Cytokinetic activities in a human cell line: the midbody and intracellular bridge

TISSUE & CELL 1973 5(1) 47-61 Published by Longman Group Ltd. PriJlted in Great Britain

d. M. MULLINS and d. d. BIESELE

CYTOKINETIC ACTIVITIES IN A HUMAN CELL LINE' THE MIDBODY AND INTERCELLULAR BRIDGE ABSTRACT. The midbody and intercellular bridge of D-98S cells were studied by time lapse cinemicrography. Before separation interconnected daughter cells tended to move in opposition to each other, maintaining tension across the bridge and, in some cases, lengthening the bridge. Cell separation was preceded by a reduction in width of the bridge, on one or both sides of the midbody, to a cytoplasmic strand less than 0-5 F wide, which was then stretched and broken by movements o[" the daughter cells, completing cytokinesis. Wave-like movements were observed to pass along longer intercellular bridges from the midbody to the daughter cells. This activity was judged to be involved with the retraction of cytoplasm from the bridge into the daughter cells. Wave activity was disrupted by the drug cytochalasin B.

involved a n d interpreting t h e m in the p r o p e r chronological sequence. Byers a n d A b r a m s o n (1968) employed time lapse cinemicrography to follow cytokinesis in HeLa cells a n d reported several interesting events, including a consistent lengthening of the intercellular bridge before cell s e p a r a t i o n a n d the seemingly c o o r d i n a t e d passage of wave-like movements along it. T h e present study utilized this technique to follow cytokinetic activities in the h u m a n cell line D-98S which is morphologically similar to H e L a a l t h o u g h derived from n o n - m a l i g n a n t tissue. In addition, cells were treated with the fungal metaholite cytochalasin B to determine its possible effects on the m i d b o d y a n d bridge.

Introduction

THE presence Of the m i d b o d y in the intercellular bridge which c o n n e c t s d a u g h t e r cells after telophase has been n o t e d frequently in the literature for b o t h n o r m a l tissues a n d cultured cells. Little insight, however, has been gained regarding the significance of these structures or the m a n n e r in which cytokinesis is completed. Because the midb o d y a n d intercellular bridge are c o n s t a n t features of cell division in b o t h invertebrate a n d vertebrate tissues (Fry, 1937) it is desirable to k n o w more a b o u t their function in the division process. The f o r m a t i o n o f the m i d b o d y has been well d o c u m e n t e d t h r o u g h several electron microscope studies (Buck a n d Tisdale, 1962a, b; R o b b i n s a n d G o n a t a s , 1964; K r i s h a n a n d Buck, 1965; E r l a n d s o n a n d d e H a r v e n , 1971). The events following the f o r m a t i o n of the m i d b o d y have n o t been clarified t h r o u g h investigations using the electron microscope, however, due to the p r o b l e m s of o b t a i n i n g properly aligned, thin sectioned p r e p a r a t i o n s of the structures

Materials and Methods Cell cultures

Stock cultures of D-98S ceils ( A m e r i c a n Type Culture Collection C C L 18.1) were grown as m o n o l a y e r s in m i l k dilution bottles. Cultures were m a i n t a i n e d at 37'~C in a growth m e d i u m consisting of Eagle's Basal M e d i u m , Earle's Base (Bioquest), s u p p l e m e n t e d with 10°,~ calf serum, 1°..~i 2 0 0 r a M L-glutamine a n d 100 units per ml penicillin-streptomycin.

The Department of Zoology, The University of Texas, Austin, Texas 78712. Received 12 October 1972. 47

48

MULLINS AND BIESELE

Chwmicrography F o r time lapse p h o t o g r a p h y cells were g r o w n in S y k e s - M o o r e chambers. W h e n n o t being used for p h o t o g r a p h y these preparations were incubated in a n a t m o s p h e r e of 10°..~ CO-, to prevent a rise in p H over long time intervals due to the loss of CO~, t h r o u g h the silicone r u b b e r gaskets sealing the c h a m b e r s (Freed, 1963). T h e g r o w t h m e d i u m was replaced at 24 h r intervals. A K o d a k 16 m m c a m e r a equipped with an E m d e c o intervelometer was used to film the time lapse sequences at speeds of 2, 4, 8 or 16 frames per minute o n K o d a k Plus-X reversal film. A Zeiss microscope equipped with p h a s e optics a n d long working distance condenser was housed in a n i n c u b a t o r c h a m b e r m a i n t a i n e d at 37°C. M o s t filming was d o n e with the 100x oil immersion objective, s u p p l e m e n t e d with a Zeiss Optov a t a t t a c h m e n t when additional magnification was desired, T h e microscope light source was filtered t h r o u g h a heat a b s o r b i n g glass a n d green interference filter. Cells were followed either f r o m different stages of mitosis or f r o m various phases of postmitotic activity.

sequences. A Zeiss Microflash unit was utilized as the i l l u m i n a t i o n source so t h a t sequence p h o t o g r a p h s could be o b t a i n e d at 10 sec intervals.

