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The effect of visible light on the mitotic spindle
Exprim~nlnl
Cell Research 21, 261-273 (1960)
THE EFFECT
261
OF VISIBLE LIGHT MITOTIC SPINDLE MRIDULA
DATTA
Bose Institute, Received
ON THE
Calcutta,
September
111&a
5, 1959
R I.c~.\ : 1.1..I
investigations on mitosis in living tissues have helped to dra\\ attention to the question of the nature of the spintlle body. Follo\ving the use of special optical techniques, the structure has been viewed in as nearly the natural undisturbed state as possible, care being taken at the same time to eliminate from the procedures, treatments lvhich might least tend to alter the arrangement of spintlle proteins. In thr search for material, it \\-as necessary to consider the property of its hein, 0 able to divide normally in a fluid medium and in this respect animal material, though restricted as to source, generally proved suitable [8-l 3, 1 (i-1 81.
The study of mitosis in the higher plants was pOSSibk in a few instances. Wada [19, ‘LO] used the strands of single cells of the stamina1 hairs of Tradescanfia with the view to minimizing cell wall interference. Rajer [l, 2, 3, 41 successfully cultivated the technique of mounting unwalled cells of the endosperm of several flowering plants, using agar and sugar solution as sealing media. The author [5,7] reported the occurrence of mitotic division in nuclei of the endospermal milk of some Palmae fruit. These nuclei, which are suspended in the watery sap of the embryo sac, are entirely free of cytoplasm and cell wall. The cytoplasm is confined to the syncitium layer, later becoming walled, which lines the walls of the endocarp. Nuclei breaking away from the syncitium are suspended and eventually undergo mitosis in this sap or milk as it is called, the morphological nature of which has yet to be determined. This sap is of low viscosity and its refractive index is lower than that of cytoplasm, whereas that of cytoplasm and nucleoplasm are almost equal. Cell inclusions and mitochondria occur in the milk. Nuclei are more sharply defined in this medium than when seen against cytoplasm, helped by the presence of the’ persistent nuclear membrane which can be demonstrated to be present at all the stages of mitosis with the micromanipulator probe and by the action of coumarin [6] which in certain concentrations has the effect of making the entire spindle shrink in size and draw away from the membrane as it does so. The spindle is structureless under the light microscope and can be detected mainly due to the cytoplasmic granules outlining it. This circumstance has led Wada [20] to suppose that the nucleus never loses its membrane. This enables it to exclude cytoExperimental
Cd
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Datta
plasmic matter from the nucleoplasm which pervades the spindle. Observations to the contrary, and in support of the general belief, are to the effect that the membrane dissolves at early prophase. As it is not possible to visually differentiate nucleoplasm from cytoplasm, it is not known with certainty as to out of the following two possibilities which actually occurs. In the course of mitosis the nucleoplasm and cytoplasm may remain distinct from each other if always separated by a membrane, otherwise with membrane dissolution at prophase they may mingle. Also, both might combine to form the spindle figure; alternatively this latter might be formed out of nucleoplasm alone. Evidence from different sources shows that the mechanism differs according to the material. In the Palmae fruit under discussion, however, it is clear that the nucleoplasm is at all stages separated from the surrounding fluid and that spindle formation is strictly It will be noted later that the spindle breakdown occurs and the intranuclear. products of that breakdown remain, within the nuclear membrane. Under phase contrast the outline of the living spindle may be discerned [l, 2,3,4, 9, 10, 191. The property of birefringence of the spindle fibres has made it possible to study some aspects of spindle structure with the help of the polarization microscope [S, 11, 16, 17). The orientation pattern of fibrils in fixed preparations has been deduced from electronmicrographs (10, 151. These studies have helped to establish Bajer [2] demonstrated that spindle formacertain facts as to spindle structure. tion began at the time of, if not before, membrane dissolution, the orientation proceeding from the polar caps towards the equator. Mazia [lo, 111 and Swann [ 16,171 have established the reality of the spindle fibres and the fact that they are orientated with reference to the mitotic centres which are the centrioles and centrosomes.They have shown that spindle elements are orientated parallel with reference to the long axis of the spindle and that individual elements may be integrated into fibres. In fact it is due to the diffusion of certain structural agents released by the mitotic centres at the laying down of that poles and at the anaphase movement of the chromosomes, that spindle fibres are aggregated out of the unorientated plasm. The arrest of the smooth flow of the mitotic movement by the action of colchicine has been explained by \Vada (191 and InouC [S] as attributable to the