PERTURBATION OF MAMMALIAN CELL DIVISION III. THE TOPOGRAPHY AND KINETICS OF EXTRUSION SUBDIVISION ANN M. MULLINGER AND R. T. JOHNSON Department of Zoology, University of Cambridge, Downing Street, Cambridge, England
SUMMARY If mitotic-arrested, cold-stored HeLa cells are incubated at 37 °C a proportion of the population divides by an aberrant process which we have called subdivision by extrusion. This process has been studied by time-lapse photography and shown to differ from normal cleavage in several respects. The cell surface becomes more generally mobile and, instead of producing the precisely localized furrowing activity of cytokinesis, gives rise to multiple surface protrusions. These protrusions enlarge at the expense of the parent cell and develop into a cluster of small daughter cells (mini segregants). The surface structure of the cell, as seen by scanning electron microscopy, also changes; the microvilli characteristic of interphase, metaphase and cleaving HeLa cells are lost during extrusion and the cell surface becomes smooth. Extrusion activity is much more variable than division by cleavage in terms of both topography and kinetics, and in general takes longer to complete. Some cells in the cold-treated populations divide by mixtures of cleavage and extrusion or by cleavage alone. The relative numbers of cells dividing in different ways vary with the conditions of pretreatment and incubation of the mitotic cells. The greater the perturbation (e.g. longer cold storage), the greater the proportion of extruding rather than cleaving cells. Human diploid cells can also be induced to subdivide by extrusion. Possible mechanisms underlying the different types of division activity are discussed.
INTRODUCTION
We recently described methods of perturbing cell division in HeLa cells which lead to abnormalities of both cytokinesis and chromosome segregation (Johnson, Mullinger & Skaer, 1975). The outcome of these disturbances is a highly aberrant process during which a mitotic cell subdivides into a cluster of many small daughter cells (referred to as mini segregants) which vary in size, composition and DNA content (Schor, Johnson & Mullinger, 1975). Although almost any perturbation of mitotic cells produces some kind of division abnormality, such as enhanced surface bubbling, multiple furrowing and non-disjunction (for review and references see Mazia, 1961), the partitioning of a mitotic cell into mini segregants probably results from a more severe disturbance of cytokinesis, similar in nature to that previously recorded by Hughes (1950) for primary chick cell cultures. It appears that there exists an alternative pattern of cell behaviour which can occur if the organization for classical mitosis is sufficiently disturbed. We propose to call this process subdivision by extrusion.
244
A
-
M
- Mullinger and R. T. Johnson
In the present study we examine, by time-lapse photography and scanning electron microscopy, the topographic changes involved in extrusion subdivision and the processes by which mini segregants are produced. These events are compared with normal division by cleavage and also with other abnormal types of division found in populations of HeLa cells which have been subjected to treatments which induce the formation of mini segregants. MATERIALS AND METHODS Cell culture and synchronization HeLa cells with a doubling time of 17 h were grown in suspension culture in Eagle's Minimum Essential Medium supplemented with 5 % foetal bovine serum, non-essential amino acids and sodium pyruvate (MEMFC, Rao & Johnson, 1970). Synchronized populations in which over 95 % of cells were in mitosis were produced by the following treatment: 2-5 mM thymidine in MEMFC, 20-24 h in suspension; normal MEMFC, 4 h in suspension; nitrous oxide at 5 atmospheres (5066 x io5 N m~2) for 9 h, in MEMFC in plastic dishes (Falcon Plastics Inc.) (Rao, 1968). Diploid human fibroblastic cells from a patient with Fanconi's anaemia (kindly supplied by Dr K. Sperling) were grown in monolayer in MEM supplemented with 20 % foetal bovine serum. They were arrested in mitosis by means of a nitrous oxide block. Time-lapse photography Nitrous oxide-arrested mitotic HeLa cells were stored at 4 °C in MEMFC for periods ranging from 5 to 12 h. After cold storage the cells were spun down at 4 °C and resuspended in one of the following media at 4 °C: (i) MEMFC, pH 7-2; (ii) MEMFC buffered to pH 72 with 20 mM HEPES (iV-2-hydroxyethylpiperazine-JV-2-ethanesulphonic acid); (iii) Hanks' basal salt solution (Hanks & Wallace, 1949) with 20 mM HEPES (BSSH) pH 72 and 80; (iv) MEMFC plus o-i /
Extrusion subdivision in human cells
245
were dehydrated in a graded series of ethanols, transferred via amyl acetate to liquid carbon dioxide and critical-point dried according to the method of Anderson (1951). After drying, the cells attached to the coverslips were sputter coated with a 50-nm layer of gold palladium and examined in a Cambridge S-4 scanning electron microscope operated at 19-20 kV with a beam current of 130—150 fiA. Stereo pairs were taken at speciment tilt angles differing by 6°, normally 390 and 45°. Mitotic diploid fibroblasts were stored at 4 °C for 12 h, plated out on polylysine-coated coverslips and incubated for 3 h at 37 °C in BSSH, pH 80, with 2 mM DTT and were prepared for scanning electron microscopy by the same methods as for HeLa. All chemicals were obtained from Sigma Ltd, except Colcemid which was obtained from Gibco Ltd, Inc. RESULTS
When populations of synchronized HeLa cells are released from a nitrous oxide mitotic block and incubated in normal growth medium (MEMFC) at 37 °C, the majority of cells complete division by cleavage (into two, or sometimes three, daughter cells) within the next hour of incubation (Fig. 1 A). If, however, the cells are stored at 4 °C for 6-12 h immediately after release of the mitotic block, and are subsequently incubated at 37 °C, division is delayed for periods of up to several hours from the start of incubation (Fig. IB-D). The cells in such cold-treated populations exhibit many different patterns of activity, ranging from normal cleavage into 2 daughters to an extremely aberrant type of behaviour (extrusion subdivision), resulting in the formation of clusters of small cells that we have called 'bunches of grapes' (BOGs). (For a glossary of terms used in this paper see Table 1.) The type of division activity can be modulated by varying the length of cold treatment and the composition of the incubation medium, but even under one set of conditions there is considerable variation within a population with respect to both the nature and the time course of division activity in different cells. We have recorded by time-lapse photography the activities of several different populations of HeLa cells during incubation periods of 4-8 h following the release of a mitotic block, both with and without prior cold storage. We have also examined by scanning electron microscopy the surfaces of dividing cells at various times during the incubation. The methods used in these studies are described in the Materials and methods section. For scanning microscopy cells were incubated on polylysine-coated surfaces so that they could be processed in situ: unlike cells in interphase, neither cleaving nor extruding HeLa cells adhere to otherwise untreated tissue culture surfaces. We now describe characteristic features of the different types of division observed and also report on their relative frequency of occurrence and kinetics in several differently treated populations. Subdivision by extrusion: formation of BOGs Time-lapse photography. An example of a cell which subdivided by extrusion into a cluster of mini segregants, or BOG, is shown in Fig. 3 (p. 265) and described in detail in the legend. Briefly, the process in this case occurred as follows. At the start of incubation at 37 °C the cold-stored mitotic cell was approximately spherical and this
A. M. Mullinger and R. T. Johnson
246
3 27
II 25
3 24 D23 3 22 3 21 3 20
- ^ . • ^ " T r . •••.,?—119
3 3 3 D 3
18 17 16 15 14
D 13 J 12 u 3 11
mo 3 9 D H D D D Zl H D
8 7 6 5 4 3 2 1
2 3 4 Time, h, from start of incubation at 37 °C
i I I
1
1
i 4
3
•tiC
2 2 2
•
•_
3
I
1
3 3
I
B
I
18 17 116 115 1 14 1 13 1 12 1 11
I 3 I 21
1 10
2
o
' — i.-.-.::---:-.--:-
V. : "'\.'.V!A.-.-.:.
^— 3
H
2
I
2 2
4 1
B 3
1 1
1
1
2 •
4 1
)
1
| 1
3 I
8 U
t
I
_
1
7 6 5 14
2
I
1 2 1 1
2 I
2 4 3 Time, h, from start of incubation at 37 °C
I
1
5
6
Fig. 1. Diagrams to show the kinetics of division of individual cells in 4 different populations of mitotic HeLa cells, incubated at 37 °C in various media, A, MEMFC no pretreatment at 4 °C. B, MEMFC after 6 h storage at 4 °C. C, BSSH pH 80 with o-i /tg/ml Colcemid after 10 h storage at 4 °C. D, BSSH pH 8-o with 2 niM DTT after 6 h storage at 4 °C. # , cells shown on plates, as follows: C2, 10, 17, 20, 30, Figs. 15, s, 3, 4, 9, respectively; DI, 2, 6, Figs. 8, 11, 13, respectively.
Extrusion subdivision in human cells
247
=1 Z3 JD Z) U ED
1 1
1 -1
r
26 25 24 23 22 21
Zl'20 Z] 19 ID 18 =3*17 o 1ZJ 16 =
I T 14 1
~ hi
3
1
1
I111!
