* Department of Cell Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6058; and Department of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria
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 1 and
2 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.
Excitation contraction (e-c)1 coupling in muscle
cells comprises a series of events linking depolarization of the plasma membrane to the release of
calcium from the sarcoplasmic reticulum (SR; Schneider,
1981 The DHPR is an L-type calcium channel that is responsible for initiating e-c coupling events by acting as a voltage sensor (Rios and Brum, 1987 Recently a third component of the junction, called triadin, has received considerable attention (Caswell et al.,
1991 BC3H1 is a nonfusing cell line derived from a mouse
brain tumor (Schubert et al., 1974 The present results show a striking correlation in the expression of DHPRs, RyRs, and triadin in the coclustering
of these three junctional proteins and in the specific association of feet (RyRs) and tetrads (DHPRs) during development. The extensive junctional domains of BC3H1 cells
allow, for the first time, the use of optical diffraction to determine the spacing and orientation of tetrads, confirming
that the disposition of tetrads is closely related to that of
alternate feet. Interestingly, incomplete arrays of tetrads
that are in the process of formation also show the alternate positioning of tetrads relative to feet, thus indicating that intrinsic molecular properties determine this arrangement.
Cell Culturing and Fixation
The BC3H1 cell line was bought from American Type Culture Collection
(Rockville, MD). The cells were grown in a growth medium containing
low glucose DME medium (GIBCO BRL, Gaithersburg, MD), 20% fetal
bovine serum, 0.5% chicken embryo extract, 100 U/ml penicillin, 100 mm/
ml streptomycin, and additional 2 mM l-glutamine. Cells were plated on
aclar (Pro-Plastics, Linden, NY), thermanox (Nunc Inc., Naperville, IL),
or glass coverslips. Some coverslips were covered with matrigel (Collaborative Biomedical Products, Bedford, MA). BC3H1 cells grow slightly
faster on matrigel and they are better attached to the coverslip. This helps
during fixation in which cells cultured directly on the coverslip tend to detach. The medium was changed every 2-3 d. At ~70% confluence, the
growth medium was replaced by a low serum medium containing 0.5% fetal bovine serum and no chicken embryo extract (differentiation medium) to induce differentiation, and the cells were fixed 3-8 d later.
Immunohistochemistry
Cultures grown on glass coverslips were fixed and immunostained as previously described (Flucher et al., 1993b Table I.
Antibodies Used and Their Specificity
; Rios et al., 1991
). Specific structures, named calcium
release units, perform this functional interaction between SR and plasma membrane (Franzini-Armstrong and Jorgensen, 1994
; Flucher and Franzini-Armstrong, 1996
). Calcium release units are formed by the close apposition of
specialized junctional domains of the SR on one side and
of the plasma membrane, including its invaginations, the
transverse (T) tubules, on the other. The junctional domains contain two key proteins involved in e-c coupling:
the ryanodine receptor (RyR) of the junctional SR (for reviews see Sorrentino and Volpe, 1993
; Meissner, 1994
; and
Coronado et al., 1994
) and the dihydropyridine receptor
(DHPR) located in the junctional domains of plasma membrane and T tubules (Jorgensen et al., 1989
; Flucher et al.,
1990
; Yuan et al., 1991
). The RyR is the SR calcium release channel (Imagawa et al., 1987
; Inui et al., 1987
; Lai et
al., 1988
). This molecule is composed of two different domains: the channel domain, inserted into the SR membrane, and the cytoplasmic domain, called the "foot." Feet
form extensive ordered arrays (Franzini-Armstrong, 1970
)
and span the narrow gap between the membranes of SR
and plasma membrane-T tubules (Block et al., 1988
; Radermacher et al., 1994
).