Cytoehalasin B treatment T r e a t m e n t m e d i u m containing 5 or 10 t~g per ml cytochalasin B (Imperial Chemical Industries Ltd) was prepared by diluting a stock solution in growth medium. The stock solution consisted of 100 tzg per ml cytochalasin B (CCB) in 10°~, aqueous dimethylsulfoxide ( D M S O ) , the solubilizer for the drug (Carter, 1967). The final p r e p a r a t i o n s thus c o n t a i n e d 0.5 °.o and 1.0~.'; D M S O respectively. A control m e d i u m of 1 °.o D M S O in growth m e d i u m was similarly prepared. I n t r o d u c t i o n of d r u g - c o n t a i n i n g m e d i u m into S y k e s - M o o r e c h a m b e r s was accomplished during filming by perfusion. A 2 . 5 m l v o l u m e of t r e a t m e n t m e d i u m was used to assure a d e q u a t e replacement of the 0 . 7 m l of n o r m a l m e d i u m present in a c h a m b e r (Sykes a n d Moore, 1960). Treatm e n t media were b r o u g h t to 37=:'C and, w h e n necessary, were gassed with 5~.o CO,. to adjust p H p r i o r to use.

Still photography Cells were plated o u t onto sterile coverslips in Petri dishes a n d i n c u b a t e d at 37~C in a n a t m o s p h e r e of 10°.o CO,,. T h e growth m e d i u m was replaced every 2 4 h r . F o r p h o t o g r a p h y the coverslips were inverted o n t o sterile glass slides to which a ring of stopcock grease h a d been applied to act as sealant. P h o t o g r a p h s were t a k e n with a Zeiss 35 m m camera a t t a c h e d to the same microscope assembly used for the time lapse

Analysis The 1 6 m m films were projected o n t o a quadrille-ruled screen for basic analysis. M e a s u r e m e n t s were m a d e from the projected images of single frames on a Craig film editor. M e a s u r e m e n t s of still m i c r o g r a p h s were m a d e directly f r o m p h o t o g r a p h i c prints. Calculations were based o n p h o t o graphs o f a calibrated stage m i c r o m e t e r m a d e with each p h o t o g r a p h i c apparatus.

Fig. 1. D-98S daughter cells connected by an intercellular bridge of approximately 13 t~ overall length. The right half bridge is folded into a wave near the periphery of its daughter cell. >~2000. Inset : The same intercellular bridge at a different time, showing a wave on the left half bridge. × 3900. Abbreviations used for Figs. 1 9: mb, midbody; w, wave; and tp, trailing process. Fig. 2. Daughter ceils connected by a short, 5/*, intercellular bridge. Note tim wide phase halo (arrows) near the bridge attachment point of the cell on the left, indicating a retraction of peripheral cytoplasm toward the center of the cell. × 2100.

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Observations T h e f o r m a t i o n of the intercellular bridge a n d m i d b o d y in telopbase, a n d the subseq u e n t n a r r o w i n g o f the bridge on each side of the m i d b o d y , are in D-98S ceils essentially as r e p o r t e d previously for H e L a (Byers a n d A b r a m s o n , 1968) a n d so will n o t be described here. Following these events the m i d b o d y is characteristically d i a m o n d or hexagonally shaped a n d 1"5 to 2 rt wide, projecting b e y o n d either side of the bridge (Fig. 1 a n d inset). The intercellular bridge has a m a x i m u m width of a b o u t 1 F* a n d m a y a p p e a r to be s o m e w h a t tapered, n a r r o w i n g f r o m the m i d b o d y t o w a r d each d a u g h t e r cell. T h e point at which the bridge joins a d a u g h t e r cell, to be referred to as the bridge a t t a c h m e n t point, is typically located at the periphery of the cell, a l t h o u g h in some cases it m a y be f o u n d near the nucleus with the bridge extending a b o v e the spread cytoplasm of the cell. In a few time lapse sequences of the final separation of two d a u g h t e r cells the m i d b o d y has been observed to r o t a t e a b o u t the long axis o f the bridge (Fig. 7). T h e changes in shape n o t e d during such r o t a t i o n indicate the m i d b o d y to be flattened in the plane of the coverslip w h e n it is oriented as in Fig. 1. D e p e n d i n g u p o n the activities of the i n t e r c o n n e c t e d cells the m i d b o d y m a y be positioned at any point a l o n g the bridge between them. F o r purposes of discussion the term halt bridge will be used to denote t h a t p o r t i o n of the intercellular bridge extending f r o m either edge of the m i d b o d y to the respective d a u g h t e r cell, regardless o f the relative physical lengths o f the two segments.

