A M . ZOOLOCIST, 7:483-498 (1967).
Evidence for Superthin Filaments
PATRICIA A. MCXEILL AND GRAHAM HOYLE
Department of Biology, University of Oregon, Eugene 97403
SYNOPSIS. We have examined a variety of invertebrate and vertebrate muscles in
the electron microscope and have seen evidence o£ a third, very thin (25 A) filament in all of them. This filament has been seen in H zones, A bands, and I
bands, as well as bridging the gap between actin and myosin filaments in greatly
stretched muscle. It, thus, seems likely that, if a third filament exists, it is elastic
and extends throughout the sarcomere from Z disc to Z disc, unlike the hypothetical actin-actin (s) filament of Hanson and Huxley (1955), or the actinmyosin gap filaments of Sjostrand (1962) and Carlsen, et al. (1965).
These filaments therefore provide an answer to some of the present paradoxes of
muscle ultrastructure.
In the introduction to this symposium,
several pieces of evidence were mentioned
which suggest that the simple version of
Huxley's sliding-filament model of muscle
does not fully explain certain physical and
physiological phenomena, such as: passive
elasticity; the long-series elasticity of certain
muscle fibers; how muscles, which are
stretched to a point where the filaments of
actin and myosin no longer interdigitate,
hold together and develop some stiffness
and tension. If a third, elastic, and possibly also contractile, filament is present in
the fibril, some of these problems could
be simply explained. With this in mind
we began examining a wide variety of
muscle fibers with the electron microscope.
We now have data from over 40 different
muscles from 19 species and 13 genera
(Hoyle, this symposium). We shall present
results from only six of these, the extensor
tibiae of the locust, Schistocerca gregaria;
the rostral depressor of the giant barnacle,
Balanus nubilus; the eye-stalk raiser of the
crab, Podophthalmus vigil; the antennal
muscle from the larva of the copepod,
Doropygus seclusus; the muscles of the
body wall of the garter snake, and, finally,
rabbit psoas muscle.
The small arthropod muscles were fixed
in situ, but in the case of the larger animals, fresh bundles of fibers or single
The work was supported by research grants
GB-3I6O from the National Science Foundation
and GM-33605 to Graham Hoyle.
fibers were excised and fixed, either at rest
length or under different degrees of stretch,
in wax troughs. The fixative used was a
4% solution of glutaraldehyde in a Millonig buffer. The material was then washed
in buffer solution and post-fixed in 1%
buffered osmium tetroxide. The fibers were
dehydrated in acetone and embedded in
Epon. In all cases the fibers were held at
a constant length during fixation and the
preliminary stages of dehydration. Sections
were cut with glass and diamond knives on
a Porter-Blum MT2 microtome, and were
stained with a saturated solution of uranyl
acetate in 50% ethanol followed by
Reynolds' lead citrate. The sections were
examined in a Siemens EIrniskop 1A, the
magnifications being checked by calibration
against a diffraction grating replica with
spacing of 0.88 p.
Figure 1 shows a series of micrographs of
the metathoracic extensor tibiae of the
locust, Schistocerca gregaria. These fibers
have long (9 /j) sarcomeres and no M
band. At the top are transverse sections
through the region of actin-myosin overlap
(Fig. la) and through the H zone (Fig.
lb). In the overlap region clear orbits of
10-12 actin filaments can be seen, surrounding each myosin. In the H zone the
myosin filaments are closer together. Dots
may be seen around the thick filaments of
a kind which would occur if filaments
which are very much thinner than actin
occupied the spaces between them. Two
(483)
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PATRICIA A. M C N E I L L AND GRAHAM HOYLE
VtMa^SSU
SUPERTHIN FILAMENTS IN MUSCLE
485
FIG. 1. Fine structure of slightly stretched fiber
from extensor tibiae of Schistocerca gregaria. a.
Transverse sections (T.S.) through overlap zone.
Note orbits of 10 thin filaments. X 120,000. b. T.S.
through H zone. Note orbits of about 10 very thin
filaments. Two sets are circled. Thick filaments
lie close together as sarcomere bows inwards.
