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

Protasi F, Franzini-Armstrong C, Flucher BE - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6058, USA. protasi@mail.med.upenn.edu

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.

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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.
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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.

Mentions: The analysis of subdomains with many incomplete tetrads provides further information about the relationship between the organization of individual tetrads and their arrays. Using a procedure similar to the one described above allowed us to designate particles belonging to a tetrad by their position adjacent to dots of an orthogonal array with a spacing of 41 nm (Fig. 8, legend). Two examples of small arrays with few particles are shown in Fig. 8, A and B. The great majority (96%) of large membrane particles in the clusters was located in correct positions of putative tetrads regardless of how complete the tetrads were (Fig. 8). The incidence of free particles apparently not part of a tetrad was low and independent of the particle density (or occupancy), which ranged from 15 to 89% of the maximal possible number of tetrad particles in 88 analyzed subdomains. We conclude that particles in the subdomains are predominantly positioned at the sites of tetrads, that is, in correspondence to alternate feet, even when the arrays of tetrads are quite incomplete. The same analysis applied to randomly disposed particles in peripheral clusters of cardiac myocytes, which do not form tetrads at all (Sun et al., 1995; Protasi et al., 1996), gives a high number of particles that could not be associated with putative tetrad centers. In addition, the frequency of these random particles is strongly dependent on the overall density of particles (Fig. 8), indicative of an accidental positioning near tetrad centers. Thus, the analysis procedure used can distinguish between randomly disposed particles and incomplete arrays of tetrads and therefore provides a quantitative measure for the degree of tetrad assembly.


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

Protasi F, Franzini-Armstrong C, Flucher BE - J. Cell Biol. (1997)

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.
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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.
Mentions: The analysis of subdomains with many incomplete tetrads provides further information about the relationship between the organization of individual tetrads and their arrays. Using a procedure similar to the one described above allowed us to designate particles belonging to a tetrad by their position adjacent to dots of an orthogonal array with a spacing of 41 nm (Fig. 8, legend). Two examples of small arrays with few particles are shown in Fig. 8, A and B. The great majority (96%) of large membrane particles in the clusters was located in correct positions of putative tetrads regardless of how complete the tetrads were (Fig. 8). The incidence of free particles apparently not part of a tetrad was low and independent of the particle density (or occupancy), which ranged from 15 to 89% of the maximal possible number of tetrad particles in 88 analyzed subdomains. We conclude that particles in the subdomains are predominantly positioned at the sites of tetrads, that is, in correspondence to alternate feet, even when the arrays of tetrads are quite incomplete. The same analysis applied to randomly disposed particles in peripheral clusters of cardiac myocytes, which do not form tetrads at all (Sun et al., 1995; Protasi et al., 1996), gives a high number of particles that could not be associated with putative tetrad centers. In addition, the frequency of these random particles is strongly dependent on the overall density of particles (Fig. 8), indicative of an accidental positioning near tetrad centers. Thus, the analysis procedure used can distinguish between randomly disposed particles and incomplete arrays of tetrads and therefore provides a quantitative measure for the degree of tetrad assembly.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6058, USA. protasi@mail.med.upenn.edu

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.

Show MeSH
Related in: MedlinePlus