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

Abstract

The sarcoplasmic reticulum (SR) of skeletal muscle cells is a complex network of tubules and cisternae that share a common lumen delimited by a single continuous membrane. The SR contains longitudinal and junctional domains characterized by distinctive patterns of protein localization, but how SR proteins reach and/or are retained at these sites is not known. Here, we report that the organization of longitudinal SR proteins is a slow process characterized by temporally distinct patterns of protein localization. In contrast, junctional SR proteins rapidly and synchronously assembled into clusters which, however, merged into mature triadic junctions only after completion of longitudinal SR protein organization. Fluorescence recovery after photobleaching experiments indicated that SR organization was accompanied by significant changes in the dynamic properties of longitudinal and junctional proteins. The decrease in mobility that accompanied organization of the longitudinal SR proteins ank1.5-GFP and GFP-InsP3R1 was abrogated by deletion of specific binding sites for myofibrillar or cytoskeletal proteins, respectively. Assembly of junctional SR domains was accompanied by a strong decrease in mobility of junctional proteins that in triadin appeared to be mediated by its intraluminal region. Together, the data suggest that the organization of specific SR domains results from a process of membrane reorganization accompanied by the establishment of multiple protein–protein interactions with intrinsic and extrinsic cues.

The sarcoplasmic reticulum (SR) of muscle cells is a specialized form of the endoplasmic reticulum (ER) dedicated to storage and release of Ca²⁺. Assembly of the SR starts in embryonic muscle cells, where an apparently disorganized accumulation of membranes is observed (1, 2). Following a series of modifications occurring throughout embryonic life and the first postnatal weeks, the SR develops in a highly structured 3D network of tubules and cisternae arranged in close association with the contractile apparatus (2–5). From a structural and functional point of view, 2 regions are identified in the SR: the longitudinal and the junctional SR. The longitudinal SR is morphologically characterized by a series of parallel tubules and is specialized in Ca²⁺ uptake, a process operated by the SR/ER Ca²⁺-ATPase (SERCA) pumps (6, 7). The longitudinal SR corresponds to the larger part of the SR and covers most of the sarcomere. However, a major density of membrane is observed in correspondence with the Z disk, where some ER domains may 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, including SERCA pumps and the inositol-trisphosphate receptors (InsP3R), are detected predominantly in correspondence with the Z disk and with a weaker signal over the M band (10). In contrast, the small muscle-specific isoform of ankyrin 1 (ank1.5), a transmembrane protein of the longitudinal SR known to interact with the myofibrillar protein obscurin, is preferentially localized near the M band and, to a lesser extent, to the Z disk (11–16).

The longitudinal SR tubules regularly merge into large structures named terminal cisternae. The region of the terminal membrane cisternae that faces the T tubule/plasma membrane represents the junctional SR domain, where the Ca²⁺-release channels of skeletal muscle cells (ryanodine receptor type 1; RyR1) and other junctional SR proteins localize (17–19). The voltage-activated Ca²⁺ channels, dihydropyridine receptors (DHPRs), are organized in tetrads on the plasma membrane/T tubule system that faces the junctional SR (2, 20, 21). The juxtaposition of junctional SR and T tubule/plasma membranes is essential for a functional coupling between the RyR1 and the α_1S subunit of the DHPR (22, 23). In contrast to longitudinal SR proteins, junctional proteins are initially imaged as clusters with no apparent relationship to the contractile apparatus. At a later stage, these clusters converge into triadic structures at the I-A band borders (2, 20).

It appears, therefore, that although the SR is a unique organelle delimited by a single continuous membrane, the longitudinal and junctional SR domains are characterized by specific patterns of protein localization (2). However, little is known about the mechanisms that mediate sorting or retention of SR proteins at these specific sites (5). To address these points, the temporal and spatial organization of longitudinal and junctional SR membrane proteins was followed in differentiating primary myotubes. Additionally, dynamics of GFP-tagged SR proteins was analyzed by using fluorescence recovery after photobleaching (FRAP) and inverse FRAP (iFRAP) techniques.

Results

Localization Kinetics of Longitudinal and Junctional SR Proteins in Differentiating Myotubes.