X 120,000. c. Longitudinal section (L.S.) through
overlap region showing ordinary thin (actin) filaments. X 80,000. d. L.S. through H zone showing
very thin (T) filaments. A few especially clear
ones are indicated by arrows. X 80,000.
particularly clear groups are indicated. The
number of these filaments is apparently less
than the number of actins, which are
present in a ratio of 5:1. The comparable
regions are shown in longitudinal sections
(the H zone, Id being contrasted with a
similar section through the region of overlap, 1c). The difference in spacing of the
myosin filaments in the two regions is
again apparent. Very thin filaments, 20-25
A in diameter, may be discerned lying
parallel to the myosin in the H zone. Observations of very thin filaments in the H
zone of muscles such as this, with very long
sarcomeres and no M line bridges, are
hindered by the inward bowing of the
central region of the sarcomere, resulting
in close proximity of the myosin filaments
in this central portion. This may also explain why the H zone is so poorly marked.
This particular specimen was stretched
somewhat, and had an H zone of approximately 2.2 fj,, the length of the sarcomere
being about 9 ^,.
Figure 2 shows a longitudinal section
through a stretched rostral depressor muscle fiber of the giant barnacle, Balanus
nubilus. The single fiber was fixed at approximately 2 1/4 times rest length—at
which it could still develop active tension
upon depolarization. At this length the
actin and myosin filaments are completely
disengaged. Unfortunately, the Z disc is
broken up and the filaments are staggered,
but not sufficiently to permit overlap of
actin and myosin at any point, unless this
can occur across the apparent boundaries
between non-aligned, adjacent "fibrils".
There appear to be very thin filaments
crossing the gap, but the details are
obscured by a large amount of glycogen in
this region. Also, these thin "gap" filaments
seem to clump together. A micrograph at
high magnification of a transverse section
through the gap region (Fig. 2b) shows
single, or clusters of, very thin filaments
(arrows) bridging the gap, in addition to
the glycogen. Also, small dots of nearly
uniform size which may represent very
thin filaments cut transversely can be seen
everywhere between the myosins. It seems
reasonable to assume that these are extensions of the equally thin "gap" filaments. They stain more densely here,
probably because the stain is less readily
washed away from between the myosin filaments.
The next muscle we shall describe is
the white portion of the eye-stalk raiser of
the crab, Podophthalmus vigil. As in the
extensor tibiae of the locust, this muscle
has very long sarcomeres which, when
stretched, show a concavity in the central
region with the result that the H zone is
hard to distinguish. Figure 3c, d shows
longitudinal sections at different places
along a stretched sarcomere. 3c is through
the H zone, where the myosins are close
together. Some very thin lines, significantly
thinner than the actins (seen in Fig. 3d)
can be seen clearly between them.
A comparison of cross sections through
similar regions to those shown in Fig. 3c, d
suggests another reason why the H zone is
so obscure in this muscle. The myosin filaments are thicker and more dense in the
central region (Fig. 3a), having diameters
up to 250 A compared with about 160 A in
the overlap zone (Fig. 3b). Sections
through possible very thin filaments may
be seen among the myosins, together with
a few extra-long actin filaments (Fig. 3a).
The next muscle to be considered is
the first antennal muscle from the larva of
the copepod, Doropygus seclusus, (Fig.
4a,b). This material was provided by Dr.
Patricia Dudley, who discovered the muscle
to be of particular interest because there
are no conspicuous Z discs in the greater
portion of the fiber. The muscle has very
PATRICIA A. M C N E I L L AND GRAHAM HOYLE
SUPERTHIN FILAMENTS IN MUSCLE
487
FIG. 2. Fine structure of barnacle fiber stretched
more than 100%. a. L.S. Note that actin filaments
(I band) are removed from overlap with myosin
(A band) filaments. X 10,000. b. T.S. Note orbits
of very fine filaments in H zone. Gap regions con-
tain granules, but also very fine filaments staining
weakly (arrows point to good examples). X 10,000.
Note small filaments in H zone, I-band filaments,
and patches of Z band.
FIG. 3. Fine structure of a stretched fiber from
the white portion of the eye-stalk raising muscle
of the crab, Podophthalmus vigil, a. T.S. through
H zone showing incomplete orbits of very thin
filaments (some examples are circled), b. T.S.
through overlap zone showing orbits of 10-12 ordinary thin filaments, c. L.S. H zone (cf. a), d. L.S.
overlap zone (cf. b). X 120,000.
FIG. 4. Fine structure of a copepod larval muscle,
the attennule rotator of Doropygus seclusus (Block
courtesy of Dr. Patricia Dudley). L.S. Note alter-
nating ordinary thin (actin) and very thin (T)
filaments in I band, and also crossing the H zone.
X 120,000.
FIG. 5. Same as Figure 4 but T.S. a. Overlap zone.