; Tanabe et al., 1988
; Adams et al., 1990
). According to the mechanical coupling
hypothesis, interaction between the voltage sensor and the
SR calcium release channel in skeletal muscle involves a
direct functional link between the two proteins (DHPRs
and RyRs; Schneider and Chandler, 1973
). Strong support for this hypothesis comes from the observation that junctional plasma membrane and T tubules are occupied by tetrads, groups of four integral membrane proteins, that are
located exactly in correspondence to the four feet subunits
(Block et al., 1988
). If tetrads correspond to groups of four
DHPRs, their alignment with the feet constitutes the basis
for an interaction between DHPRs and RyRs. The lack of
tetrads in dysgenic myotubes carrying a mutation of the
DHPR (Franzini-Armstrong et al., 1991
) and their reappearance after transfection with cDNA encoding for the
DHPR (Takekura et al., 1994a
) provided initial evidence
for the identification of tetrads with DHPRs. One puzzling
observation, however, is that tetrads are associated only
with alternate feet, thus creating two categories of feet:
those that are linked to tetrads and those that are not
(Block et al., 1988
; Franzini-Armstrong and Kish, 1995
).
; Knudson et al., 1993a
,b). This 95-kD SR protein is
involved either in the interaction between RyRs and DHPRs
(Brandt et al., 1990
; Kim et al., 1990
; Fan et al., 1995a
,b) or
in the association between RyRs and calsequestrin (Knudson et al., 1993a
,b; Guo and Campbell, 1995
). The latter is
the calcium binding protein located in the lumen of the
terminal SR cisternae (Meissner, 1975
; MacLennan et al.,
1983
; Ikemoto et al., 1989
; Pozzan et al., 1994
).
). Withdrawal of growth
factors induces these cells to differentiate by expressing
several skeletal muscle-specific proteins (Taubman et al.,
1989
), including RyRs (Marks et al., 1989
; Airey et al.,
1991
), and functional L-type calcium channels identified as
DHPRs (Caffrey et al., 1987
; Caffrey and Farach, 1988
;
Morton et al., 1988
; Rampe et al., 1988
). These cells were chosen for the study of calcium release unit development
because they form extensive peripheral couplings, junctions between the SR and the plasma membrane (Marks et
al., 1989
, 1991). We sought to confirm that tetrads are
composed of DHPRs and explored how tetrads and feet
are assembled into coextensive arrays.
Materials and Methods
). Methanol-fixed cultures were incubated with 10% normal goat serum in PBS containing 0.2% BSA (PBS/
BSA) for 30 min and then incubated in primary antibodies for at least 2 h
at room temperature or overnight at 4°C. After washing in five changes of
PBS/BSA the cultures were incubated in fluorochrome-conjugated secondary antibodies (Cappel Laboratories, Malvern, PA) for 1 h at room
temperature and washed again. Controls were performed in which the primary antibodies were omitted or were composed of an inappropriate antibody combination (mouse primary with anti-rabbit secondary and vice
versa). The glass coverslips were then mounted in 90% glycerol, 0.1 M Tris,
pH 8.0, with 5 mg/ml p-phenylenediamine to retard photobleaching. The specimens were viewed and photographed with black and white film on a
light microscope (Axioskop or Axiovert; Carl Zeiss, Thornwood, NY)
equipped with epifluorescence optics. Pictures were digitized by scanning
the negative film, and the contrast and density of the pictures were optimized with image processing software (Adobe Photoshop, Adobe Systems
Inc., Mountain View, CA). The working dilutions and the sources of primary antibodies are listed in Table I. All antibodies have been fully characterized and used on cultured skeletal myotubes in the quoted literature.
Electron Microscopy
Cells grown on either type of plastic coverslip were washed twice in PBS at 37°C, fixed in glutaraldehyde, and kept in fixative for up to 1-4 wk before further use. For thin sectioning, two different protocols were used: (a) 3.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, followed by 2% OsO4 for 2 h at room temperature and saturated uranyl acetate for 4 h at 60°C; (b) 2.5% glutaraldehyde and 1% tannic acid for 1 h followed by extensive washes in buffer; 1% OsO4 for 2 h at 4°C; and 0.5% uranyl acetate for 2 h at room temperature. The samples were embedded in Epon 812 and the sections stained either in saturated aqueous uranyl acetate or in 1% uranyl acetate followed by lead salts, both for 8 min.
