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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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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.
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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.

Mentions: Cultured BC3H1 cells were immunolabeled with antibodies specific for two proteins of the junctional SR: the RyR and triadin (95-kD protein); for proteins of the junctional T tubules or plasma membrane: the DHPR (α1 and α2 subunits); and for a general T tubule antigen of unknown identity (Flucher et al., 1991). Before change to low serum medium, BC3H1 cells were negative for all antibodies used (not shown). 4 d after serum withdrawal (D4), numerous spindle-shaped differentiated cells reacted with the antibodies against the junctional proteins (Figs. 1 and 2). About 40% of the cells differentiate, as indicated by the expression of junctional proteins (115 out of 283 cells in 15 randomly chosen fields from two coverslips; ∼0.5 mm2 area). The α1 and α2 subunits of the DHPR, the RyR, and triadin are located in numerous discrete clusters at the periphery of the cell, whereas focusing up and down through the cells showed that little to no specific immunolabel was found in the cytoplasm. Double immunolabeling of RyRs with either α1 and α2 subunits of DHPRs or triadin shows the colocalization of all four proteins within surface clusters (Figs. 1 and 2). The immunofluorescent clusters are variable in size, some of the aggregates being quite large compared to those seen in normal myotubes in vitro (Flucher et al., 1994) and occasionally appear to be composed of several subdomains (Fig. 1 C, inset). In double labeling experiments, the sizes and shapes of corresponding RyR/DHPR or RyR/triadin clusters agree well with one another, indicative of a parallel incorporation of triad proteins into SR–surface membrane junctions.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Cultured BC3H1 cells were immunolabeled with antibodies specific for two proteins of the junctional SR: the RyR and triadin (95-kD protein); for proteins of the junctional T tubules or plasma membrane: the DHPR (α1 and α2 subunits); and for a general T tubule antigen of unknown identity (Flucher et al., 1991). Before change to low serum medium, BC3H1 cells were negative for all antibodies used (not shown). 4 d after serum withdrawal (D4), numerous spindle-shaped differentiated cells reacted with the antibodies against the junctional proteins (Figs. 1 and 2). About 40% of the cells differentiate, as indicated by the expression of junctional proteins (115 out of 283 cells in 15 randomly chosen fields from two coverslips; ∼0.5 mm2 area). The α1 and α2 subunits of the DHPR, the RyR, and triadin are located in numerous discrete clusters at the periphery of the cell, whereas focusing up and down through the cells showed that little to no specific immunolabel was found in the cytoplasm. Double immunolabeling of RyRs with either α1 and α2 subunits of DHPRs or triadin shows the colocalization of all four proteins within surface clusters (Figs. 1 and 2). The immunofluorescent clusters are variable in size, some of the aggregates being quite large compared to those seen in normal myotubes in vitro (Flucher et al., 1994) and occasionally appear to be composed of several subdomains (Fig. 1 C, inset). In double labeling experiments, the sizes and shapes of corresponding RyR/DHPR or RyR/triadin clusters agree well with one another, indicative of a parallel incorporation of triad proteins into SR–surface membrane junctions.

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