J. Cell Sci. i, 217-222 (1966)
Printed in Great Britain
217
A NOTE ON THE STRUCTURE OF
SPINDLE FIBRES
N. A. BARNICOT
Department of Anthropology, University College London
SUMMARY
Single mitotic cells from cultures of newt heart tissue or from human fibroblast cultures
were burst on a dilute calcium chloride solution and negatively stained on electron-microscope
grids. The electron-microscopic appearance of spindle fibres negatively stained with uranyl
acetate and with ammonium molybdate is described. As in the case offibresfrom sperm tails,
the spindlefibreswere found to be tubular and the wall was seen to be composed of longitudinal
fibrils about 35 A in diameter.
INTRODUCTION
The structure of spindle fibres as seen in electron micrographs of ultrathin sections
has been described in a wide variety of organisms and tissues (see Harris, 1962). After
fixation in buffered osmium tetroxide or glutaraldehyde, followed by staining with
heavy metals, the fibres have the appearance of tubes, about 250 A in diameter, with
a relatively dense wall. It has often been remarked that spindle fibres resemble the
fibres of cilia and flagella in size and structure and the association of all these fibre
types with centriolar bodies is a striking feature in common. The fine structure of
the fibres of flagella and cilia has proved difficult to elucidate in sectioned material,
though Gibbons & Grimstone (i960) thought that the wall of the central fibres in the
flagella of certain protozoa might have a helical structure, and Grimstone & Cleveland
(1965) saw a similar appearance in the fibres of contractile axostyles. Andre (1961) and
Afzelius (1959) found indications of two concentric dense layers in the walls of
transversely sectioned flagellar fibres. The problem was clarified by the work of Pease
(1963) and of Andre & Thiery (1963), who applied the technique of negative staining
with phosphotungstate to partially disrupted sperm tails. They were able to see that
the negative-staining reagent penetrated the axial region of the fibres, thus establishing
their tubular nature, and they also found that the wall was composed of longitudinal
fibrils, about 30 A in diameter, arranged in parallel.
In the course of studies on mitotic newt cells, burst on a water surface so as to
liberate and spread the chromosomes, the writer obtained negatively stained preparations of spindle fibres. The appearance of the fibres after various negative-staining
procedures is described below.
218
N. A. Barnicot
METHODS
Details of the method of selecting and bursting single mitotic cells will be given in
a later paper dealing with the chromosomes. The cells were obtained by culturing
pieces of newt heart tissue as described by Barnicot & Huxley (1965). In one experiment human fibroblasts growing as a monolayer were used and one preparation containing spindle fibres was obtained from this material. Cells were burst on the surface
of distilled water containing 0-002 M CaCl2. The solution was not buffered and the
pH was about 5-2. It may be that the presence of calcium ions in the spreading solution
is important for the preservation of spindle fibres, since it has been noted (Harris
& Mazia, 1962; Barnicot & Huxley, 1965) that in sectioned material they are preserved satisfactorily only if the buffered osmic fixative contains calcium and is somewhat acid (pH 6-5 or less). However, for other reasons, very few successful preparations
of the mitotic apparatus were obtained when water alone was used for spreading, so
that it has not yet been possible to investigate this point adequately. The favourable
effect of low pH in stabilizing the isolated mitotic apparatus has been noted by Kane
The burst cells were picked up on copper grids on which a collodion (Pyroxylin,
B.D.H.) film had been laid and coated with carbon. The greater strength of this double
film was found to be an advantage, especially when grids with a single diameter hole
of 800 [i were used. The carbon-coated surface with the adherent cell material was
immediately touched on the surface of the negative-staining reagent and held there for
15-30 sec. The excess reagent was then removed with a fine glass capillary under a
binocular dissecting microscope and the grid allowed to dry.
The negative-staining reagents, all freshly made before use, were as follows:
(1) 2 % uranyl acetate in water adjusted to pH 4-8 with N sodium hydroxide; (2)
1 % ammonium molybdate in water either adjusted to pH 6-5 with sodium hydroxide
or without the addition of alkali, in which case the pH was about 5-2; and (3) 1 % phosphotungstic acid in water adjusted to pH 6-5 with N sodium hydroxide.
In some cases the material was fixed briefly before negative staining. The grid was
floated for 10-30 min on 5% glutaraldehyde buffered with O'lM phosphate buffer,
rinsed in the buffer alone, then in distilled water, and finally touched on the uranyl
acetate or ammonium molybdate solution and dried as for unfixed cells. The grids
were examined in a Siemens Elmiskop 1B electron microscope using double-condenser
illumination. The higher resolution photographs of spindle fibres were taken at
instrumental magnifications of 40000 or 60000.