Length o f the hltercelhdar bt'idge Intercellular bridges of 3 to 5/,, as in Fig. 2, to greater than 20 t~ in overall length h a v e been observed. While n o counts were m a d e of the frequencies o f different lengths, general observations indicate p r o p o r t i o n a t e l y fewer examples of increasingly longer bridges. Time lapse sequences show t h a t bridge length is n o t static a n d m a y change considerably over a period of time, primarily in the direction of increased length. N o consistent pattern o f increased bridge length was f o u n d to occur in D-98S cells following mitosis, so that bridge length could not be used as an indicator for different stages of cytokinesis. W h e r e observed, increases in the length of the intercellular bridge prior to the events of final separation coincided with cytoplasmic m o v e m e n t s of the d a u g h t e r cells, associated either with active motility or with changes in cell shape. While D-98S cells are characteristically epithelial in general m o r p h o l o g y , they are capable of a certain degree of motiIity. F o r m a n y pairs of d a u g h t e r cells such activity is negligible or, where present, may be limited to small m o v e m e n t s a n d changes in shape which do not alter bridge length. In some cases, however, one or b o t h d a u g h t e r cells m a y display sufficient directed motility to increase the length of the bridge. I n the m a x i m u m displacement of this type observed b o t h d a u g h t e r cells exhibited motility, m o v i n g away from each other upon flattening o n t o the coverslip after telophase. F r o m a n initial length of 3 ~ the bridge was increased to greater t h a n 21 > in length over a 129 m i n interval. Actively motile cells a p p e a r more r o u n d e d

Fig, 3. The formation and movement of a wave is shown on tile left half bridge, beginning with the initial bending of the half bridge ill 3a. Note that the left half bridge becomes shortened through this activity, while the right half bridge, previously reduced in width, is stretched and lengthened. The time intervals for this and other sequences are relative to the first micrograph of the sequence. 3b, 420 sec; 3c, I 2 rain 30 sec; 3d, I 3 rain 30 sec; 3e, -~ 3 rain 60 sec: 3f, q 4 min 20 sec. ×4500. Fig, 4. Two stages in the reduction in widtb of the left half bridge. In this example the length of the ball bridge remained essentially unchanged during the process. 4b, ~ 7 min 45 sec. .: 2600.

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in shape and less flattened onto the substrate than do more stationary cells. They also display more locally organized leading edges of ruffled membrane activity, consistent with their direction of movement. Cytoplasmic movements which do occur tend to increase the distance between daughter cells and movements of all or a part of one cell toward the other prior to separation are rare. Cytoplasmic movements which increase the length of the intercellular bridge without a net displacement of either daughter cell are similar to those discussed under the topic of Cell separation.

Wave activity A series of movements occurs along half bridges of approximately 3 t~ or greater length. On the shortest of such half bridges these appear as distortions in the basic shape of the bridge which originate at, or close to, the midbody and pass along the length of the half bridge to its attachment point with the cell. The movements are similar on longer half bridges (for example 8 tt or longer) with the exception that the point of origin appears to be at approximately the midpoint of the half bridge. The basic features of this movement resemble those of the passage of 'thickenings' along the intercellular bridge of HeLa cells reported by Byers and Abramson (1968). Their use of the term 'wave' seems appropriate and it will be used to describe the similar event observed in this study. Close examination of individual 16 m m frames and still micrographs indicates that for D-98S cells a wave is formed not from a thickening of the bridge but rather f r o m a folding of it. This is most c/early seen near the attachment point of the bridge where the extent of folding for each wave is greatest (Fig. I and inset). The bridge itself appears to be flattened in the plane of the coverslip and is, thus, ribbon-like in shape with the folds directed toward and at approximately right angles to the surface of the coverslip. Individual waves can be seen to differ considerably in height or 'amplitude' on different half bridges. Up to two waves have been observed to be present at one time on longer half bridges (Fig. 4) although where the largest waves are formed only a single wave is present at a time. The number of waves present simultaneously on shorter