fact that spindle structure has suffered alteration. Inoue believed that a longitudinal contraction of the fibres was taking place in the spindle, which under the action of colchicine was rapidly decreasing in length, although negligibly so in width. In certain concentrations the spindle ultimately disappeared and the chromosomes scattered. Certain concentrations of coumarin ]6] have the effect of breaking down the spindle mechanism in coconut, so that it ultimately assumes a round shape. In areca it is seen that the action is on the spindle but not on the metaphase plate and at anaphase, that portion of the spindle between the two plates remains unaltered. The above observations pertain to the orientated part of the nucleoplasm, which alone is visible. The term mitotic apparatus as applied to the organized spindle along with centrioles, asters and chromosomes [12] is not applicable to the present material, as all of these structures are not observable. The spindle changes its shape and volume in the usual course of mitosis. The spindle is likewise alterable by many external factors which may leave the chromosomes undisturbed. A spindle which has disappeared may reappear in the same place after these external factors are removed [S]. The balance between the orientated and Experimentd
Cell Research 21
Effect of light on mitotic spindle unorientated plasm is seen to be capable of constant variations. The breakdown of spindle structure has been more often assumed than demostrated. The growing tissue is known to prefer the dark, and to avoid the light. It was Bajer’s experience that some endosperm material would divide on the microscope whereas some would not. Thus light was specifically noted to interfere with mitosis. In the case of materials less sensitive to light, the exposure had to be kept to the minimum in respect of both intensity and duration. In the course of observations on Palmae endosperm, it was possible to determine that the breakdown of mitosis was caused by light, that specifically the action was on the spindle mechanism, that the damage was proportional to duration and intensity of illumination and that within limits the process of breakdown could be reversed in the darkness. The pattern of breakdown could to a certain extent be followed, which gave some indications as to spindle structure.
MATERIAL
AND
METHODS
The fruit of two species were used, namely coconut (Cocos nucifera) and betel or areca nut (Areca cafe&u). Both showed a deterioration of spindle structure under illumination. In areca, division was immediately arrested and subsequent changes were due to light interference. Division in coconut progressed unchecked, but the illuminated spindles showed more and more the altered structure till well within I+ hours all the nuclei were showing the photoeffect. Structural changes in coconut were complicated by the alterations in spindle shape occurring as a natural consequence of mitosis (Figs. 19-22). Therefore data on the light effect were mainly taken from areca. The coconut spindle is comparatively stable at telophase and the changes due to light effect measured at this stage approximate those of areca (Table II). The method of obtaining preparations has been previously described 15, 71. Briefly, a drop of endosperm milk, taken up with a glass rod from a freshly opened fruit, was touched to a coverslip, which was then inverted over a grooved slide and at once placed under observation. Fruits before being used were stored for at least 24 hours in a sterile thermostatic chamber maintained at 26 +l”C. All glassware and apparatus were kept in this chamber in which mounting and observations were carried out. Each fruit was discarded after fresh mounts had been made, and each mounted slide was kept under observation for a maximum period of 15 minutes, before being discarded. Fresh nuclei were thus mounted straightaway in their original medium without any change in the environment, except that of the entry of light from the microscope light source, and such heat as might result from the same. The heat from the light source could not be entirely eliminated but was considerably reduced. Microscope, glassware and the material itself were kept at a constant low temperature throughout operations. Blue filters were placed in the light source. Light was used at a comparatively low intensity and for not more than 15 minutes at a time. Under these conditions and at the maximum period of illumination, the rise in temperature on the mounted slide was determined by means of a Beckmann’s differential thermometer, not to exceed 0.2”C. A Leitz ortholux microscope fitted with a built-in light source and a daylight filter, was used for observations, measurements and photomicrography. The condenser Experimental
