LAMELLAE
CELLS
IN
THE
OF WALKER
ROBERT
SPINDLE
OF MITOTIC
~56 C A R C I N O M A
C. B U C K ,
M.D.
From the Department of Microscopic Anatomy, University of Western Ontario, London, Canada
ABSTRACT
In mitotic cells of Walker 256 carcinoma some four-layered lamellar structures were observed
which had the appearance of paired cysternae of the ER. Two inner membranes were
regular, smooth surfaced, and closely applied to each other. The two outer membranes
were somewhat irregularly placed in relation to the inner pair; they showed attached R N P
particles and connections with cysternae of the ER. The membranes often appeared to
radiate from the region of the centrosphere towards the compact mass of chromosomes.
Thus, they lay amid the spindle fibres and are referred to as "spindle lamellae." They
approached the centrioles closely but were not observed to be continuous with them. They
appeared to terminate in the pole of the spindle by joining smooth surfaced membranes in
the centrosphere. Their equatorial termination was in relation to the chromosomes. At
the surface of the chromosome mass they frequently split into two double membranes,
which were closely applied to the chromosome substance. The most prominent and complicated membranes were seen in anaphase cells. An hypothesis is advanced which ascribes
the development of the nuclear membrane to the spindle lamellae.
INTRODUCTION
In mitotic cells of different kinds of animal tissues
Porter (9, 10) observed some double tubular
structures which, because of their tendency to
be associated with other spindle structures, he
called "spindle filaments." They were thought to
be equivalent to long, slender filaments observed
earlier in whole cell mounts of cultured cells (12).
A careful description was provided of the appearance of these structures in sections but no study
of them seems to have been made by other workers.
Chentsov (5) has independently and very briefly
referred to some equivalent "five-layered" structures in cells of an induced rhabdomyoblastoma.
Recently, the finding of the "spindle filaments"
in animal cells was briefly reviewed by Porter
and Machado (13) who compared the cytoplasmic
structures of mitotic animal and plant cells. A
component of the spindle in the latter was found
to be certain membranes of the endoplasmic
reticulum (ER) which were suspended, like
drapes, among the other parts of the spindle.
With regard to the double tubules of the animal
cell the authors stated, " I t is entirely possible
that they are the animal cell equivalent of the
E R elements noted here in the plant cell, but it
must be admitted that the aspect of doubleness
has not been seen with certainty in Allium cells
fixed in either OsO4 or KMnO4."
M y observations support their suggestion and
indicate that these structures, like the E R of the
plant cells, are important in the development of
the nuclear membrane of certain mammalian
cells.
MATERIALS
AND METHODS
Most of the observations have been made on cells of
the Walker 256 carcinoma after growth intramuscu-
227
larly in the leg of Sprague-Dawley rats for a period
of 4 to 8 days. Tissues were fixed in 1 per cent buffered
osmium tetroxide at p H 7.3 for 1 hour at 4°C, treated
for another hour at the same temperature with 1 per
cent aqueous uranyl acetate, dehydrated with
ethanol-water up to 70 per cent ethanol, and then
with acetone. They were embedded in Vestopal W
(15), and sections mounted on uncoated grids were
examined in an RCA E M U 3 D electron microscope.
Sections of about l # thickness stained with hot 1 per
cent methylene blue were studied with the light
microscope for the purpose of locating cells in
mitosis and determining, whenever possible, the
phase of the mitotic cycle and the orientation of the
cell.
OBSERVATIONS
In mitotic cells of the Walker tumor, profiles were
seen consisting of four dense n a r r o w lines separated
from each other by lighter material (Fig. 1). T h e
profiles, when studied in serial sections, were observed to persist in the third dimension, sometimes for several microns (Figs. 2 and 3). Usually
only relatively short profiles were found but occasionally they were tortuous, b r a n c h i n g a n d
several microns in length (Fig. 4). Since profiles
showing branching, tortuosity, and considerable
length would not likely be produced by the sectioning of t u b u l a r structures, they were interpreted as representing broad m e m b r a n e s or
lamellae. T h e shorter profiles m i g h t represent
either short lamellae or tongue-like extensions
from the margins of larger ones.