For freeze fractures, the cells were fixed in glutaraldehyde as in (a)
above and infiltrated in 30% glycerol. A small piece of the coverslip was
mounted with the cells facing a droplet of 30% glycerol, 20% polyvinyl alcohol on a gold holder and frozen in liquid nitrogen-cooled propane (Osame et al., 1981). The coverslip was flipped off to produce a fracture that
followed the culture surface originally facing the coverslip. The fractured
surfaces were shadowed with platinum either at 45° unidirectionally or at
25°C while rotating and replicated with carbon in a model BFA 400 Balzers freeze fracture. Sections and replicas were photographed in an electron microscope (410; Philips Technologies, Cheshire, CT).
Immunohistochemistry
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 (1 and
2 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
1 and
2 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
1 and
2 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.
Immunostaining for a general T tubule protein gave negative results (Fig. 2 D) even in cells that are differentiated, as indicated by the presence of DHPR-positive clusters. The great majority of the junctional protein clusters appear on or near the surface membrane, both on the ventral, substrate-facing and the dorsal sides of the cells.
Electron Microscopy
Thin Sections: Arrays of Feet.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).
Freeze Fracture: Clustered Tetrads.
The fracture plane follows the cell membrane facing the substrate (the same as shown and analyzed in the immunofluorescence experiments; Figs. 1 and 2). In undifferentiated cultures, the cells are odd shaped and smaller; after withdrawal of growth factors, larger, spindle-shaped cells similar to those positive for antibodies against junctional proteins (Figs. 1 and 2) become numerous, but undifferentiated cells are still present.
The cytoplasmic leaflet in undifferentiated cells is characterized by randomly disposed intramembrane particles
and lack of caveolae or other membrane invaginations
(Fig. 4 A). Only 1 out of 138 observed cells in the growth
phase, or 0.7% (from three coverslips, two cultures),
had few shallow plasma membrane mounds where large
particles were clustered at a somewhat higher density (Fig. 4 B). The clusters in this single cell had a low density, and they contained few large particles; and it is questionable whether occasional groups of large particles are
equivalent to tetrads (Fig. 4 B, circle). Upon differentiation, numerous cells show two characteristic changes: the
presence of frequent clusters of tall particles with large diameters and openings of membrane invaginations (compare Fig. 4, A, undifferentiated, with C, differentiated). About 40% of the cells (41.3% of the 804 cells from 19 replicas of 9 cultures) have clusters of large particles in
cultures exposed to differentiation medium for 3-8 d. This
is in agreement with immunofluorescence.
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.
The distortions of tetrads and the lack of one or more particles may either arise from common freeze fracture artifacts or reflect an incomplete or imperfect molecular assembly. To interpret our data in terms of junction assembly we need to distinguish between these two possibilities. The distortion and slight misplacement of particles (Fig. 5 E, 1 and 2) are similar to artifacts often seen in freeze fracture images, and we assume that they do not reflect any structural defect of the proteins. Missing particles are often substituted by short stumps (Fig. 5, C and D), which may represent proteins broken during fracturing. In areas where the arrays appear most complete, small stumps are almost invariably present, indicating that these arrays were more complete than they may seem on a superficial examination. Some missing particles are not substituted by stumps (Fig. 5 E, 3 and 4), and thus the protein was probably absent from that position. In areas where the arrays appear most incomplete, missing particles are less frequently substituted by stumps (Fig. 5 E, 3 and 4). In these areas the tetrads may be in the process of assembling and thus may lack many components, or, less likely, be in the process of disassembling (see Discussion).
Analysis of Tetrad Arrays.Large clusters of tetrads are often made up of several subdomains with distinct orientations of their arrays. This could result from multiple and independent initiation sites for the formation of arrays within one cluster. We first analyzed the parameters of well differentiated tetrad arrays. Centers of tetrads could easily be identified and marked with a dot in areas with fairly complete clusters, even though some tetrads were partially distorted and missed some components (Fig. 6, C and E). This generated orthogonal arrays of dots with a spacing of 41.2 ± 3.1 nm (mean ± 1 SD) along the orthogonal axes (Fig. 6 E, dashed lines) and 58.4 ± 4.7 nm along the diagonals (Fig. 6 E, arrows). The latter spacing is approximately twice the spacing between the feet measured in thin sections.