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(n.a. 1.3) and diaphragm, were adjusted to allow of maximum illumination, the intensity of which was controlled by varying the current passing through the filament of the lamp of the microscope light source by means of a variable resistance connected to it in series. The meter incorporated in the filament circuit indicated the amount of current passing through the lamp, an Osram G\‘, 5A tungsten filament bulb. This contained the entire visible region of the spectrum. Due to the incorporation of the daylight filter in the optical path, the light beam was found to be rich in the 4500 A wavelength component. The intensities of other componcnls such as red, yellow etc. were reduced but not eliminated. Experiments were conducted by noting the time taken to complete the reaction at the different intensities of illumination. Maximum bright field was obtained at ‘4.2 A. This was reduced each time to at least 4.1 A to discern1 the material, which was sharply definable at 4.0 A and just visible at 3.3 A. l>ata were taken at 3.0 A and 4.2 A, the latter being preferred as reaction then took place at a faster rate. Immediately upon mounting, a suitable spindle figure was selected in the field and exposed to standard illumination till the completion of reaction, readings being taken at intervals. Data were taken with an achromatic / 9 micrometer eyepiece in combination with the achromatic objective / 10 (n.a. 0.25) and ,‘:42 (n.a. 0.85). Photomicrographs were taken with a Leitz attachment and Leica miniature camera on Adox KB14 film, at exposures of 5 to 14 seconds and printed on glossy Agfa Brovira paper at the magnifications indicated. Under these conditions the reaction which took place could have been due to the two causes mentioned, namely heat or light. The absence or presence of the reaction and the rate at which it occurred could be connected directly with the absence or presence of the light factor as well as of its intensity. Such a direct relationship was not found with temperature alone in the absence of light. Coconut nuclei divide at lower temperatures, but in the case of areca the limits are sharply defined. Thus at 25°C not a single division will occur, while from 26°C to 37°C many division figures will be found. In an intact fruit maintained in and opened at any temperature within this range, the spindle figures will be perfect, but they will begin to show a deterioration the moment they are exposed to light. This deterioration will be at a faster rate when exposed to the microscope light, in which case the question of a rise in temperature will be involved, but it will occur at a very slow rate if left on the laborator?, table exposed to room light, in which event there will have been no rise in temperature. IJurthermore, in material on the microscope showing the reaction at 26”C, if the temperature is increased to 2X%, the rate of reaction will slow down. These several instances illustrate that it is not the heat which brings about and enhances the reaction. Thus, if there is a rise in temperature only, in the absence of light there is no reaction, whereas a reaction occurs in light without attendant increase of temperature, which latter seems rather to retard than to enhance the reaction. The observed reaction therefore appears to demonstrate the photosensitivity of the spindle. It was necessary to isolate the factor responsible for the reaction of the spindle and to establish the optimum conditions for its action. This being completed, the pattern of reaction in the visible spindle was studied, with reference to the entire nuclear content as demarcated by the membrane, with a view to gathering evidence of structure in the living untreated spindle, under optimum conditions.
Effect of light on mitotic spindle
265
RESULTS The areca nucleus forms a perfectly shaped spindle at metaphase (Fig. 10) within the size range (36-44 X lCZ2) p, being broadest at the equator and tapering to the two poles where it may he dra\vn out at the apices. The shape at the prophase and cleavage stages is oral anti polar caps are rounded. At anaphase and telophasc the sides of that part of the spindle between the t\vo ro\vs of chromosomes are parallel, \vhile the spindle caps taper to the t\vo poles (Text-Fig. S). \\‘hen about to divide, the nucleus elongates, appearing clear and structureless and nucleoli disappear. Chromosomes are
15 Time
in minutes
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10
15
20
in minutes
2
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,‘.’ ,” : : 1 !““““,“‘,“““I’
(II:;
5 Time
1 /‘... ::* /:-
0
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".;.,~: .. '..:
,.--
10 mr 6 mt Omt
3 Text-Figs. 1, 2.-The mean alterations in linear length and breadth undergone by Areca spindle figures at metaphase, taking into account both the half spindles at the same time, works ont to be at the rate of a little over 0.9 p along the long axis (Text-Fig. 1) and 0.5 p across the equator (Text-Fig. 2). Text-Figs. 335 ( x 3000).-Diagrammatic representation of changes in shape of actual spindle figures of Areca nuclei, at respectively metaphase, early anaphase and late anaphase, showing the successive changes in shape undergone by each figure under continuous illumination. In Text-Figs. 3 and 4, the final form is a true sphere, but in Text-Fig. 5 due to the chromosome plates being at a distance from each other, the decrease in length of the figure is partial and the final figure is elongated. Although drastic changes are taking place in the spindle, the chromosome plates remain unaltered in dimensions, positions and relative distance from each other. Experimental
Cell Research 21
Mridula TABLE
I. Measurement Light
Record
Stage of division
tl0.
