A M . ZOOLOCIST, 7:465-481 (1967).
Gross-Bridges and Periods in Insect Flight Muscle
MICHAEL K. REEDY
Department of Physiology, School of Medicine, University of
California, Los Angeles 90024
SYNOPSIS. The periodic structure of the cross-bridge lattice of glycerinated
Lethocerus Might muscle has been studied in sections by electron microscopy, assisted
by optical diffraction, and in unfixed fiber bundles by X-ray diffraction. Diffraction
patterns exhibit first through ninth orders of 1166 A, virtually all of which were
found to arise from the lattice of cross-bridges. Diffraction and inspection show
that "horizontal" cross-bridges of relaxation become slanted in rigor, and may push
acting toward the M line in producing the increase in tension seen with the induction of rigor.
Myosin filaments contain unexpected structural features. Cross-bridge origins
form opposed pairs repeating every 146 A and rotating 67.5 degrees with each
repeat, thus defining twin, left-handed, helical tracks which require l]/2 turns (or
8 x 146 A) to establish a meridional repeat of 1166 A. Each origin is dual and
gives rise to two bridges; thus, the unit grouping of paired origins involves four
bridges. One half-turn of the myosin helix requires 388 A, matching the actin
helix exactly in pitch. (Actin is, however, right-handed.) The resulting match
seems awkward azimuthally (sixteenfold myosin distributes bridges to a sixfold
envelope of actin filaments), but minimizes axial mismatching between subunits
of the myosin and actin and lends credence to the theory that all bridges may
swing synchronously during typical, low-amplitude, oscillatory contractions.
Dr. Riiegg (1967) has just described the
relationships between the splitting of ATP
and the production of work during oscillatory contraction of insect flight muscle.
The glycerol-extracted flight muscle fibers
of the giant waterbug, Lethocerus, up to 1.5
cm long, which lend themselves so well to
chemical and mechanical studies, have
proven no less suitable for structural work.
I will describe electron microscopic and
X-ray diffraction studies carried out during
my period of postdoctoral research in Dr.
H. E. Huxley's laboratory in Cambridge,
using samples of the muscle preparation
provided by Dr. J. W. S. Pringle. The first
stage of this work has been reported earlier
(Reedy, Holmes, and Tregear, 1965), and
the remainder is in preparation as a series
of papers to be submitted elsewhere
(Reedy, 1967, and Huxley, Reedy, Holmes,
and Tregear, 1967). Since these papers
will be lavishly illustrated (as my 60 slides
today might lead you to suspect), I am
providing relatively scanty illustration for
the published version of my talk. In particular, no X-ray pictures will be found
here, partly because the two optical dif-
fraction pictures convey the same arguments. Nevertheless, I hope to make this
text a fair summary of the major findings,
conclusions, and implications of this whole
investigation.
Muscle fibers, either relaxed or in rigor
(see footnote, page 467), were serially fixed
in aqueous solutions containing glutaraldehyde, osmium tetroxide, and (usually)
uranyl acetate, then dehydrated through
acetone, and embedded in Epon or
Araldite. I should mention four departures
from currently conventional electron
microscope methods. Using X-ray diffraction, we compared unfixed muscle with
fixed and embedded muscle, which enabled
us to identify the magnitude and character
of preparative artifacts, as described later.
Using optical diffraction (Klug and Berger,
1964), we could carry such analysis of
periodicity to electron micrographs of small
regions of one sarcomere in one section.
Measurement of section wrinkles enables
me to support a claim to thinner sections
(down to 170 A) than it has recently been
credible or fashionable to claim; these were
cut with a Dupont diamond knife and a
(465)
466
MICHAEL REEDY
Actlh Uyer
FIGS. 1 and 2. Unbranched cylindrical fibrils of
glycerinated Lelhocerus flight muscle are 3 /t in
diameter, with 2.6 p sarcomeres.
FIG. 3. Periodicity (380 A) and cross bands are
seen in thick section (1000-1500 A) of rigor sped-
men. 36,000 X.
FIG. 4. Thick cross section shows gray bridge material connecting dense filaments in regular array.
Derivation of the most useful thin longitudinal
sections is indicated (see Fig. 6).
CROSS-BRIDGES AND PERIODS IN MUSCLE
Porter-Blum MT-1 ultramicrotome. The
infrequently-used section-staining sequence
of potassium permanganate-lead citrate
provided better contrast than I have ever
achieved otherwise.