M U L L t N S A N D BIESELE half bridges was not determined due to the difficulty of distinguishing individual waves on them. The manner in which waves form is best followed in fihn sequences of the formation of large waves on a longer half bridge such as in Fig. 3. The initial step appears to consist of a movement of the half bridge in the direction of its respective daughter cell. Whether this m o v e m e n t originates in the cell or the bridge is not apparent. It does, however, bring about a bending of the half bridge at about its midpoint (Fig. 3a) and may also result in a m o v e m e n t of the midbody toward the daughter cell (up to 6/~ has been observed) and a corresponding increase in the length of the opposite half bridge. That these events are due to a force originating at the half bridge on which the wave is formed, or at its daughter cell, and not to a pushing force originating at the opposite cell or half bridge is indicated by the fact that they continue to occur after one half bridge has been essentially eliminated during cell separation (as in Fig. 3) and no significant cytoplasmic movements are observed in its daughter cell. The bend that is formed in the half bridge provides the folding which constitutes a wave. The wave then progresses along the length of the half bridge to the attachment point with the cell (Figs. 3b-f). Where measurements were possible the rate of this motion was found to vary from about 1 to 4/* per minute oll different half bridges. In cases where the midbody is noticeably shifted during the formation and movement of a wave the folded portion of the half bridge seems to be taken up by the cell as the wave reaches the bridge attachment point. The midbody does not shift back to its original position and the half bridge remains shortened while the opposite half bridge retains its increase in length (compare Figs. 3a and f). Where shifts such as these are observed only the half bridge which is shortened displays pronounced wave activity. Wave activity also takes place when no overall changes in half bridge length or position of the midbody occur. Waves are smaller under these conditions and appear to be equally present on both half bridges. The midbody typically undergoes small, back and forth displacements which might

C Y T O K I N E T I C A C T I V I T I E S IN H U M A N C E L L L I N E be accounted for by the unfolding of waves as they reach the attachment point so that the half bridge would straighten and resume its original position or by alternate, small changes in length of the two half bridges with wave activity.

Sel)aratio#l qf' da~lghter cells In contrast to the HeLa ceils studied by Byers and Abramson (1968), D-98S cells undergo neither a consistent, gradual increase ill overall bridge length nor a postmitotic rounding tip from the flatlened state prior to the completion of cytokinesis, although motile cells are somewhat rounded as has been noted. The event which precedes separation in all cases is the reduction of one or both half bridges to a cytoplasmic strand less than 0-5 F~ in width. On the shortest half bridges, such as that of Fig. 2, this is achieved by a constriction of the bridge next to the midbody. F o r bridges of greater length the reduction in width is associated with a movement of cytoplasm from the half bridge into its respective daughter cell. This may take place in essentially two ways. Following the formation of a constriction at one side of the midbody most of the cytoplasm of that half bridge may be moved back into the daughter cell with no changes in the length of either half bridge. The cytoplasm moves away from the point of constriction leaving a thin strand extending to the midbody as in Fig. 4. Wave activity takes place during these movements and may be part of the mechanism involved. Alternatively, a half bridge may first become progressively shortened with the passage of successive waves along it before any reduction in its width takes place. In this case a constriction develops after the midbody has m o v e d to the periphery of the daughter cell and is adjacent to the bridge attachment point (Fig. 6). This results in the situation illustrated in Figs. 6b and 3a where one half bridge has been reduced to a short, thin strand and the opposite half bridge has become correspondingly lengthened. The two processes described here are not always separate. A constriction may develop after a half bridge has become partially retracted through wave activity, allowing the first process to occur from that point. Final separation is brought about through

53

cell activities that stretch a ihalf bridge which has become reduced in width, causing it to become thinner and break. As a widthreduced half bridge is stretched and its length increases, it does appear to become visibly thinner in projected sequences, although the images of such thin objects are not sufficient for accurate measurements from individual frames. The thinning of the bridge is also shown, however, by the fact that small irregularities on it can be seen to become spaced progressively farther apart as it is stretched, indicating the attenuation of the bridge between them. Several cytoplasmic movements, either singly or in combination, may contribute to this stretching. A frequently observed movement results when a portion of the peripheral cytoplasm of a daughter cell, including the bridge attachment point, is drawn back toward the center of the cell, increasing the distance between the bridge attachment points and stretching the bridge (Fig. 5). The parts of lhe cell away from the bridge remain stationary, so that the cell is roughly semicircular or crescent shaped at the completion of the movement. The edge of the cell which has been withdrawn is smooth, with no ruffling activity, and is characterized by a pronounced phase halo, indicating its greater thickness compared to the remainder of the cell periphery (Fig. 5c). Similar profiles are also found before separation as seen in Figs. 2 and 9. W h e n a given daughter cell displays this type of movement either of the two half bridges may be stretched providing a reduction in width has occurred. In cases where a bridge attachment point is located on an extended cell process retraction of the process may stretch and break a reduced half bridge. Where one half bridge has been reduced to a short strand and the other remains extended and unreduced in width, separation may come about through the retraction of the extended half bridge. Wave motion is associated with the retraction as has been described. This type of activity is seen in the sequence of Fig. 3. Another variation of the separation process is seen when both half bridges are reduced in width and no cytoplasmic movements sufficient to stretch and break one of them take place. Alternate changes in the