H.|
I I
15
I )
J3I 14 _U 13 _ J 12 ID 11 __!• 10 _ID 9 _J 8 ^J 7 6 141 5
n
1413 1
m 4 ^Zl 3 ZZ3' 2 JH 1
1
2 3 4 Time, h, from start of incubation at 37 °C
5
Key • Cleavage J
7 -3T":--.---:;g.- .- . 2 T
H'.l-t '
I •
: r : i
•• ?
R
l~i >
1
' . • • • •
~B
I
0
I
I
• 6
1
I
1 2 3 Time, h, from start of incubation at 37 CC
4 = 3 U • 2
CD Extrusion Kim Long phase with active balloon CJ Bubbling CD 1 cell L2J 2 daughters GD 3 daughters DO 4 daughters GO Bunch of grapes (more than 4 daughters) ES Incomplete division into 3 daughters B3J Incomplete division into 4 daughters Ci-1 Cell illustrated in time-lapse series
Fig. ic and D. For legend see opposite.
shape was maintained for about 70 min. Extrusion started with the formation of small surface blebs. These appeared asynchronously during the course of about 2 min and were distributed non-uniformly over the visible cell surface. Within the next few min many blebs increased in size and changed shape, some forming elongated structures, or fingers (Fig. 3H). Such enlargements occurred rapidly, although often asynchronously at different points on the cell surface; individual blebs developed into
248
A. M. Mullinger and R. T. Johnson Table 1. Glossary of terms used in the paper
Term
Definition
Bleb
Generally small, localized protrusion approximately 0-5-2 yttm in diameter but sometimes larger, e.g. Fig. 3E.
Finger
Elongated, localized protrusion, approximately 7—10 /«m in length, e.g. Fig. 3H.
Balloon
Large protrusion of localized origin. Approximately 10-25 /*m i n length, e.g. Figs. 7G, 8 and o,F.
Schematic representation
Mini segregants Products of extrusion subdivision of mitotic cell. Variable in size (< i - > 10 /an) and with or without DNA.
BOG
' Bunch of grapes'. group of attached mini segregants derived from a single parent cell by extrusion subdivision, e.g. Figs. 3N-T and 4J-K.
Bubbling
Widespread and prolonged surface activity with the production of numerous small blebs, without further development into large protrusions or BOG, e.g. Fig. 13G-L.
, fj?yW}y%$2?
._....
Extrusion subdivision in human cells
249
fingers within 40-90 s. As extrusion continued the protrusions enlarged, coalesced and changed shape before finally developing into approximately spherical mini segregants. Simultaneously, the parent cell decreased in size and was eventually obscured by the cluster of closely adhering daughter mini segregants. The time taken to reach this stage (BOG), from the appearance of the first bleb, was about 10 min. During a subsequent 3 h of observation the appearance of the BOG continued to change, although more slowly than before, as some mini segregants fused or divided further. Most extruding cells divided into BOGs in a broadly similar manner, but there were considerable differences in details. Examples illustrating the variations in extrusion activity seen under the range of conditions used in this study (see Materials and methods) are shown in Figs. 3-9 which are described in the accompanying figure legends. Many protrusions started as small blebs in the manner described above, but others originated from a less localized region of the cell surface (e.g. Fig. 7). Most changed in appearance as they enlarged, often assuming a wide variety of different shapes. Some, usually those with a more regular outline, enlarged without much change in shape (Fig. 4A-E). Most eventually became spherical or divided further into smaller spherical segregants, although a few appeared to regress into the parent cell. The maximum size attained by protrusions was variable. The largest (balloons), accounting for some 25-75 % of the total cell volume, developed on only a small proportion of extruding cells. In some instances these balloons were the first protrusions to develop (Fig. 23); alternatively, they developed at a later stage from one or two of a number of blebs or fingers (Fig. 9). The range of shapes and sizes and also the total number of protrusions per cell varied, although there were never more than 2 balloons per cell at any stage. The time taken for a protrusion to reach maximum size was also variable but even in the case of large balloons was not usually longer than 2-3 min. On any particular cell different protrusions appeared asynchronously, though usually within 10-20 min; in extreme cases the interval between the appearance of the first and last protrusion was as long as 2 h. There was marked variation in the degree of activity associated with extrusion in different cells and in the time for which individual protrusions continued to change in appearance before becoming spherical. The most pronounced movements and changes were associated with balloons, some of which were retained for periods of up to 3 h before subdividing into mini segregants. Many discrete waves of contraction passed along elongated balloons, from tip to base at a rate of 15-20 fimjram (Fig. 8), while more bulbous balloons often pulsated (Fig. 7). There were frequent changes in the orientation of balloons relative to their parent cell. The initial BOG was usually further modified, though the rate of change was low when compared with the initial extrusion activity. Fusion and further division of individual mini segregants resulted in the transformation of BOGs into various products ranging from clusters of more than 100 mini segregants to single re-formed cells (Figs. 3-5, and later section on scanning electron microscopy). Changes in the cluster occasionally lead to the complete or partial separation of daughters. For example, in the cell shown in Fig. 6, several mini segregants fused to form a larger cell which subsequently elongated and developed into a fine process with a distal cell body. 17-2
250
A. M. Mullinger and R. T. Johnson
Scanning electron microscopy. Various stages of extrusion subdivision have been identified in scanning electron micrographs of populations of HeLa cells fixed at different times after the start of incubation in BSSH. In the earliest samples the points of adhesion to the polylysine-coated surface were not visible. In later samples many of the cells were attached to the surface by long microvilli (up to 5 /on in length) both before, during and after extrusion activity (Figs. 16-31). Undivided spherical cells were found in samples fixed from 2 min to 4 h from the start of incubation, although in gradually decreasing numbers in later samples. These cells, in most respects identical to HeLa described by Paweletz & Schroeter (1974a, b) and Porter, Fonte & Weiss (1974), had many microvilli (o-i /im in diameter, and a fraction of a micrometre to 1-5 /an in length) as shown in Figs. 16-18. The density of microvilli varied from cell to cell but they were usually distributed uniformly over the cell surface. From 1-4 smooth-surfaced blebs (about 0-5-2 /«m in diameter) were also present on some cells. After 4 h of incubation the appearance of the majority of cells could be correlated with stages in extrusion activity seen by time-lapse photography. (Those cells which did not extrude either remained spherical or came to resemble flattened interphase cells; Fig. 37.) Cells with blebs,fingers and balloons could be recognized (Figs. 19-24, 28, 29). Manyfingersand balloons were of highly irregular shape and variable diameter, and in some cases there was a constriction at the junction between protrusion and parent cell (Fig. 22). Whatever their size, protrusions had a smooth surface. In contrast, the appearance of the parent cell surface was variable. Extruding cells with small protrusions or with a small number of protrusions generally retained microvilli ; in these cases there was an abrupt discontinuity in the surface features of cell and protrusion (e.g. Figs. 19-21, 23, 24). In cells with proportionately more surface involved in extrusion, microvilli usually became sparser. In one extruding cell half the surface was smooth and half bore microvilli; the larger protrusions were concentrated on the smooth half (Fig. 22). In addition to cells in the early stages of extrusion, there were also clusters of mini segregants where the parent cell was either small or not visible (Figs. 29-31, 36). Such BOGs were heterogeneous with respect to total number, size and arrangement of their mini segregants. Most mini segregants were spherical, ranging in diameter from about 6 to less that 1 /im (i.e. to below the resolution of the time-lapse films); others were more irregular (Figs. 31, 36). Occasionally, mini segregants were detached from the main cluster (Figs. 26, 30, 36). All elements of the fully formed BOG had smooth surfaces, although at higher magnifications, surface irregularities (e.g. tiny blebs) were occasionally observed. Division by cleavage
After cold storage a number of mitotic cells divided by normal cytokinesis. The cells first elongated slightly and a single cleavage furrow developed which separated 2 daughter cells of similar size (Fig. 10). Fusion of daughter cells was observed in only one instance. Scanning electron micrographs of cleaving cells in control populations
Fig. 2. Highly schematic drawings of sequential stages of 2 dividing HeLa cells to highlight the progressive differences in the surface topology between cleavage division (A) and extrusion subdivision (B).
