CONTORTED MITOSIS AND T H E SUPERFICIAL PLASMAGEL
LAYER
WARREN H. LEWIS
(From the Department of Embryology, Carnegie Institution of Washington)
A peculiar type of mitosis, characterized by extreme contortions, lobulations, and constrictions of the dividing cell, and for a short period thereafter of
the two daughter cells, has been observed in tumor C37, obtained April 23,
1936, 159 days after a single injection of 0.8 mg. of 1 :2:5:6-dibenzanthracene
dissolved in lard into the axilla of a BA strain mouse. The tumor, a spindlecell sarcoma (Fig. 1) yields 100 per cent takes in the strain in which it origi-
TUMOR
OF THE FOURTEENTH
BA GENERATION
OF TUMORC37,
SPINDLE-CELL
SARCOMA
The cells are arranged in bands. Fat holes are seen at the left. X 200.
FIG.1. SECTION
OF
A
nated and has now been carried for over 50 generations in mouse-to-mouse
transfers. The cells (Fig. 2 ) have remained practically the same since the
first generation and the unique type of mitosis has been noted in practically
every series of cultures made from tumors of various generations. Many of
the cells in cultures from different generations also showed pinocytosis or
drinking by cells (Lewis, 1937 ) .
The contortions usually begin in metaphase (Fig. 9) and increase during
anaphase, telophase, and early daughter cell stages (Figs. 10 and l l ) , They
may, however, begin in late prophase or not until after division. They last
1
Aided by a grant made by the International Cancer Research Foundation.
408
. CONTORTED
MITOSIS AND THE SUPERFICIAL PLASMAGEL LAYER
409
for some time after division or until the daughter cells, if they are attached to
the cover-glass, have spread out somewhat on the latter. The most extreme
contortions are seen in the daughter cells just after division (Figs. 3 and 1 1 ) .
During the period when the cells are in the contorted condition they are con-
AND MACROPIIAGES
I N CULTURE FROM A C37 TUMOR
OF THE
FOURTEENTH
GENERATION
From left to right, macrophage, malignant cell, macrophage, malignant cell. All cells show
pinocytosis. X 1100.
FIG.3. Two CONTORTED
DAUGHTER
CELLSIMMEDIATELY
AFTER DIVISION
FROM A c37 TUMOR
OF
THE SEVENTEENTI1 GENERATION,
EXCISED
N O V . 12, 1936
Two-day hanging-drop culture in medium 221 (chicken plasma 2 parts, Locke solution 2 parts,
and beef embryo extract 1 part). X 1100.
MALIGNANT
CELLS
FIG.2. TYPICAL
tinually changing shape. Constrictions appear and disappear in different regions, squeezing the cells into lobulated and elongated forms. Some of the
lobulations project into the medium, away from the cover-glass. The phenomenon is somewhat different from the usual one involved in the formation
of pseudopodia in that the latter are thin and partly free from mitochondria
FIGS.4 TO 9. VARIOUSSTAGES
OF A DIVIDING
CELLOF TEE FORTIETII
GENERATION
FROM A TUMOR
EXCISED
Nov. 2, 1937, ADOUTA YEARAFTE,R FIG.1 WASTAKEN,MEDIUM221. X 1100
[Legend cont. at foot of p . 4111
410
CONTORTED MITOSIS AND THE SUPERFICIAL PLASMAGEL LAYER
41 1
(Fig. 2 ) while the lobulations of the contorted cells are filled with them. The
worm-like squirming of the contorted cells shows especially well in motion
pictures.
Figs. 4 to I 5 show various stages of a dividing cell from a 40th generation
tumor excised Nov. 2, 1937, about a year after Fig. 3 was taken. I t was not
possible to determine the exact moment when the various stages of mitosis
began and ended, but it is evident that anaphase and telophase take considerably longer than in normal embryonic cells (Levi, 1916; Lewis and Lewis,
1917). The total time involved from the first observation of the prophase
stage at 9.48 to the completion of cleavage was about one hour and forty
minutes. Prophase must have started some minutes before the first photograph (Fig. 4 ) was taken, since considerable change from the ordinary resting cell, (Fig. 2 ) has occurred. The time involved from the completion of
cleavage until the daughter cells attained the usual resting cell size (Figs. 11 to
15) was about two hours. The contortions continued, though gradually decreasing, for about half an hour during this growth period.