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.
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.
Correspondence between Arrays of Tetrads and Feet.
Numerous similarities between arrays of feet seen in thin sections and the arrays of tetrads detected by freeze fracture
suggest that both molecular assemblies belong to a single
underlying cytoplasmic structure and that they develop
concomitantly. (a) Both arrays are rarely seen in undifferentiated cells but frequently observed in differentiated
cells. (b) Both structures occur in variable sizes. (c) Particle clusters occupy a patch of membrane that is slightly
raised into a shallow, flat mound indicative of the apposition of an SR cisterna underneath. (d) In some junctions,
feet and tetrad particles occupy only a portion of the junction (Fig. 9, A and B). (e) Multiple subdomains of feet in
individual peripheral couplings and multiple subdomains
of tetrads in the large particle clusters indicate that both
proteins become organized in the junctions starting from
multiple seed points. This is illustrated in Fig. 9, C and D,
showing a large cluster of particles composed of several minidomains with different orientations of tetrad arrays
(Fig. 9 C, arrows) and a junctional gap containing two separate groups of feet with different orientations (Fig. 9 D,
arrows). (f) The diffraction pattern from arrays of tetrads
and the spacing between tetrads are entirely compatible
with a model of tetrad disposition based on a 1:2 ratio of
tetrads to feet. The measured spacing between tetrads
along the diagonal of the orthogonal array is twice the
measured spacing between feet, as predicted by this model.
Correspondence of Clusters of Feet and Tetrads with Clusters of Immunolabeled RyRs and DHPRs
Immunoreactive clusters of RyRs and DHPRs share several characteristics with clusters of feet and tetrads as seen
in electron microscopy. First, all are absent in cells during
the proliferating phase, but they appear simultaneously in
an increasing portion of BC3H1 cells upon differentiation.
Secondly, DHPR/RyR immunoclusters are located almost
exclusively at the periphery of the cells and occur at similar densities as clusters of tetrads. For example, the cells
shown in Figs. 1 C and 2 B, which are good examples of
well differentiated cells with numerous clusters, have cluster densities of 314 and 213/1,000 µm2, respectively. These
clusters mainly represent peripheral couplings, since EM
of cells at comparable stages of differentiation shows very
few primitive T tubules forming junctions that are located immediately below the cell surface. The density of immunoclusters is in good correspondence with densities of tetrad clusters ranging between 183 and 278/1,000 µm2 measured in freeze fracture replicas of well differentiated cells from cultures of the same age. Thirdly, with both techniques the clusters in BC3H1 cells are found to be variable
in size and often considerably larger than the corresponding clusters in normal myotubes developing in vivo and in
vitro (compare with Pincon-Raymond et al., 1985; Flucher
et al., 1993b
, 1994
; Takekura et al., 1994b
). And finally,
large DHPR/RyR clusters are sometimes composed of several smaller subdomains that may correspond to tetrad
clusters with widely spaced subdomains.
Assembly of the e-c coupling apparatus is an early event
during skeletal muscle differentiation (Flucher, 1992).
Studies with BC3H1 cells have shown that transcription of
mRNAs for RyR1 and the skeletal form of DHPR is initiated briefly after withdrawal of growth factors (Rampe et
al., 1988
; Marks et al., 1989
). The present study adds triadin to the list of junctional proteins that are concomitantly
expressed in the early differentiation process. Furthermore, all three proteins (RyR, DHPR, and triadin) assemble into junctional complexes essentially as soon as they are synthesized. Parallel appearance during development of
DHPR clusters detected by immunocytochemistry and of
arrays of tetrads as seen by freeze fracture analysis, as well
as similar densities of both structures in differentiated myocytes, gives strong support to the identification of the tetrads as groups of four DHPRs.
Development of calcium release units in the nonfusing
cell line BC3H1 cells is independent of myoblast fusion,
and in this sense it resembles myofibrillogenesis, which has
also been shown to be independent of the fusion process.