These q u a d r i l a m i n a r m e m b r a n e s were composed of an inner pair, closely applied to each
other, a n d a n outer pair. T h e inner m e m b r a n e s
measured a b o u t 70 A in width a n d were separated
from each other by a faMy uniform light space
a b o u t 40 A to 70 A wide. Both surfaces of the
i n n e r m e m b r a n e s were smooth,
T h e outer pair of membranes, separated from
the i n n e r pair by a somewhat irregular space
varying from 60 to 350 A, frequently showed
saccular or fusiform dilatations. In m a n y places
a continuity of the outer m e m b r a n e with cysternae
of the E R was seen (Fig. 4). T h e outer m e m b r a n e s
often showed a small n u m b e r of a t t a c h e d R N P
particles, comparable in size a n d n u m b e r to
those seen on the cysternae of the E R in the interphase state of the Walker t u m o r cell.
By serial sectioning it was established t h a t a
particular q u a d r i l a m i n a r m e m b r a n e was in
continuity with other similar membranes. In
228
FIGURE ]
Profiles of slender structures show an inner pair
of parallel lines close together and an outer pair
separated a variable distance from the inner ones.
Continuity between the inner and outer lines
exists at the ends of the profiles where, in this
micrograph, are found expansions which resemble
the membranous sacs of the endoplasmic reticulum
(ER). X 103,000.
fact, it is possible that the q u a d r a l a m i n a r m e m branes constitute a continuous system, a l t h o u g h
this would be very difficult to establish.
Each of the inner m e m b r a n e s was continuous
with its homolateral outer m e m b r a n e at the ends
of the profile of a particular q u a d r i l a m i n a r m e m b r a n e b u t not with the other inner m e m b r a n e
(Fig. 1). T h u s the four layers were considered to
THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 11, 1961
FIGURES ~ AND 3
A cell, which is probably in metaphase, cut obliquely at two levels. A system of four-layered profiles is
seen near the surface of the chromosome mass (CH) in both sections. The profiles are thought to represent lamellae having appreciable width. The tendency for the lamellae to radiate towards the surface
of the chromosomal mass is also apparent. X 8,000.
represent a n association between two very flat
cysternae. T h e adjoining m e m b r a n e s of the
cysternae were smooth, regular, a n d closely
applied to each other. T h e outer m e m b r a n e s h a d
the general c h a r a c t e r of the m e m b r a n e s of the
ER, with which they were continuous.
A complete description of the distribution of
the m e m b r a n e s within the dividing cell would be
difficult to provide as they seemed to be found, in
one section or another, in almost every conceivable site. T h e obvious reason for this is t h a t
one is not always aware of the plane of sectioning
a n d relations are then difficult to establish. F r o m
the study of favourably sectioned material a n d
from serial sections, it was established t h a t the
m e m b r a n e s were most frequently found in the
region between the centrosphere a n d the mass of
chromosome material. I n m a n y sections they
a p p e a r e d to fan out from the region of the centrosphere (Figs. 2, 3, 5). However, they were not
observed extending all the way to the centrioles
No direct connection between the tubules of the
centrioles a n d these m e m b r a n e s could be found,
a l t h o u g h in places they a p p r o a c h e d each other
very closely (Fig. 6).
O n the other h a n d , a continuity with some
delicate, irregular, smooth m e m b r a n e s of the
centrosphere was sometimes seen (Fig. 6). T h e
latter m e m b r a n e s outlined a n irregular network
of saccular spaces between m a n y small spherical
vesicles of the centrosphere. T h e y resembled the
smooth surfaced c o m p o n e n t of the E R a n d were
considered to represent the polar termination of
the q u a d r i l a m i n a r membranes.
