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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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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.
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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.

Mentions: Consistent with the immunolocalization, BC3H1 cells have few internal junctions but numerous peripheral couplings (SR–surface junctions) of variable size, many larger than those seen during differentiation of normal myotubes (Fig. 3, compare with Pincon-Raymond et al., 1985; Flucher et al., 1993b, 1994; Takekura et al., 1994b). Some junctional gaps have none or few identifiable feet, many have small arrays of regularly spaced feet occupying only part of the gap (Fig. 3 A, arrows), and others are entirely filled with arrays of feet (Fig. 3, B and C, arrows). The average distance between feet measured in thin section images showing very distinct profiles is 31.0 ± 3.5 nm (mean ± 1 SD; number of junctions = 47).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139832&req=5

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.
Mentions: Consistent with the immunolocalization, BC3H1 cells have few internal junctions but numerous peripheral couplings (SR–surface junctions) of variable size, many larger than those seen during differentiation of normal myotubes (Fig. 3, compare with Pincon-Raymond et al., 1985; Flucher et al., 1993b, 1994; Takekura et al., 1994b). Some junctional gaps have none or few identifiable feet, many have small arrays of regularly spaced feet occupying only part of the gap (Fig. 3 A, arrows), and others are entirely filled with arrays of feet (Fig. 3, B and C, arrows). The average distance between feet measured in thin section images showing very distinct profiles is 31.0 ± 3.5 nm (mean ± 1 SD; number of junctions = 47).

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