Orbits of filaments which are thinner than the
ordinary (actin) thin filaments may be discerned.
b. H. Zone. Note numerous small dots among thick
filaments. Arrows point to fine bridges. X 180,000.
FIG. 6. L.S. of heavily stretched fiber from, garter
snake (ribs-skin). Note that the zone of termination of I band filaments (arrows) is well separated
from the A band. Fine filaments (T) may be seen
in the gap. X 80,000.
FIG. 7. T.S. of the same material shown in Figure
6. The regions marked T correspond to the gap
zones. They are filled with some glycogen and a
mass of small dots which we consider represent
transverse sections through very thin filaments.
Dots of similar size can be seen between thick
filaments in the H zone. X 80,000.
FIG. 8. T.S. of rabbit psoas, near the center of a
sarcomere. Note cross-bridges between thick filaments in the M band (M) region. In several instances, small dots, which could represent sections
across very thin filaments, may be seen in the
centers of the triangles formed by the cross-bridges
between the thick filaments. X 120,000.
FIG. 9. L.S. thiough extremely heavily stretched
fiber from rabbit psoas. Note that I band (actinAc) filaments have been completely removed from
overlap by a length of about I p. The resulting
"gap" is bridged by very thin filaments (T).
X 30,000.
FIG. 10. L.S. at higher power through extremely
heavily stretched psoas fiber of the rabbit. Scratch
mark indicates line of transect used for making
automatic-recording densitometer measurements between arrows (see Fig. 11). X 60,000.
FIG. 11. Selected sections from automatic-recording
densitometer tracing through regions of the T filament and actin filament shown in Figure 10 (psoas
fiber of rabbit), a. Region of the T filament. The
regions marked (t) correspond to places where a
fine filament can be seen on the photomicrograph,
b. Region of the A filament. The regions marked
(a) correspond to places where an ordinary thin
(actin) filament can be seen on the photomicrograph. The arrows indicate sharp peaks where
a possible T filament overlaps an actin filament.
In a and b, the smoothed baseline (minimum
transmittance) is indicated (B).
FIG. 12. Superimposed tracings of selected peaks
from complete densitometric tracing (part of which
is given in Fig. II) of greatly stretched rabbit psoas
fiber. Effective width, allowing for densitometer
tracing speed, is indicated. Baselines (minimum
transmittance) indicated by B.
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PATRICIA A. MCNEILL AND GRAHAM HOYLE
SUPERTHIN FILAMENTS IN MUSCLE
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PATRICIA A. M C N E I L L AND GRAHAM HOYLE
short sarcomeres, approximately 1.5 /x, and
is neatly organized, having well-marked A
and H regions. In several places very fine
filaments can be seen extending across the
H zone (arrows). Similar filaments are
noticeable in the I bands, in which an
alternation of ordinary thin (actin) and
very thin filaments occurs. These very
thin filaments appear to be continuous with
the myosin filaments, reminiscent of fine
filaments connecting the thick ones with
the Z discs in insect fibrillar flight muscle
(Auber and Couteaux, 1963). The appearance may well be spurious in the copepod,
due to the filaments lying above each
other.
Transverse sections through overlap and
H regions are given in Figure 5a,b. Very
thin filaments may be seen clearly in the
H zone and also in the overlap regions.
Note that they can be distinguished clearly
from cross-bridges. Most of these filaments
are closely associated with myosin filaments.
Some are indistinct, and relatively thick,
perhaps because at the A-I junction two
or more of these very thin filaments clump
together, giving the appearance of a single
thicker filament. A count in the H zone
of this muscle gives an approximate ratio
of two very thin filaments to each myosin;
this could be a common feature, even in
muscle fibers with higher ratios of actin
to myosin, although in insect leg muscle
fibers the ratio is close to 4:1, which is
the ratio for ordinary thin (rather than
superthin) to thick filaments.
All the fibers considered so far were from
invertebrates. The possibility had to be
envisaged that very thin filaments have
been secondarily lost in the evolution of
vertebrate muscle, in which they have not
been found (Huxley, 1957). However, since
most of the published work on the ultrastructure of vertebrate striated muscle was
done before the introduction of glutaraldehyde, which is such an excellent fixative,
we decided to examine also some vertebrate material. We began with the garter
snake, using the muscles between the ribs
and the skin (Hess, 1963). This muscle
showed evidence of superthin filaments,
similar to those observed in the invertebrate muscles, both in the H zone and
in the gap region produced when the
muscle was stretched 100-120%. In the
greatly stretched muscle (Fig. 6) the actin
and myosin filaments have remained more
completely in register than in the stretched
barnacle fiber, and a slightly less dense,
fuzzy region can be seen between the ends
of the actin and myosin filaments. This
fuzzy region has a longitudinal, filamentous structure but it is not clear enough
to permit observation of a fine filament
for any distance. Also, as with the barnacle,
much glycogen is found in this region.