Time
of light intensity Length of spindle in p
Datta effect in Areca catechu. at 4.2 A. Breadth of spindle in p
Diameter of chromosome plate in p
Distance between chromosome plates in ,u
2.55 p.m. 3.00 p.m. 3.05 p.m.
42 32 28
22 26 27
Metaphase
12.45 p.m. 12.49 p.m. 12.53 p.m.
44 36 29
18 30 29
18 18 18
3
Rletaphase
6.30 p.m. 6.33 p.m. 6.37 p.m.
38 32 27
20 24 25
20 20 20
4
Metaphase
1.20 p.m. 1.25 p.m.
26 19
16 18
16 16
5
Metaphase
3.20 p.m. 3.25 p.m. 3.30 p.m.
40 34 30
24 26 29
24 24 24
6
Metaphase
2.50 p.m. 2.55 p.m. 3.00 p.m.
28 26 21
16 18 20
16 16 16
7
Anaphase
2.00 2.10 2.20 2.25
p.m. p.m. p.m. p.m.
44 37 33 30
1x 20 22 22
IX 18 plates bending plates bending
16 16 16 16
8
Anaphase
3.30 p.m. 3.35 p.m. 3.40 p.m.
34 30 28
20 22 26
20 20 plates bending
12 12 12
9
Cleavage
5.40 p.m. 5.45 p.m. 5.48 p.m.
34 30 29
25 28 29
20 20 20
10
Cleavage
11.35 a.m. 11.40 a.m. 11.50 a.m.
34 22 21
16 18 21
14 14 14
1
Late prophase
2
Experimental
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22 26 chromosomes disappear
Effect of light on mitotic spindle hydrated prior to metaphase, but from this stage to telophase, condense and are clearly visible. Whatever the stage of mitosis, the elongated spindle commences to alter its shape instantaneously upon exposure to light. At metaphase a well-defined sequence of changes is witnessed (Table I). The acute polar tips retract and TABLE
II. Measurements of light Light
Record no.
Stage of division
Time
1
Metaphase
12.07 p.m. 12.17 p.m. 12.32 p.m.
2
Xnaphase
3
intensity
Spindle length in ,u
effect in Cocos nucifera. at 4.2 A.
Spindle breadth in p
Diameter of chromosome plate in p
Distance between chromosome plates in p
42 36 32.5
22 24 27
22 23 24
4.00 p.m. 4.10 p.m.
36 40
30 24
20 plates bending
10 18
Anaphase
3.40 3.50 4.00 4.10
p.m. p.m. p.m. p.m.
36 31 31 31
22 23 25 26
22 22 -
10 16 18 -
4
Cleavage
4.55 p.m. 5.00 p.m. 5.10 p.m.
52 44 42
20 26 30
20 20 20
5
Cleavage
3.15 p.m. 3.25 p.m. 3.37 p.m.
