Coordinated incorporation of skeletal muscle dihydropyridine receptors and ryanodine receptors in peripheral couplings of BC3H1 cells

Abstract

Rapid release of calcium from the sarcoplasmic reticulum (SR) of skeletal muscle fibers during excitation-contraction (e-c) coupling is initiated by the interaction of surface membrane calcium channels (dihydropyridine receptors; DHPRs) with the calcium release channels of the SR (ryanodine receptors; RyRs, or feet). We studied the early differentiation of calcium release units, which mediate this interaction, in BC3H1 cells. Immunofluorescence labelings of differentiating myocytes with antibodies against alpha1 and alpha2 subunits of DHPRs, RyRs, and triadin show that the skeletal isoforms of all four proteins are abundantly expressed upon differentiation, they appear concomitantly, and they are colocalized. The transverse tubular system is poorly organized, and thus clusters of e-c coupling proteins are predominantly located at the cell periphery. Freeze fracture analysis of the surface membrane reveals tetrads of large intramembrane particles, arranged in orderly arrays. These appear concomitantly with arrays of feet (RyRs) and with the appearance of DHPR/RyS clusters, confirming that the four components of the tetrads correspond to skeletal muscle DHPRs. The arrangement of tetrads and feet in developing junctions indicates that incorporation of DHPRs in junctional domains of the surface membrane proceeds gradually and is highly coordinated with the formation of RyR arrays. Within the arrays, tetrads are positioned at a spacing of twice the distance between the feet. The incorporation of individual DHPRs into tetrads occurs exclusively at positions corresponding to alternate feet, suggesting that the assembly of RyR arrays not only guides the assembly of tetrads but also determines their characteristic spacing in the junction.

Figure 1

Double-immunofluorescence labeling of triad proteins in differentiated BC₃H1 cells. 4 d after change to low serum medium (D4) many cells have assumed a spindle shape and express triad proteins, whereas other cells remain undifferentiated (asterisks). Both the DHPR α₂ subunit (B) and triadin (E) are colocalized with the RyR (C and F) in clusters at or close to the cell surface, indicative of plasma membrane–SR junctions. The shapes and the distribution of the immunolabeled clusters correspond highly with one another in the double- labeled pairs (examples indicated by arrows in B, C, E, and F). Some clusters are unusually large and are composed of multiple subdomains (see inset in C at 4-fold higher magnification). (A and D) Phase contrast images of fields shown in B, C, E, and F, respectively. Bar, 10 μm.

Figure 2

Double-immunofluorescence labeling of DHPR α₁ and α₂ subunits with the RyR and a general T tubule marker. In differentiated BC₃H1 cells (D4), clusters of α₁ DHPRs (A) and RyRs (B) are colocalized at or close to the cell surface (examples indicated by arrows). The colabeled clusters most likely represent peripheral couplings between the SR and either the plasma membrane or short invaginations. Cells that express clusters of α₂ DHPR (C) do not express the nonjunctional T tubule proteins labeled with the antibody TT2 (D), indicating that a mature T system is not present in differentiated BC₃H1 cells. Bar, 10 μm.

Figure 3

Thin sections showing the periphery of differentiated BC₃H1 cells (D3–D8). Peripheral couplings are formed by an SR cisterna associated with the plasma membrane. Arrays of feet, positioned at regular intervals (arrows), occupy only part of the junction in A but the whole junction in B and C. Bar, 0.1 μm.

Figure 4

Freeze fracture replicas of the plasmalemma from cells in growth medium (A and B) and from a differentiated cell (D6; C). Undifferentiated cells have a smooth surface with uniform distribution of intramembrane particles (A), while ∼41% of cells in differentiation medium have numerous clusters of large intramembrane particles (C, semicircles). Within the clusters, the particles form groups of four (tetrads). Only 1 of the 138 cells examined in growth medium contained small clusters of large particles, occasionally grouped as in a tetrad (B, circle); these may be precursors of the larger, more crowded clusters of differentiating cells. Openings of surface invaginations (arrows) are present only in differentiated cells. Smaller openings probably belong to primitive T tubules and larger ones to shallow membrane invaginations. Invaginations are infrequent, particularly in early (D3–D5) cells. Bars: (A and C) 0.5 μm; (B) 0.2 μm.

