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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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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.
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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.

Mentions: The ordered disposition of tetrads in arrays was confirmed by optical diffraction analysis. In images of rotaryshadowed replicas (Fig. 7 A), the position of each particle is precisely marked by the symmetric ring of platinum shadow around it, thus making the array an appropriate object for optical diffraction. The pattern of reflections in the diffraction pattern (Fig. 7 B) was indexed by two orthogonal lattices with spacings of 1/18.4 ± 0.4 and 1/41.9 ± 1.6 nm (mean ± 1 SD, the average of 5 to 6 patterns from different particle clusters). The two values correspond, respectively, to the distance between particles in the tetrad and to the center-to-center distance between tetrads, as measured in the micrographs. The angle between the two lattices, 65.5 ± 0.5°, corresponds to the skew angle between lines joining the centers of tetrads and those joining the centers of particles within a tetrad (Franzini-Armstrong and Kish, 1995). The significance of the two lattices was determined by comparison with diffraction patterns derived from a model of tetrad arrays (Fig. 7 C). The model is built by exactly superimposing tetrads over alternate feet in an array constructed as in Takekura et al. (1994a) and Franzini-Armstrong and Kish (1995). The main feature of the model is the skew angle between the orthogonal array formed by tetrad centers and that of the four tetrad subunits, which is determined by the underlying disposition of feet. The diffraction pattern of this model (Fig. 7 D) indexes on two orthogonal lattices with spacings inversely proportional to the center-to-center distances between the adjacent tetrads and between the particles composing the tetrads. The two lattices in the diffraction pattern are skewed by an angle of 71.5°. Thus the array of tetrads in the freeze fracture images is composed of groups of square (or quadrate) units disposed in an orthogonal array with a skewed disposition, just as in the model.


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)

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.
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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.
Mentions: The ordered disposition of tetrads in arrays was confirmed by optical diffraction analysis. In images of rotaryshadowed replicas (Fig. 7 A), the position of each particle is precisely marked by the symmetric ring of platinum shadow around it, thus making the array an appropriate object for optical diffraction. The pattern of reflections in the diffraction pattern (Fig. 7 B) was indexed by two orthogonal lattices with spacings of 1/18.4 ± 0.4 and 1/41.9 ± 1.6 nm (mean ± 1 SD, the average of 5 to 6 patterns from different particle clusters). The two values correspond, respectively, to the distance between particles in the tetrad and to the center-to-center distance between tetrads, as measured in the micrographs. The angle between the two lattices, 65.5 ± 0.5°, corresponds to the skew angle between lines joining the centers of tetrads and those joining the centers of particles within a tetrad (Franzini-Armstrong and Kish, 1995). The significance of the two lattices was determined by comparison with diffraction patterns derived from a model of tetrad arrays (Fig. 7 C). The model is built by exactly superimposing tetrads over alternate feet in an array constructed as in Takekura et al. (1994a) and Franzini-Armstrong and Kish (1995). The main feature of the model is the skew angle between the orthogonal array formed by tetrad centers and that of the four tetrad subunits, which is determined by the underlying disposition of feet. The diffraction pattern of this model (Fig. 7 D) indexes on two orthogonal lattices with spacings inversely proportional to the center-to-center distances between the adjacent tetrads and between the particles composing the tetrads. The two lattices in the diffraction pattern are skewed by an angle of 71.5°. Thus the array of tetrads in the freeze fracture images is composed of groups of square (or quadrate) units disposed in an orthogonal array with a skewed disposition, just as in the model.

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