42 36 29
22 25 27
14 14 14
become rounded. From then on the figure continues to decrease in length and at the same time increase in girth. This gradual modification continues till the figure turns into a sphere, when no further changes are witnessed. Measurements show that in no case is the full girth attained before the decrease in length is completed. The alteration along the two directions is synchronous (TextFigs. 3, 4; Figs. 3, 4, 5; Table I). The two halves of the spindle alter at an equal rate. Variations, such as the bending of the metaphase plate occur rarely, but are corrected if the light is cut off. The decrease in length takes place at the average rate of 0.9 ,U per minute (Text-Fig. 1) and the increase in breadth at 0.5 ,U per minute (Text-Fig. 2). Experimental
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A similar sequence of changes is followed at early anaphase and cleavage stages (Text-Fig. 4; Figs. 14, 15, 16). At late anaphase and telophase the initial reaction is the same. The final shape, however, is not that of a sphere, for the spindle remains more or less elongated, according to the stage of mitosis attained at the moment of exposure to light (Text-Fig. 5). The chromosome plate appears to he inert to the action of light; as seen from side vie\\-, it alters neither position nor dimensions. At mctaphase, the chromosome plate remains unchanged, in the centre of a mitotic figure that is undergoing a drastic alteration, although the spindle membrane moves away from the plate. The cleavage plate behaves in the same \vay as the metaphase plate. At anaphase not only do the t\\-o plates remain unaltered as to relative positions, hut the portion of spindle between them remains apparently unaltcrctl as well. l’rior to metaphase the plate is less rigid and may extend hreadthwise as the figure increases in girth (Fig. 0). Often under strong illumination, the \vcakl\ defined chromosomes of late prophase may disappear altogether (Fig. 2). The reaction is considered to be complete when the figure turns into a sphere. The spherical shape is comparatively stable. This is evident in hoth the resting nuclcolated nuclei and in the altered ones, which have been found to remain unaffected by subsequent illumination for a period of at least one hour. Figs. I-li.-The nuclei of Areca undergoing mitosis in endospermal sap are generally free of cytoplasm. Division is arrested in the illuminted figure which within the permanent nuclear membrane begins at once to undergo a simultaneous decrease in length and increase in girth till finally it turns spherical. Late Prophase.-Figs. l-2 ( x 665). The reaction is completed in 2 minutes while the scattered chromosomes, barely visible at first (l), finally disappear completely (2). Figs. 3-5 ( y 460). In this already rounded figure (3) after 4 minutes of light treatment (4) and again 3 minutes later (5), the chromosome plate is not rigid but extends along with the waxing figure. Fig. 9 ( x 475) depicts an almost completed sphere in which chromosomes stretch right along the equator. Metaphase.-Figs. 6-S (X 523). The spindle almost rounds out within 12 minutes, but the chromosome plate does not extend. It bends slightly (8). Fig. 10 ( x 582). The sharply defined spindle at 1.30 p.m. progressively shows the reaction in Figs. 11, 12 ( x 650), at respectively 1.40 p.m. and 1.43 p.m. Fig. 13 ( x 600). An almost spherical spindle in which the unchanged plate has been left clear in the centrc. Chromosomes are placed linearly along the long axis of the spindle. Lnle l’elophuse and Cleavage.--Pigs. 14-16. These are different nuclei placed at the successive stages of photoreaction. Fig. 14 ( x 600). The untreated cleavage figure with the plate touching the membrane. At later stages the figure reduces in length (Fig. 15, x 560) ancl the membrane moves away from the plate. In Fig. I6 ( x 620) some cytoplasmic gel may bc noticed. Resting nucleus.-Fig. 17 ( x 600). The resting nucleus is round in shape and nucleoli are visible. Figs. 18-22 ( x 200).-Coconut nuclei undergoing mitosis in endospermal sap, in the illuminated mount. Fig. 18. A perfectly shaped untreated metaphase figure. Figs. 1%22.-The nucleus undergoing mitosis is partly rounded out in a mount illuminated for 40 minutes. Fig. 19, Metaphase, 1.40 p.m.; Fig. 20, Anaphase, 1.57 p.m. IJig. 21, Telophase, 2.7 p.m.; Fig. 22, Cleavage, 2.25 p.m. Apart from the light effect the achromatic figure undergoes alterations in shape and volume in the course of mitosis. Experimental
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Effect of light on mitotic spindle
3
6
269
4
7
5
8
10
lb 15
18
19
20
Experimental
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270
Mridula
Datta