Labeling of 2-day differentiated myotubes with a polyclonal antibody against ank1.5 revealed, in most cells, a diffused signal throughout the entire SR (Fig. 1A). However, in a small fraction of cells, ank1.5 immunostaining was concentrated in a region of the SR near the M band (Fig. 1D). All cells labeled with an antibody against SERCA presented a diffused staining (Fig. 1B). Staining with antibodies against RyR and triadin yielded a partially superimposable punctate pattern (Fig. 1 G and H), in agreement with previous data (4, 21). In 4-day differentiated myotubes, ank1.5 was organized in bands in ≈55% of the myotubes. In approximately half of these cells, ank1.5 was only found at the level of M bands (Fig. 1J), whereas in the remaining cells, a second band was detected at the level of the Z disk (Fig. S1 A–C). In approximately 1/3 of 4-day differentiated myotubes, SERCA staining formed a striated pattern close to the Z disk (Fig. 1K). It is noteworthy that SERCA started to be organized at the Z disk only in those myotubes where ank1.5 was organized in a banded pattern. After 4 days of differentiation, RyR and triadin colocalized in clusters in >90% of the myotubes (Fig. 1 M and N). After 6 days of differentiation, immunostaining for ank1.5 revealed a localized signal in ≈70% of the myotubes. In approximately half of these myotubes, ank1.5 was organized at the level of the M band (Fig. S1 D–F), whereas in the remaining cells, it was localized both at M bands and Z disks (Fig. 1 P, S, and V). At this stage, SERCA staining was organized in bands at the level of the Z disk in approximately half of the myotubes (Fig. 1Q), and again, organization of SERCA at the Z disks was detected only in those myotubes where ank1.5 was organized at least at the M band. RyR staining was still found in distinct clusters in the majority of 6-day differentiated myotubes (Fig. 1T), although in ≈10% of these myotubes, it was detected at the border between A and I 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 Z disk regions (Fig. 1 V–X). The relative percentages of myotubes 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 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 differentiation. The majority of 2-day differentiated myotubes showed a diffused staining for ank1.5 (A) and SERCA (B). Only in a minority of myotubes ank1.5 was observed at the level of the M band (D), in an alternating pattern with α-actinin. (E) In all 2-day differentiated myotubes, RyR (G) and triadin (H) formed clusters. Improved organization of ank1.5 (J) and SERCA (K) was observed in 4-day differentiated myotubes, whereas RyR (M) and triadin (N) were still organized in clusters. In 6-day differentiated myotubes, a further improvement in ank1.5 (P, S, and V) and SERCA (Q) localization was observed. RyR staining was still observed in clusters in the majority of the myotubes (T), although in a fraction of cells triadic structures were formed (W). (Scale bar: 5 μm.)

Table 1.

Percentages of myotubes presenting SR proteins organized at specific sites

Days of differentiation	ank1.5 at M band	ank1.5 at M band and Z disk	SERCA at Z disk	RyR and triadin in clusters	RyR and triadin in triads
2	7.4 ± 3.3	0	0	100	0
4	31.8 ± 12.4	24.0 ± 15.2	32.0 ± 30.7	93.7 ± 12.5	6.3 ± 12.5
6	32.2 ± 22.7	39.2 ± 14.0	49.5 ± 15.7	82.2 ± 16.5	17.8 ± 16.5

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

Dynamics Studies of Longitudinal and Junctional SR Proteins in NIH 3T3 Cells.

The specific localization of SR proteins observed in mature myotubes may result from the reorganization of the SR membrane around the developing myofilaments (2, 8, 24) and from the establishment of interactions with intracellular structures, like the cytoskeleton, the myofilaments, and the plasma membrane (25). To address this question, a set of transmembrane proteins representative of longitudinal and junctional SRs were cloned in frame with the GFP (Table 2). The dynamic properties of these GFP-fusion proteins were analyzed by FRAP experiments to calculate the diffusion constant (D), which measures the rate of protein movement, and the mobile fraction (M_f), which measures the fraction of fluorescent proteins that are able to move. At first, experiments were performed in NIH 3T3 cells to analyze the properties of the GFP proteins in the ER of nonmuscle cells. In NIH 3T3 cells, all GFP-tagged proteins studied showed a reticular pattern, except Junctophilin 1 (JP1) GFP-fusion protein (GFP-JP1) which, in addition to a reticular organization, also formed discrete clusters (Fig. S2). Fluorescence recovery measurements revealed that all GFP-tagged proteins with a reticular pattern had diffusion constant values in the range of those reported for membrane proteins (25) and were highly mobile (Table 2). FRAP experiments on GFP-JP1 organized in clusters revealed a significantly lower mobility compared with that observed for GFP-JP1 organized in a reticular network, although no difference was observed in the diffusion constants (Table 2).