54

M U L L I N S AND B I E S E L E

lengths of the half bridges and associated m o v e m e n t s of the m i d b o d y back a n d forth between the daughter cells may occur under these circumstances (Fig. 7). Displacements o f the m i d b o d y up to 12 tz have been observed. Such m o v e m e n t s are discontinuous a n d no constant rate can be obtained for them. The overall appearance suggests first one and then the other cell to be pulling m o r e strongly on the midbody. At some point during this activity one of the half bridges, the longer o f the two at that time, will break, completing separation. N o wave m o t i o n has been detected on the thinned half bridges during this type o f activity. It is during the m o v e m e n t s outlined here that the previously mentioned r o t a t i o n of the m i d b o d y may occur (Fig. 7). While before final separation almost all cytoplasmic movements of both daughter cells are away from their bridge a t t a c h m e n t points, this polarity is not maintained once separation is completed, and cytoplasmic m o v e m e n t s o f one cell toward the o t h e r do occur. N o consistent time factor has been noted for the completion of cytokinesis. Some cells have been observed to separate

within 3 5 hr after a n a p h a s e while others have been followed for periods up to 8 h r without separation.

Breakage of the midbody During the filming of one time lapse sequence the breaking apart of the m i d b o d y was recorded. Single frames from that sequence are included in Fig. 8. At the start of filming both half bridges had undergone the redtict i o n in width which precedes the completion o f cytokinesis. The m i d b o d y was m o v e d repeatedly back and forth between the daughter cells due to cytoplasmic movements which p r o d u c c d changes in length o f the two half bridges. During this activity the m i d b o d y a p p e a r e d to break into two pieces, one on each side o f the bridge's long axis (Fig. 8b). Subsequent to the breaking apart of the m i d b o d y the daughter cells ceased pulling against each other across the bridge, The bridge itself, which before was less t h a n 0.5 ~ in width on either side o f the m i d b o d y , b r o a d e n e d to a width of more t h a n 10 t~ by 214rain following breakage (Fig. 8d), a n d the cytoplasm o f the two cells became con-

Fig. 5. Separation of daughter cells. Following time formation of a constriction on timeleft half bridge (arrow, 5a) the right daughter cell retracted its half bridge and then withdrew the peripheral cytoplasm around its bridge attachment point. This resulted in the stretching and breaking of the left half bridge at the point of constriction. In 5d the withdrawn cytoplasm is seen lmmovingback toward its original position following the completion of cytokinesis. 5b, +8 rain; 5c, I 12 rain 15 sec: 5d, ! 28 rain 30 sec. 3. 1600. Fig. 6. Reduction in width of the right llalf bridge Following its retraction into the right daughter cell. 6b, " 9 rain 15 sec. " 2600. Fig. 7. Movement of the midbody between two daughter cells after botb half bridges have become reduced in width. The different profiles of the midbody in 7a and 7b resulted from the rotation of the midbody about the long axis of the bridge. 7b, t lOmin3Osec. :,2100, Fig. 8. Breakage of the midbody. In 8a the midbody is seen before breaking. In 8b-d the breaking of the midbody and merging of the daughter cells are shown. The midbody appears to be in two separate pieces in 8b (two arrows), but these are seen to be joined together in 8c and d (arrows).8b, +180min30sec;8c, ! 197min45sec: 8d, i 213 rain 45 sec. 7~1500. Fig. 9. Trailing processes extend from both daughter cells. Timearrows on 9a lmmrk foldings of a process which were seen to move from the tip of the process toward the cell. In 9b, taken 17 min later, the same process is seen to be reduced in width, as are others near it. 7 2200.

