CELL MITOSIS BY WILDER D. BASCROFT .4ND CHARLES GURCHOT
Some years ago, our attention was called by Professor Chambers and M r . I. H. Page of the Cornel1 Medical College t o the fact that the physical cheinistry of cell division was in a rather confused stat,e, and we were asked to give our opinion as t o the general theory. All the experiments were done by Mr. Gurchot . The general problem can be stated in a very simple manner. If we have a drop of liquid and if we lower the surface tension locally a t two opposite poles, d l the drop constrict and tend to break into two drops a t the equator where the surface tension is relatively high or along a line passing through the poles, where the surface tension is relatively low? In 1876 Biitschli stated that such a drop would split along the equator, it being pinched in two by the action of t,he relatively high surface bension along that line. T. Brailsford Robertson’ has put forward and defended the opposing view t’hat the drop pulls apart where the surface tension is the lowest,. “In a continuous fluid surface of variable tension the regions of high superficial tension must be convex to the outer medium while those of low superficial tension must be concave to the outer medium. If this concavity occurs all round an equatorial circumference a dumb-bell shaped surface mill be produced of which the equatorial (approximately cylindrical) portion may be short or long according to whet,her the slope of the cleft’ is steep or gradual. If this process (reduction of tension) proceeds sufficiently far to cause the 1engt)h of the equatorial cylindrical portion to become equal t o its circumference, it rvill break and one drop will split into two.’’ The flaw in this reasoning is the assumption that an area of relatively high surface tension must be convex to the outside medium. It is true that a drop of one liquid at the bottom of a beaker filled with another liquid will be more nearly spherical, the higher the surface tension. With a lower surface tension the drop will flatten out more under the influence of gravit.y. The flattened drop is less convex than the spherical one on the top and more convex a t the sides-a point which Robertson has overlooked. Robertson then introduces the tacit, assumption that a further lowering of the surface tension would cause a greater effect in the same direction and that, having gone from convex to flat on the upper side, it will then go from flat to concave. In the first place, it mill not do that. The drop will spread out t,hinner and thinner, until it forms a layer one, or at most two, molecules thick, after iyhich it will spread no more, so far as we now know. In the second place, this has very little to do with the case in question, because we are not dealing with localized differences of surface tension except in so far as the action of gravity sets up different surface tensions in different parts of the drop. .irchiv Entwicklungsmechanik, 27,
29
(1909); 32, 368
(1911);
35, 692 i I 9 r l :
CELL MITOSIS
431
Robertson offered some experiments with olive oil in support of his views.
He placed a thread soaked in alkali round a drop of oil submerged in water by addition of chloroform. Since the alkali reacts with the olive oil to form soap, this lowers the surface tension. The drop separates into two drops along the line of t,he thread, as though it had been cut in two by the thread. At first sight this experiment seems conclusive. McClendonI supports Butschli’s views that a drop of liquid will tend t o split into two drops along the line where the surface tension is relatively highest. He considers “that in Robert,son’sexperiment the oil drop is divided by the gravitation of the thread which has been soaked in an alkaline solution. The alkali reduces the surface tension, t,hus reducing the resistarce t’o division, and thus allows division by a thread of less weight than would otherwise be necessary. This seems to be a just criticism because Robertson? admit,s that “light threads of sewing-cotton cannot be employed with success as they float upon the top of the oil-droplet without sinking into it; the diminution in tension is, consequently, not equatorial. I obt’ain the best results with linen threads 0.4 mm. in diameter. Linen threads 0.2 mm. in diameter only, float upon the surfaces of the drops and rarely cause division.’’ Robertson accounts for this fact in a different, way. He says, when discussing the weight of the thread, t,hat “it is not sufficient that a small sector of an equatorial circumference of the drop be touched by alkali; it, is necessary that soap should be formed in the periphery of an equatorial plane. This cannot occur unless the weight of the thread is sufficient to cause it t o sink a little way into the drop.” It apparently did not occur t o Robertson that he could soak the thread in soap instead of in alkali. McClendon did a set of experiments of his own. “It is not my ambition to imitate cell division exactly with a model; but an analysis of t,he following experiment may throw some light on what’ happens when a cell divides. X stender dish is half filled with distilled water and a funnel inserted so that the stem reaches the bottom. A saturated solut,ion of pure sodium chloride in distilled water is poured very slowly into the funnel so that the salt solution comes to lie beneath the pure water without much mixing. About I cc. of the chloroform-oil mixture is poured in, so that it forms a drop suspended in the liquid. Two pipettes are filled with fifth-normal sodium chloride solution and quickly introduced into the water, and the alkali allowed t o flow gently against opposite poles of the drop. If the alkali reaches both poles a t the same time and in the same quantity, the drop elongates along the polar axis and usually constricts into an hour-glass shape. I t often divides into two at the constriction. The conditions necessary for complete division are very limited, as is explained in the following analysis of the experiment. “The alkali reduces the surface tension at the poles and the pressure [?I inside the drop causes a bulging of these regions. At the same time the polar Am. J. Phgsiol., 27, 240 (1910);Archiv Entwicklungsmechanik, 34, 263 (1912); 37, 233 (1913). Archiv Entwicklungsmechanik 35, 695 (1913)
43 2
WILDER D. BASCROFT AND CHARLES GURCHOT
surface films spread and the equatorial surface film contracts, producing vortex movements in the drop and in the medium. These movements may he demonstrated by mixing a little soot with oil and a few carmine grains with the water. The surface currents carry the alkali toward the equator and enlarge the polar regions of reduced tension. The rapid fall in the internal pressure caused by the rapid enlargement of the surfaces of low tension allows a constriction of the equatorial region. The equatorial surface is not concave (as Robert,son intimates) but remains convex until the meridional concaveness equals the equatorial convexness. When the surfaces of reduced tension have espanded until the area of high tension forms but a narrow band around the constricted equator, this band acts like an isolated band of a tension equal t o the tension of its surface minus tke tersion of the surface of t,he polar regions. The entire force of this residual tension is exerted toward the division of the drop. However. the completion of the division takes time. and the band is reduced rapidly in jvidth i:y the spreading of the alkaline surfaces. If the constrict,ion is not conipleted before t,he alkali spreads over the whole drop. t,he drop returns t o a spherical shape. Therefore it is unusual that a complet,e division of the drop occurs. Failure t o perform this experiment, is due t o lack of dexterity. The smaller the drop, the (relatively) greater is the dexterity required. I’ipett,es with capillary openings are more easily controlled, and the concentration of alkali may be increased to compensate for reduced quantity. but it should not be heavier than the salt solution.” Robertson says: “I have repeated this experiment of 3IcClendon’s a number of times and under a variety of conditions and I regret t o have t o admit that I have hitherto been unahle t o ohtain precisely the appearance which he illustrates. I?oubtless t,his is due to some defect in my technique; but, granting this t o be the case, the phenomenon tiescrilied by McC’lendon is only indirectly clue to molecular forces and does not bear upon the mechanics of cell division at all. “Briefly, what I observe in repeating ,\Ic(.’lentlon’s experiment is the following:-as soon as the alkali touches the drop of oil (which is resting upon the flat bottom of a glass vessel). the drop ceases to be spherical and flattens quite suddenly. Transiently, the flattened mass may bulge in irregular shapes towards the pipettes; but the usual effect (if the alkali be not too strong) which immediately succeeds this ii: a gat,hering up of one or other or both of the equatorial extremities of the mass into a more or less globular form and an elongation of the inass equatorially in a direction perpendicular t o the line joining the openings of the pipettes. If this takes place a t both equatorial extremities at once, division of the mass into two may actually occur in a plane perpendicular t o t,he plane of division which is depicted by McClendon. “It is easy t o understand what is really taking place under these circumstances. The forces acting upon a drop which is resting upon a flat surface are not purell- molecular: gravity is also acting and tending t o flatten the drop and cause flowing in a lateral direction. This tendency is resisted by the surface tension of the tlrop : but, if the superficial tension lie reduced over any
CELL MITOSIS
43 3