Table 2.

Mobility of longitudinal SR and junctional GFP-protein constructs expressed in NIH3T3 cells

	D (μm2/s)	Mf (%)	n
GFP-SERCA2a	0.17 ± 0.07	92.2 ± 9.2	13
GFP-InsP3R1	0.12 ± 0.10	72.2 ± 16.0	18
ank1.5-GFP	0.37 ± 0.16	87.1 ± 9.7	20
GFP-RyR1	0.20 ± 0.18	75.3 ± 11.5	17
GFP-α1S	0.17 ± 0.10	67.1 ± 15.7	12
GFP-JP1 reticular	0.06 ± 0.02	72.3 ± 19.4	14
GFP-JP1 clusters	0.06 ± 0.05	51.8 ± 11.0	19
Triadin-GFP	0.22 ± 0.15	87.1 ± 8.9	15
Junctin-GFP	0.39 ± 0.29	92.8 ± 7.1	18

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

Dynamics of Longitudinal SR Proteins in Primary Skeletal Muscle Cells.

The dynamics of 3 GFP-fusion proteins representative of the longitudinal SR, SERCA2a, InsP3R1 Ca²⁺-release channel, and ank1.5 were then analyzed in muscle cells. In undifferentiated myotubes, GFP-SERCA2a, GFP-InsP3R1, and ank1.5-GFP were diffused throughout the SR (Fig. 2 A, D, and J), whereas in differentiated myotubes they organized predominantly in transverse bands (Fig. 2 B, E, and K). Counterstaining of these differentiated cells with an antibody against α-actinin showed that the fluorescent signals of GFP-SERCA2a and GFP-InsP3R1 colocalized with α-actinin at the Z disk (Fig. 2 B Inset and E Inset), whereas ank1.5-GFP signal was alternating with that of α-actinin in a pattern compatible with localization at the M band (Fig. 2K Inset). Therefore, the transfected GFP-fusion proteins distributed in a manner similar to that of endogenous proteins. It has to be noted that in differentiated myotubes, a small fraction of GFP-SERCA2a and GFP-InsP3R1, but not ank1.5-GFP, remained diffused over the SR or formed brief longitudinal striations (Fig. 2 B, E, and K), which are likely to reflect saturation of the membrane system by transfected proteins.

Fig. 2.

Dynamics of longitudinal SR proteins in differentiating myotubes. Localization of GFP-SERCA2a (A and B), GFP-InsP3R1 (D and E), GFP-InsP3R1Δ14 (G and H), ank1.5-GFP (J and K), and ank1.5Δ-GFP (M and N) in undifferentiated (A, D, G, J, and M) and differentiated (B, E, H, K, and N) myotubes. Insets in B, E, H, K, and N show the relative position of the bands observed for the GFP-proteins (in green) in differentiated myotubes, with respect to the staining of α-actinin (in red) that labels the Z disks. Graphic representations of the mobile fraction (M_f in %) of GFP-SERCA2a (C), GFP-InsP3R1 (F), GFP-InsP3R1Δ14 (I), ank1.5-GFP (L), and ank1.5Δ-GFP (O). (Scale bar: 5 μm.) **, Statistical significance with P ≤ 0.01; all means in the graphs are indicated with SDs.