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CYTOKINETIC ACTIVITIES IN HUMAN CELL LINE tinuous. By the time of Fig. 8d the continuity between the cells was definite enough that they could be considered a single binueleate cell The midbody, as seen in Figs. 8c and d, did not actually break into two separate pieces. The two portions of it which seemed separate are actually connected, The shape of the broken midbody is somewhat suggestive of the ring-shaped midbodies reported by Buck (1963) for cultured rat erythroblasts. Trailing processes When an extended portion of cytoplasm is retracted in D-98S cells, as in the movements noted during cell separation, the peripheral cytoplasm is not uniformly withdrawn. At what are apparently points of contact between the cell and the coverslip the cytoplasm remains stationary and processes extend from the retracting edge to these stationary points. Structures of this type, as seen in Figs. 1, 5 and 9, will be referred to as trailiHg processes. The same type of structure is found extending from the trailing edge of motile cells. Trailing processes may be as much as 1 t~ in width and may exceed 20 t~ in length in cases of cell motility. As it is formed the process is usually not pulled taut by the retracting cytoplasm. Pronounced undulations of the process take place and the alternate thick and thin profiles observed for individual processes suggest them to have a flattened, ribbon-like shape. The tip of the process remains stationary during these undulations. Following its formation a trailing process undergoes a gradual reduction in width until it becomes so thin it is barely resolvable (compare Figs. 9a and b) although the tip may retain its original dimensions. Coinciding with this reduction in width the slack is removed from the process and it assumes a more linear appearance. With the loss of slack the undulations of the processes are considerably reduced and can be seen to resemble somewhat the wave activity of the intercellular bridge. The undulations appear to be loops, or foldings, of the process which form near the tip and pass along the length of the process to the cell periphery (Fig. 9a). Once the process has become reduced in width the undulations cease and it is retracted into the cell.

57

Cytochalasin B treatment Cells were filmed for periods up to 3.5 hr following the introduction of CCB-conraining medium into culture chambers. The effects of the drug at the 10 tLg per ml concentration were noticeable within 15 min after administration. At a concentration of 5 t~g per rnl the early effects of the drug were less pronounced but the results were generally similar to those at 10/~g- The general morphology of D-98S cells after treatment resembled that reported by Goldman (1972) for BHK-21 ceils. Some cells became rounded, often with extended processes, while others remained spread. Nuclear extrusion was common. The control medium, containing 1°.~i DMSO, did not appear to affect cells treated with it. Following CCB treatment the undulations of trailing processes ceased and normal ruffled membrane activity was disrupted, although movements at the cell periphery continued throughout the periods of observation, Where short processes projected from the edge of a cell they were seen to undergo changes in shape involving alternate increases and decreases in length and width. Cytoplasmic granules near the cell periphery were observed to move in and out of the processes coincident with these changes in shape, indicating a pulsating movement of the cytoplasm might be taking place at the periphery. The structural integrity of the midbody did not appear to be altered by the presence of CCB. The progression of waves on the intercellular bridge, however, was completely disrupted by the drug. Although typical wave movement ceased, what appeared to be outpocketings of the bridge, rather than foldings of it, appeared and moved along the lengths of the ball bridges. In some instances the outpocketings appeared to originate at the daughter ceils and in others on the bridge. The rates of their movements varied considerably, even for individual outpocketings at different times. Movements of the outpocketings both toward and away from the midbody occurred and in some cases single outpocketings were observed to change their direction of movement. As noted for the short processes, cytoplasmic granules were observed to move into and out of the half bridges after CCB treatment.

58

MULLINS AND BIESELE Discussion

There appear to be three constant features involved with the events by which cytokinesis is completed in D-98S cells: (1) before separation cytoplasmic movements of daughter cells are directed away from their bridge attachment points, which tends to lengthen the intercellular bridge or at least maintain tension across it; (2) separation is preceded by the reduction in width of one or both half bridges to a strand less than 0"5 ~¢ wide; and (3) separation is completed by cytoplasmic movements of the daughter cells which stretch and break a reduced half bridge. Where the intercellular bridge is longer than about 5 t~ the reduction in width of a half bridge is accompanied by the withdrawal of cytoplasm from the half bridge into its daughter cell. The cytokinetic process in D-98S cells thus differs considerably from that reported for HeLa by Byers and Abramson (1968). In contrast to HeLa, D-98S ceils undergo neither a postmitotic rounding up from the flattened state nor a consistent increase in the length of the intercellular bridge prior to separation.