considerable area, bulging will occur and the fluid will flow towards the side of reduced tension because the centre of gravity of the fluid mass is lowered thereby. ?rTolecular forces, although reduced, are still effective, however, and, if the mass be not too large or the t,ension too much reduced. the region of high tension will become highly curved and the regions of low tension indented in accordance with the principles already made clear, so that t’he fluid mass becomes elongated equatorially in a direction perpendicular t o t’he line joining the openings of the pipettes. “That t,he flowing and bulging of the drop towards the region of reduced tension is really due t o gravity and not to molecular forces is readily seen from the following experiment, Saturated sodiuni chloride solut’ionis poured into a glass cylinder until it approaches near to the top, and then the cylinder is inclined and distilled water poured gently down the side so as to avoid so far as possible the mixing of the two fluids. I n this way we obtain a cylinder of fluid of continuously varying density increasing from above down. On no\v introducing into this, drops of t,he chloroform-oil mixture described above, we find that t,hey float in the fluid a t a little distance tielow the surface, where the fluid is of the same density as the drop. Under these circumstances the drop hehaves as if it were weightless; only molecular forces affect it and gravity has no action upon it.’ On now applying tenth-normal caustic soda t o tn-o poles of such a drop by means of pipettes, we find that no bulging of the drop towards the pipettes appears a t all: on the contrary, the poles flatten and the equatorial region becomes highly curved, so that the drop is converted into a disk of which the long diameters are perpendicular t o the line joining the orifices of the pipettes. If tenth-normal caustic soda be employed. the result, is in no way different,. The rationale of this phenomenon will be apparent from what has preceded.” This apparently brings the matter t o the point that a mixture of olive oil and chloroform behaves one way for AIcClendon and another way for Itobertson, in each case behaving as t,he experimenter thinks it should theoretically. It is a beaut,iful instance of how difficult it, is to tell just what an experiment really shows. Some light has been thrown on the subject by the experiments of Spekj2 who agrees with McClendon that, a drop tends to split into two drops along the line at’ which the surface tension is relatively highest. Spek worked both with mighted oil drops which lay on the bott,om of the beaker and with drops which stayed suspended. While both kinds of drops give the same results in general, he thinks that it is more satisfactory to work with drops on the bottom of the beaker. I n his first experiment he let, a caustic soda solution flow out of two pipettes and impinge on opposite sides of an oil drop which had been colored with alkanna. Under these conditions the formation of a film of solid soap on the poles of the drop becomes visible, and he considers this objectionCf. J . Plateau: “Statique des liquides sournis aux seules forces rnoleculilires,” 2 (1873); Mach: “The Science of LIechanics,” 384 (1902). Archiv Fhtwicklungsmechanik, 44, 5 (1918).
43 4
WILDER D. BANCROFT AND CHARLES GURCHOT
able. He therefore touched opposite sides of the drop with crystals of sodium carbonate. In this way he was able to make drops of bergamot oil, of an oil used for night lights and mixed with olive oi!, of castor oil, of cedar wood oil, and of pure oleic acid divide at the equator, the best results being obtained with the mixture of the night-light oil and olive oil, and with bergamot oil, When drops of mercury in dilute acid were touched a t opposite points with crystals of potassium bichromate, the mercury divided along the line of highest (relative) surface tension. Spek was also able to confirm Robertson’s apparently contradictory results. “I was never able to obtain a constriction a t the equator with drops of pure olive oil suspended in water. Regardless whether one touched the drop with one or with two crystals of soda, the drop always separated into a lighter and a heavier half. If one touches the drop on the right and left, with soda crystals, the plane of division was always a horizontal one passing through the two poles, just as Robertson describes it. If the differences in the densities happen not t o be sufficient to cause a splitting, the drop stretches in a vertical direction. The explanation of this contradictory result is undoubtedly to be found in a special behavior of the olive oil [or of the olive oil soap]. Xone of the other oils studied by me and not even mixtures of olive oil with other oils showed this separation into a higher half. With the other oils the effects due to differences of surface tension were not obscured by disturbing circumstances as in the case of olive oil. “I