"V
?'•"'*€
a a-
S
s
252
A. M. Mullinger and R. T. Johnson
show that they retained microvilli throughout division. Initially the cleavage furrow was also covered with microvilli (Figs. 32, 33) but later, as it deepened and the cells began to separate, they were connected by smooth bridges (Figs. 34, 35). At all stages during cytokinesis the cells were attached to the polylysine substrate by microvilli and/or broader structures. During division into three, the parent cell bulged and cleavage furrows separated daughters, which were often unequal: usually furrows formed somewhat asynchronously and fusion back to 2 cells often occurred, sometimes before the second furrow was complete (Figs. 11 and 14, cell 2), though more commonly from 5 min to 1-5 h later (Fig. 1). Cleavage into 4 cells was commonly followed by fusion to 3 cells (Fig. 12) or, rarely, two. Pronounced surface activity sometimes occurred after the completion of both normal and multiple cleavage: this we have called bubbling. It appears to involve the formation and withdrawal of small blebs over the entire cell surface (cf. Fig. 13G-L) and differs from extrusion activity since the majority of protrusions remain small, do not transform into mini segregants, are often short-lived and are more mobile than most of the smaller protrusions that give rise to mini segregants. Bubbling occurred at various times after plating out at 37 °C and continued for periods of up to 1 h, finally subsiding in intensity and leaving the original cell or group of cells (Fig. 1). Bubbling was not seen in all preparations and was particularly common and active in cells treated with 2 n w DTT in BSSH, pH 8-o (Fig. 1 D). Division showing elements of both cleavage and extrusion In addition to cells which divided either by cleavage or extrusion, others showed a complex sequence of activities which we judge to have involved elements of both processes (Figs. 13, 14, cell /, and 15). These ranged from cleavage-dominant divisions to those in which extrusion was the overriding behaviour with an element of cleavage only just recognizable. In some cells the predominant behaviour switched from one type to the other (Fig. 15). Although it is difficult to generalize about mixed divisions, the following patterns emerge. First, extreme extrusion activity (as indicated in Fig. 15L-P) did not precede or accompany furrow formation. Second, when extrusion and cleavage activities occurred simultaneously they were both separately localized and mild. The products of mixed divisions ranged from 3 daughters to BOGs. Kinetics of division by cleavage and extrusion In control mitotic populations the onset of cleavage was relatively synchronous in comparison with cells that had been cold-stored (Fig. 1). In cold-stored populations there was no significant difference in the time of initiation of either cleavage or extrusion activity; both were similarly asynchronous in their time of onset. In general, division was completed more quickly by cleavage than by extrusion, and the range of durations was greater for extrusion or mixed divisions than for cleavage (Table 2). Cleavage took longer when 3 or 4 rather than 2 daughters were formed. The more prolonged subdivisions by extrusion were often associated with long-lived balloons. The time course of transformations after the initial division (e.g. fusion) also varied (Fig. 1).
Extrusion subdivision in human cells
253
Comparison of division patterns produced by different treatments
Table 3 shows the types of activities observed in several differently treated populations of HeLa cells during 4-h periods of incubation at 37 °C. In control populations, incubated in MEMFC without prior cold storage, division was entirely by cleavage. In populations which had been stored at 4 °C for 6 h before incubation at 37 °C the
Table 2. Duration of the different types of division Time for completion of division, min A
Treatment of mitotic cells
Type of division
Shortest
Longest
Mean
No. of cells
Cleavage 8 12 5 4 180 68 12 Extrusion 3 12 Mixed cleavage 4 19 7 and extrusion — — —. — 8 h at 4 °C Cleavage 21 6 48 Incubation in Extrusion 9 — 2 BSSH pH 80 Mixed cleavage 17 24 and extrusion 11 12 6 h at 4 °C Cleavage 9 5 20 27 2 — Incubation in Extrusion 12 MEMFC pH Mixed cleavage 32 5 17 7-2 and extrusion The times relate to the interval between the first external sign of division and the formation of the initial group of daughter cells (i.e. subsequent re-fusion or further subdivision were excluded). In some cases the point of completion of division by extrusion was not clear cut. Data refer to divisions taking place during the first 4-8 h of incubation at 37 °C. For details of incubation media, see Materials and methods. 6 h at 4 °C Incubation in BSSH pH 8 0
proportion of cells dividing into 3 or 4 by cleavage increased and the number giving rise to doublets was reduced. Moreover, some cells subdivided by extrusion or by a mixture of extrusion and cleavage. This applied to cells incubated in either MEMFC pH 7-2 or BSSH pH 8-o. Cold storage for longer periods (8-10 h) promoted subdivision by extrusion or by mixed cleavage-extrusion rather than by cleavage, regardless of whether the cells were in MEMFC, BSSH or BSSH plus Colcemid. Given the same period of cold storage the proportion of extruding cells was greater in BSSH than in MEMFC. No division solely by cleavage occurred in cells cold-stored for 10 h. The patterns of cleavage and extrusion division were similar in populations subjected to the different treatments. The presence of Colcemid (o-i /
IO
0
s
o
0
0
2
o
0
o o
o
o
O
O
O
2
i
o
o
3
i
3
0
4
0
3
6
I
0
2
5
19
3
S
4
9
2
2
2
A
Cleavage into
I
2O
6
o
o
3
0
9
3
I
o
>4
0
o
o
o
o
4
o
o
o
o
o
3
Extrusion into
o
0
o
0
0
O
O
2
o
I
I
O
2
I
o
3
A
0
0
I
O
O
2
o
4
5
I
2
2
i
i
o 7
3i
IO
i7
is
18
18
2
16
20
29
27
5°
II
7
o
0
6o 68
18
6o
o 29
17
6
o
i7
6i
93
16
2O
24
13
17
22
o
Mixed cleavage Total no. Un- Clea- Extru- and exof cells divided vage trusion trusion (%) (%) > 4 observed (%) (%)
Mixed cleavage and extrusion into
* Only the initial behaviour of the cells is indicated in this Table (i.e. subsequent re-fusion or further subdivision are omitted). f Some of these cells divided later. For details of incubation media, see Materials and methods.
BSSH pH 8-o BSSH pH 8-o BSSH pH 8-o -t-Colcemid, (o-i tfg/ml)
PH7-2
MEMFC + 20 m M HEPES pH 7 -2 MEMFC
PH7-2
MEMFC
P H 7 -2
MEMFC
Incubation medium at 37 °C
Remained un- r dividedf
s
s
°3 5
' and R.
6 8
IO
8
6
o
Time of storage at 4°C, h
Behaviour of cells*
Table 3. The behaviour of nitrous oxide-arrested mitotic HeLa cells during a \-h period of incubation at 37 °C in various media after different periods of cold-storage
A. M. Mu
Extrusion subdivision in human cells
255
Extrusion subdivision in a diploid human fibroblast
Extrusion activity is not restricted to aneuploid tissue culture cells. Under appropriate conditions human diploid fibroblasts can be induced to form BOGs, although they do so less readily than HeLa cells. In the case of Fanconi cells extrusion subdivision is produced in a majority of mitotic cells only after prolonged cold storage followed by incubation in the presence of DTT (see Materials and methods). Under these conditions the range of protrusions observed was smaller than for extruding HeLa and no large balloons were seen. In other respects the BOGs produced from diploid cells were essentially similar to HeLa BOGs in surface topography (Fig. 39).