A cross section of the same muscle fiber
(Fig. 7) shows clear A, I, and T (for superthin) filament regions. Here again, it is
difficult to distinguish the profiles of individual superthin filaments, but it is clear
that this region contains some material
which is much finer than actin. Encouraged by these findings, and since most of
the classical work on muscle ultrastructure
has utilized rabbit psoas, we decided that
our next step should be to examine this
muscle further. Cross sections through the
M line and the overlap regions may be
compared in Fig. 8. The arrangement of
the M line bridges in hexagonal stars is
clearly visible, and in several places small
dots which could represent sections through
very fine filaments passing across the H
zone can be seen in the centers of the
triangles formed by the bridges. In the
lower part of the picture is a typical array
of actin filaments around the myosins in
the overlap zone.
A longitudinal section of a rabbit psoas
fiber which we were able to stretch remarkably to more than 100% of its rest
length during fixation is shown in Fig. 9.
The actins have been pulled well out from
between the myosins, leaving gaps of approximately 0.5 ji, across which extend
many fine filaments closely resembling the
gap filaments of Sjostrand (1962) and
Carlsen, el al. (1965).
Figure 10 shows, at higher magnification, some particularly clear superthin filaments. This micrograph was used to obtain
SUPERTHIN FILAMENTS IN MUSCLE
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PATRICIA A. MCNEILL AND GRAHAM HOYLE
SUPERTHIN FILAMENTS IN MUSCLE
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SuPERTHIN FILAMENTS IN MUSCLE
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iv.y
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PATRICIA A. M C N E I L L AND GRAHAM HOYLE
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SUPERTHIN FILAMENTS IN MUSCLE
recording densitometcr measurements across
two gap regions and an I band (adjacent
t t t
t t
t
t/|,t
to the scratch). The slit was adjusted to
the equivalent of a 20 A gap.
t
t t t .
t t . t At A t
-B
a a
a a a a a
a a a a a a
B
a a a a
a
B
superthin
actin
a
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PATRICIA A. MCNEILL AND GRAHAM HOYLE
Parts of the densitonieter tracing are
given in Fig. 11. The absorption peaks
are downwards. The lower portion shows
the tracing across the I band, where several
of the broad peaks can be directly correlated with actin filaments on the previous
figure. Some finer peaks are also observed
which could be attributed to the presence
of thinner filaments in parallel with them.
The top trace was taken across the gap
region where several very thin filaments
could be easily recognized and compared
with the trace. It is composed of many
narrow peaks of less density than peaks
due to actin filaments. In order to compare the diameters of the actins and the
superthin filaments, we superimposed several tracings of each, giving the results
shown in Fig. 12a,b. The difference in
width of peaks is striking; the mean width
of actin is about 75 A, as frequently confirmed by many authors. The other peaks
lie between 25-40 A. We believe these represent a new type of filament, the super-
thin filaments which may be present in all
kinds of muscle.
Note added in proof. Recent evidence obtained by
Hoyle and Jensen (in preparation), using negative
staining, strongly suggests that the T filaments are
polymeric chains of actin monomers, i.e., single actin
strands. Ordinary actin filaments, which are shorter,
are double strands, or polymers o£ actin dimers.
REFERENCES
Auber, J., and R. Couteaux. 1963. Ultrastructure
de la slrie Z dans des muscles de Dipteres. J.
Microscopie 2:309-324.
Carlsen, F., F. Fuchs, and G. Knappeis. 1965. Contractibility and ultrastructure in glycerol-extracted muscle fibers. I. The relationship of contractibility to sarcomere length. J. Cell Biol. 27:
25-34.
Hanson, J., and H. E. Huxley. 1955. The structural
basis of contraction in striated muscle. Symp.
Soc. Exptl. Biol. 9:288-264.
Huxley, H. E. 1957. The double array of filaments
in cross-striated muscle. J. Biophys. Biochem.
Cytol. 3:631-648.
Sjostrand, F. 1962. The connections between Aand I-band filaments in striated frog muscle. J.
Ultrastruct. Res. 7:225-246.