RESULTS
Spindle fibres were found in four newt and one human unfixed cell negatively
stained with uranyl acetate. Very often they occurred as somewhat disorganized
bundles of more or less parallel fibres scattered in various parts of the field (Fig. 2).
These aggregates were probably spindle fibre bundles torn and disarranged during
the bursting and spreading of the cell. In this form the fibres were often unsuitable
Structure of spindle
fibres
219
for higher-resolution work, but more isolated fibres lying in thin deposits of negativestaining reagent could generally be found. In only one instance has the attachment of
a bundle to a chromosome been seen (Fig. 1). In this case the chromosome itself was
unstretched and very dense, so that details of the junctional region could not be discerned. The negatively stained image of the spindle fibre stained with uranyl acetate
corresponds in many respects to that described by Pease (1963) and by Andre & Thiery
(1963) for the sperm-tail fibre. The individual fibres have a diameter ranging from
about 250 to 270 A. Even in fairly dense bundles of fibres the stain penetrates the
axial core. As seen in uranyl acetate preparations the fibre is bounded on either side
by a layer about 50 A thick with an irregular, ragged outer surface (Figs. 4, 5, 7 and 8).
In the central zone between these bounding layers two (Fig. 5) or three (Fig. 4) fine
longitudinal fibrils could often be seen. These fibrils, like those described by Pease,
had the appearance of rows of longitudinally connected granules about 35 A in
diameter. In many instances the granules appeared to be connected to each other
laterally so that they formed a regular square lattice, with the staining reagent filling
the interstices (Figs. 4, 5). The distance between the centres of the granules in both
longitudinal and transverse directions was about 50 A and this agrees with Pease's
figure for the transverse spacing in sperm-tail fibres but seems to be distinctly smaller
than his value of 88 A for the longitudinal periodicity. As found by Pease in the case
of flagellar fibres, the spindle fibres are usually straight or gently curved suggesting
considerable rigidity, and when fragmentation has occurred the breaks are usually
quite sharp (Fig. 8), suggesting that they have little extensibility. The broken ends
sometimes provide useful information. In the fibre shown in Fig. 5 it appears that the
upper part of the wall has broken away, exposing a short length of the lower surface
in which at least six fibrils can be counted. This interpretation is supported by some
preliminary observations on stereo-electron micrographs of other specimens taken by
Mr R. Willis. One of the fibres shown in Fig. 4 seems to be a more extreme example
of such a break; the upper wall is broken away at a point where another fibre crosses
it and a long stretch of the lower wall containing six fibrils is exposed. Short pieces
of fibrils which are probably derived from fragmented fibres (Fig. 4) are quite often
seen. Occasionally a fibre embedded in thick uranyl acetate deposit showed a series
of nodules apparently lying within the wall (Fig. 7). It has not been possible to determine the number of fibrils in the fibre wall with certainty, but it is likely to be in the
range 9-12. Spindle fibres from a human fibroblast stained with uranyl acetate are
shown in Fig. 8, They appear to be indistinguishable in size and structure from those
of newt cells.
In preparations stained with ammonium molybdate adjusted to pH 6-5 very few
spindle fibres were found and these were so much damaged that they were hardly
recognizable as such, but the same solution at pH 5-2 yielded fairly well-preserved
fibres in the two cells so treated. Nevertheless one has the impression that even the
more acidic ammonium molybdate solution is rather more damaging than uranyl
acetate. The fibres tended to be scattered at random (Fig. 3) and less often aggregated
into bundles, perhaps because the reagent not only weakens the fibres but disperses
other material which tends to gum them together. Individual fibres were often seen to
220
N. A. Barnicot
be disrupted into short segments by local dissolution of the wall and at points of
breakage the wall sometimes appeared to be split and the frayed ends curved outwards
(Fig. 3). The wall itself, as seen in projection, was distinctly thinner than in uranyl
acetate preparations and less ragged on its external surface (Fig. 6). This suggests
that in uranyl acetate preparations there may be an external layer which is partially
removed in ammonium molybdate; if so it remains unclear whether it is an integral
part of the fibre structure or is composed of adventitious material adhering to the
external surface. Two or three fibrils (Fig. 6) were often visible in the central zone
of fibres negatively stained with ammonium molybdate but, in contrast to uranyl
acetate preparations, granularity of the fibrils was indistinct, and there was less
evidence of cross-linkage. This, together with the appearance of the broken ends,
suggests that in ammonium molybdate the bonding of the fibrils may be considerably
weakened.