FRAP experiments revealed that the dynamics of GFP-SERCA2a, either when diffused throughout the SR or when localized near the Z disk, did not show any difference in the mobile fraction and the diffusion constant (M_f of 88.8% ± 9.1%, n = 16, and 93.2% ± 4.0%, n = 23; D of 0.22 ± 0.1 μm²/s and 0.20 ± 0.1 μm²/s, respectively), as shown in Fig. 2C. The diffusion constant of GFP-InsP3R1 was similar in undifferentiated and differentiated myotubes (D of 0.13 ± 0.07 μm²/s and 0.07 ± 0.05 μm²/s, respectively). However, the M_f of GFP-InsP3R1 was reduced from 79.1% ± 11.2% (n = 20) to 62.8% ± 11.3% (P ≤ 0.01, n = 14) when the protein was organized at the Z disk level (Fig. 2F). InsP3R1 has been described to associate with the actin 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-InsP3R1Δ14). In differentiated myotubes, the GFP-InsP3R1Δ14 was still observed in bands near the Z-disk region (Fig. 2H; Inset shows overlay with α-actinin staining). However, the mobile fraction and the diffusion constant of GFP-InsP3R1Δ14, 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 ± 0.1 μm²/s and 0.16 ± 0.2 μm²/s and M_f of 75.7% ± 15.3% (n = 19) and 76.2% ± 8.9% (n = 18), 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 observed less frequently. Photobleaching of selected areas of undifferentiated myotubes expressing ank1.5-GFP where the protein was homogeneously distributed in the SR (Fig. 2J) revealed a high diffusion coefficient (D of 0.20 ± 0.12 μm²/s), with a mobile fraction of 88.0% ± 6.6% (n = 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 in the diffusion constant and mobile fraction was observed (D of 0.07 ± 0.06 μm²/s, P ≤ 0.01 and M_f of 55.9% ± 13.8%, n = 21, P ≤ 0.01), indicating that the mobility of ank1.5-GFP became reduced when the protein was organized at the M band. Localization of ank1.5 has been shown to depend on its interaction with obscurin (12–16). Therefore, to further understand the molecular basis of ank1.5 localization and dynamic properties, an ank1.5-GFP construct mutated in the binding site for obscurin (ank1.5Δ-GFP) was characterized. In differentiated myotubes, ank1.5Δ-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 with α-actinin staining). Furthermore, the mobility of ank1.5Δ-GFP localized at the Z disk was similar to that measured for the diffused protein (D of 0.4 ± 0.2 μm²/s, M_f of 92.1% ± 7.2%, n = 21; D of 0.4 ± 0.3 μm²/s and M_f of 92.2% ± 7.8%, n = 22, respectively), as shown in Fig. 2 M–O. Thus, deletion in ank1.5 of the binding site for obscurin resulted in mislocalization of the mutant protein near the Z disk and in the absence of localization-dependent changes in protein dynamics.

Dynamics of Junctional Proteins.

The key players in the process of Ca²⁺ release in skeletal muscle cells are RyR1 on the junctional SR and DHPR on the T tubule/plasma membrane. A protein important for the docking of the SR to the T tubule/plasma membrane is JP1, a membrane protein of the junctional SR able to bind lipid components of the plasma membrane (19). In differentiating myotubes, GFP-RyR1 was initially diffused in the SR but rapidly formed clusters that later assembled at the border between the A and I bands (Fig. 3A). Fluorescence recovery after photobleaching in selected areas of the SR of undifferentiated myotubes, where GFP-RyR1 was homogeneously distributed, revealed a rapid recovery of fluorescence (D of 0.06 ± 0.05 μm²/s) and an M_f of 72.0% ± 12.8% (n = 13), as shown in Fig. 3B. When GFP-RyR1 was organized in clusters and in triadic structures, the mobile fraction decreased significantly (M_f = 43.9% ± 12.1%, n = 17, and 41.9% ± 13.2%, n = 16, respectively; P ≤ 0.01), whereas the diffusion constant did not change (D = 0.09 ± 0.10 μm²/s and 0.07 ± 0.08 μm²/s, respectively). These data indicate that the initially highly mobile pool of GFP-RyR1 present in the undifferentiated SR becomes partially immobilized once organized in clusters and triadic junctions.

Fig. 3.

Dynamics of triadic proteins in differentiating myotubes. (A) Subcellular localization of GFP-RyR1 as a representative triadic protein. In poorly differentiated myotubes, GFP-RyR1 presented a diffused distribution (i), whereas in more differentiated myotubes, GFP-RyR1 was organized in clusters (ii), and eventually in triadic structures (iii). Graphic representations of the mobile fraction (M_f, in %) of GFP-RyR1 (B) and GFP-α_1S (C) measured in a diffused distribution, organized in clusters and in triads (D). Graphic representations of the mobile fraction of GFP-JP1 organized in clusters and in triadic structures. (Scale bar: 5 μm.) **, Statistical significance with P ≤ 0.01; all means in the graphs are indicated with SDs.