Wave activiO' and bridge length The event of primary importance in HeLa would seem to be the lengthening of the intercellular bridgc through the passage of waves along it from the midbody to the daughter cells. Wave activity is also observed in D-98S cultures but only in cases where a half bridge has already been elongated by cell movement. Byers and Abramson described the waves in HeLa as 'thickenings' of the bridge, whereas for D-98S cells waves are clearly formed from foldings of a flattened, ribbon-like bridge. Examination of Figs. 8 and 9 from Byers and Abramson's paper, however, would seem to indicate that for HeLa the waves might not be thickenings, but rather undulations of the bridge, actually similar to waves as reported here for D-98S cells. While wave motion in D-98S cells was observed to result in a shortening of the half bridge on which a wave formed, and a lengthening of the opposite half bridge, the passage of a wave along a half bridge in HeLa was reported to bring about a lengthening of the half bridge. Byers and Abramson interpreted the increased bridge

length in HeLa as being due to a wave actually moving its respective daughter cell along the sheaf of microtubules extending from the bridge into each daughter cell. F o r this to occur it would be necessary for a wave to be of sufficient strength to overcome not only the inertial mass of its daughter cell but also the resistance arising from the cell's adhesion to the substrate. In addition, it is not clear why a wave with enough force to accomplish this would not also cause a displacement of the midbody toward the opposite daughter cell and a decrease in length of the opposite half bridge. it is possible to interpret the data for HeLa in a manner consistent with the observations obtained for D-98S cells in this study. Where pronounced increases in the length of the intercellular bridge of D-98S cells occur the cause seems to be that of active cell motility. This is consistent with the observations of McQuilkin and Earle (1962), who reported bridges stretched as long as 125/x for motile L-929 fibroblasts. As motile D-98S cells are more rounded in shape it might be suggested that the postmitotic rounding noted for HeLa is also associated with cell motility and that the increased length of the intercellular bridge is brought about by cell motility rather than by wave motion. While a completely rounded, spherically shaped cell would not be expected to exhibit motility, Figs. 4 and 5 from the Byers and Abramson paper show cells with ovoid, rather than circular proflies, indicating some flattening onto the substrate. The reported movement of a HeLa daughter cell with the arrival of a wave at its periphery might then be the result of a release of tension as the bridge became folded adjacent to the cell, allowing the cell to move away a short distance with the arrival of each wave. With regard to this idea it should be noted that wave activity continued on bridges being elongated by the active movement of D-98S daughter cells. The most reasonable assumption regarding the significance of D-98S wave activity would seem to be that it serves primarily to retract the cytoplasm of extended bridges into the daughter cells. In addition to the observations of bridge activity which have been given, the likelihood that this is the case is emphasized by the similarities in general form and activity between longer bridges

CYTOKINETIC ACTIVITIES IN H U M A N CELL LINE and trailing processes. Both are flattened extensions of cytoplasm resulting from cell movements, both display wave-like activity, and both undergo a reduction in width involving a movement of cytoplasm into the cell before the breaking of the bridge or the release of the attached tip of the process. In each case wave activity ceases once the reduction in width is achieved. The events seen in connection with the trailing process are most likely involved with its retraction into the main body of the cell, and it is reasonable to assume that the similar events on the intercellular bridge are involved with bridge retraction. In a sense, a long intercellular bridge might be regarded as a special case of the trailing process in which two processes from difl'erent cells each terminate at a common point, the midbody, rather than in separate attachment points on the substrate. Another cytoplasmic movement having resemblances to wave activity is that of membrane ruffling. Ingrain (1969) analyzed the ruffling cycle of cultured chick fibroblasts by filming the cells from a lateral view. He found the basic movements to consist of an extension of the leading edge, followed by an upward flexion of the extended cytoplasm and its subsequent withdrawal by the cell. The retraction of a half bridge through wave activity is similar and might be interpreted as repeated cycles of flexion and withdrawal of a previously extended portion of the cell's peripheral cytoplasm. The similarities in form of these three cytoplasmic activities, coupled with their common disruption by CCB, suggests that they may be different manifestations of the basic contractile properties of the cell rather than truly distinct processes.

Functional significance of the midbody It seems likely that the midbody has some functional significance for cell activities, most likely those of cytokinesis. It is a relatively large structure and its appearance follows a constant pattern in the events of cell division. The formation of the midbody is coordinated with the development of the division furrow and the midbody remains as a central element in the intercellular bridge which persists between daughter ceils for some time after furrowing. The questions which seem reasonable to ask here are those