In two cells prepared with phosphotungstate (pH 6-5) no spindle fibres were
detected after prolonged searching. This may, of course, be due to chance but it is
worth noting in view of the fact that this reagent gave satisfactory preservation of
sperm-tail fibres (Pease, 1963; Andre & Thiery, 1963). It may be that, despite similarities in morphology, the fibres from different sources differ in their resistance to disruptive reagents.
Spindle fibres were found in preparations fixed in glutaraldehyde and subsequently
stained with ammonium molybdate (pH 5-2). In these preparations, the contrast of
negatively stained structures was somewhat impaired. The wall of the fibres, as seen
in projection, was distinctly thicker than in unfixed fibres stained in the same way, but
little detail could be seen and the fine-fibrillar components of the wall were not
detected.
The author is grateful to Professor J. Z. Young and Dr E. G. Gray for allowing him to use the
facilities of the electron-microscope laboratory in the Department of Anatomy, University
College. The help of Mr R. Willis, Mrs M. Roman, Miss T. Charlton and Mr P. O'Hara in
various aspects of the work, and of Miss P. Aylott and Mr S. Waterman with the photography is
also gratefully acknowledged.
REFERENCES
B. A. (1959). Electron microscopy of the sperm tail. Results obtained with a new
fixative. J. biophys. biochem. Cytol. 5, 269-278.
ANDRE, J. (1961). Sur quelques details nouvellement connus de l'ultrastructure des organites
vibratiles. J. Ultrastruct. Res. 5, 86-108.
ANDRE, J. & THIERY, J. P. (1963). Mise en Evidence d'une sous-structure fibrillaire dans les
filaments axone'matiques desflagelles.J. Microscopie 2, 71-80.
BARNICOT, N. A. & HUXLEY, H. E. (1965). Electron microscope observations on mitotic chromosomes. Q. Jlmicrosc. Set. 106, 197-214.
GIBBONS, I. R. & GRIMSTONE, A. V. (i960). On flagellar structure in certain flagellates.
J. biophys. biochem. Cytol. 7, 697-715.
GRIMSTONE, A. V. & CLEVELAND, L. R. (1965). The fine structure and function of the contractile axostyles of certainflagellates.J. Cell Biol. 24, 387-400.
HARRIS, P. (1962). Some structural and functional aspects of the mitotic apparatus in sea
urchin embryos. J. Cell Biol. 14, 475-488.
AFZELIUS,
Structure of spindle
fibres
221
P. & MAZIA, D. (1962). The fine structure of the mitotic apparatus. In The Interpretation of Ultrastructure (ed. R. C. J. Harris), pp. 279-305. New York: Academic Press.
KANE, R. E. (1965). The mitotic apparatus. Physical-chemical factors controlling stability.
J. Cell Biol. 25, 137-144.
PEASE, D. C. (1963). The ultrastructure offlagellarfibres.J. Cell Biol. 18, 313-326.
HARRIS,
{Received 19 January 1966)
222
N. A. Barnicot
Fig. i. Spindle-fibre bundle attached to an unspread chromatid. Negative-stain uranyl
acetate, x 37 500.
Fig. 2. Disrupted spindle-fibre bundle passing across axis of a chromatid (left).
Negative-stain uranyl acetate, x 77000.
Fig. 3. Scattered fragments of spindle fibres. Negative-stain ammonium molybdate,
pH 5-2. Note local disruption of fibre wall, and fraying of broken ends of some fibres
(arrows), x 95 000.
Fig. 4. Spindle fibres adjacent to dense patch of uranyl acetate (top right). Note ragged
surface of fibre wall in the two lower fibres (left) and presence of thin cross-linked
fibrils in their axial region. The top fibre (left) appears to be split exposing the lower
part of the wall. Fibrillar fragment of fibre wall (arrow), x 285 000.
Fig. 5. Spindle fibre showing break in wall exposing its lower surface (right). Note also
ragged surface of fibre wall. Two fibrils are visible in the axial region in the intact
portion to the left. Negative-stain uranyl acetate. X 285 000.
Fig. 6. Spindle fibres negatively stained with ammonium molybdate, pH S'2. The
wall is thinner than in the uranyl acetate specimens shown above and appears to be
disrupted in some places. Three fibrils are seen in the axial region of the upper fibre
(right), x 285000.
Fig. 7. Spindle fibres in dense uranyl acetate deposit. The fibre running horizontally
shows nodules which appear to lie within it. x 240000.
Fig. 8. Spindle fibres from a human fibroblast negatively stained in uranyl acetate.
Note sharply broken end of lower fibre, x 190000.
Journal of Cell Science, Vol. i, No. 2
N. A. BARN I COT
(Facing p. 222)
Journal of Cell Science, Vol. i, No. 2
For legends see preceding page.
N. A. BARNICOT