Given the functional association between RyR1 and the DHPR on the T tubule, we next extended our analysis to the dynamics of the DHPR α_1S subunit. In undifferentiated myotubes, homogeneously distributed GFP-α_1S moved rapidly (D of 0.40 ± 0.24 μm²/s) and presented a relatively high mobility, with an M_f of 74.8% ± 13.7% (n = 14), as shown in Fig. 3C. However, when GFP-α_1S was organized in distinct clusters and in triadic junctions, the mobile fraction was reduced to 42.2% ± 13.3% (n = 19) and 40.8% ± 12.9% (n = 12, P ≤ 0.01), respectively, although no significant change in D was observed (0.39 ± 0.29 μm²/s and 0.21 ± 0.21 μm²/s, respectively). Overall, these results indicated that the dynamics of GFP-α_1S on the T tubule were comparable to those of GFP-RyR1 on the junctional SR, with almost 60% of the GFP-fusion proteins becoming immobile once recruited into junctional SR domains.

To complete our analysis of the dynamic properties of triadic proteins, we studied the GFP-JP1 fusion protein. GFP-JP1 immediately assembled in clusters that later in differentiation converged into triadic junctions (Fig. S3). FRAP experiments on GFP-JP1 organized in distinct clusters or at triads showed that more than half of the fluorescent molecules were immobile, with an M_f of 40.7% ± 10.4% (n = 25) and 34.0% ± 14.1% (n = 18), respectively (Fig. 3D). This indicates that at least 60% of GFP-JP1 was strongly associated with these structures. No difference was observed in the diffusion constant (D of 0.07 ± 0.1 μm²/s and 0.07 ± 0.07 μm²/s, respectively).

Dynamic Properties of Triadin and Junctin.

Triadin and junctin are 2 structurally related proteins that form a macromolecular complex with RyR1 and calsequestrin (18, 23, 29, 30). Triadin-GFP presented an organization pattern similar to that of other junctional proteins (Fig. 4A). Fluorescence recovery measurements following photobleaching of areas of myotubes where triadin-GFP was diffused (Fig. 4Ai) indicated that the protein was highly mobile (D of 0.42 ± 0.30 μm²/s and M_f of 70.7% ± 29.0%, n = 22). A major change in triadin-GFP mobility was observed when this protein was organized in clusters, because the M_f decreased to 20.9% ± 8.7% (n = 23; P ≤ 0.01), as shown in Fig. 4B. The mobility of triadin-GFP was further reduced in triadic junctions, because only in 50% of the myotubes was it possible to measure a recovery in fluorescence after photobleaching (M_f of 15.6% ± 6.4%, n = 8, and D of 0.23 ± 0.24 μm²/s). In the remaining 50% of myotubes (n = 8), no recovery could be measured, as if all triadin-GFP molecules were irreversibly bound to triadic structures. Qualitative iFRAP experiments confirmed these findings, because no evident decrease in fluorescence in the unbleached structures was observed, even when the acquisition was prolonged up to 20 min (Fig. S4A).

Fig. 4.

The luminal region of triadin is responsible for its strong association to junctional domains. (A) Representative images of myotubes where triadin-GFP was observed in a reticular diffused state (i), in clusters (ii), and in triadic structures (iii). Graphic representations of the mobile fraction (M_f in %) of triadin-GFP (B), junctin-GFP (C), a triadin-junctin-GFP chimera (D), and a junctin-triadin-GFP chimera (E). (Scale bar: 5 μm.) *, Statistical significance with P ≤ 0.05; **, statistical significance with P ≤ 0.01; all means in the graphs are indicated with SDs.

In differentiating myotubes, junctin-GFP followed an organization pattern similar to that of all other junctional proteins (Fig. S5A). As shown in Fig. 4C, fluorescence recovery measurements in myotubes where junctin-GFP was diffused revealed a high mobility (D of 0.28 ± 0.24 μm²/s and M_f of 88.9% ± 9.3%, n = 16). Junctin-GFP organized in clusters or triadic structures and showed a significantly reduced diffusion constant (D of 0.06 ± 0.06 μm²/s and 0.03 ± 0.02 μm²/s, respectively; P ≤ 0.01) but, in contrast to other junctional proteins, only a moderate reduction in the mobile fraction was observed (M_f of 77.7% ± 16.4%, n = 43; P ≤ 0.05; and 72.8% ± 11.6%, n = 22; P ≤ 0.01, respectively). In agreement, iFRAP experiments showed that equilibration of fluorescence intensity between bleached and un-bleached regions was rapid (Fig. S4B).