DISCUSSION
The division process in eukaryotic cells can be disturbed in many ways. Most commonly the patterns of cleavage and chromosome segregation are affected (Mazia, 1961; Stubblefield, 1964; Cox & Puck, 1969). A more extreme form of mitotic disturbance involving the complete inhibition of furrow formation has recently been investigated (Johnson et al. 1975). In this process, protrusions arise at multiple sites on the cell surface and develop at the expense of the parent cell to give rise to mini segregants. We have termed the activity subdivision by extrusion and in this paper have examined the topography and kinetics of the process in human cells. We believe that extrusion constitutes a form of derestricted cortical activity and that its analysis may provide insight into the ways in which elements of the normal mitotic cortex are assembled and operated. Because blebs are the first sign of extrusion activity it is pertinent to examine their occurrence and modulation on the surface of other tissue culture cells, most of which have surface blebs (e.g. Boss, 1955; Price, 1967; Puck, Waldren & Hsie, 1972). The appearance and frequency of such blebs depends on the cell type, the conditions of culture and the stage in the cell cycle (e.g. Porter, Prescott & Frye, 1973; Enlander, Scott & Tobey, 1974; Rubin & Everhart, 1973; Hale, Winkelhake & Weber, 1975). They are most frequently found in mitosis, particularly towards anaphase and telophase. In HeLa cells, grown either in suspension or monolayer, they are present in all phases of the cycle (Porter et al. 1973; this paper). In mitotic HeLa, blebbing is localized in space and time; it occurs during late anaphase and telophase and is less common in the region of the division furrow (Robbins & Gonatas, 1964a; Byers & Abramson, 1968; Erlandson & de Harven, 1971). In both mitotic and interphase cells blebbing can be modulated by certain treatments, and agents that affect microtubules are particularly effective: for example, blebs are induced in interphase Chinese hamster ovary cells by vinblastine and Colcemid and this effect is reversed by cyclic AMP and testosterone, 2 agents associated with rearrangement and increased numbers of microtubules (Puck et al. 1972; Porter, Puck, Hsie & Kelley, 1974; Borman, Dumont & Hsie, 1975). Similarly, Colchicine increases surface blebbing in mitotic cells (Bucher, 1939). Agents which lead to the de-
256
A. M. Mullinger and R. T. Johnson
polymerization of microtubules (e.g. Colcemid, vinblastine sulphate, low temperature and high hydrostatic pressure) are also associated with enhanced cytoplasmic churning activity and displacement of cell organelles (Robbins & Gonatas, 1964&; Stubblefield & Brinkley, 1966; Woodcock, 1971; Aronson, 1971; de Brabander & Borgers, 1975; Inoue & Ritter, 1975; Krishan & Frei, 1975; and Rebhun, 1975). The internal state of disorganization is often accompanied by cortical changes. Mechanical disturbance, narcotic agents such as chloral hydrate, and sulphydryl reactants and reagents, can also affect blebbing activity in dividing cells (Chambers, 1938; von Mollendorf, 1939; Hughes, 1950, 1952 a; Byers & Abramson, 1968). The involvement of actin in surface blebbing is implicated by several pieces of evidence. For example, the application of Cytochalasin B leads to bleb formation in Chinese hamster ovary cells (Puck et al. 1972) and Cytochalasin D has a similar effect on a number of different cell lines (Miranda, Godman, Deitch & Tanenbaum, 1974; Godman, Miranda, Deitch & Tanenbaum, 1975). Phalloidin, an agent that increases the number of actin filaments, also causes massive blebbing in liver cells (Govindan, Faulstich, Wieland, Agostini & Hasselbach, 1972; Lengsfeld, Low, Wieland, Dancker & Hasselbach, 1974; Frimmer, Kroker & Porstendorfer, 1974; Weiss, Sterz, Frimmer & Kroker, 1973), while the blebbing of Ilyanassa eggs, induced by exogenous isotonic calcium, is associated with rearrangement of microfilaments in the cortical cytoplasm in the region of the bleb constriction. The abnormally high (0-34 M) calcium required to induce bleb formation in these eggs suggests the induction and operation of a calcium-dependent contractile ring, in which activity is inhibited by Cytochalasin B but not by Colchicine (Conrad & Williams, 1974). It is possible that the predisposition of somatic cells to bleb at mitosis may be one outcome of the radical morphogenesis of microfilaments into meshworks, bundles or rings during this period of intense contractile activity. It may help to explain the ease with which blebbing can be induced in mitotic rather than interphase cells. In the attached interphase cell in tissue culture, the majority of actin filaments are aggregated into longitudinal bundles, except in local areas of movement where they occur as a meshwork (Goldman, Schloss & Starger, 1976). With the onset of prophase and cell rounding, the actin bundles are dispersed into a uniform meshwork and finally into the contractile ring of anaphase. Cytokinesis completed, the actin filaments are again dispersed and reassembled at opposite poles of the daughter cells (Sanger, 1975). Some agents that promote blebbing in interphase and mitotic cells are also those which induce extrusion, the first sign of which is augmented blebbing. This suggests that severe disturbance to both microtubules and microfilaments precedes extrusion activity. The promotion of extrusion by cold storage is probably explained in part by depolymerization of microtubules (e.g. Tilney & Porter, 1967) and by changes in the contractile and structural properties of actin (Pollard, 1976; Stossel & Hartwig, 1976). It is, however, also possible that for extrusion to begin, other elements of the cell must be additionally disrupted. For example high pH promotes extrusion, as does trypsinization, although we do not yet understand why this should be so (Johnson et al. 1975; Downes, Johnson & Mullinger, in preparation). The final proportion of extruding cells in a given population may therefore be a result of interplay
Extrusion subdivision in human cells
257
between disturbances which affect different aspects of the molecular organization of the dividing cell. In this connexion it is worth noting that the shorter periods of cold storage employed by Lesser & Brent (1970) to hold and thus accumulate mitotic HeLa cells in metaphase before synchronous release into interphase, are alone insufficient to induce extrusion activity. Some further indication of the mechanism of extrusion and the role of different factors comes from a consideration of the variability of division patterns in populations subjected to different treatments. The cells we observed had been accumulated in nitrous oxide and the state of contraction of the chromosomes in these cells resembled that of normal metaphase (Rao, 1968) although the organization of microtubules was probably disrupted (Brinkley & Rao, 1973). Subsequent cold storage would, presumably, have led to rapid depolymerization of microtubules and also some loss of organization of actin microfilaments (Tilney & Porter, 1967; Pollard, 1976; Stossel & Hartwig, 1976). During the subsequent incubation, whether preceded by cold storage or not, at least 95 % of cells divided during an 8-h period. The types of division varied according to the degree of recovery from the perturbations of cold, nitrous oxide and other treatments. Cells which divided into two by cleavage must have attained a normal condition and been able to assemble a spindle and contractile band (Schroeder, 1970). In this connexion it has been shown that the position of the contractile band in normal cytokinesis is determined by the position of the asters of the spindle in relation to the equatorial cell surface (Chambers, 1951; Rappaport, 1968). This interaction is dependent on inducing factors of equal but opposite polar origin and is critically timed, localized and rapid in its effect (Rappaport, 1971, 1975). The contractile force required for cleavage is probably achieved by a muscle-like actin-myosin interaction, although the distribution of myosin in the furrow region is not yet known. In cells dividing by multiple cleavage the normal interactions of cytokinesis are clearly disturbed. Multiple furrowing in other systems has been shown to arise either by the movement of a fixed number of organizing centres with respect to the cortex (as in echinoderm eggs, Rappaport, 1973), or by the simultaneous activity of multiple organizing centres (Heidemann & Kirschner, 1975). In HeLa cells, prolonged periods in nitrous oxide at high pressure are alone sufficient to increase the number of multiple division figures and subsequent cleavages (Brinkley & Rao, 1973). Abnormal anaphase figures and aberrant cytokinesis are described in Chinese hamster cells incubated in the presence of Colcemid (Stubblefield, 1964), and also in mouse L cells released from a vinblastine block (Krishan, 1968). In the present study a number of HeLa cells displayed both extrusion activity and also elements of normal cytokinesis. These mixed divisions suggest the operation of contractile band(s) in cells which also extrude, although furrowing does not occur when extrusion activities are at their most intense. Furrow development is, of course, precluded in the final stages of extrusion. Cells in which extrusion activity appears to be the sole manner of cytokinesis are presumably unable to assemble any elements of a normal contractile band before they are committed to subdivide by extrusion. The contractile machinery is so disseminated that the cell is now capable of prolonged and uncontrolled activity. It is probable that the onset of extrusion is preceded by local cortical weakening asso-
258
A. M. Mullinger and R. T. Johnson
ciated with changes in the organization of the cortex; this could involve both changes in the state of polymerization of actin molecules and also the morphogenesis of filaments into cortical structures (cf. Godman et al. 1975). Initial extrusion of cytoplasm through the points of weakness could either be the result of internal hydrostatic pressure or active contraction. As the protrusions enlarge their junction with the parent cell generally remains narrow, often for considerable time, arguing in favour of some remaining cortical structure. As the protrusions grow larger there is evidence of displacement of contractile activity into them at the expense of the parent cell. Further growth may result from contractile forces within the protrusions rather than in the parent cell. Such intrinsic contractility probably accounts for the mobility of protrusions. This is most clearly seen in the multiple constriction waves that pass along balloons, always from tip to base (cf. Dornfeld & Owcarzak, 1958). The rate of movement of the constriction waves is similar to that of the peristaltic contractions in the fertilized eggs of Pollicipes polymerus (Lewis, Chia& Schroeder, 1973). In this connexion it is worth noting that the induction, morphogenesis and activity of contractile bands are not uniquely associated with mitotic organizing centres (Conrad, 1973; Schroeder, 1973), although the complete separation of 'daughter' regions may be. Among cells subdividing by extrusion there is variation in the number of protrusions, their size, shape and rate of development or regression. It is probable that the greater the number of protrusions the greater the initial cortical disturbance and weakening. For example, in cells with a single, large protrusion the remaining surface does not generally extrude until later. The fact that all protrusions have a smooth surface suggests that their enlargement is associated with loss of microvilli from the parent cell and that this generates additional smooth surface membrane. The eventual size of any one protrusion is probably determined by the availability of cytoplasm, cortex and membrane accumulated from the parent body in competition with other sites of extrusion. The irregular shape of protrusions suggests a considerable cortical architecture. The extremely mobile surface of the extruding cell and also the loss of microvilli and transformation to the smooth surface of the final BOG probably indicate marked changes in the properties, spatial organization and composition of the membrane. Such changes have been observed in surface membranes of certain other cells undergoing gross cortical activity, either natural or induced. For example, surface blebbing and rearrangement of receptor sites are associated with disruption of microfilaments and microtubules (Godman et al. 1975; Poste, Papahadjopoulos & Nicolson, 1975). In interphase HeLa suiface blebbing, produced by insertion of electrodes, is followed by changes in membrane resistance and capacitance and also by the development of faint electrical coupling between cells (Hiilser, 1974). Similarly, during the induction of blebbing in phalloidin-treated cells, there is increase in the efflux rate of potassium (Frimmer, Gries, Hegner & Schnorr, 1967). Finally, in rabbit erythroid precursor cells the surface protrusions containing partly expelled nuclei have a lower density of electric charge than the rest of the cell (Skutelsky & Danon, 1969). Consideration of membrane properties during extrusion subdivision is closely related to the question of whether there are changes in cell volume and/or surface area.