doubt, therefore, whether McClendon used pure olive oil. If he didand I don’t know what really pure olive oil is-it had different properties from the oil used by Robertson and by me.” Without making special experiments, it is impossible to say what the difference was between the olive oil used by Robertson and Spek on the other hand and by McClendon on the other hand. I t may have been a lack of free acid or a difference in stearin content’. It seems t o us that Spek has settled the matter satisfactorily and that a liquid drop does tend to divide along the line of the highest (relative) surface tension. Since the theory of the matter did not seem clear to Professor Chambers nearly seven years after the publication of Spek’s paper, it seemed desirable t o repeat some of Robertson’s work. Before describing the experiments done in the Cornel1 laboratory, there is another point of disagreement between Robertson and 11cClendon which should be mentioned. McClendon’ says: “The mechanics of cell division may be illustrated by a more tangible model. A rubber balloon is inflated with air and attains a spherical shape. The rubber may represent the surface film and wit,h uniform thickness of rubber we obtain. uniform tension and spherical shape. If the equator of the balloon is re-inforced with a rubber band, the tension along the equator is increased and the balloon is constricted equatorially .” Robertson2 says that McClendon forgets that a rubber balloon can in n o way afford an analogy t o a fluid surface because, in the first place, adjacent Archiv Entwicklungsrnechanik, 34, 265, ( 1 9 1 2 ) . Archiv Entwicklungsmechanik, 34, 701 (1918).
CELL MITOSIS
43 5
non-continuous portions of its surface do not attract' one another and, in the second place, the tension of rubber varies with the degree t o which it, while that of a surface does not. The falsity' of this analogy has repeatedly been insisted upon by physicists." An analogy may not be complete in every detail and perhaps never is; hut that, is no reason why an analogy may not' be valuable. I t is a reason why one must guard against falling into error when making use of an analogy; hut it is quite unjustifiable t o say that a rubber balloon can in no way afford an analogy t o a fluid surface. I n most of the experiments done in the Cornell laboratory, advantage was taken of the technique developed by Robertson and by McClendon, but with modifications ITherever necessary. For instance, McClendon used rancid olive oil, which is a bit indefinite. When olive oil has been allowed to become rancid spontaneously, one cannot be sure about the amount of free acid in a drop of oil. An insufficient amount will keep the drop from dividing rapidly; it may divide very slowly or even not a t all. Consequently the oil used was made rancid artificially by adding oleic acid. Ten percent free acid was found to work very satisfactorily, Chloroform was also added, to increase the density of the drops of oil. h drop of oil was placed in a large dish of distilled water and enough sodium chloride solution added to enable the drop to be suspended. The salt solution was mixed thoroughly with the water so that there was no concentrated bottom layer. Robertson's experiment of splitting a drop with a thread impregnated with alkali was perfornied first. As Robertson does it, a thread of given diameter and impregnated with a sodium hydroxide solution of a given concentration is looped around the middle of an oil drop situated a t approximately half a centimeter below the surface of the water solution. The experiment is not an easy one to do, and it is rather unfortunate that such a painstaking technique v a s developed, for it will be shown that Robertson's experiment proves nothing whatever. This conclusion is based partly on the conviction that any experiment which can be interpreted from several antagonistic points of view is valueless in proving one to the exclusion of the others. I n the first place, if on coming in contact with the inipregnated thread, the drop contracts to some extent, the loop of thread must be made smaller to keep pace with the contraction or the contraction will cease and the drop will not divide. T o do this and at the same time to be sure that the drop is not being cut by the thread is something of an achievement. If Robertson's technique is followed strictly, the case is much worse. On beginning the experiment Robertson tightens the loop just sufficiently to einbetl the inipregnated thread in the drop. Inevitably a soap film is formed along the path of the thread and also below by diffusion of the alkali toward the center of the drop. The soap film dissolves and, obviously, the drop splits at least as far down as the thread. If the thread is pulled tight, so as to embed it in the drop once more, the splitting will continue and eventually the drop will split completely ~.
' Cf. for example,
338 !1892).
