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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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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.
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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.

Mentions: The particle clusters in differentiated cells contain regular arrays of tetrads (Figs. 5 and 6). Three criteria define tetrads: (a) a complete tetrad is composed of four large membrane particles positioned at the corners of squares with a distance of 17 to 18 nm between the centers of adjacent particles (Fig. 5, lines A and B); (b) tetrads always occur in groups forming orthogonal arrays with a distance of ∼41 nm between the centers of adjacent tetrads (Fig. 6, C and E, and see below); and (c) particles outside their designated positions at the corners of the squares are usually excluded from the membrane within tetrad arrays (Fig. 6). These characteristics allow the unequivocal recognition of tetrads even if one criterion is not fully met. For instance, the “square” can become slightly distorted during fracturing (Fig. 5 E, 1 and 2), or some of the particles may be missing from the corners (Fig. 5, C–E). Complete or incomplete (three large particles) tetrads are practically never found in cells in growth medium (see above) and are not found in cells that, although grown in differentiation medium, appear undifferentiated, as indicated by the absence of membrane invaginations and overall scarcity of membrane particles. Arrays of tetrads mostly occur on plasma membrane mounds that presumably represent the areas of close SR apposition.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: The particle clusters in differentiated cells contain regular arrays of tetrads (Figs. 5 and 6). Three criteria define tetrads: (a) a complete tetrad is composed of four large membrane particles positioned at the corners of squares with a distance of 17 to 18 nm between the centers of adjacent particles (Fig. 5, lines A and B); (b) tetrads always occur in groups forming orthogonal arrays with a distance of ∼41 nm between the centers of adjacent tetrads (Fig. 6, C and E, and see below); and (c) particles outside their designated positions at the corners of the squares are usually excluded from the membrane within tetrad arrays (Fig. 6). These characteristics allow the unequivocal recognition of tetrads even if one criterion is not fully met. For instance, the “square” can become slightly distorted during fracturing (Fig. 5 E, 1 and 2), or some of the particles may be missing from the corners (Fig. 5, C–E). Complete or incomplete (three large particles) tetrads are practically never found in cells in growth medium (see above) and are not found in cells that, although grown in differentiation medium, appear undifferentiated, as indicated by the absence of membrane invaginations and overall scarcity of membrane particles. Arrays of tetrads mostly occur on plasma membrane mounds that presumably represent the areas of close SR apposition.

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