Extrusion subdivision in human cells
259
Cleavage of a cell into 2 daughters with a combined volume equal to that of the parent cell requires a 26% increase in surface area, assuming that all the cells are smooth spheres. Relatively little is known about the changes in the cell surface or the origin of surface membrane in normal cleavage. In some cases membrane components are synthesized during interphase and the ' extra' membrane is stored in microvilli which are mostly lost at cytokinesis (Knutton, Sumner & Pasternak, 1975). The surface area of a BOG exceeds not only that of a smooth sphere of the same volume but probably also one covered with microvilli of the size and density observed in mitotic HeLa (preliminary calculations). If, however, there is a substantial change in volume of the magnitude described for normal cytokinesis by Zwaan & Hendrix (1973) and Cone (1969) (but not by Jung & Rothstein, 1967) then increase in surface area need not accompany extrusion. It might be predicted that only those cells with microvilli are inducible if new membrane assembly and/or change in volume are not involved in the process. We do not exclude the possibility that conformational changes in the membrane lead to local stretching or expansion in extruding cells. Another factor in BOG formation may be disturbance of the normal control of membrane assembly or synthesis. We find, for example, that considerable amounts of internal membranes are rapidly assembled in some HeLa cells during the induction of extrusion (unpublished observations). Disruption of mitosis by Colcemid and vinblastine also results in hypertrophy of internal membranes (Erlandson & de Harven, 1971; de Brabander & Borgers, 1975; Krishan, Hsu & Hutchins, 1968). Extrusion subdivision is not restricted to HeLa cells since it can be induced in other human cell types, although with much greater difficulty. The ease of induction is closely related to whether the cells are heteroploid tissue culture lines (HeLa, Hep 2, KB) - all easily induced, or diploid fibroblast cultures (4 diploid lines of independent origin tested, unpublished observations) - all induced with difficulty. The ease of extrusion may, of course, be associated with epithelioid rather than fibroblastic cells as is the case for cytochalasin D-induced zeiotic blebbing (Godman et al. 1975). Spontaneous activity at very low frequency (io~6 or less) has also been observed in our cultures of HeLa cells (e.g. Fig. 38). The BOGs that arise spontaneously cannot be distinguished from those which are induced. The present studies indicate that during the process of extrusion there are major changes in the cortical contractile activity of mammalian cells and that these can be induced by relatively simple procedures. Extrusion-like behaviour, often called zeiosis or zeiotic blebbing, has previously been recorded in other cells both in mitosis and interphase (e.g. Hughes, 1950, 1952a, b; Dornfeld & Owczarzak, 1958; Rose, 1966; Shepro, Belamarich, Merk & Chao, 1969). Our work has indicated that the process of extrusion can be modulated and that the underlying mechanisms are now amenable to analysis.
260
A. M. Mullinger and R. T. Johnson
We gratefully thank Miss M. Duller, Miss J. Kibble, Mr J. Rodford and Mr G. Runnals for assistance; Professor Daniel Mazia for kindly supplying both polylysine and a copy of his (then) unpublished manuscript on polylysine; and also Andrew Collins and Stephen Downes for their helpful comments. The work was supported by the Cancer Research Campaign. R.T.J. is a Cancer Campaign Research Fellow.
REFERENCES T. F. (195 I). Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. Ser. II13, 130-134. ARONSON, J. F. (1971). Demonstration of a colcemid-sensitive attractive force acting between the nucleus and a center. J. Cell Biol. 51, 579-583. BORMAN, L. S., DUMONT, J. N. & HSIE, A. W. (1975). Relationship between cyclic AMP, microtubule organization and mammalian cell shape. Studies on Chinese hamster ovary cells and their variants. Expl Cell Res. 91, 422-428. Boss, J. (1955). Mitosis in cultures of newt tissues. IV. The cell surface in late anaphase and the movements of ribonucleoprotein. Expl Cell Res. 8, 181-187. BRINKLEY, B. R. & RAO, P. N. (1973). Nitrous oxide: effects on the mitotic apparatus and chromosome movement in HeLa cells. J. Cell Biol. 58, 96-106. BUCHER, O. (1939). Zur Kenntnis der Mitose. VI. Der Einfluss von Colchicin und Trypaflavin auf den Wachstumsrhythmus und auf die Zellteilung in fibrocyten Kulturen. Z. Zellforsch. mikrosk. Anat. 29, 283-322. BYERS, B. & ABRAMSON, D. H. (1968). Cytokinesis in HeLa: post-telophase delay and microtubule-associated motility. Protoplasma 66, 413-435. CHAMBERS, R. (1938). Structural and kinetic aspects of cell division. J. cell. comp. Physiol. 12, 149-165. CHAMBERS, R. (195 I). Micrurgical studies on the kinetic aspects of cell division. Ann. N. Y. Acad. Sci. 51, 1311-1326. CONE, C. D. (1969). Electroosmotic interactions accompanying mitosis initiation in sarcoma cells in vitro. Trans. N. Y. Acad. Sci. 31, 404-427. CONRAD, G. W. (1973). Control of polar lobe formation in fertilized eggs of Ilyanassa obsoleta Stimpson. Am. Zool. 13, 961-980. CONRAD, G. W. & WILLIAMS, D. C. (1974). Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions. Devi Biol. 37, 280—294. Cox, D. M. & PUCK, T. T. (1969). Chromosomal non-disjunction: the action of Colcemid on Chinese hamster cells in vitro. Cytogenetics 8, 158-169. DE BRABANDER, M. & BORGERS, M. (1975). The formation of annulated lamellae induced by the disintegration of micro tubules. J. Cell Sci. 19, 331-340. DORNFELD, E. J. & OVVCZARZAK, A. (1958). Surface responses in cultured fibroblasts elicited by ethylene diamine tetraacetic acid. J. biophys. biochem. Cytol. 4, 243-250. ENLANDER, D., SCOTT, T. & TOBEY, R. A. (1974). Observations on the surface of synchronized Chinese hamster ovary cells in suspension culture. Scanning Electron Microscopy 1974, 573580. ERLANDSON, R. A. & DE HARVEN, E. (1971). The ultrastructure of synchronized HeLa cells. J. Cell Sci. 8, 353-397FRIMMER, M., GRIES, J., HEGNER, D. & SCHNORR, B. (1967). Untersuchungen zum Wirkungsmechanismus des Phalloidins. Freisetzung von lysosomalen Enzymen und von Kalium. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 258, 197-214. FRIMMER, M., KROKER, R. & PORSTENDORFER, J. (1974). The mode of action of phalloidin: demonstration of rapid deformation of isolated hepatocytes by scanning electron microscopy. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 284, 395-398. GODMAN, G. C , MIRANDA, A. F., DEITCH, A. D. & TANENBAUM, S. W. (1975). Action of Cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface. J. Cell Biol. 64, 644-667. ANDERSON,
Extrusion subdivision in human cells
261
R. D., SCHLOSS, J. & STARGER, J. (1976). Structure and possible physiological functions of actin-like microfilaments. Abstracts of papers presented at the meeting on Cell Motility. Cold Spring Harbor Laboratory. GOVINDAN, V. M., FAULSTICH, H., WIELAND, Th., AGOSTINI, B. & HASSELBACH, W. (1972). In-vitro effect of phalloidin on a plasma membrane preparation from rat liver. Natiirwissenschaften 59, 521-522. HALE, A. H., WINKELHAKE, J. L. & WEBER, M. J. (1975). Cell surface changes and Rous sarcoma virus gene expression in synchronized cells. J. Cell Biol. 64, 398-407. HANKS, J. H. & WALLACE, R. E. (1949). Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Soc. exp. biol. Med. 71, 196-200. HEIDEMANN, S. R. & KIRSCHNER, M. W. (1975). Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies. J. Cell Biol. 67, 105-117. HUGHES, A. F. W. (1950). The effect of inhibitory substances on cell division. A study on living cells in tissue cultures. Q. Jl microsc. Sci. 91, 251-278. HUGHES, A. F. W. (1952 a). The Mitotic Cycle: the Cytoplasm and Nucleus during Interphase and Mitosis. London: Butterworth. HUGHES, A. F. W. (19526). Some effects of abnormal tonicity on dividing cells in chick tissue cultures. Q. Jl microsc. Sci. 93, 207-219. HULSER, D. F. (1974). Ionic coupling between non-excitable cells in culture. In Methods in Cell Biology, vol. 8 (ed. D. M. Prescott), pp. 289-317. New York and London: Academic Press. INOUE, S. & RITTER, H. JR. (1975). Dynamics of mitotic spindle organization and function. In Molecules and Cell Movement (ed. S. Inoue & R. E. Stephens), pp. 3-31. New York: Raven Press. JOHNSON, R. T., MULLINGER, A. M. & SKAER, R. J. (1975). Perturbation of mammalian cell division: human mini segregants derived from mitotic cells. Proc. R. Soc. Land. B 189, 591GOLDMAN,
602.
