 12.9% (n ϭ 12, P Յ 0.01), respectively, possible to measure a recovery in fluorescence after photo- Ϯ although no significant change in D was observed (0.39 0.29 bleaching (Mf of 15.6% Ϯ 6.4%, n ϭ 8, and D of 0.23 Ϯ 0.24 ␮m2/s and 0.21 Ϯ 0.21 ␮m2/s, respectively). Overall, these results ␮m2/s). In the remaining 50% of myotubes (n ϭ 8), no recovery ␣ indicated that the dynamics of GFP- 1S on the T tubule were could be measured, as if all triadin-GFP molecules were irre- comparable to those of GFP-RyR1 on the junctional SR, with versibly bound to triadic structures. Qualitative iFRAP experi- almost 60% of the GFP-fusion proteins becoming immobile once ments confirmed these findings, because no evident decrease in recruited into junctional SR domains. fluorescence in the unbleached structures was observed, even To complete our analysis of the dynamic properties of triadic when the acquisition was prolonged up to 20 min (Fig. S4A). proteins, we studied the GFP-JP1 fusion protein. GFP-JP1 In differentiating myotubes, junctin-GFP followed an organ- immediately assembled in clusters that later in differentiation ization pattern similar to that of all other junctional proteins converged into triadic junctions (Fig. S3). FRAP experiments on GFP-JP1 organized in distinct clusters or at triads showed that (Fig. S5A). As shown in Fig. 4C, fluorescence recovery measure- ments in myotubes where junctin-GFP was diffused revealed a high more than half of the fluorescent molecules were immobile, with 2 mobility (D of 0.28 Ϯ 0.24 ␮m /s and Mf of 88.9% Ϯ 9.3%, n ϭ 16). an Mf of 40.7% Ϯ 10.4% (n ϭ 25) and 34.0% Ϯ 14.1% (n ϭ 18), respectively (Fig. 3D). This indicates that at least 60% of Junctin-GFP organized in clusters or triadic structures and showed GFP-JP1 was strongly associated with these structures. No a significantly reduced diffusion constant (D of 0.06 Ϯ 0.06 ␮m2/s difference was observed in the diffusion constant (D of 0.07 Ϯ and 0.03 Ϯ 0.02 ␮m2/s, respectively; P Յ 0.01) but, in contrast to 0.1 ␮m2/s and 0.07 Ϯ 0.07 ␮m2/s, respectively). other junctional proteins, only a moderate reduction in the mobile fraction was observed (Mf of 77.7% Ϯ 16.4%, n ϭ 43; P Յ 0.05; and Dynamic Properties of Triadin and Junctin. Triadin and junctin are 72.8% Ϯ 11.6%, n ϭ 22; P Յ 0.01, respectively). In agreement, 2 structurally related proteins that form a macromolecular iFRAP experiments showed that equilibration of fluorescence

4698 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810243106 Cusimano et al. Downloaded by guest on October 1, 2021 intensity between bleached and un-bleached regions was rapid M-band represented the first longitudinal SR protein organized (Fig. S4B). to a specific site. This agrees with recent results that revealed an early localization of ank1.5 with respect to other SR proteins and Differences in the Dynamic Properties of Triadin and Junctin Are its early colocalization with obscurin during in vivo muscle Dictated by the Intraluminal C-Terminal Tail. Comparison of the development (32). These observations further support a possible amino acid sequences of triadin and junctin revealed some involvement of ank1.5 and obscurin in the association of the differences in the luminal tails of these proteins. Two GFP- developing SR with the myofilaments (32). Moreover, FRAP proteins were generated, one containing the cytosolic and experiments performed in cultured myotubes revealed a reduc- transmembrane region of triadin and the intraluminal domain of tion in the dynamics of ank1.5 following localization at the M junctin (triadin-junctin-GFP), and the other containing the band in differentiated myotubes. This reduction in ank1.5 mo- cytosolic and transmembrane region of junctin and the intralu- bility can be explained by previous indications of a direct minal domain of triadin (junctin-triadin-GFP). No significant interaction between ank1.5 and the myofibrillar protein obscurin differences were observed in the dynamics of these 2 proteins in (12–16). Accordingly, ank1.5⌬-GFP, an ank1.5 mutant lacking NIH 3T3 cells (Table S1). In undifferentiated myotubes, triadin- the binding site for obscurin (12–16), instead of localizing near junctin-GFP was diffused and showed an Mf of 81.10% Ϯ 17.28% the M band region of the SR localized near the Z disk and, more (n ϭ 20) and a D of 0.15 Ϯ 0.15 ␮m2/s, as reported in Fig. 4D. significantly, did not present a localization-dependent change in When this protein was organized in clusters or in triadic struc- mobility in FRAP experiments. Thus, these data suggest that, if tures (Fig. S5B), a slight decrease in the Mf was observed unable to bind obscurin at the M band, the ank1.5⌬-GFP mutant (68.91% Ϯ 17.11%, n ϭ 39, P Յ 0.05; and 59.96% Ϯ 12.71%, n ϭ remains free to move in the SR membrane where, similarly to 28, P Յ 0.01, respectively), as shown in Fig. 4D. This was accom- what was previously proposed for SERCA pumps, it is observed panied by a reduction of the D to 0.06 Ϯ 0.06 ␮m2/s and 0.04 Ϯ 0.02 near the Z-disk, likely because of the higher density of SR mem- ␮m2/s, respectively (P Յ 0.01). In undifferentiated myotubes, the branes at this region. This mechanism could also explain why junctin-triadin-GFP protein was diffused and showed an Mf of GFP-InsP3R1⌬14, although unable to interact through protein 88.95% Ϯ 11.08% (n ϭ 22) and a D of 0.24 Ϯ 0.15 ␮m2/s. However, 4.1N to the actin cytoskeleton, was still localized at the Z disk. once organized in clusters or in triadic structure (Fig. S5C), the Mf of junctin-triadin-GFP was dramatically decreased to 24.30% Ϯ Junctional Protein Organization and Mobility. Immunostaining ex- 7.59% (n ϭ 20) and 23.60% Ϯ 8.99% (n ϭ 15), respectively (P Յ periments indicated that the organization of the junctional SR 0.01), whereas no change was observed in the diffusion constant domains occurred in 2 steps, which appeared to simultaneously values (D of 0.15 Ϯ 0.12 ␮m2/s and 0.22 Ϯ 0.29 ␮m2/s, respectively), involve all junctional proteins. Within the first 2 days after induction as shown in Fig. 4E. These data indicate that amino acid residues of differentiation, junctional SR proteins formed a large number of in the C-terminal tail of triadin mediate the decrease in mobility clusters that did not show a specific spatial relationship with the observed for triadin-GFP and junctin-triadin-GFP at junctional sarcomere. This organization in clusters preceded the organization sites. of longitudinal SR proteins. During the second step, junctional SR proteins became organized at the border between A and I bands. Discussion It is worth noting that the organization of junctional proteins in Organization and Mobility Properties of Longitudinal SR Proteins. triadic structures was a late event in myotube differentiation and Longitudinal SR proteins were randomly distributed over the was only observed in myotubes where the longitudinal SR proteins, entire SR membrane in the first days of differentiation and like ank1.5 and SERCA, were already organized. This suggests that became organized within 6 days to specific regions of the SR the assembly of SR proteins at longitudinal and junctional SR following distinctive temporal kinetics. In differentiated myo- domains may initially be independent of each other, but become tubes, SERCA pumps, the most abundant longitudinal SR interconnected at a later stage. membrane proteins, were observed in a region of the SR close Available evidence suggests that junctional proteins form a to the Z disk. Analysis of the dynamics of a GFP-SERCA2a multiprotein complex of high molecular mass, with the RyR1 fusion protein revealed that following myotube differentiation, channel interacting with several junctional proteins, including no change was observed in GFP-SERCA2a mobility. This is triadin, JP1, and DHPR (29, 30, 33). Analysis of the dynamic compatible with the behavior of a transmembrane protein that properties of junctional SR membrane proteins revealed that is free to laterally diffuse in the lipid bilayer. In fact, electron assembly into clusters is accompanied by a strong reduction of microscopy studies had provided evidence that SERCA particles their mobile fraction. Among the junctional proteins analyzed, are widely distributed in the longitudinal SR (6), and no evidence triadin-GFP presented the most drastic reduction in mobility that SERCA pumps are associated with other cellular structures upon association to junctional domains, whereas junctin-GFP has been reported (31). Thus, the SERCA immunofluorescence retained a relatively high mobile fraction also after its segrega- signal observed in bands at the Z disk, and to a lesser extent to tion to the junctional SR domain. A