In brief, we have discovered something
about how cross-bridges move, and a good
deal about the way in which they are disposed in a lattice. This lattice-model promises to help us to understand how actinmyosin interaction may be maximized and
even synchronized throughout active fibrillar insect muscle during the contractile
phase of each oscillatory cycle. (See Pringle,
1967)
Previous ultrastructural work on asynchronous flight muscle had characterized
the regular hexagonal array of myosin and
actin filaments (Fig. 4) and the presence
of periodically distributed interfilament
cross-bridges (Hodge, 1955; Hanson and
Huxley, 1957; Worthington, 1962). A
straightforward application of the sliding
filament model of contractility (Huxley
and Hanson, 1960) to this type of muscle
is complicated by the fact that the myosin
filaments appear to be connected directly
to the Z bands (Aubert and Couteaux,
1963). No more will be said of this complication; the results I wish to speak of
seem very congenial to a sliding filament
interpretation of such muscle, whatever the
role of such series-connections.
Three bits of information came out of
the first stage of ultrastructural study of
glycerinated Lethocerus muscle. The first
bit concerned the helically structured actin
filaments (Hanson and Lowy, 1963), whose
exact pitch in Lethocerus was found to be
2 X 388 A, or such that the two-stranded
helix completes a half turn in 388 A.
(This unpublished result of Brown et al.
(1965), was based on X-ray diffraction pictures in which exact measurements of the
very strong actin layer lines at 51 A and
59 A were possible.) Second, X-ray diffraction indicated a structural difference between the two mechanical states of rigor
and relaxation1, in that rigor specimens
produced a powerful off-meridional 388 A
layer line, while relaxed specimens pro-
467
duced instead an intense meridional 146 A
spot. Third, we used electron microscopy
and X-ray diffraction in a complementary
fashion that produced a satisfactory and
convincing account of the chief rigorrelaxation difference (Reedy, Holmes, and
Tregear, 1965). Shrinkage artifacts due to
processing reduced myosin-to-myosin filament side-spacing by about 10% to 480 A,
and reduced all axial periods by 2%.
Nevertheless, X-ray diffraction monitoring
proved that the major periodic characteristic of each state was preserved in
muscle specimens which had been fixed
and embedded for electron microscopic
study. Next, electron micrographs demonstrated that the diagnostically dominant
period of each state was associated with
a particular appearance of the cross-bridge
lattice.
In both states, the bridges were found to
be grouped in symmetrical pairs along
myosin filament profiles, and in lateral
register across the fibril. In relaxed muscle
they were predominantly at right angles
to the filaments and were axially spaced
so as to express a period of 143 A (note
2% reduction from the prefixation value
of 146 A). In rigor, the bridges were angled
like barbs on a harpoon, about 45° to
the filament-axis, so that the actin end of
each bridge was about 150 A closer to the
center of the sarcomere than the myosin
end, and they clearly expressed an axial
period of 380 A (Fig. 5). In very thin
sections which included single layers of
actin filaments only (Figs. 6-8) the included
cross-bridge ends appeared clearly and exl Relaxation (muscle extensible) requires ATP
with Mg** (preventing firm or lasting cross-bridge
linkages to actin). Rigor (muscle tears when
stretched) exists when ATP is absent (cross-bridges
attached, cross-link lattice o£ actin and myosin
filaments, prevent sliding o£ filaments). One synopsis o£ this work confusingly referred to rigor as
"contraction". Huxley and Hanson (1960) have
stressed that the mechanical and elastic features
which identify rigor, relaxed, or active states of
muscle do not depend on length of sarcomere,
and can be explained in terms of cross-bridge behavior. This behavior is controlled by ATP and
divalent cations, Mg*+ and Ca" (Hasselbach, 1964).
This is the model I have assumed in discussing our
work on rigor and relaxed fibrillar muscle.
468
MICHAEL REEDY
clusively expressive of this period, which we
knew reflected the half-pitch of the actin
helix. We reasoned, therefore, that the
binding of cross-bridges to actin somehow labeled the actin period so that it
came to dominate the rigor cross-bridge
lattice. The major diffraction findings
could be explained. The angling of bridges,
so that the actin end of one bridge very
often overlapped the myosin end of the
next, tended to weaken severely the transverse lattice planes repeating at 146 A. At
the same time, the angulation meant that
the bridges were aligned along diagonal
lattice planes coordinated to the 388 A
repeat, hence intensifying the off-meridional scattering on this layer line. Putting
tension on these muscle bundles, actually
stretching them by up to 5%, failed to
alter the dimensions or the intensity of
the X-ray diffraction patterns from either
relaxed or rigor specimens. Since, as this
indicates, the angled rigor cross-bridges
seem able to bear tension without "unbending" (a supposition also supported by
electron microscopy), we may begin to
consider a model in which the micro-event
producing contractile tension involves a
pushing movement by a cross-bridge which
attaches to actin at some non-rigor angle
and then swings to approximate the rigor
angle. Indeed, David White's finding
(1967, Ph. D. thesis) that Lethocerus fibers
develop a marked rise in tension during
isometric rigor induction, (quickly produced by washing ATP out of a relaxed
fiber bundle), and our finding concerning
the change in cross-bridge angle during
rigor induction, are experimental facts
which converge rather insistently towards
such a pushing model of cross-bridge activity.