F r o m this point the m e m b r a n e s r a n towards
the surface of the compact chromosome mass
taking, at first, a relatively straight course between
the spindle fibres. O n a p p r o a c h i n g the chromosomes they became tortuous a n d branching. I n
some sections the four m e m b r a n e s a p p e a r e d to
pass into the chromosome mass, presumably between chromosomes. No unequivocal instances
were found in the Walker t u m o r of the m e m b r a n e s
passing from one chromosome set to the other.
Usually, m e m b r a n e s appeared to extend over a
p a r t of the surface of the chromosomes. Sometimes
all four layers were seen r u n n i n g parallel to a n d
against the chromosomes. M o r e often, however,
the four-layered system became split at the chromosome surface into two separate double mere-
R. C. BUCK Lamellae in Spindle of Mitotic Cells
229
FmV~E 4
Part of Fig. 2 showing the tortuosity of lamellae and their connections with cysternae of the ER (arrows). A few RNP granules are attached to the outer membranes of the lamellae. In places, two-layered membrane (NM) lies against the chromosomal substance (CH). X 38,000.
branes, the splitting taking place between the
inner pair of membranes (Figs. 4 and 7). Frequent
examples were found of profiles of short, and
apparently separate, pieces of double membrane
on the surface of the chromosomes, especially
on the polar surface (Fig. 8). These images were
interpreted as representing a progression of the
process illustrated in Fig. 7.
Although the phase of the mitotic cycle in many
sections could not be established with certainty,
particularly when, as in the Walker tumor,
multipolar mitoses occurred (Fig. 6), my opinion
is that in anaphase the membranes reached their
greatest degree of development. Short profiles
were still seen in telophase (Fig. 9). They have
not been found in interphase nor in prophase cells.
Some of the cells showing the membranes were
230
probably in metaphase, but on this point I am
uncertain. However, examples have been observed in metaphase in another tumor (spontaneous lymphosarcoma in a Swiss mouse).
Even in anaphase it appeared that only some
of the cells possessed the membranes, although
conclusive proof for this statement would be difficult to provide. Certainly, their very extensive
development was observed only occasionally.
The configuration and relations of the membranes are shown diagrammatically in Fig. I0.
DISCUSSION
Except for the fact that the double structures of
the mitotic spindle were described as tubular,
rather than membranous, Porter's (9, 10) earlier
T H E JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 11, 1961
FIGURE 5
This polar view shows the radial arrangcmcnt of spindle lameUae from the centrospherc (centriole at
X) toward the chromosomes (CH). In this location they run between the spindle fibrcs (SF) and some
irregular, smooth surfaced membranes of the ER. X 33,400.
account is confirmed in all important respects.
Because they appeared to him as double tubules
and because they formed part of the spindle, he
investigated the possibility that they might join
the double tubules of the centrioles. Porter was
unable to find any conclusive evidence for this
interesting possibility but the suggestion was put
forward that they might, at any rate, act as centres
or axes of spindle fibre organization. Their
continuity with the cysternae of the E R and their
association with R N P particles were seen.
If my interpretation is correct, that these
structures represent profiles of flat cysternae and
not tubules, then Porter's term "spindle filaments"
should properly be changed to "spindle lamellae."
Except for their double nature the spindle
lamellae are similar to parts of the E R described
by Porter and Machado (13) in plant cells, and
these authors, w h o m I quoted earlier, suggested
that this might be the case.
The observations show that in Walker tumor
cells in anaphase the spindle lamellae come into
relation with the surface of the chromosomes and
appear to be involved in the formation of the
nuclear membrane. The studies of Watson (16,
17) clearly showed that in the interphase cell the
nuclear envelope is a specialized part of the ER.