The described changes are dependent on the intensity of light, being completed at 4.2 A illumination in 5-10 minutes, at 4.0 A in 15-20 minutes and at 3.3 A or under, not only taking a longer time but often not being completed at all. Data were taken at 26”C, at 4.2 amp intensity of illumination. Any other spindle figures that happened to be in the treated mount, were found also to have undergone photoreaction. The degree of reaction was related to the duration of illumination. In the metaphase figure of areca, if the light vvas cut off for 10 minutes just as the polar tips were being drawn in, they could be reformed within this period of darkness. If the figure was illuminated until it became oval and then placed in darkness for 10 minutes, the process of rounding up continued in the dark until the figure turned into a sphere. Coconut nuclei are similarly affected but at a slower rate. Division The reaction will not be completed in progresses despite the photoreaction. the division cycle of one nucleus, but in illuminated material, the nuclei which enter mitosis at later stages will be progressively more altered in shape. This process will continue till within l+ hours all the mitotic figures will have been completely rounded and no more divisions will occur. Apparently spindles that yet deviate from spherical form may enter division, but once the figure has turned spherical, the spindle mechanism can no longer function. DISCUSSION
Mazia [lo], Swann [17] and InouC [8] demonstrated the reality of the highly orientated continuous fibres connecting the mitotic centres and stretching at least from pole to equator. While conceding that these elements are parallel, Wada [ 191 and Sato [15] tend to believe that they are discontinuous. It is accepted generally that the sub-microscopic structure of the achromatic figure is composed for the most part of protein micelles which are extended and linked to form long chains, and such fibrils may be integrated into hbres. If these links are caused to be broken by some agency, the figure will revert to the amorphous gel state and will no longer be capable of its mechanical function. The disturbing factor being removed, the orientation may be reimposed upon the gel and the bipolar spindle figure re-emerge [8, 11, 191. The spindle can retain its structural integrity despite a considerable amount of distortion, in material which has been centrifuged or otherwise subjected to mechanical pressure. Mitosis was seen to progress normally in spindles flattened gradually by bajer [l] to almost chromosome thickness. Experimental
Cell Research 21
Effect of light on mitotic spindle It can be noticed that in these cases the polar caps remained undamaged. Rajer could destroy the spindle by sudden pressure. In this case, the bipolarity of the figure was probably destroyed. The functional spindle must necessarily have an elongated form and intact polar extremities. In the present work nuclei are observed in almost their natural state, except for one factor, namely light, the action of which differs according to the material. On the whole, animal material appears to be more resistant [9] as ill effects of illuminution are mentioned but rarely. Even under illumination the nuclear membrane remains as a comparatively indestructible elastic structure, its outer shape reflecting the orientation of the nuclear micelles within it, elongating when they are longitudinally extended and assuming the static spherical form when the latter lose their linear orientation. An aspect of mitotic structure that becomes strikingly apparent as such a dissociation of micelles takes place due to the effect of light, is the firm bonding established between kinetochore and spindle fibre at metaphase, which is known to be a critical phase [ 131 with respect to spindle structure. Spindle dissolution stops short of the chromosome plate, resulting in undamaged preservation at metaphase of the chromosome plate, and at anaphase of the two plates as well as of the spindle area bounded by them (Text-Fig. 5). This behavior may be explained in two ways; either the spindle dissolution proceeds in the direction of pole to equator or else the passage of kinetochores at the anaphase movement alters spindle structure. The bonding agents that diffuse from the kinetochores at metaphase permeate that area, with the result that the so-called plate behaves as a unit in being structurally differentiated from the rest of the spindle. Prior to metaphase the plate is not rigid and the chromosomes are sensitive to the action of light. As the chromosome plate remains unaltered from metaphase onwards the site of spindle dissolution may possibly be situated at the poles, the plasm which retracts from the poles increasing the girth of the figure. This process has to be considered as a reversal of the work carried out by the mitotic ccntres at prophase. In the altering spindle the polar orientation is possibly maintained, as borne out by Inoub’s diagrams and the preliminary observations with coumarin. This accounts for the continuation of mitosis in the partially rounded nucleus of coconut. Inoue explained the decrease in length of the spindle figure during colchirine treatment as due to the lengthwise contraction of spindle fibres, although it occurred to him that this would not be a satisfactory explanation when spindle size prior to disappearance approached zero; therefore some Experimental
Cell Research 21
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Datta