A more detailed model of the crossbridge lattice required more information
about the distribution of cross-bridges along
the myosin filament. Figure 6 shows a thin
section of rigor muscle, in which an actin
layer, a "myac" layer (alternating myosin
and actin filaments), and the transitional
"split myosin" region between the two are
all visible. My first preconception of my-
osin filament structure was difficult to reconcile with the apparent spacing of
bridges and with the apparent numbers of
bridges recognized in rigor. According to
the preconceived model, successive crossbridge pairs repeated at axial intervals of
146 A (143 A) and at azimuthal intervals
of 60°. This should have produced an obvious longer period corresponding to 3 X
146 A which would develop an appreciable
vernier mismatch with the 388 A (380 A)
actin period after just a few repeats. However, the cross-bridges mark 27 to 30 repeats of the 380 A period in each half-sarcomere with no sign of such a mismatch;
and even in relaxed muscle no 430 A
period is found. Then there was the discrepancy in apparent numbers of bridges.
The bridge groupings which formed the
chevrons in rigor muscle and marked the
380 A period apparently involved twice as
many bridges as expected, for the typical
repeating configuration was a double
chevron rather than a single chevron. I
had to abandon my preconceptions and let
the evidence suggest a different model.
Three different trails finally led to the
model of the surface lattice of a myosin
filament portrayed in Figures 11 and 12.
One trail involved extracting a maximum
of objective periodic information (by
optical diffraction) from electron micrographs. Another involved analyzing the
screw sense or absolute hand of the helical
arrangement of cross-bridges along the myosin filaments. A third involved validating
the doublet structure that produced the
double chevrons by coming to recognize its
manifestations in different views of muscle.
Most of the information came from my
electron micrographs of rigor muscle, because these showed much more regularity
of detail than I ever found in relaxed muscle. However, X-ray diffraction from relaxed muscle (Huxley, et al., 1967) uniquely supports this model, so it should be
understood that I intend it for both rigor
and relaxation.
Returning to Figure 6, one notes prominent oblique striping in the split myosin
region. Here, a grazing section of the myac
CROSS-BRIDGES AND PERIODS IN MUSCLE
469
— Z —
EM-36OA
Xray388A
FIG. 5. Characteristic cross-bridge positions and
dominant periods of rigor and relaxed specimens
are seen here in single layers of filaments (myac
layers) at various magnifications.
layer samples a one-sided aspect of the
helical system of cross-bridges. These diagonal stripes are coordinated perfectly with
the 380 A period. By considering these
stripes together with the symmetry of the
chevrons in a myac layer, I began to think
of a myosin-filament model in which
bridges were disposed along twin helical
tracks which completed one half-turn every
380 A (388 A). However, one would have
470
MICHAEL REEDV
S
CROSS-BRIDGES AND PERIODS IN MUSCLE
471
FIG. 6. Thin section (about 200 A) oE rigor muscle
passes from actin layer on left through obliquelystriped, split-myosin region to myac layer to second
split-myosin region at upper right corner.
FIG. 7. Actin layer, in slightly thicker section than
Figure 6 (here about 300 A), shows staggered bead-
ing in addition to transverse striping.
FIG. 8. 380 A cross-bridge period of rigor is marked
in thin sections by lateral projections and beads.
The beads represent end-on views of cross-bridge
segments, and are not prominent in thinnest sections (Fig. 6).
to sacrifice either the 146 A feature or the
60° feature of the cross-bridge repeat on
the preconceived model.
Since relaxed muscle showed a clear
143 A repeat but no sign of the 3 X 143 A
period which should arise from a 60°
repeat, the latter feature had to go, to be
replaced by a 67.5° azimuthal repeat. The
"Relaxed" diagram in Figure 5 is based
on an incompletely worked out version of
this model which I had in mind in the summer of 1965. At about that time, optical
diffraction came to my aid and helped
me toward a much more rapid and confident solution than would otherwise have
been possible. This technique allows analysis of periodic patterns in electron micrographs by using a transparency of the micrograph just as if it were a diffraction
grating, through which monochromatic
coherent light can be passed so as to produce a diffraction pattern, which we call
the optical transform of the micrograph.