C. 11. llinchin: "Hydrostatics and I'.lernentar?- IIyrlrokinetics,"
43 6
JVILDER D. BASCROFT A S D CHARLES CCRCIIOT
in two. Therefore Rohertson’s method of decreasing the surface tension of a drop at the equator may very likely cause a drop to divide; but probably surface tension has nothing whatever to do with it. Or if it has, there is no way of finding it, out. But suppose the drop should divide when the threat1 merely touches the equator; and one is sure that the drop has not been strangled. Here an antagonist to the “equatorial theory” can suggest that a momentary bulging of the drop in the region of the equator has caused the thread to become ever so slightly embetided and the drop will divide in the manner shown above. As a matter of fact about one per cent of the number of drops experimented upon were observed to tlividr. McC’lendon’s exprriment does not seem to be open to serious objections and it was the next to be tried. Here the surface tension is lowered in the region of the poles. Instead of resorting to pipettes which allow dilute caustic soda to flow against the opposite poles of the drop, glass tubrs were used (inside bore z mm) bent into right angles antl pluggwl with cotton at the end intended to come in contact with the drop. The tubes were filled to the same level with concentrated caustic soda solution. The ends were brought barely in contact with the poles. The caustic soda solution oozed out slo~vlythrough the cotton plugs and the drop divicletl, almost immediately in the majority of cases, into two equal or unequal halves without any appendages remaining as a reminiscence of a former attachment. This experiment can be performed with comparative ease once everything has been properly adjusted. There can be no question concerning its significance. A rather amusing, even perhaps a little malicious, variation of the above experiment, is interesting. If a bare thread is looped around the equator of the drop and if, at the same time, the surface tension is retluced a t the poles the drop will divide in the usual way. The paradox here is that if some oppofients of the “equatorial theory” feel impelled not to recognize the validity of the foregoing experiment they are called upon to explain the fact that an ordinary thread looped about the equator of an oil-drop causes the latter to divide. But everything does not rest hrre. If a drop divides because the surface tension is reduced at the poles there is yet a phenomenon which remains to be demonstrated. If the drop really divides, it does so by virtue of the fact that the surface skin is stretched toward the equator since the tension is raised there. Eventually the drop is squeezed in two. Consequently, a point on the surface near a pole should be seen to move toward the equator during the process of division. If hIcClendon’s experiment is performed in a medium containing commercial sodium chloride instead of the pure salt the phenomenon is demonstrated very easily. The commercial salt contains calcium or magnesium salts or both. These react with the caustic soda antl enough calcium or magnesium base io produced to form a cap of insoluble soap at the poles of the drop. V’hen caustic soda is applied at the poles as before, t h e two insoluble soap films arr pulled instantly over the surface of the drop antl inert in the initltlle to fori11 a white equatorial band. This effect takes place :o the same extent xvhether the drop is touched at the poles only momentarily,
CELL MITOSIS
43 i
so as to prevent complete division, or whether the process is allowed to go to completion. S o such bad effect was observed when pure sodium chloride was used in the immersing medium. Here it may he well to recall an experiment performed before all the others and n-hich, although perhaps not conclusive in its results, nevertheless showed interesting possibilities. thin-walled rubber balloon was filled with about I O O cc of distilled water. This simulated a large drop or a large cell covered xith a membrane. The balloon lyas immersed in a three-layer liquid contained in a tall vessel. The bottom layer was carbon bisulphide, the middle one tlistillecl water and xylene piade up the top layer. The south pole of the balloon touched the carbon bisulphide layer and the north pole touched the xylene layer. Since xylene and carbon bisulphide both eniulsify rubber there resulted a lowering of the surface tension at the poles of the balloon. TJnfortunately, carbon bisulphide emulsifies rubber too quickly and the balloon burst. The procedure was repeated but this time xylene alone was used and noTv the surface tension was reduced a t one pole only. After having stood fur about fifteen minutes the once spherical balloon now assumed an oval shape. The apex of the oval rested on the bottom of the vessel and the wide portion rested against the xylene layer-part of the kalloon being actually immersed in the layer. I t should be pointed out that, notwithstanding the hydrostatic pressure, the water in the balloon was squeezed toward the region of lessened surface tension. Here was an indication that the bulging of a drop in the direction of lessened surface tension is at least not an untenable hypothesis. If this last experiment is only an indication of where the truth may lie, the insoluble soap film experiment leaves no room for doubt about where the truth does lie. 1he experimental part of this manuscript x a s sent to Professor Chambers and 1 I r . Page early in 1 9 2 j and it is a pleasure to add a few paragraphs written by 1Ir. Page. "We are now in a position to examine the evidence as demonstrated by observation of the living cell during the act of cleavage. for and against the two theories of cellular division. "-A hrief description of the process will offer a basis for comparison with the facts observed in the dividing oil drop. Kot long before division occurs, the viscosity of the protoplasm decreases markedly as demonstrated hy Chambers' with the microdissection needle, and by Heilbrunn* using the centrifuge method. Active Erownian movpment is set up in the region of the furrow which is yet another indication of this decrease in viscosity. That this viscosity change is important as a precursor to interfacial tension changes cannot be doubted. Further, Just3 has shown hy the use of cyt,olysis by hypotonic solutions that the polar region becomes the point of least resistance to the osmotic bulging of the egg. Here lye have an indication that the pole is the region of low resistivity. Tan Beeden' made the interesting observation J. Exp. Zool., 23, 483 (191j:.