possible explanation for the the M band, rather than reflecting a specific retention mecha- behavior of junctin is that this protein may only establish nism, may result from the higher density of SR membranes in transient interactions with the junctional multiprotein complex these regions (9, 24). Similarly to SERCA, InsP3R1 also was (25). Results obtained with chimeric fusion proteins containing localized at the Z disk region of differentiated myotubes. How- different regions of triadin and junctin indicated that the region ever, the mobility of GFP-InsP3R1was reduced after localization responsible for the high affinity of triadin for the junctional to the Z disk region, as if this protein were anchored to a fixed domains is the intraluminal tail, which contains binding sites for structure. Indeed, deletion in InsP3R1 of the binding site for other junctional proteins, like RyR1 and calsequestrin (18). protein 4.1N, which has been reported to mediate an interaction between InsP3R1 and the actin cytoskeleton (26, 28), resulted in Longitudinal and Junctional SR Assembly: A Model. The reported a mutant protein that, although localized at the Z disk in results let us propose a working model for SR domain assem- differentiated myotubes, presented a mobility similar to that of bly, as reported in Fig. S6. Anchorage of the SR to the diffused GFP-InsP3R1. This suggests that interaction with pro- myofibrils, likely due to the binding between ank1.5 and the tein 4.1N may control the mobility of GFP-InsP3R1 by linking myofibrillar protein obscurin, appears to be an early event in this protein to the actin cytoskeleton upon differentiation. At the developing longitudinal SR. The next step is the redistri-

variance with SERCA and InsP3R1, ank1.5 was predominantly bution of longitudinal SR membrane along the sarcomere, CELL BIOLOGY localized at the M band. Furthermore, localization of ank1.5 to with major membrane densities at the level of the Z disk and

Cusimano et al. PNAS ͉ March 24, 2009 ͉ vol. 106 ͉ no. 12 ͉ 4699 Downloaded by guest on October 1, 2021 M band. Longitudinal SR proteins might then remain rela- Materials and Methods tively free of moving in the SR membrane (e.g., SERCA) or, Detailed materials and methods are provided as SI Materials and Methods. if able to anchor to external clues, like the actin cytoskeleton and the myofibrils, they can be selectively retained in a specific Cultures of Primary Skeletal Myoblasts. Cultures of primary rat myoblasts were region, as proposed for the InsP3R1 and ank1.5. The junc- performed as reported previously (14). tional SR domain appears to be the first SR domain to Immunofluorescence and Antibodies. Details on immunofluorescence and an- assemble, forming distinct clusters with no spatial relationship tibodies are described in SI Materials and Methods. to the myofibrillar apparatus. This process is probably in part regulated by internal clues through formation of a multiprotein GFP Fusion Proteins. A detailed description of GFP-fusion protein cloning complex of junctional proteins. Interestingly, according to strategies is reported in SI Materials and Methods. dynamic properties measured in FRAP experiments, the de- gree of association with this supramolecular complex appears FRAP Experiments and Statistical Analysis. Details on FRAP experiments and to differ among junctional proteins (i.e., triadin Ͼ RyR Ͼ statistical analysis are described in SI Materials and Methods. junctin). Only in a second step, which follows the organization ACKNOWLEDGMENTS. We thank members of our laboratory for suggestions of the longitudinal SR, junctional SR domains will align with and critically reading this manuscript. We also thank E. Carafoli, K. Mikoshiba, the contractile apparatus at the A-I border by establishing P.D. Allen, M. Grabner, B.E. Flucher, and I. Marty for providing valuable yet-unidentified interactions with the contractile apparatus. reagents, and Dr. Rainer Pepperkok (EMBL, Heidelberg, Germany) for helpful suggestions on FRAP techniques. This work was supported in part by grants Future work will help further extend our knowledge on the from ASI (Agenzia Spaziale Italiana), the University of Siena (PAR 2006 and molecular interactions proposed in this model. PAR 2007), and Telethon (GGP08153).

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