The first few flight muscle transforms
were made for me by Berger and Klug
(who introduced this application of
optical diffraction, 1964) and clearly demonstrated two periods in rigor (143 A and
570 A) which had escaped the eye in these
micrographs, and had yet to be detected
by X-ray diffraction.
Figures 13 and 14 show the appearance
of thick sections of rigor and relaxed muscle, and are accompanied by optical transforms which reflect the periodic structure
of each image. Various layer lines are
labeled with the value of the corresponding
axial periods. The thick section of rigor
shows the strong 380 A period typical of
that state, most obviously as beading along
thin filament profiles, beading which is
lined up in very straight rows transversely.
Every third row shows increased intensity,
indicating a superperiod of 3 X 380 A or
1140 A. Note that the rigor transform ex-
hibits layer lines corresponding to first,
second, third, fifth, sixth, eighth, and ninth
orders of 1140 A. The eighth order is, in
fact, 143 A.
The surface lattice of the myosin filament diagrammed in Figure 9 or 11 was
originally generated from the two parameters assumed earlier, namely, that crossbridge pairs repeat every 146 A, and that
they are rotated by enough (67.5°) to develop helical tracks that twist through 180°
in 388 A. Note how this develops a "beat"
period of 1166 A (1140 A) corresponding
to 3 X 388 A and 8 X 14 6 A, and note
also how helical tracks are set up which
slant or twist in the opposite direction to
complete a half-turn every 233 A (fifth
order of 1166 A). The fact that 1140 A and
228 A periods can be detected experimentally in micrographs argues very strongly
for this model, which appears uniquely
capable of developing all four periods. The
228 A (233 A) layer line, though not
usually clear on transforms of relaxed
muscle, is very satisfyingly indicated (accompanied by the strong 146 A period and
a weak 388 A layer line) in X-ray diffraction pictures of relaxed preparations,
including some non-glycerinated material
examined immediately post-mortem (Huxley, et al, 1967).
Some features of the rigor diffraction pattern have not yet been mentioned. The 570
A layer line is probably wholly derived as
second-order diffraction from the lattice of
points marking the 1140 A period, and the
190 A layer line is at least partly derived
in this way from the 380 A lattice. However, part of the 380 A layer line, part of
the 190 A layer line, and probably all of
the 127 A layer line can be attributed
to the lattice set up by the actin ends of
the cross-bridges. Figure 15 diagrams the
way in which cross-bridge attachments must
be distributed along the sixfold actin en-
472
MICHAEL REEDY
velope which surrounds each myosin, in
order to satisfy all the evidence collected on
this point, and the derivation of the three
r
388 A
periods just mentioned can be appreciated
from this diagram. An interesting result
from more recent X-ray studies (Huxley,
I46A
o o
oo
G .O
gb
O O
O O
O.
o Q o oXX° ° fj ° ° t
"66 A
5 oo ' O
5
O
O
7
o
o
8 O
o o
\ r
O°
6
36O°
9o o
10
»
\
\
«•
<
\
\
\
388
\
%
\
A_
\
\
*
-
146
"A
\
1 • •
\
2 /%
3• *
Ai •
6 • i
7 lm
8»
\
\
\
" \
" \
\
9
\
\
%
«»
\
i
\
233A J_/
\
•
\
«•
\
m
••
m
9 • •
/
p
m
t
36O°
11
11
FIGS. 9-10 and 11-12. These represent two stages in
the development of a model of the myosin filament.
(Fig. 9, left, is a partial radial projection of actin
filament structure.) Cross-bridge origins on the
surface of the myosin shaft are displayed in radial
projection in Fig. 9, center, and Fig. 11. Fig. 9,
right, and 10 indicate the effects of azimuthal re-
peats of 67.5° on the profile of the filament and
on its relation with the sixfold actin envelope.
Doubling of bridge numbers without altering the
surface lattice leads to configurations of crossbridges as in Fig. 12, including many "flared X"
conformations (see Fig. 21).
473
CROSS-BRIDGES AND PERIODS IN MUSCLE
t) ft *
»
..'.; 1 ':
• *' t
'.',
• * *fi •
• < f|'»
V
• Mr »
•i
' • ' • * • '
.: •; \; ]; .: : ,
T
•
'
•
(1 i' :; 1
.
*
*
*
,
\
*
*
i
•:
•
t
<
<* •
;
'
•
«
•
•
^
•
•
»
.