C. & ROTHSTEIN, A. (1967). Cation metabolism in relation to cell size in synchronously grown tissue culture cell. J. gen. Physiol. 50, 917-932. KNUTTON, S., SUMNER, M. C. B. & PASTERNAK, C. A. (1975). Role of microvilli in surface changes of synchronized P815Y mastocytoma cells. J. Cell Biol. 66, 568-576. KRISHAN, A. (1968). Time-lapse and ultrastructure studies on the reversal of mitotic arrest induced by vinblastine sulfate in Earle's L cells. J. natn. Cancer Inst. 41, 581-595. KRISHAN, A. & FREI, E., III. (1975). Morphological basis for the cytolytic effect of Vinblastine and Vincristine on cultured human leukemic lymphoblasts. Cancer Res. 35, 497JUNG,
501.
A., Hsu, D. & HUTCHINS, P. (1968). Hypertrophy of granular endoplasmic reticulum and annulate lamellae in Earle's L cells exposed to vinblastine sulfate. J. Cell Biol. 39, 211—
KRISHAN, 216.
A. M., LOW, I., WIELAND, T., DANCKER, P. & HASSELBACH, W. (1974). Interaction of phalloidin with actin. Proc. natn. Acad. Sci. U.S.A. 71, 2803-2807. LESSER, B. & BRENT, T. P. (1970). Cold storage as a method for accumulating mitotic HeLa cells without impairing subsequent synchronous growth. Expl Cell Res. 62, 470-473. LEWIS, C. A., CHIA, F-S. & SCHROEDER, T. E. (1973). Peristaltic constrictions in fertilized barnacle eggs (Pollicipespolynterus). Experientia 29, 1533-1535. MAZIA, D. (1961). Mitosis and the physiology of cell division. In The Cell, vol. 3 (ed. J. Brachet & A. E. Mirsky), pp. 77-412. New York and London: Academic Press. MAZIA, D., SCHATTEN, G. & SALE, W. (1975). Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. J. Cell Biol. 66, 198-200. MIRANDA, A. F., GODMAN, G. C , DEITCH, A. D. & TANENBAUM, S. W. (1974). Action of Cytochalasin D on cells of established lines. I. Early events. J. Cell Biol. 61, 481-500. PAWELETZ, N. & SCHROETER, D. (1974a). Scanning electron microscopic observations on cells grown in vitro. I. HeLa cells in interphase. Cytobiologie 8, 228-237. PAWELETZ, N. & SCHROETER, D. (19746). Scanning electron microscopic observations on cells grown in vitro. II. HeLa cells in mitosis. Cytobiologie 8, 238-246. POLLARD, T. D. (1976). The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba. J. Cell Biol. 68,- 579-601. LENGSFELD,
262
A. M. Mullinger and R. T. Johnson
PORTER, K. R., FONTE, V. & WEISS, G. (1974). A scanning microscope study of the topography of HeLa cells. Cancer Res. 34, 1385-1394. PORTER, K. R., PUCK, T . T., HSIE, A. W. & KELLEY, D. (1974). An electron microscope study
of the effects of dibutyryl cyclic AMP on Chinese hamster ovary cells. Cell 2, 145-162. PORTER, K., PRESCOTT, D . & FRYE, J. (1973). Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J. Cell Biol. 57, 815-836. POSTE, G., PAPAHADJOPOULOS, D. & NICOLSON, G. L. (1975). Local anaesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors. Proc. natn. Acad. Sci. U.S.A. 72, 4430-4434. PRICE, Z. (1967). The micromorphology of zeiotic blebs in cultured human epithelial (HEp) cells. Expl Cell Res. 48, 82-92. PUCK, T . T., WALDREN, C. A. & HSIE, A. W. (1972). Membrane dynamics in the action of
dibutyryl adenosine 3': 5'-cyclic monophosphate and testosterone on mammalian cells. Proc. natn. Acad. Sci. U.S.A. 69, 1943-1947. RAO, P. N . (1968). Mitotic synchrony in mammalian cells treated with nitrous oxide at high pressure. Science, N.Y. 160, 774-776. RAO, P. N . & JOHNSON, R. T . (1970). Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature, Lond. 225, 159-164. RAPPAPORT, R. (1968). Geometrical relations of the cleavage stimulus in flattened, perforated sea urchin eggs. Embryologia 10, 115-130. RAPPAPORT, R. (1971). Cytokinesis in animal cells. Int. Rev. Cytol. 31, 169-213. RAPPAPORT, R. (1973). Cleavage furrow establishment- a preliminary to cylindrical shape change. Am. Zool. 13, 941-948. RAPPAPORT, R. (1975). Establishment and organization of the cleavage mechanism. In Molecules and Cell Movement (ed. S. Inoue & R. Stephens), pp. 287-304. New York: Raven Press. REBHUN, L. I. (1975). Induction of amoeboid movement in marine eggs. In Molecules and Cell Movement (ed. S. Inoue & R. Stephens), pp. 233-238. New York: Raven Press. ROBBINS, E. & GONATAS, N. K. (1964a). T h e ultrastructure of a mammalian cell during the mitotic cycle. J. Cell Biol. 21, 429-463. ROBBINS, E. & GONATAS, N. K. (19646). Histochemical and ultrastructural studies on HeLa cell cultures exposed to spindle inhibitors with special reference to the interphase cell. J. Histochem. Cytochem. 12, 704-711. ROSE, G. G. (1966). Zeiosis. I. Ejection of nuclei into zeiotic blebs. Jl R. microsc. Soc. 86, 87-102. RUBIN, R. W. & EVERHART, L. P. (1973). The effect of cell-to-cell contact on the surface morphology of Chinese hamster ovary cells. J. Cell Biol. 57, 837-844. SANGER, J. W. (1975). Changing patterns of actin localization during cell division. Proc. natn. Acad. Sci. U.S.A. 72, 1913-1916. SCHOR, S. L., JOHNSON, R. T . & MULLINGER, A. M. (1975). Perturbation of mammalian cell
division. II. Studies on the isolation and characterization of human mini segregant cells. J. Cell Sci. 19, 281-303. SCHROEDER, T . E. (1970). The contractile ring. I. Fine structure of dividing mammalian (HeLa) cells and the effects of Cytochalasin B. Z. Zellforsch. mikrosk. Anat. 109, 431-449. SCHROEDER, T . E. (1973). Cell constriction: contractile role of microfilaments in division and development. Am. Zool. 13, 949-960. SHEPRO, D., BELAMARICH, F. A., MERK, F. B. & CHAO, F. C. (1969). Changes in thrombocyte
ultrastructure during clot retraction. J. Cell Sci. 4, 763-779. SKUTELSKY, E. & DANON, D . (1969). Reduction in surface charge as an explanation of the recognition by macrophages of nuclei expelled from normoblasts. J. Cell Biol. 43, 8-15. STOSSEL, T . P. & HARTWIG, J. H. (1976). Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. J. Cell Biol. 68, 602-619. STUBBLEFIELD, E. (1964). DNA synthesis and chromosomal morphology of Chinese hamster cells cultured in media containing AT-deacetyl-AT-methyl Colchicine (Colcemid). In Symp. int. Soc. Cell Biol. (ed. R. J. C. Harris), pp. 223-248. New York and London: Academic Press. STUBBLEFIELD, E. & BRINKLEY, B. R. (1966). Cilia formation in Chinese hamster fibroblasts in vitro, as a response to Colcemid treatment. J. Cell Biol. 30, 645-652.