59

of why furrowing halts after progressing to a certain point, and what is the relationship of the midbody to this activity. A considerable amount of electron microscope work has recently been offered as evidence for the hypothesis that the division furrow is formed by the activity of a contractile ring of microfi[aments located beneath the plasma membrane of the furrow (see Schroeder, 1970, 1972, for review). If such a ring is present it is likely that the extent of its contractility is limited, and that this limit is marked by time maximum penetration of the furrow. The midbody, then, might stabilize the division process at this point, possibly maintaining the furrow and serving as an effective point of separation between daughter cells until the completion of cytokinesis. The idea of the midbody as a division point between daughter ceils prior to their final separation is given some support by our observations. On long intercellular bridges wave activity moves along the half bridges from each side of the midbody towards the respective daughter cells, and it has been noted that any movements of one daughter cell toward the other are rare before separation but do occur afterwards. The sequence of Fig. 8 suggests that this tendency of interconnected daughter cells to be distinguished from each other and to move away from each other may be mediated by the midbody. When the midbody was intact the cells pulled actively against each other across the intercellular bridge. With the breakage of the midbody the cells ceased to move in opposition to one another and cytoplasmic continuity between them was re-established. While no general conclusions can be drawn from a single observation the possibility certainly exists that the midbody provides a structural means by which one daughter cell is effectively distinguished from the other and by which the activities leading to separation are organized. In fact, this might be accomplished simply by contact of the midbody and plasma membrane, as is indicated by an isolated observation made during a treatment with colchicine. What was judged to be an intercellular bridge with midbody was selected for filming. Wave motion was present on the bridge. During filming the presumed midbody detached from the

60 bridge and floated free. With this occurrence wave activity on the bridge ceased. It does not seem likely that a midbody, which is normally within the plasma membrane, would detach in this manner, nor that colchicine would induce such an event. What was thought to be the midbody may have been only a particle of debris attached to the cytoplasmic bridge. If so, it appeared to function as a midbody by providing a division point on the bridge in terms of wave progression, and after its loss, no more waves were formed. Cytochalasin B At the concentrations used CCB did disrupt wave motion on the intercellular bridge, the undulations of trailing processes and membrane ruffling; the similarities among these activities has already been noted. We had thought that CCB might disrupt the midbody in some way, but no visible changes in its structure were observed, and recent work by Krishan (1972) has shown that midbodies do form in L cells after several hours in CCB at concentrations to 10 p~g per ml. There is still a controversy as to whether the effects of CCB are on groups of contractile microfilaments (Wessels et al., 1971) or on other properties of cells (Estensen, Rosenberg and Sheridan, 1971). The references given outline the various arguments well, so the different hypotheses will not be entered into here. If the work indicating that contractile microfilament activity is important in cell flmctions is correct, particularly the work of Spooner, Yamada and Wessels (1971) on membrane ruffling and cell locomotion, then it would seem very likely that similar networks of microfilaments would be present beneath the plasma membrane in the intercellular bridge and trailing processes to account for wave-like movements in these structures. Some of our observations on cytokinesis in untreated D-98S cells would seem to have some relevance toward certain arguments as to the manner by which CCB inhibits the separation of daughter cells, Estensen (1971) suggested that since furrows often form and then regress in the presence of CCB that the drug might exert its effect by inhibiting the fusion of mcmbranes. The only point, however, at which membrane

MULL1NS AND BIESELE fusion would seem to be of importance in D-98S cells is at the breakage of the intercellular bridge, an event which follows furrowing by 3 or more hours and which is preceded by a reduction in bridge width and by cell movements which stretch the bridge. /t might be suggested that a more likely point for the action of CCB would be in the events that lead to the stretching and breaking of the intercellular bridge. It is possible that in the case of the suspension cultures used by Estensen, where the factor of cell motility would be eliminated, that the important mode of action for CCB might be inhibition of membrane fusion, but no evidence was offered to support this idea. Krishan (1972) filmed L cells in CCB and concluded that the drug prevented separation by inhibiting the movement of daughter cells away from each other: he did not believe the drug to affect furrowing since division furrows formed and then regressed in its presence. This is in agreement with our observations that D-98S daughter cells do move away from each other before separation and that various cytoplasmic movements are important in stretching and breaking the intercellular bridge. It is not clear, however, why inhibition of cell motility should cause the division furrow to regress and lead to the fusion of daughter cells. The fact that Estensen (1971) found CCB to inhibit cytokinesis effectively in suspension cultures of Novikoff hepatoma cells argues against the importance of cell motility. The events of Fig. 8 in this study have led us to suggest that the midbody may be of importance in cytokinesis, and it is possible that CCB may act by preventing an association between the midbody and plasma membrane or cortical cytoplasm of the furrow base as furrowing ceases. Certainly the manner by which CCB inhibits cytokinesis is not yet resolved and will require further investigations of several cell functions.

Acknowledgements This investigation was supported by N I H Training Grant No. 5 TO1 GM 00337-12 from the National Institute of General Medical Sciences. J. J. Biesele is the recipient of research career award 5-K6-CA-18366 from the National Cancer Institute.

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References

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