-ii
tj-,
i ; ; s : ! ;:
•LL
•' i ? i i r
/
nt* * 'V
HI! 1 [1
1
i
i
4-
•
•
•• 38OA
•
•
• I/YOA
13
FIG. 13. Rigor, thick section (as Fig. 3), with optical transform oC same. Layer line positions from
0 to 9th orders of 1140 A are indicated; transform
shows all but 4th and 7th orders. Inspection of
micrograph fails to detect or discriminate the
weaker periods shown by diffraction. Periods labelled are those associated with the surface lattice
of the myosin (Fig. 11).
474
MICHAEL REEDY
FIG. 14. Relaxed thick section with optical transform. Xote more intense period at 143 A and
weaker other periods, especially 380 A and 190 A,
as compared with rigor in Figure 13. (Horizontal
and sloping off-vertical spikes are transforms ot
edges of aperture used to mask micrograph for
optical diffraction.)
CROSS-BRIDGES AND PERIODS IN MUSCLE
1 2 3 4 5 6 1
360°
FIG. 15. Distribution of actin ends of cross-bridges
to six actins around each myosin is seen here in a
radial projection of the actin envelope. Circles
represent optimum sites for bridge attachment,
repeating with each half-twist of the actin helix.
This distribution can be inferred from patterns of
bridge periods in Figures 6 and 7. [Each circle
indicates the target segment straddled or approximated by two successive pairs of cross-bridges
(double chevrons, etc.)] It is interesting to compare this with the surface lattice of the myosin.
et al.j 1967) concerns the behavior of these
three layer lines during glutaraldehyde
fixation of relaxed muscle. Each gains in
intensity during fixation. The most interesting interpretation of this holds that
the influence of the positions of actin
filaments on the ordering of the relaxed
cross-bridge lattice is increased by fixation;
in other words, relaxed cross-bridges are
largely unattached to actin before fixation,
but become so attached by the action of the
fixative. Prompting this interpretation is
the fact that all of the cross-bridges actually
seen in sections of relaxed muscle do appear
to be joined to actin filaments (e.g., Fig.
4 is from relaxed muscle).
Thus diffraction studies can be largely
rationalized by a model of the insect crossbridge lattice in which the arrangement of
scattering centers at the myosin surface
contributes to layer lines corresponding to
first, second, third, fifth, and eighth orders
of 1166 A, while the arrangement, at a
greater radius, of bridge-related scattering
centers along the sixfold actin envelope
contributes to third, sixth, and ninth order
layer lines of 1166 A. I also did experiments in which India ink was used to paint
out selected features (myosin shafts, or
cross-bridges) in micrographs, and the
475
optical transforms obtained from such selective ablation experiments have supported this idea of slightly different lattices
from "myosin ends" and "actin ends" of the
same set of cross-bridges.
Some problems of specific detail within
the bridge lattice remain. Regarding the
screw sense of the cross-bridge helix along
the myosin filaments, three different strategies were devised for diagnosing this. The
first method involved obtaining serial, thin,
longitudinal sections (like Fig. 16) and following a given myac layer through two successive sections, with particular attention to
the split myosin region bordering this filament layer. It was found that the oblique
striping which runs 10 o'clock-4 o'clock
(filament axis lying 12 o'clock-6 o'clock)
was produced in grazing the near side of
the myac layer, while the striping that runs
2 o'clock-7 o'clock was produced where the
section grazed tangentially along the far
side of the myac layer.
A second method of screw diagnosis is
based on transverse sections thin enough
(150 A-200 A range) to show a well developed herringbone mosaic type of pattern
(Fig. 17). These sections are extremely interesting and important. For one thing,
they confirm an inference which may be
drawn from longitudinal sections, to the
effect that all myosin filaments in a sarcomere are in helical register, so that all
cross-bridges at the level of any given transfibrillar plane extend along the same
azimuthal direction. They do not include
all bridges which are distributed to actin
filaments along one half-turn of the myosin cross-bridge helix, because the myosin
segment sampled in such sections is only
about one quarter-turn in length. The
plane of section is not precisely parallel to
the transfibrillar planes of helical register.
Rather, it transects the lattice with a slight
obliquity, sampling successive levels and
orientations of cross-bridges as it traverses
the fibril. Thus the herringbone effect is
produced, as the direction of included
cross-bridges changes in 60° steps and lends
prominence to different planes of the array
of hexagonal myofilaments.
MICHAEL REEDY
FIG. 16. This is one of an actual pair of thin
serial sections used to diagnose the left-handed
screw sense of myosin cross-bridge twin helix, by
relating each direction of oblique striping to grazing section o£ near or far sides of myac layers.
These sections are about 250 A thick.