micelles should have to be excluded. The areca records show this expulsion actually taking place and that the characteristic pattern of spindle reduction, leading to a turnover of nuclear gel to the unorientated state, as far as can be followed, is similar to that observed for colchicine in sea urchin egg and for coumarin in coconut. This entire pattern of changes cannot be followed with the polarizing microscope. There is this difl’erencc in the degrees of reaction observed that whereas the light intensities employed are not sufficient to break the bonding at the metaphase plate, the colchicine solntion used is of sufficient strength to break it as a result of lvhich the chromosomes scatter. ostergren [l-r] listed a number of diverse substances having the ell’ect of colchicine on mitosis and was led to deduce that spindle destruction appeared to be a non-specific reaction to any ill treatment. Present observations indicate that the arrest of mitosis is related to the breakdolvn of the spindle mechanism and moreover that the breakdown pattern is similar in the cases of such unrelated causative factors as colchicine and roumarin on the one hand and light on the other, which latter is incidentally a natural agent in the environment of the plant. SUMMARY
In coconut and arecanut endosperm, it is possible to observe the mitotic spindle in its natural state; the only and unavoidable disturbing factor being the light from the microscope light source. The nuclear membrane is permanent, carrying within it, at all stages of division and reduction, the total content of nuclear matter. The bipolar highly orientated state of the achromatic figure is changeable by visible light, the whole mitotic figure assuming a spherical shape at a certain intensity and duration of the illumination. The visible light has probably the effect of dissociating the linked chains of extended protein micelles which compose the bulk of spindle substructure, resulting in their return to the amorphous gel. The bonding of chromosomes at their kinetochores with the spindle fibres has the effect of altering spindle structure both in the areas occupied by them and that over which they have passed. As a result, at metaphase and anaphase the chromosome plates and that area of the spindle bounded by them remain inert to the action of light. The metaphase spindle remaining unaltered at the equator, the dissociating plasm is released at the polar ends. The spindle retains its bipolar orientation while it is undergoing reduction and can remain functional as long as it has the elongated shape. Experimenful
Cell Research
21
Effect of light on mitotic spindle
273
This pattern of breakdown under illumination compares favourably in under colchicine points of similarity with the observed pattern of breakdown and coumarin treatment, and may well take place in all cases of arrested mitosis destruction has had to be deduced.
reflect the sequence of events which in which, usually, the fact of spindle
It is a pleasure to acknowledge my indebtedness to Dr. D. M. Bose, Director, Bose Institute, for kindly extending laboratory facilities, to Sri Jyoti Dutt of the Chemistry Department, Bose Institute, for checking the temperature on the mounted slide, as well as to Dr. T. C. Bhadra of the Physics Department, Bose Institute, for determining the spectral properties of the light source. This work has been aided in part by the grant of an I.C.I. fellowship by the National Institute of Sciences, India. REFERENCES 1.
A., Ezperientiu 2, 221 (1955). Expff. Cell Research 13, 493 (1957). ibid. 14, 245 (1958). BAJER, A. and MoLF: BAJER, J., Chromosoma 7, 558 (1956). D.zw.\, M., I’runs. Llose Inst. 19, 117 (1952). --Ann. Rep. Bose Inst. 47 (1957). DIXT, iU., lVntwe 171, 799 (1953). Isouk, S., Ezptf. Cell Research, Suppf. 2, 305 (1952). K.~wanfun.~, K., Cyfofogiu 22, 347 (1957). iWiz~.i, D., Symposi” Sot. &pi/. Rid. 9, 335 (1955). -Advances in Hiof. Med. Phys. 4, 69 (1956). hJER,
2. ~ 3. ~ 4.
5. 6. i. 8. 9.
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11. and DAN K.,
Sat!. kd Sci. 38, 826 (1952). A, Ezpff. Cd Research 15, 138 (1958). 30, 429 (1911). SiTO, S., Cytoloyia 23, 383 (1958). SWINX, M. M., Intern Reu. Cyfof. 1, 195 (1952). --Symposia Sot. E&pff. Riof. 6, 89 (1952). TARI.WI, S. and T.AKAHISIIA, O., Ezpff. Cell Research 11, 346 (1956). \Vma, B., Cyfoloyia 15, 88 (1929). PTOC.
13. hlhm, D. and %mERmNN 14. ~TISRGREX, G., Heredifas 15. 16. 17. 18. 19.
20. --~~
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7, 389 (1955).
Experimenfaf
Cell Research 21
Cell Research 21, 261-273 (1960)
THE EFFECT
261
OF VISIBLE LIGHT MITOTIC SPINDLE MRIDULA
DATTA
Bose Institute, Received
ON THE
Calcutta,
September
111&a
5, 1959
R I.c~.\ : 1.1..I
investigations on mitosis in living tissues have helped to dra\\ attention to the question of the nature of the spintlle body. Follo\ving the use of special optical techniques, the structure has been viewed in as nearly the natural undisturbed state as possible, care being taken at the same time to eliminate from the procedures, treatments lvhich might least tend to alter the arrangement of spintlle proteins. In thr search for material, it \\-as necessary to consider the property of its hein, 0 able to divide normally in a fluid medium and in this respect animal material, though restricted as to source, generally proved suitable [8-l 3, 1 (i-1 81.