Figure 5

Montage of tetrads either unidirectionally (A, C, and E) or rotary (B and D) shadowed. A tetrad is composed of four equal intramembrane particles disposed at the corners of a square with a center-to-center spacing of 17 to 18 nm between particles. The particles have a large diameter, and the elongated platinum-free “shadow” indicates that they are tall. Incomplete tetrads apparently miss one or more particles, but short, distorted stumps in place of the missing particles indicate that the protein was present before fracturing (C and D). Some tetrads truly miss a component, since no stump is visible in the place of a missing particle (E, 3 and 4). The whole tetrad may become distorted during fracturing, resulting in an asymmetric shape (E, 1 and 2). Bar, 0.1 μm.

Figure 6

Tetrads, whether perfectly preserved or distorted during fracturing, are identified on the basis of their position in arrays. Once a tetrad is identified in A (square, and at higher magnification in inset) it is easy to see that equally oriented adjacent tetrads are located in an orthogonal arrangement around it. Marking the tetrads by putting a dot in their centers helps in identifying the pattern. In B–E the tetrads (including those that miss one or two components) are marked in the second of the two identical images (C and E). The dots define an orthogonal arrangement with a center-to-center spacing between adjacent tetrads of ∼41 nm (E, along the dashed line). The spacing along the diagonals (E, arrows) is ∼58 nm. Bars, 0.1 μm.

Figure 7

Images and optical diffraction patterns of a rotary-shadowed tetrad cluster (A and B) and of a model array, constructed by exactly positioning tetrads over an array of feet, in correspondence of every other foot (Franzini-Armstrong and Kish, 1995; C and D). The position of each tetrad particle is marked by a small ring of platinum shadow in A and modeled by a filled circle in C. Alignment of the particles is visible by holding the micrograph at eye level and glancing along the axes indicated by the arrows. Small, winged arrows in A and C indicate alignment of tetrad centers along the sides of an orthogonal array with a spacing of ∼41 nm. See also Fig. 6. The diffraction pattern of the freeze fracture (B) indexes on two orthogonal lattices skewed relative to each other, with spacings of ∼1/42 (small, winged arrows) and ∼1/18 nm (large arrows), corresponding to the distance between the centers of adjacent tetrads and of the particles within the tetrads, respectively. The diffraction pattern from the model also indicates two orthogonal lattices with spacings corresponding to those between the centers of tetrads (small, winged arrows) and the centers of tetrad subunits (large arrows). The angle between the two lattices in the diffraction pattern from the tetrad arrays (65–66°) differs only slightly from that of the model array (71.5°). Bar, 0.1 μm.

Figure 8

Arrangement of particles in tetrad arrays is not dependent on degree of completeness of tetrads. (Graph) Comparison of particle clustering in cardiac junctions (filled circles, randomly distributed particles) and BC₃H1 junctions (open circles, particles forming tetrads); each circle represents data from one continuous cluster of particles. (Abscissa) Relative frequency of particles that were closely associated to an orthogonal array of dots at a spacing of 41 nm. (Ordinate) Relative frequency of particles not associated with the array. Values are expressed as percentage of the maximal possible number of particles constituting tetrads within the junction area (4 × the number of dots). In BC₃H1 cells, the percentage of nonassociated particles is constant, and on average only 4% of the particles cannot be assigned to a tetrad position, regardless of the degree of filling of a junction (15– 89%). In the random clusters of cardiac muscle, the frequency of particles not positioned at tetrad locations is higher and increases with the overall density of particles. Analysis procedure: subdomains of large clusters of particles in micrographs from cells at D3–D7 were overlaid with the array of dots and rotated to achieve maximal proximity of particles and dots. Particles were scored as either clustered around the dots, corresponding to the expected position in a tetrad (abscissa), or as distant to dots without apparent relationship to a tetrad (ordinate). Fig. 8, A and B shows two examples of incomplete arrays, with “dotted” tetrads. In A there are 18 particles clustered near the dots, or 37% of the 48 particles needed for a complete tetrad array. Arrows indicate four misplaced particles (or 8%). The values for Fig. 8 B are 50 and 2%. Particles at the edge of the array are not counted. Dotted arrays in Fig. 6 E fell in the 80–90% complete category. Data for cardiac junctions were obtained from micrographs constituting that data base in Sun et al. (1995) and Protasi et al. (1996). Bar, 50 nm.