Differences in the Dynamic Properties of Triadin and Junctin Are Dictated by the Intraluminal C-Terminal Tail.

Comparison of the amino acid sequences of triadin and junctin revealed some differences in the luminal tails of these proteins. Two GFP-proteins were generated, one containing the cytosolic and transmembrane region of triadin and the intraluminal domain of junctin (triadin-junctin-GFP), and the other containing the cytosolic and transmembrane region of junctin and the intraluminal domain of triadin (junctin-triadin-GFP). No significant differences were observed in the dynamics of these 2 proteins in NIH 3T3 cells (Table S1). In undifferentiated myotubes, triadin-junctin-GFP was diffused and showed an M_f of 81.10% ± 17.28% (n = 20) and a D of 0.15 ± 0.15 μm²/s, as reported in Fig. 4D. When this protein was organized in clusters or in triadic structures (Fig. S5B), a slight decrease in the M_f was observed (68.91% ± 17.11%, n = 39, P ≤ 0.05; and 59.96% ± 12.71%, n = 28, P ≤ 0.01, respectively), as shown in Fig. 4D. This was accompanied by a reduction of the D to 0.06 ± 0.06 μm²/s and 0.04 ± 0.02 μm²/s, respectively (P ≤ 0.01). In undifferentiated myotubes, the junctin-triadin-GFP protein was diffused and showed an M_f of 88.95% ± 11.08% (n = 22) and a D of 0.24 ± 0.15 μm²/s. However, once organized in clusters or in triadic structure (Fig. S5C), the M_f of junctin-triadin-GFP was dramatically decreased to 24.30% ± 7.59% (n = 20) and 23.60% ± 8.99% (n = 15), respectively (P ≤ 0.01), whereas no change was observed in the diffusion constant values (D of 0.15 ± 0.12 μm²/s and 0.22 ± 0.29 μm²/s, respectively), as shown in Fig. 4E. These data indicate that amino acid residues in the C-terminal tail of triadin mediate the decrease in mobility observed for triadin-GFP and junctin-triadin-GFP at junctional sites.

Discussion

Organization and Mobility Properties of Longitudinal SR Proteins.

Longitudinal SR proteins were randomly distributed over the entire SR membrane in the first days of differentiation and became organized within 6 days to specific regions of the SR following distinctive temporal kinetics. In differentiated myotubes, SERCA pumps, the most abundant longitudinal SR membrane proteins, were observed in a region of the SR close to the Z disk. Analysis of the dynamics of a GFP-SERCA2a fusion protein revealed that following myotube differentiation, no change was observed in GFP-SERCA2a mobility. This is compatible with the behavior of a transmembrane protein that is free to laterally diffuse in the lipid bilayer. In fact, electron microscopy studies had provided evidence that SERCA particles are widely distributed in the longitudinal SR (6), and no evidence that SERCA pumps are associated with other cellular structures has been reported (31). Thus, the SERCA immunofluorescence signal observed in bands at the Z disk, and to a lesser extent to the M band, rather than reflecting a specific retention mechanism, may result from the higher density of SR membranes in these regions (9, 24). Similarly to SERCA, InsP3R1 also was localized at the Z disk region of differentiated myotubes. However, the mobility of GFP-InsP3R1was reduced after localization to the Z disk region, as if this protein were anchored to a fixed structure. Indeed, deletion in InsP3R1 of the binding site for protein 4.1N, which has been reported to mediate an interaction between InsP3R1 and the actin cytoskeleton (26, 28), resulted in a mutant protein that, although localized at the Z disk in differentiated myotubes, presented a mobility similar to that of diffused GFP-InsP3R1. This suggests that interaction with protein 4.1N may control the mobility of GFP-InsP3R1 by linking this protein to the actin cytoskeleton upon differentiation. At variance with SERCA and InsP3R1, ank1.5 was predominantly localized at the M band. Furthermore, localization of ank1.5 to M-band represented the first longitudinal SR protein organized to a specific site. This agrees with recent results that revealed an early localization of ank1.5 with respect to other SR proteins and its early colocalization with obscurin during in vivo muscle development (32). These observations further support a possible involvement of ank1.5 and obscurin in the association of the developing SR with the myofilaments (32). Moreover, FRAP experiments performed in cultured myotubes revealed a reduction in the dynamics of ank1.5 following localization at the M band in differentiated myotubes. This reduction in ank1.5 mobility can be explained by previous indications of a direct interaction between ank1.5 and the myofibrillar protein obscurin (12–16). Accordingly, ank1.5Δ-GFP, an ank1.5 mutant lacking the binding site for obscurin (12–16), instead of localizing near the M band region of the SR localized near the Z disk and, more significantly, did not present a localization-dependent change in mobility in FRAP experiments. Thus, these data suggest that, if unable to bind obscurin at the M band, the ank1.5Δ-GFP mutant remains free to move in the SR membrane where, similarly to what was previously proposed for SERCA pumps, it is observed near the Z-disk, likely because of the higher density of SR membranes at this region. This mechanism could also explain why GFP-InsP3R1Δ14, although unable to interact through protein 4.1N to the actin cytoskeleton, was still localized at the Z disk.