* J. Exp. Zool.,
34, 41; ! 1 9 2 1 ~ .
Am. J. Physiol., 61,505 ' 1 ~ 2 2 ) . Bull. Acad. roy. Belg., 41 (1tih3 I .
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WILDER D. BASCROFT A S D CHARLES GURCHOT
that the Ascaris egg before division shows an outward bulging t o form more or less distinct lobes! almost amoeboid in aspect ab the two poles. Preceding and concommitant with these phenomena the development of the tnFo asters and spindle forming the amphiaster occurs. The egg elongates (“karyokinetic elongation”) as the amphiaster attains maximum size. The cleavage furrow now cuts the karyokinetic axis at right, angles. Ho-xever, it does not appear essent’ial that the aster be present inasmuch as constriction division may occur in anastral types such. for example, as noted by Farr‘ in pollen forming divisions of various dicotyledonous plants. Particularly significant to note is that a peripheral floiv occurs towards and not away from the equator. Thus in Arbncia eggs pigment granules collect around the cleavage furrow in such a way as to demonstrate strikingly this type of viscous flow. Spek2 especially has studied these vortical currents in many types of living eggs on division. We see that the flow is away from the area of low to one of higher tension precisely as in the oil experiment in which an insoluble calcium soap was formed at the poles. It will be recalled that in this experiment the soap cap migrated to the equator to form a white band around that region, the flow being toward the area of greater tension, namely the equator. “Butschli3 in 18;6 suggested that division was brought about by an increase in interfacial tension subsequent to nuclear division in the equatorial region of the egg. He subsequently elaborated this thesis with little change in the principles involved. ‘ W e are forced to the conclusion from the preceding experimental work that the lowering of interfacial tension at the poles accounts for the division of an oil drop under the stated experimental conditions. Insofar as the dividing oil drop parallels the cleavage of the cell we may believe that similar forces are involved in this latter process. W t h i n defined limits this physical system does mimic fission division.” The general conclusion to be drawn from this paper is that Butschli, McClendon, and Spek were right and that Robertson was wrong. When the surface tension of a drop of liquid is lowered locally a t two opposite points or poles, the drop tends to divide equatorially, along the line of highest (relative) surface tension. I t is literally pinched in two by the contraction along the line of t,he highest (relative) surface tension and it consequently bulges a t the poles, which are, by definition, the points of lowest (relative) surface tension. In view of the abnormal results which Robertson and Spek obtained with their so-called pure olive oils and in view of the apparent effect which the nature of the soap has, these experiments should be repeated with caustic potash substituted for caustic soda or potassium carbonate substituted for sodium carbonate. I t would also be well t o t r y substituting a solid soap or a soap solution for the corresponding alkali. Although these experiments are both interesting and desirable, they are not necessary to the purpose of this paper and consequently have not been done by us. Cornell Cnirersity. Am. J. Bot., 5 , 3;9 (1918). Archiv. Entnlcklungsmechanik, 44, 5 (1918). Abh. Senckenberg naturforsch. Gas., 10, (1876)