Figure 8

Arrangement of particles in tetrad arrays is not dependent on degree of completeness of tetrads. (Graph) Comparison of particle clustering in cardiac junctions (filled circles, randomly distributed particles) and BC₃H1 junctions (open circles, particles forming tetrads); each circle represents data from one continuous cluster of particles. (Abscissa) Relative frequency of particles that were closely associated to an orthogonal array of dots at a spacing of 41 nm. (Ordinate) Relative frequency of particles not associated with the array. Values are expressed as percentage of the maximal possible number of particles constituting tetrads within the junction area (4 × the number of dots). In BC₃H1 cells, the percentage of nonassociated particles is constant, and on average only 4% of the particles cannot be assigned to a tetrad position, regardless of the degree of filling of a junction (15– 89%). In the random clusters of cardiac muscle, the frequency of particles not positioned at tetrad locations is higher and increases with the overall density of particles. Analysis procedure: subdomains of large clusters of particles in micrographs from cells at D3–D7 were overlaid with the array of dots and rotated to achieve maximal proximity of particles and dots. Particles were scored as either clustered around the dots, corresponding to the expected position in a tetrad (abscissa), or as distant to dots without apparent relationship to a tetrad (ordinate). Fig. 8, A and B shows two examples of incomplete arrays, with “dotted” tetrads. In A there are 18 particles clustered near the dots, or 37% of the 48 particles needed for a complete tetrad array. Arrows indicate four misplaced particles (or 8%). The values for Fig. 8 B are 50 and 2%. Particles at the edge of the array are not counted. Dotted arrays in Fig. 6 E fell in the 80–90% complete category. Data for cardiac junctions were obtained from micrographs constituting that data base in Sun et al. (1995) and Protasi et al. (1996). Bar, 50 nm.

Figure 9

Comparison between arrays of particles and of feet. (A) An array of particles covers approximately half (between arrows) of a domed membrane domain (between arrowheads). (B) This corresponds to peripheral couplings (between arrowheads) in which only a portion of the junctional gap is occupied by an array of feet (between arrows). (C) A very large cluster of particles contains several subdomains of tetrads with different orientations (arrows), separated by spaces with fewer particles (asterisk). (D) A peripheral coupling containing two separate arrays of feet (arrows) with slightly different orientations. In fact, feet are better delineated at right than at left of the junction, indicating different orientations of the two arrays relative to the plane of the section. Bar, 0.2 μm.

Figure 10

Formation of tetrad arrays in calcium release units. (A) Superimposed arrays of feet (squares) and tetrads (groups of four circles) represent the regular association of tetrads with every other foot. To emphasize this arrangement, adjacent rows of feet are drawn in different shades of gray. Dashed lines in A show the axes of the orthogonal array and the skew of the orientation of tetrads. Since the size of a tetrad is slightly larger than that of a foot, the association of tetrads with alternate feet (B) represents the densest packing possible. However, steric hindrance alone would not prevent either the positioning of tetrads at larger intervals (D) or the association of incomplete tetrads with neighboring feet (C, bottom). Such patterns are not observed; rather, complete and incomplete tetrads are always associated with alternate feet (C, top row), suggesting that the alternating association is determined by other factors. Formation of extensive arrays of feet before the incorporation of tetrads by centripetal diffusion and subsequent immobilization of tetrads opposite feet should result in arrays with unoccupied centers (E) that are never seen. The observations of multiple microarrays of tetrads and feet with different orientations within the larger junctions and the alternate disposition of tetrads are more consistent with a concomitant centrifugal growth of both arrays (F).