Extrusion subdivision in human cells
263
L. G. & PORTER, K. R. (1967). Studies on the microtubules in Heliozoa. III. The effect of low temperature on these structures in the formation and maintenance of axopodia. J. Cell Biol. 34, 327-343VON MOLLENDORF, W. (1939). Zur Kenntnis der Mitose. VIII. Zur Analyse des pathologischen Wachstums, hervorgerufen durch Chloralhydrat, Geschlechtshormone und cancerogene Kohlenwasserstoffe. Z. Zellforsch. mikrosk. Anat. 29, 706-749. WEISS, R., STERZ, I., FRIMMER, M. & KROKER, R. (1973). Electron microscopy of isolated rat hepatocytes before and after treatment with Phalloidin. Beitr. path. Anat. 150, 345-356. WOODCOCK, C. L. F. (1971). The anchoring of nuclei by cytoplasmic microtubules in Acetabularia.J. Cell Set. 8, 611-621. ZWAAN, J. & HENDRIX, R. W. (1973). Changes in cell and organ shape during early development of the ocular lens. Am. Zool. 13, 1039-1049. (Received 14 April 1976) TILNEY,
IS
264
A. M. Mullinger and R. T. Johnson
Figs. 3-15. Frames from time-lapse films of cold-stored mitotic HeLa cells incubated at 37 CC in various media. Figs. 3, 4, 5, 9, 15: 10 h storage at 4 °C and incubation in BSSH pH 8 0 with o-i fig/ml Colcemid. Figs. 6, 7: 8 h storage at 4 °C and incubation in BSSH pH 8 0 . Figs. 8, 11, 13 : 5 h storage at 4 °C and incubation in BSSH pH 8-o with 2 mM D T T . Figs. 10, 12, 14: 6 h storage at 4 °C and incubation in BSSH pH 8-o. Fig. 3. Typical extrusion subdivision into a BOG. Blebs and fingers are formed and the BOG, once produced, is further subdivided, A-T, 196 min 24 s. x 560. (cf. Fig. i c , cell 17.) A-D, cell bulges and blebs appear, E, further development of blebs, F-G, some blebs elongateinto fingers, H—1, asynchronous and varied development of surface protrusions: some blebs remain, others elongate into fingers, j , protrusions enlarge and some coalesce. Parent cell smaller, K-L, protrusions becoming spherical. Parent cell much smaller. By this stage one area of the cell surface (arrow) has shown no apparent surface activity, M, typical BOG composed of a tight cluster of mini segregants. Parent cell obscured, N - T , further development of BOG. Rearrangement of cluster: some mini segregants are subdivided, (A, 74 min 16 s from start; A-B, 33 min 4 s; B-C, 10 min 8 s; C-D, 1 min 12 s; D-E, I min 12 s; E-F, 32 s; F-G, 40 s; G-H, 24 s; H - I , I min 4 s; I - J , 1 min 20 s; J-K, I min 12s; K - L , 40 s; L-M, 56 s; M - N , I min 4 s; N-o, 5 min 4 s; O - P , 35 min 36 s; P - Q , 24 min 16 s; Q-R, 33 min 52 s; R-S, 27 min 36 s; S-T, 16 min 32 s.) Fig. 4. Extrusion subdivision into a BOG. The initial protrusions formed have a more regular shape than those in Fig. 3. A-L, 164 min 32 s. x 560. (cf. Fig. 1 c, cell 20.) A-B, 2 protrusions appear, C-D, first 2 protrusions enlarge. Third and fourth protrusions appear, E-F, protrusions enlarge further, particularly the first and fourth. Parent cell simultaneously becomes smaller, G-J, contraction wavesonlargestprotrusion (balloon) which is then withdrawn partially and subdivided. Parent cell much smaller and finally no longer visible. BOG formed, K-L, further development of BOG. Rearrangement of cluster and, finally, fusion of some mini segregants. (A, 143 min 4 s from start; A-B, I min 52 s; B-c, 1 min 4 s; C-D, I min 44 s; D-E, I min 4 s; E-F, 40 s; F - G , 24 s; G-H, 6 min o s; H - I , 13 min 20 s; I - J , 22 min 8 s; J-K, 35 min 52 s; K - L , 80 min 24 s.)
Extrusion subdivision in human cells
265
18-2
266
A. M. Mullinger and R. T. Johnson
Fig. 5. Reconstitution of parent cell after extrusion subdivision into a small number of mini segregants. A-F, 68 min 16 s. x 560. (cf. Fig. i c , cell 10.) A-B, blebs appear, enlarge and become approximately spherical. Parent cell becomes smaller, c, BOG with about 6 mini segregants. D-F, further development of BOG: mini segregants reconstituted into a single cell. No further surface activity was observed after frame F. (A, 78 min 56 s from start; A-B, 6 min 56 s; B-c, 36 min 16 s; C-D, 16 min o s; D-E, 2 min 48 s; E-F, 6 min 16 s.) Fig. 6. Post-extrusion changes in a BOG. Some mini segregants fuse together and elongate. (Several other BOGs in this population behaved similarly.) A-L, 251 min 12 s.
X460.
A-B, blebs and fingers appear, c, protrusions now approximately spherical and parent cell smaller. D, BOG formed, E-F, further development of BOG. Rearrangement of cluster. One mini segregant starts to elongate and to develop a fine process (arrow). G-L, fine process (arrows) elongates and by frame I is no longer visible. Elongating mini segregant fuses with others and is separated from the main cluster by a narrow stalk, (A, 55 min 28 s from start; A-B, 2 min; B-C, 6 min; C-D, 9 min 36 s; D-E, 7 min 28 s; E-F, 13 min 36 s; F-G, 12 min 56 s; G-H, 5 min 36 s; H - I , 46 min24 s; I-J, 22 min 16 s; J-K, 63 min 4 s; K - L , 62 min 16 s.) Fig. 7. Active movement of balloon during extrusion subdivision, A-L, 55 min 4 s. X460. A-B, blebs appear, C-D, blebs enlarge into a balloon (arrow), E-G, balloon highly motile and grows at the expense of the parent cell, H - J , balloon still motile. Blebs appear on small parent cell, K-L, balloon subdivided, apparently not by extrusion, and BOG formed, (A, 142 min 24 s from start; A-B, 40 s; B-c, 8 s; C-D, 16 s; D-E, 16 S; E - F , 24 s; F - G , 104 s; G-H, 24 s; H-l, 112 s; I-J, 16 s; J-K, 27 min 36 s; K - L , 21 min 28 s.) Fig. 8. Contraction waves on an extruded balloon, A-H, I min 56 s. x 580. (cf. Fig. I D , cell 1.) A, extruding cell (arrow) with many small blebs and one large balloon, B-F, contraction wave passes along balloon from tip to base, G-H, second contraction wave appears from tip of balloon and passes back, (A, 95 min 56 s from start; A-B, 8 s; B-c, 8 s; C-D, 12 s; D-E, 16 s; E - F , 8 s; F-G, 12 s; G-H, 52 s.)
Extrusion subdivision in human cells
267
268
A. M. Mullinger and R. T. Johnson
Fig. 9. Extrusion subdivision into a BOG with formation of balloons, A-J, 106 min 40 s. x 560. (cf. Fig. 1 c, cell 30.) A-C, blebs appear. D, blebs at opposite sides of parent cell elongate into fingers synchronously, E-F, both fingers enlarge into balloons. G, balloons withdrawn towards parent cell which is now subdivided, H-J, BOG formed and rearranged, (A, 344 min 48 s from start; A-B, 3 min 4 s; B-C, 24 s; C-D, 48 s; D-E, 24 s; E-F, I min 28 s; F-G, 4 min 16 s; G-H, I I min 28 s; H-l, 9 min 44 s; I-J, 75 min 4s.) Fig. 10. Cleavage division into 2 daughters, A-E, 9 min o s. x 530. A—B, cell (arrow) elongates, c, cleavage furrow starts, D, cleavage furrow deepens, E, 2 daughters, (A, 132 min from start; A-B, I min o s; B-c, 2 min 12 s; C-D, 2 min 36 s; D - E , 3 min 12 s.) Fig. 11. Cleavage division into 2 daughters with the formation of one complete and one incomplete furrow, A-F, 23 min 8 s. x 580. (cf. Fig. 1D, cell 2.) A-C, cell (arrow) becomes trilobed and progressively constricted, D-E, only one of the 2 cleavage furrows deepens sufficiently to separate a daughter cell, F, incipient cleavage furrow of larger cell regresses, (A, 53 min 44 s from start; A-B, 7 min 32 s; B-C, 3 min 4 s; c—D, 3 min 12 s; D—E, 6 min 4 s; E—F, 3 min 16 s.) Fig. 12. Multiple cleavage division followed by reconstitution. A-H, 63 min 24 s. X53°A-c, development of 4-lobed figure by multiple constrictions, D - F , constriction furrows deepen to produce 4 daughter cells, G—H, reconstitution of 3 daughters. (A, 26 min 48 s from start; A-B, 20 min 44 s; B-c, 1 min 52 s; C-D, 2 min 16 s; D-E, 3 min o s; E - F , 3 min 32 s; F-G, 18 min 16 s; G-H, 13 min 44 s.) Fig. 13. Mixed extrusion and cleavage division into about 4, followed by reconstitution to 3, daughters and, finally, general surface bubbling, A-L, 48 min 48 s. x 650. (cf. Fig. 1 D, cell 6.) A, blebs appear, B, blebs enlarge into fingers and simultaneously the parent cell is constricted in a similar manner to normal cleavage, c, development of extrusion and cleavage activities: extruded fingers enlarge (arrows) and cleavage furrow deepens. D-E, further development by extrusion and cleavage into a group of 4 daughter cells. F, reconstitution to 3 daughters, G-L, simultaneous surface bubbling on all 3 cells, (A, 137 min 56 s from start; A-B, 4 min 8 s; B-C, 2 min 32 s; C-D, 2 min 56 s; D-E, 7 min 52 s; E-F, 6 min 12 s; F-G, 10 min 12 s; G-H, 24 s; H - I , 36 s; i-J, 32 s; J-K, 2 min 40 s; K - L , 10 min 44 s.)