It is, therefore, not surprising that recent studies
of dividing ceils have already demonstrated that,
in the dissolution of the nuclear membrane,
vesicles are formed which are finally indistinguishable from those of the ER, and during its
R. C. BUCK Lamellae in Spindle of Mitotic Cells
231
232
THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 11, 1961
FIGURE 7
Spindle lamellae at the chromosome (CH) surface. The upper part of the field shows many vesicles
and membranes of the centrosphere and spindle region. In some places the spindle lamellae are applied to the chromosome surface as a four-layered membrane. In other places the lamellae divide so
that they are continued as two-layered membranes. X 43,000.
reformation the membranes of the E R are involved (1-3, 8, l l , 13, 18). Multiple membranes
(parafusorial lamellae) are seen in spermatogenic
cells of Drosophila (6). It is thought that the nucleolus may be implicated in the formation of the
inner layer of the nuclear membrane in Amoeba
proteus (14). Although Bernhard (4) has suggested
the possibility of the de novo origin of the nuclear
membrane, Jones (7) appears to be alone in
supporting it.
O n the basis of the present observations the
hypothesis is advanced that in the Walker tumor
cells, at least, a membrane destined to become the
nuclear membrane forms in the region of the
FIGURE 6
Polar view of an anaphase cell in which multipolar mitosis is occurring. Spindle lamellae
show a general tendency to radiate from the centrosphere towards the surface of the
chromosomes (CH). They have not been found in continuity with the centrioles (X).
Their polar extremity appears to be by their becoming continuous with the numerous
smooth surfaced cysternae of the centrosphere. X 39,800.
R. C. Buck
Lamellae in Spindle of Mitotic Cells
233
FIGURE 8
Late anaphase cell showing a section through one
chromosome set. Short, apparently separate,
pieces of nuclear membrane cover part of the
surface of the chromosome mass (CH). The polar
surface shows a more complete covering than the
equatorial surface. X 12,400.
spindle during anaphase. This preformed n u c l e a r .
membrane (the spindle lamellae) may become
very extensive, in fact, more extensive than the
membranes of the interphase cell except the plasma
and nuclear membranes. It is suggested that the
formation of the spindle lamellae begins by the
apposition of cysternae of the E R and that the
lamellae grow both interstitially and by the
incorporation of more cysternae of the ER. In
late anaphase the spindle lamellae begin to
unfold and separate into double membranes which
spread over the surface of the compact chromosome mass. The " s m o o t h " inner and " r o u g h "
outer layers of the nuclear envelope are thus
derived, respectively, from the " s m o o t h " inner
and " r o u g h " outer membranes of the spindle
lamellae. The process is not completed until late
telophase, parts of the spindle lamellae retaining
their integrity, yet connected to the nuclear membrane already laid down. How such a process
might be accomplished is shown diagrammatically
in Fig. I 1.
The question of the prevalence of the spindle
lamellae in different types of mitotic mammalian
cells cannot be answered yet. Porter's observations,
those of Chentsov (5), and my own have been
made principally on tumor cells. Perhaps fewer
normal somatic cells in mitosis have been studied.
Yet, in collaboration with Mr. J. M. Tisdale,
I examined several hundred mitotic cells in the
FIGURE 9
234
TtlE JOURNAL OF BIOPHYSICALAND BIOCHEMICALCYTOLOGY •
VOLUME ]1,
1961
FIGUR]~ l0
FIGURE 11
Diagram to show the structure and relation of the
spindle lamcllae. A lamella consists of four layers of
membrane, the outer ones being continuous with
cystcrnae of the Et( at various points on their surface
and at their polar extremity. From the region of the
centrospherc the lamellae radiate towards the chromosome surface, sometimes branching before reaching it. At the chromosome surface the lamellac are
split into two double mcmbranes.
Schema for the development of the nuclear membrane in Walker tumor cells. According to an hypothesis advanced in the text, the spindle lamellae
develop by the close apposition of cysternae of the
ER (1), which are part of a continuous membranous
system (dotted lines). The lamellae grow during
anaphase (2, 3). In late anaphase (4, 5) they begin
to separate at the chromosome surface (shaded
mass) and a double nuclear membrane is laid down
(6) from the preformed spindle lamellac.
e p e n d y m a of I0- to 12-day-old rat embryos with-
spindle lamellae exist in normal mitotic cells,
I am grateful to Dr. Keith R. Porter for helpful comments and for providing unpublished findings from
his own work. Technical assistance was provided by
Messrs. William Daniels and Charles E. Jarvis.