Junctional Protein Organization and Mobility.

Immunostaining experiments indicated that the organization of the junctional SR domains occurred in 2 steps, which appeared to simultaneously involve all junctional proteins. Within the first 2 days after induction of differentiation, junctional SR proteins formed a large number of clusters that did not show a specific spatial relationship with the sarcomere. This organization in clusters preceded the organization of longitudinal SR proteins. During the second step, junctional SR proteins became organized at the border between A and I bands. It is worth noting that the organization of junctional proteins in triadic structures was a late event in myotube differentiation and was only observed in myotubes where the longitudinal SR proteins, like ank1.5 and SERCA, were already organized. This suggests that the assembly of SR proteins at longitudinal and junctional SR domains may initially be independent of each other, but become interconnected at a later stage.

Available evidence suggests that junctional proteins form a multiprotein complex of high molecular mass, with the RyR1 channel interacting with several junctional proteins, including triadin, JP1, and DHPR (29, 30, 33). Analysis of the dynamic properties of junctional SR membrane proteins revealed that assembly into clusters is accompanied by a strong reduction of their mobile fraction. Among the junctional proteins analyzed, triadin-GFP presented the most drastic reduction in mobility upon association to junctional domains, whereas junctin-GFP retained a relatively high mobile fraction also after its segregation to the junctional SR domain. A possible explanation for the behavior of junctin is that this protein may only establish transient interactions with the junctional multiprotein complex (25). Results obtained with chimeric fusion proteins containing different regions of triadin and junctin indicated that the region responsible for the high affinity of triadin for the junctional domains is the intraluminal tail, which contains binding sites for other junctional proteins, like RyR1 and calsequestrin (18).

Longitudinal and Junctional SR Assembly: A Model.

The reported results let us propose a working model for SR domain assembly, as reported in Fig. S6. Anchorage of the SR to the myofibrils, likely due to the binding between ank1.5 and the myofibrillar protein obscurin, appears to be an early event in the developing longitudinal SR. The next step is the redistribution of longitudinal SR membrane along the sarcomere, with major membrane densities at the level of the Z disk and M band. Longitudinal SR proteins might then remain relatively free of moving in the SR membrane (e.g., SERCA) or, if able to anchor to external clues, like the actin cytoskeleton and the myofibrils, they can be selectively retained in a specific region, as proposed for the InsP3R1 and ank1.5. The junctional SR domain appears to be the first SR domain to assemble, forming distinct clusters with no spatial relationship to the myofibrillar apparatus. This process is probably in part regulated by internal clues through formation of a multiprotein complex of junctional proteins. Interestingly, according to dynamic properties measured in FRAP experiments, the degree of association with this supramolecular complex appears to differ among junctional proteins (i.e., triadin > RyR > junctin). Only in a second step, which follows the organization of the longitudinal SR, junctional SR domains will align with the contractile apparatus at the A-I border by establishing yet-unidentified interactions with the contractile apparatus. Future work will help further extend our knowledge on the molecular interactions proposed in this model.

Materials and Methods

Detailed materials and methods are provided as SI Materials and Methods.

Cultures of Primary Skeletal Myoblasts.

Cultures of primary rat myoblasts were performed as reported previously (14).

Immunofluorescence and Antibodies.

Details on immunofluorescence and antibodies are described in SI Materials and Methods.

GFP Fusion Proteins.

A detailed description of GFP-fusion protein cloning strategies is reported in SI Materials and Methods.

FRAP Experiments and Statistical Analysis.

Details on FRAP experiments and statistical analysis are described in SI Materials and Methods.