FIG. 17. Herringbone mosaic effect was well develo|>ed only when sections were thinner than 200
A. Myosin filaments require 380 A for a half
twist of screw; sections this thin sample only 1/
turn of screw, and seem to sample cross-bridges
lying along only one of three bridging directions
(actually, two of three; see Fig. 21). Slight obliquity
causes the section to sample successive levels and
directions of bridging as the fiber is transected.
Text explains how serial sections enable the diagnosis of myosin screw sense.
CROSS-BRIDGES AND PERIODS IN MUSCLE
In order to use such sections for screw
diagnosis, I employed serial sections which
partly intercept a landmark such as the Z
band or M band. Serial sections enable
us to learn whether the slightly oblique
plane of section is sloping into the landmark from the nearside or the farside. This
in turn enables the observer to determine
whether traversing the fibrillar transection
from A band to (e.g.) Z band constitutes a
withdrawal or an advance along the myosin
helices. In the example shown in Figure
17, this method has been employed to
learn that progress from left to right
represents an "advance" (i.e., away from
the observer) along the myosins. As can be
seen, this corresponds to a sequence of
anticlockwise 60° steps in cross-bridge prominence and direction.
A third method of screw diagnosis required neither serial sections nor especially
thin sections. It is enough to have one section about 1000 A thick. Here I obtained
oblique sections, sliced at an angle of 45°
(or better, 38°) from the fiber axis. Figure
20 displays part of one fibril encountered
in such a section. Useful in this instance is
the finding that the cross-bridges appear
more prominent, more dense, and more
regularly spaced on the right side of the
thick filament profiles than they do on the
left side. The photograph of a model
(Fig. 18) illustrates why this indicates a
left-handed screw when the doubly helical
filaments are tilted "top end toward the
viewer", as they are known to be in the
micrograph shown. A moment's review will
show that the results I quoted from the
other two methods also indicate a lefthanded screw for the twin helical arrangement of cross-bridge groupings along the
myosin filaments.
The last property of the model (Fig. 11)
which remains to be accounted for is the
assignment of two cross-bridge origins to
each scattering center in the myosin surface
lattice. This solution to the doublet problem (double chevrons, etc.) emerged from
ultrathin transverse sections of rigor muscle. If cross-bridge groupings are spaced at
axial intervals of 143 A, then sections just
477
thinner than 2 X 143 A should sample
sometimes one, and sometimes two, levels
of bridge origins. Sections thinner than 1.5
X 143 A should sample one level at a time
more often than two levels at once. Such
sections were obtained, and their thickness
was verified to be 170 A-200 A by measuring the thickness of wrinkles in the sections. (We shall soon see why this was the
thickness required in order to get the herringbone mosaic effect discussed earlier in
connection with screw diagnosis.) In these
sections, the pattern of cross-bridges associated with the majority of profiles of
myosin filaments turned out to be the
"flared X" configuration of four bridges illustrated in Figures 21 and 12. Once this
was accepted as the true unit grouping
which repeated along myosin filaments, it
provided satisfactory interpretation for
various regular but mystifying features of
longitudinal sections, including the double
chevrons in myac layers and the mixture of
beading and lateral projections found in
layers of actin filaments.
It came as a surprise to recognize that
there are twice as many cross-bridges as
there are scattering centers on the surface
lattice of the insect myosin filament. It is
important to realize that if the number of
scattering centers were somehow doubled,
so as to provide one for each cross-bridge,
then the diffraction patterns would be different, for the lattice would be different.
Again, this is important in comparing the
rigor and relaxed states of the cross-bridge
lattice which look so different in micrographs; for the diffraction evidence does not
point to any fundamental re-ordering of
the surface lattice of the myosin when relaxation replaces rigor. It is therefore still
a bit puzzling that visible manifestations of
the flared X grouping cannot be demonstrated with any confidence in sections of
relaxed muscle.
The cross-bridge arrangments already
described in rigor enable us to diagnose
the screw sense of actin's double helix.
Thin sections showing the flared X forms
are typically slightly oblique, producing
patterns related to the herringbone mosaic
478
MICHAEL REEDY
CROSS-BRIDGES AXD PERIODS IN MUSCLE
479
FICS. 18-20. Oblique thick sections of cylindrical
fibrils produce an elliptical fibrillar profile (Fig.
19), where seemingly continuous profiles of filaments are actually slanted segments which overlap,
as seen in side view to the left of Figure 20. The
diagnosis of myosin screw sense uses the principle
shown by the model (Fig. 18). In Figure 20, crossbridges are more prominent and periodic along
the right sides of profiles of thick filaments. This
is best seen by sighting along the filaments in
Figure 20 at a glancing angle to the page. The
obliquity of the filaments is known here to correspond to that of the model, which thus shows
why right-sided bridge prominence indicates lefthanded screw sense.
effect mentioned earlier. As the flared X
forms rotate in traversing the fibril, changes
in form can also be seen with respect to
the actin-centered, bridge configurations.