This study was supported by grants from the National
Cancer Institute of Canada and the Medical Research Council of Canada.
they are more transitory than in tumor cells.
Received for publication, April 15, 1961.
out finding a single instance of spindle lamellae.
O n the other hand, profiles have been seen in the
regenerating liver of rats 48 hours after partial
hepatectomy. It is possible that although the
~GURE 9
Daughter cells in telophase in which the nuclear membranes (NM) are almost reformed,
At the level of this section the plasma membranes (PM) appear to separate the cells.
Spindle lamellae are still found in the cytoplasm of both cells and in the cell on the
right a part of the nuclear membrane resembles a spindle lamella. M 24,500.
R. C. BUCK Lamellae in Spindle of Mitotic Cells
235
REFERENCE8
l. AMANO, S., and TANAKA, H., Dynamic structure
of the nuclear membrane, with special reference to its disappearance and reappearance in
mitosis as observed under the electron microscope, Acta haemat., Japan, 1957, 20, 319.
2. BARER, R., JOSEPH, S. and MEEK, G. A., Membrane interrelationship during meiosis, Proc..
4th Internat. Conf. Electron Micr., Berlin, 1958,
233.
3. BARER, R., JOSEPH, S. and MEEK, G. A., The
origin of the nuclear membrane, Exp. Cell
Research, 1959, 18, 179.
4. BERNHARD, W., Ultrastruetural aspects of
nucleocytoplasmic relationship, Exp. Cell
Research, 1958, 6, suppl. 17.
5. CHENTSOV, G. S., An electron microscopic study
of induced rhabdomyoblastoma of the rat,
Proe. Acad. Sc., U.S.S.R., 1960, 132, 447.
6. ITO, S., The lamellar systems of cytoplasmic
membranes in dividing spermatogenic cells of
Drosophila virilis, or. Biophysic. and Biochem.
Cytol., 1960, 7, 433.
7. JONES, O. P., De novo origin of the nuclear membrane, Nature, London, 1960, 188, 239.
8. MOSES, M. J., Breakdown and reformation of the
nuclear envelope at cell division, Proc. 4th
Internat. Conf. Electron Micr., Berlin, 1958, 230.
9. PORTER, K. R., Changes in cell fine structure
accompanying mitosis, Symposium on Fine
Structure of Cells, Leiden, 1954, Groningen,
Noordhoff Ltd., 1955, 236.
10. PORTER, K. R., The submicroscopic morphology
236
11,
12.
13.
14.
15.
16.
17.
18.
of protoplasm, The Harvey Lectures, 1957, 51,
175.
PORTER,K. R., Problems in the study of nuclear
fine structure, Proc. 4th Internat. Conf. Electron
Micr., Berlin, 1958, 186.
PORTER, K. R., and KALLMAN,F., Significance
of cell particulates as seen by electron microscopy, Ann. N. Y. Acad. Sc., 1952, 54, 882.
PORTER,K. R., and MACHADO, R. D., Studies on
endoplasmic reticulum. IV. Its form and distribution during mitosis in cells of onion root
tip, J. Biophysic. and Biochem. Cytol., 1960, 7,
167.
ROTH, L. E., OBETZ, S. W. and DANIELS, E. W.,
Electron microscopic studies of mitosis in
amebae. I. Amoeba proteus, J. Biophysic. and
Biochem. Cytol., 1960, 8, 207.
RYTER, A., and KELLENBERGER, E., Inclusion au
polyester, Proc. 4th Internat. Conf. Electron Micr.,
Berlin, 1958, 52.
WATSON, M. L., The nuclear envelope. Its
structure and relation to cytoplasmic membranes, J. Biophysic. and Biochem. Cytol., 1955, 1,
257.
WATSON, M. L., Further observations on the
nuclear envelope of the animal cell, J. Biophysic, and Biochern. CytoL, 199, 6, 147.
YASUZUMI,G., Electron microscopy of the nuclear
membrane in prophase and telophase in
Yoshida sarcoma cells, Z. Zellforsch., 1959, 50,
110.
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