However, BC3H1 lack actinin, a major component of the
Z lines, and I-Z-I brushes, that is, the association of thin
filaments with Z lines (Holtzer et al., 1997
). The observation that in BC3H1 cells some aspects of e-c coupling development and myofibrillogenesis, such as the formation
of peripheral couplings and A bands, occur normally whereas other aspects, like T tubule and Z line formation,
are deficient may be indicative of at least two parallel regulatory mechanisms during early myogenesis, one of which
is lacking in these cells. However, the deficiencies in the
development of the muscle-specific cytoskeleton and membrane system may also be causally related, since the e-c
coupling membranes become anchored at the Z lines during early sarcomere formation (Flucher et al., 1992
, 1993a).
Thus lacking Z lines could uncouple the internalization of
the e-c coupling apparatus leading to the exceptional development of peripheral couplings and paucity of dyads
and triads in BC3H1 cells.
Most of the peripheral couplings in BC3H1 cells do not have full assemblies of feet and tetrads. The number of cells with junctions, the density of junctions per cell, and the completeness of assemblies of tetrads and feet within the junctions all increase in parallel between days 3 and 6-7 of differentiation. Thus, junctions with incomplete assemblies most likely are in the process of formation. Whereas incomplete assemblies could also be indicative of degenerative processes, we would expect degradation to increase and not decrease with age. If assembly and disassembly of junctions occur simultaneously, the rate of assembly must exceed that of disassembly to yield an overall increase in junctions. Furthermore both processes have to proceed through structurally identical stages, since only one pattern of incomplete junctions has been observed. Therefore, we believe that the analysis of incomplete assemblies of tetrads and feet is relevant for understanding the process of junction formation.
The developmental analysis of the junctional complexes
between SR and surface membrane in BC3H1 cells is consistent with the identification of feet as RyRs and tetrads
as groups of four DHPRs. The parallel appearance during
development of several structural and molecular components , such as feet, tetrads, and immunoclusters, indicates
the coordinated assembly of RyRs and DHPRs in the adjacent membrane domains of the undifferentiated SR-surface membrane junctions. Arrays of feet in the junctional
SR membrane and of tetrads in the surface membrane often form around multiple, independent initiation points
within single large junctions, resulting in several subdomains with different orientations. This suggests a mechanism of assembly by gradual incorporation of the junctional proteins into a preformed narrow junctional gap
followed by their arrangement into extended arrays. The
assembly could occur simultaneously at several points of
one junction, and the separate arrays would eventually
converge into one large array containing several subdomains. Alternatively, this pattern could result from the assembly of junctions from several preassembled junctional
segments (Yuan et al., 1991). However, this mechanism
would also require the simultaneous fusion of the underlying SR compartment, which is not consistent with the observed SR-plasma membrane junctions without feet. Given the ability of RyRs to self assemble into ordered arrays
(Takekura et al., 1995
), their normal assembly into T tubule-SR junctions without DHPRs in dysgenic myotubes
(Powell et al., 1996
), and the dependence of DHPRs on
RyRs for achieving a related ordered arrangement (Protasi, F., C. Franzini-Armstrong, and P.D. Allen, unpublished observations; for review see Flucher and FranziniArmstrong, 1996), we would expect that the organization
of the feet array precedes and probably drives the formation of tetrads.
Previous analysis of the disposition of tetrads relied on
direct observation and identification of individual tetrads,
which was often hampered by missing components. Optical diffraction, on the other hand, detects the underlying
order even where individual components are missing. This
analysis shows that tetrads, even if incomplete, are spaced
at distances that correspond to those of alternate feet and
that tetrad position is skewed relative to the orthogonal
axes of the array, as expected from an exact superimposition of tetrad particles over feet subunits (Franzini-Armstrong and Kish, 1995).
The spacing between the centers of adjacent particles of
a tetrad (~18 nm) is larger than the center-to-center distance between foot subunits (~14.5 nm; Radermacher et al.,
1994), indicating that the centers of the DHPR particles
are closer to the corners of a foot than to their centers.