The latter are crudely sigmoidal. Two incongruent sigmoids occur. As the flared X
forms rotate by one 60° step, the sigmoids
change from one enantiomorph to the
other (Fig. 21). (This requires an advance
of about 130-200 A along the axis.) Figure
22 shows how the azimuth of cross-bridge
attachment to actin rotates in the opposite
direction from that observed for myosin.
Now, since we have already shown myosinchanges to follow a left-handed double
helix, this means that actin-changes follow
a right-handed double helix. Depue and
Rice (1964) first certified this right-handed
screw diagnosis for actin filaments prepared
by shadowcasting with heavy metals. Since
cross-bridges probably correspond to HMM
(the heavier of two "meromyosin" fragments derivable from myosin by gentle
tryptic digestion), let us not forget Huxley's
pleasing observation (1963) that HMM
particles "decorate" actin filaments so as
to express the pitch and polarity of actin
forming a serial arrowhead structure. Such
findings are now satisfyingly complemented
by the in situ relations between structure
of actin and attachment of cross-bridges
observed in insect fibrillar muscle.
I would like to conclude with a resume
of the chief ideas which emerge from this
work on the cross-bridge lattice of fibrillar
insect flight muscle.
1. X-ray diffraction and optical diffraction studies indicate no artifact other than
shrinkage in the rigor cross-bridge lattice.
In relaxed muscle, however, it seems likely
that the attachment of cross-bridges to
actin, as observed microscopically, is a
fixation artifact, of which the diffraction
studies do indeed give evidence.
2. We have shown that the transition
from relaxation to rigor is marked structurally by a change in cross-bridge angle, appropriate in direction and ..magnitude to
account for the increase in tension which
has been observed to accompany isometric
rigor induction.
3. There are four cross-bridges (the
flared X grouping) every 146 A along the
myosin filaments of insect fibrillar muscle.
4. Both actin and myosin filaments follow double helical patterns, of equal pitch,
such that one half-turn is completed every
388 A. The helix is left-handed for myosin,
right-handed for actin. Both the pitch and
screw senses permit a system where the subunits of myosin and actin filaments can remain in phase through the whole 1.2 ^
length of the half A band.
5. This structural match between filaments of myosin and actin offers strong
morphological encouragement for the
often-considered idea (in Pringle's group at
least) that during each cycle of oscillation
the cross-bridges might all fire synchronously, or nearly so. This requires 150300 A of filament travel for one crossbridge cycle, which may not be unreasonable to expect, as discussed elsewhere
(Reedy, 1967, and see Pringle, 1967).
6. We are now in a position to relate the
X-ray diffraction pattern to the detailed
structure of the cross-bridge lattice, including angular positions of the crossbridges. Diffraction studies of working
oscillating muscle will soon be attempted
to learn how cross-bridges behave in the
active state. An active muscle system in
which cross-bridge movements were synchronized should yield this information to
stroboscopic X-ray studies, where such information may not be accessible in a system
of asynchronously moving cross-bridges
(e.g., vertebrate striated muscle?).
480
MICHAEL REEDY
Acth
FIG. 21. In this transverse section of muscle, 180
A thick, unhurried inspection reveals that the
predominant cross-bridge configuration is the flared
X grouping (see Fig. 12) of four bridges. Note
how flared X's rotate counterclockwise as you
traverse from right to left, from' bottom to top,
due to a slight obliquity like that of Figure 17.
•V
Example: In the sixth myac row from the bottom,
X's are upright until they reach the left-most
myosin, where this rotation is observed. Myosinmyosin spacing varies here from less than 400 A
to nearly 500 A, depending on effects of compression on the section. X 204,000.
FIG. 22. Screw sense of actin is indicated as oppo-
CROSS-BRIDGES AND PERIODS IN MUSCLE
481
site that of myosin, because sigmoids involve
rotation opposite to that shown by flared X's.
Example: Second myac row from bottom shows
same changes as diagram, occurring within the
span of the ten right-most myosin profiles.
7. The role of the "C filaments" which
connect the ends of myosin filaments to
the Z bands is far from clear. They seem to
offer an explanation for the steep lengthtension curve of relaxed fibrillar muscle.
Again, they seem to bring us part way
toward an explanation of the role of stretch
in supporting the mechanism of active
oscillation. However, there is no current
evidence to show how or why a strain on
the shafts of myosin filaments might trigger
the enzymatic and mechanical activity of
cross-bridges.