During prophase the cells of this tumor, like those from normal tissues and
other tumors, withdraw their processes and tend to become spherical (Figs. 4
to 8). There is evidently a centripetal pull on the processes at this time, for
occasionally the tips of longer ones remain firmly attached to the cover-glass,
become entirely separated from the rest of the contracting process, and are
left as disconnected tags of cytoplasm-often far from the cell-which undergo
various contortions, degenerate, and are frequently ingested by macrophages.
Other tags that remain attached by fine threads may later become reincorporated into the daughter cells as the latter spread out after division (Figs.
13-15). Such a thin hyaline process may snap in two without any appreciable
detrimental effect to the cell. The lack of injury is probably due to the
gelated condition of the stretched process, which on breaking does not allow
any of the endoplasm to escape.
Most normal embryonic and adult cells and malignant cells exhibit during anaphase and telophase, and shortly thereafter, a series of clear blebs on
the surface which suddenly appear and disappear without much distortion of
the cell body (Levi, 1916; Lewis and Lewis, 1932). The dividing cells of
C37 show very little of this bleb formation but much distortion.
Fig. 4. 9:48: an early prophase stage, a resting cell, and a macrophage. Note partial withdrawal of processes in early prophase, large nucleus full of chromosomes, cytoplasm packed with
mitochondria.
Fig. 5 . 10:07: same cell nineteen minutes later, in prophase, showing further withdrawal of
Drocesses and general contraction or roundinn u p of the cell; the nuclear wall is gone.
Fig. 6. 15:15, eight minutes later: late-prophase stage, showing further contraction of the cell
into an irregular thick mass. Remnants of the processes remain adherent to the cover-glass.
Fig. 7. 10:35, twenty minutes later: late prophase or early metaphase, showing further withdrawal of processes and contraction of cell into a rounded mass except for the thickened process
adherent to the neighboring cell, which shows partial withdrawal of its processes; many small
mitochondria.
Fig. 8. 10:57,twenty-two minutes later: metaphase. Several remnants of the processes still
radiate out from the cell as threads with globules. There is partial withdrawal of the process from
neighboring cell.
Fig. 9. 11:13, sixteen minutes later: late metaphase. The cell has become somewhat irregu(For further stages, see Figs. 10-15)
lar. The neighboring cell has recovered.
41 2
WARREN H . LEWIS
Presumably all the cells we are dealing with have an exceedingly thin
(monomolecular) invisible interface surface membrane which continually
exerts a surface tension pressure on the cell. Its action would oppose rather
than aid any distortion of the cell. We must turn elsewhere for an explanation of the contortions.
Beneath this membrane, or at the surface so far as visibility is concerned,
one can frequently see a clear hyaline layer or ectoplasm which separates the
granule-containing endoplasm with its mitochondria from the surface of the
cell. There are many reasons for considering that this layer consists of
gelated cytoplasm that is continuously in a state of contraction. It is well
known that colloidal gels exert continuous contractile tension, sufficient sometimes to flake off part of the inner wall of a glass vessel in which the gel sets.
The assumption that gelated cytoplasm also exerts continuous contractile tension thus seems probable. In a cell that has become spherical the contractile
layer is presumably of even thickness and density over the entire surface, so
that the tension exerted in a tangential direction at any point equals that at
any other point. A thickening or a thinning of the layer in any area by additional gelation or solation would produce an irregular cell or a contorted cell,
the extent and complexity of the contortions depending upon the location or
locations and degree of the changes in the superficial contractile layer. The
evidence for such a layer depends in part on its visibility, in part upon the very
plausible explanation it offers as one of the factors concerned with changes in
form, locomotion and division of cells, and in part on the presence of a similar
layer in the ameba, the plasmagel layer of Mast. That contractile tension is
exerted by a superficial layer is clearly apparent in the behavior of migrating
lymphocytes and neutrophiles (Lewis, W. H., 1931, 1934). At the base of an
advancing pseudopod there is usually a constriction ring formed by the contraction of a thickened band of the plasmagel layer. That this exerts considerable pressure is seen in the fact that the nucleus, which fills most of
the body of the lymphocyte, is often deeply indented as it is pushed through
the ring by contraction of the superficial plasmagel layer of the posterior part
of the cell.