One consequence of this is that complete tetrads may not
be able to fit over adjacent feet (Fig. 10, A and B). However, while simple steric hindrance might contribute to the
peculiar alternate disposition of tetrads over feet in the
mature junction, it cannot by itself explain the development of this pattern. During formation of the junction,
steric hindrance would prevent neither individual DHPRs
from associating with adjacent feet (Fig. 10 C, lower rows
of feet) nor the association of tetrads with feet at intervals
>2:1 ratio (Fig. 10 D). Instead, we find that even when tetrads are incomplete, individual DHPRs occupy fixed positions in association with the subunits of alternate feet (Fig.
10 C, top row of feet). This means that even though DHPRs
appear to associate with feet subunits individually and not
in groups of four, they interact only with feet in odd positions of the array (1, 3, 5, etc.) and not with those in even
positions (2, 4, 6, etc.). This striking exclusion of DHPR
particles from half of the feet in the array suggests a molecular determination of the alternate association pattern.
Since the large majority of RyRs in mouse muscle is of a
single isoform (Takeshima et al., 1994
; Giannini et al.,
1995
), it is unlikely that this pattern arises from a direct
and preferential interaction of the DHPRs with a specific
RyR isoform in that position (Block et al., 1996
). Rather,
we must assume that DHPRs assemble into tetrads in synchrony with the formation of feet arrays. This is also supported by the following consideration. If feet first formed
extensive arrays and tetrads were acquired subsequently
as a result of the infiltration of DHPRs into the junction
from its periphery, the circular pattern shown in Fig. 10 E
would develop. But subdomains with tetrads at the periphery, as opposed to the center, are not seen. Furthermore, subdomains of tetrads would then be expected to show the
same orientation throughout a junction. The observation
of multiple subdomains of tetrad arrays and feet with different orientations is more consistent with a concomitant
radial growth of both arrays, starting from multiple seed
points (Fig. 10 F). A simultaneous assembly of the arrays
of feet and tetrads is also consistent with the joint emergence and early association of the two proteins shown by
immunolabeling of rat primary cultures (Flucher et al., 1994
)
and BC3H1 cells (present study).
DHPRs do not require the presence of RyRs for their
insertion into peripheral couplings (Protasi, F., C. Franzini-Armstrong, and P.D. Allen, unpublished observations). However, in the presence of RyRs, DHPRs associate preferentially with those parts of the junctions where
the arrays of feet are present. This was first shown in cardiac muscle (Protasi et al., 1996) and has now been confirmed for junctions assembled by skeletal muscle proteins. At present, a direct link between DHPRs and RyRs
is debated; therefore this preferred association of tetrads
and feet with each other may depend on additional junctional proteins (Caswell et al., 1991
; Marty et al., 1994
;
Guo and Campbell, 1995
). Immunolabeling of triadin
shows that this protein, which has been implicated in the
RyR-DHPR interaction, is expressed and inserted into
the junction during the critical period.
Our findings emphasize the close spatial, and probably also temporal, relationship in the gradual assembly of the corresponding arrays of feet and tetrads within preformed SR-plasma membrane junctions. The strict adherence of DHPR particles to positions of alternate feet even during early stages of junction formation suggests a framework of conditions for the molecular organization of the e-c coupling apparatus in skeletal muscle that goes beyond simple steric hindrance dictated by the position of DHPRs relative to RyR subunits.
.
1. Abbreviations used in this paper: DHPR, dihydropyridine receptor; e-c, excitation contraction; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; T, transverse.We thank Drs. A.H. Caswell, S. Fleischer, and S. Froehner for their generous gift of antibodies. This work was supported in part by National Institutes of Health grant 15835 to C. Franzini-Armstrong and the Pennsylvania Muscle Institute (CFA) and by grants from the Austrian National Bank (5353) and from the Fonds zur Förderung der wissenschaflichen Forschung (S06612-MED) to B.E. Flucher. B.E. Flucher is an Austrian Programme for Advanced Research and Technology fellow of the Austrian Academy of Sciences.