Other work on Lethocerus flight muscle
seems to be approaching agreement with
the quantitative ideas above. The data of
Riiegg and Tregear (1966) on the utilization of ATP allow for an interpretation
whereby each cross-bridge may split only
one ATP molecule per cycle of working
oscillation. The first estimates of myosin
content in relation to fibrillar structures
suggest that there are six myosin molecules
per four cross-bridges (Chaplain and
Tregear, 1966; their ratio of three myosins
per cross-bridge is based on the obsolete
filament model with two bridges per 146
A). At present, I believe that the evidence
which allows us to enumerate bridge population is more direct and free of assumptions than our first evidence on myosin
content. The idea that each cross-bridge
contains a variable or non-integral number
of myosin molecules does not yet seem the
likeliest solution.
mass of myosin per cross-bridge in insect fibrillar
flight muscle. J. Mol. Biol. 21:275-280.
Depue, R. H., and R. V. Rice. 1965. F-actin is a
right-handed helix. J. Mol. Biol. 12302-303.
Hanson, J., and J. Lowy. 1963. The structure of
F-actin filaments isolated from muscle. J. Mol.
Biol. 6:46-60.
Hasselbach, W. 1964. Relaxing factor and the relaxation of muscle. Prog. Biophysics Mol. Biol.
14:167.
Hodge, A. J. 1955. Studies on the structure of
muscle. III. Phase contrast and electronmicroscopy of Dipteran flight muscle. J. Biophys.
Biochem. Cytol. 1:361-384.
Huxley, H. E. 1963. Electron microscope studies on
the structure of natural and synthetic protein
filaments from striated muscle. J. Mol. Biol. 7:
281-308.
Huxley, H. E., and J. Hanson. 1957. Preliminary
observations on the structure of insect flight
muscle. Proc. Stockholm Conf. on Electron Microscopy, 1956 (Uppsala). Academic Press, New York.
202-204.
Huxley, H. E., and J. Hanson. 1960. The molecular
basis of contraction in cross-striated muscles, p.
183-227. In G. H. Bourne, (ed.), The structure
and function of muscle, I. Academic Press, New
York.
Huxley, H. E., M. K. Reedy, K. C. Holmes, and
R. T. Tregear. 1967. A study of muscle fixation
by X-ray diffraction. (In preparation).
Klug, A., and J. E. Berger. 1964. An optical method
for the analysis of periodicities in electron micrographs and some observations on the mechanism
of negative staining. J. Mol. Biol. 10:565-569.
Pringle, J. W. S. 1967. The contractile mechanism
of insect fibrillar muscle. Progr. in Biophys. and
Mol. Biol. 17:1.
Reedy, M. K. 1967. Ultrastructure of insect flight
muscle. I. Screw sense and structural grouping
in the rigor cross bridge lattice. J. Mol. Biol. 28:
Reedy, M. K. 1967. Ultrastructure of insect flight
muscle. II. and III. (In preparation).
Reedy, M. K., K. C. Holmes, and R. T. Tregear.
1965. Induced changes in orientation of the
cross bridges of glycerinated insect flight muscle.
Nature 207:1276-1280.
Riiegg, J. C. 1967. ATP-driven oscillation of glycerol-extracted insect fibrillar muscle: mechanochemical coupling. Am. Zoologist 7:457-464.
Riiegg, J. C, and R. T. Tregear. 1966. Mechanical
factors affecting the ATPase activity of glycerolextracted insect fibrillar flight muscle. Proc. Roy.
Soc. (London), B. 165:497-512.
White, D. C. S. 1967. D. Phil, thesis, Zoology, Oxford
University.
Worthington, C. R. 1961. X-ray diffraction studies
on the large-scale molecular structure of insect
muscle. J. Mol. Biol. 3:618-633.
I am particularly grateful to Drs. H. E. Huxley,
A. Klug, and K. C. Holmes for many stimulating
discussions and continued encouragement. For
the gifts of living and glycerinated Lethocerus flight
muscle, I thank Prof. J. W. S. Pringle. For a sizable
gift of time and patience, I thank Deirdre, my wife.
I was supported during this work by USPHS Fellowship 2-F2-NB-21075-02 and 03.
REFERENCES
Auber, J., and R. Couteaux. 1963. Ultrastructure de
la strie Z dans des muscles de Dipteres. J. Microscopie 2:309-324.
Brown, W., K. C. Holmes, H. E. Huxley, and A.
Klug. 1965. Unpublished observations.
Chaplain, R. A., and C. T. Tregear. 1966. The