Let us now consider the action of the contractile layer in cell division. We
have already noted that most cells tend to become spherical in prophase and
metaphase. During anaphase, as the chromosomes pull apart and move to the
poles, the cell usually becomes more or less elongated, the long axis passing
through the two centrioles and the two groups of daughter chromosomes, and
the short one approximately at right angles to the long one in the middle of the
now bilateral cell. A constriction or contraction ring appears around the short
axis of the cell as the chromosomes move toward the poles. This ring rapidly
deepens until the cell is divided into two parts. Frequently the two daughter
cells remain attached to each other by a short strand of clear cytoplasm (contractile layer), and the connection is often not broken until the two cells
migrate away from each other and pull the strand out into a long slender fiber
which eventually breaks.
The elongation of the cell in anaphase is probably due to an increase in
tension of a band of the superficial contractile layer which develops at right
angles to the spindle, in the region superficial to the former position of the
*
FIGS.10-13. FURTHER
STAGES
OF DIVIDINGCELLSHOWNIN FIGS. 4-9
Fig. 10. 11:19, six minutes later: beginning cleavage, chromosomes a t the poles, cell more
irregular. Anaphase about six minutes.
Fig. 11. 11:26, seven minutes later: cleavage about completed. The time required for cleavage is about seven minutes. The daughter cells are much contorted.
Fig. 12. ll:37, eleven minutes later: chromosomes clumped; daughter cells less contorted.
Fig. 13. 12:22, forty-five minutes later: daughter cells flattened out on cover-glass; small
inular nuclei; many small mitochondria; remnants of cell processes. A macrophage has mi(Fou further stages, see Figs. 14 and 15)
.led into the field.
413
FIGS.14 AND 15. FURTIiER STAGES OF DIVIDINGCELL SHOWN I N FIGS. 4-13
Fig. 14. 12:40, eighteen minutes later: some increase in size of cells and number of ITitochiondria. One cell has ruffle pseudopodia.
Fig. 15. 1:32, fifty-two minutes later: increase in size of cells and of nuclei. Some remn ants
are
Of ' processes not yet incorporated into cells appear to be still alive; others are nodular and
'ring.
The
daughter
cells
have
attained
about
the
usual
resting
cell
size.
dy
414
CONTORTED MITOSIS AND THE SUPERFICIAL PLASMAGEL LAYER
415
E
metaphase choromosome plate. We can assume that the contractile layer has
either become more viscous or has increased in thickness by the addition of
more gelated cytoplasm in the band region. As the two groups of chromosomes pull apart, there is left between them a clear area free from granules
and mitochondria. I t is possible that the peculiar material of this area brings
about in some way an increase in the viscosity or in the thickness of the equatorial band. Such a band would automatically exert more contractile tension
than the rest of the plasmagel layer and result in the constriction which divides
the cell. The connecting strand which usually unites the two daughter cells
for some time is analogous to the tail of the migrating lymphocyte, which appears to be formed by the clamping down of the constriction ring on the posterior end of the cell. The surface tension of the cell probably opposes rather
than aids the cleavage.
The peculiar contortions and lobulations observed in the dividing cells of
C37 may be explained then by irregular and shifting bands and areas of the
contractile layer, which are continually changing in viscosity. They distort
and constrict the cells here and there and squeeze them out in various directions.
SUMMARY
The assumption that cells possess a superficial layer of gelated cytoplasm
(plasmagel layer) which automatically exerts continuous contractile tension
and that this layer undergoes various local and general changes in viscosity and
thickness with corresponding variations in its contractile tension, offers a key
to one of the important factors concerned in changes of cell form, in cell
locomotion, and in cell division.
The contorted mitoses observed in the division of the cells of the spindlecell sarcoma C37 are explained by the development of changing contraction
bands of the plasmagel layer which produce constrictions that result in marked
distortions and lobulations of the dividing and young daughter cells.
BIBLIOGRAPHY
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LEWIS,W. H., AND LEWIS,M. R.: Anat. Rec. 13: 359-367, 1917.
LEWIS,W. H.: Bull. Johns Hopkins Hosp. 49: 29-36, 1931.
LEWIS,W. H., AND LEWIS,M. R.: Am. J. Cancer 16: 1153-1183, 1932.
LEWIS,W. H.: Bull. Johns Hopkins Hosp. 55: 273-279, 1934.
LEWIS,W. H . : Am. J. Cancer 29: 666-679, 1937.
MAST,S. 0.: Protoplasma 14: 321-330, 1931.