Extrusion subdivision in human cells
269
270
A. M. Mullinger and R. T. Johnson
Fig. 14. Examples of cleavage (cell 2) and mixed cleavage and extrusion (cell 1) division, A-P, 25 min 16 s. x 550. Cell 1. A-B, cell becomes tri-lobed. c-F, only one constriction furrow deepens and separates a large elongating lobe, G-N, one region of the cell (arrow) undergoes extrusion activity and is finally isolated by a second constriction furrow. Three spherical cells are formed, O-P, final reconstitution of 2 cells. Cell 2. A-P, 2 constriction furrows develop to produce 3 daughter cells which reconstitute to 2. (A, 82 min 32 s from start; A-B, 2 min 12 s; B-C, 44 s; C-D, 1 min 28 s; D-E, 24 s; E-F, 40 s; F-c, 40 s; G-H, 36 s; H - I , 44s; 1—j, 1 min 24 s; J-K, 2 min 8 s; K-L, I min 32 s; L - M , I min o s; M - N , 3 min 8 s; N - O , 3 min 56 s; O - P , 4 min 40 s.) Fig. 15. Mixed cleavage and extrusion division into a BOG. A-T, 180 min 8 s. x 560. (cf. Fig. i c , cell 2.) A-D, blebs develop on cell (arrow) and enlarge, E-G, all visible protrusions coalesce and constriction furrow appears, H, constriction furrow deepens, I-K, coalesced protrusions enlarge as parent cell grows smaller, L—o, local, followed by more general, development of extrusion activity, P-Q, one protrusion develops into a motile balloon. R-T, further subdivision into a BOG. (A, 7 min 52 s from start; A-B, 23 min 52 s; B-C, 8 min 40 s; C-D, 15 min 20 s; D-E, 13 min 44 s; E - F , 14 min 16 s; F-G, 15 min 36 s; G-H, 21 min 28 s; H - I , 18 min 16 s; I-J, 8 min 8 s; J-K, 48 s; K-L, I min 4 s; L - M , 2 min 16 s; M-N, 2 min 24 s; N-o, 3 min 36 s; o-p, 1 min 28 s; P - Q , 56 s; Q-R, 6 min o s; R-s, 9 min 28 s; s-T, 12 min 48 s.)
Extrusion subdivision in human cells
271
B
r4P H
K
oO^ N_£
272
A. M. Mullinger and R. T. Johnson
Figs. 16-39. Scanning electron micrographs of HeLa cells (Figs. 16-38) and human diploid fibroblast (Fig. 39) attached to polylysine-coated coverslips. The micrographs are oriented with the substrate below the cells except in the case of stereo pairs which are rotated anticlockwise from this position through 90 0 . Figs. 16-31, 36, 37. Nitrous oxidearrested mitotic HeLa cells, stored at 4 °C for 11 h (except Fig. 28, 8 h; Fig. 31, 4 h), plated out on polylysine-coated coverslips and incubated at 37 °C in BSSH pH 7 2 (except Figs. 28 and 31, pH 80). Fixed 2 h (Figs. 18, 20-22, 25, 28, 29, 31, 36, 37) or 3 h (Figs. 16, 17, 19, 23, 24, 26, 27, 30) after the start of incubation. Figs. 32-35. Nitrous oxide-arrested mitotic HeLa cells plated out on polylysine-coated coverslips and incubated at 37 °C in MEMFC. Fixed 2 h after the start of incubation. Fig. 38. BOG from a random suspension culture of HeLa, plated out on a polylysine-coated coverslip and incubated at 37 °C in MEMFC. Fixed 2 min after plating out. Fig. 39. Nitrous oxide-arrested mitotic human diploid cell stored at 4 °C for 12 h and plated out on a polylysine-coated coverslip and incubated at 37 CC in BSSH pH 8-o with 2 mM D T T . Fixed 3 h after the start of incubation. Fig. 16. Cold-stored mitotic cell with many microvilli and also small blebs. Long microvilli attach the cell to the substrate. Stereo pair, x 4000. Fig. 17. Enlargement of part of Fig. 16. x 7800. Fig. 18. Cold-stored mitotic cell with relatively few microvilli. x 3500. Figs. 19, 20. Early stages of extrusion subdivision; development of smooth-surfaced blebs. Fig. 19 probably corresponds to Fig. 3E. Fig. 19, x 3800. Fig. 20, x 2800.
Extrusion subdivision in human cells
273
274
A
- M- Mullinger and R. T. Johnson
Fig. 21. Multiple extrusions of both smooth-surfaced blebs and fingers of various shapes. The parent cell surface still bears some microvilli. This stage probably corresponds to Figs. 3H and 6 A. Stereo pair, x 3500. Fig. 22. Extruding cell with many protrusions of highly variable size and shape. The surfaces of the protrusions are smooth, apart from a few small blebs. One half of the parent cell surface bears microvilli, the other half is smooth. Stereo pair, x 3400.
Extrusion subdivision in human cells
275
276
A. M. Mullinger and R. T. Johnson
Fig. 23. Extruding cell with a large balloon. The surface of the parent cell, but not the balloon, bears microvilli. The stalk connecting the balloon to the parent cell has a few microvilli (see also Fig. 24). The sucker-like distal end of the balloon is attached to the substrate. (Wrinkles on the balloon may be artifacts of specimen preparation.) x 3300.
Fig. 24. Enlargement of part of Fig. 23. x 8000. Fig. 25. Extruding cell probably at a later stage than in Fig. 22 with many protrusions of approximately spherical shape. Parent cell still retains some microvilli. x 3100. Fig. 26. Final stage of extrusion subdivision, possibly corresponding to Fig. 6j. x 2800.
Fig. 27. Enlargement of part of Fig. 26 to show smooth surface, x 6100.
Extrusion subdivision in human cells
277
278
A. M. Mullinger and R. T. Johnson-
Fig. 28. Extruding cell with many smooth blebs of various shapes. Compared with the cell in Fig. 21, the parent cell surface is relatively smooth, although short microvilli and tiny blebs are still present. The whole cell is attached to the substrate by long microvilli. Stereo pair, x 4000. Fig. 29. Top: bunch of grapes (BOG). All mini segregants are smooth. Bottom: extruding cell with 2 major protrusions; one, the balloon, shows evidence of a contraction wave (arrow). This cell probably corresponds to Fig. 8. x 3100.
Extrusion subdivision in human cells
279
280
A. M. Mullinger and R. T. Johnson
Fig. 30. Cluster of typical mini segregants, or BOG. The smallest segregants tend to be concentrated in one region where they are intermixed with attenuated processes. Microvilli extend from the cluster to the substrate. This stage probably corresponds to Fig. 3T. Stereo pair, x 4700. Fig. 31. Atypical BOG. Most of the mini segregants are elongated and bear a fine process. Stereo pair, x 2600.
Extrusion subdivision in human cells
281
19-2
282
A. M. Mullinger and R. T. Johnson
Figs. 32-35. Stages in cleavage division into 2 daughter cells. The cells retain microvilli throughout and also bear a few smooth blebs. They are attached to the polylysinecoated substrate by microvilli and in some cases by larger broad-based projections. Fig. 32. Separation of daughter cells has started. The furrow is covered with microvilli. x 3400. Fig. 33. Enlargement of the furrow region of Fig. 32. Note the aligned surface ridges (arrow) running approximately normal to the furrow and giving rise to microvilli. Stereo pair, x 7500. Fig. 34. Further deepening of furrow. The cells are joined by several cytoplasmic strands, of various sizes. Some of the blebs on the daughter cells appear to have collapsed. Stereo pair, x 5300.
Extrusion subdivision in human cells
283
284
A. M. Mullinger and R. T. Johnson
Fig. 35. Late stage of cytokinesis. The daughter cells have moved apart, but are still connected by a narrow, smooth-surfaced bridge. The broad-based projections (arrow) to the substrate are particularly clear, x 3700. Fig. 36. BOG with both attached and detached mini segregants. x 1500. Fig. 37. Below: irregularly shaped cell with microvilli and smooth-surfaced blebs. This cell may possibly have beenfixedwhile dividing by mixed cleavage and extrusion. Above: (arrow) part of a flattened interphase cell with microvilli. x 2900. Fig. 38. BOG from a suspension culture of HeLa cells, x 5100. Fig. 39. Extrusion subdivision induced in a human diploid cell. The protrusions are generally smaller than in HeLa cells but show similar surface features, x 2200.
Extrusion subdivision in human cells