The study of mitosis in the higher plants was pOSSibk in a few instances. Wada [19, ‘LO] used the strands of single cells of the stamina1 hairs of Tradescanfia with the view to minimizing cell wall interference. Rajer [l, 2, 3, 41 successfully cultivated the technique of mounting unwalled cells of the endosperm of several flowering plants, using agar and sugar solution as sealing media. The author [5,7] reported the occurrence of mitotic division in nuclei of the endospermal milk of some Palmae fruit. These nuclei, which are suspended in the watery sap of the embryo sac, are entirely free of cytoplasm and cell wall. The cytoplasm is confined to the syncitium layer, later becoming walled, which lines the walls of the endocarp. Nuclei breaking away from the syncitium are suspended and eventually undergo mitosis in this sap or milk as it is called, the morphological nature of which has yet to be determined. This sap is of low viscosity and its refractive index is lower than that of cytoplasm, whereas that of cytoplasm and nucleoplasm are almost equal. Cell inclusions and mitochondria occur in the milk. Nuclei are more sharply defined in this medium than when seen against cytoplasm, helped by the presence of the’ persistent nuclear membrane which can be demonstrated to be present at all the stages of mitosis with the micromanipulator probe and by the action of coumarin [6] which in certain concentrations has the effect of making the entire spindle shrink in size and draw away from the membrane as it does so. The spindle is structureless under the light microscope and can be detected mainly due to the cytoplasmic granules outlining it. This circumstance has led Wada [20] to suppose that the nucleus never loses its membrane. This enables it to exclude cytoExperimental
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Research 21
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plasmic matter from the nucleoplasm which pervades the spindle. Observations to the contrary, and in support of the general belief, are to the effect that the membrane dissolves at early prophase. As it is not possible to visually differentiate nucleoplasm from cytoplasm, it is not known with certainty as to out of the following two possibilities which actually occurs. In the course of mitosis the nucleoplasm and cytoplasm may remain distinct from each other if always separated by a membrane, otherwise with membrane dissolution at prophase they may mingle. Also, both might combine to form the spindle figure; alternatively this latter might be formed out of nucleoplasm alone. Evidence from different sources shows that the mechanism differs according to the material. In the Palmae fruit under discussion, however, it is clear that the nucleoplasm is at all stages separated from the surrounding fluid and that spindle formation is strictly It will be noted later that the spindle breakdown occurs and the intranuclear. products of that breakdown remain, within the nuclear membrane. Under phase contrast the outline of the living spindle may be discerned [l, 2,3,4, 9, 10, 191. The property of birefringence of the spindle fibres has made it possible to study some aspects of spindle structure with the help of the polarization microscope [S, 11, 16, 17). The orientation pattern of fibrils in fixed preparations has been deduced from electronmicrographs (10, 151. These studies have helped to establish Bajer [2] demonstrated that spindle formacertain facts as to spindle structure. tion began at the time of, if not before, membrane dissolution, the orientation proceeding from the polar caps towards the equator. Mazia [lo, 111 and Swann [ 16,171 have established the reality of the spindle fibres and the fact that they are orientated with reference to the mitotic centres which are the centrioles and centrosomes.They have shown that spindle elements are orientated parallel with reference to the long axis of the spindle and that individual elements may be integrated into fibres. In fact it is due to the diffusion of certain structural agents released by the mitotic centres at the laying down of that poles and at the anaphase movement of the chromosomes, that spindle fibres are aggregated out of the unorientated plasm. The arrest of the smooth flow of the mitotic movement by the action of colchicine has been explained by \Vada (191 and InouC [S] as attributable to the fact that spindle structure has suffered alteration. Inoue believed that a longitudinal contraction of the fibres was taking place in the spindle, which under the action of colchicine was rapidly decreasing in length, although negligibly so in width. In certain concentrations the spindle ultimately disappeared and the chromosomes scattered. Certain concentrations of coumarin ]6] have the effect of breaking down the spindle mechanism in coconut, so that it ultimately assumes a round shape. In areca it is seen that the action is on the spindle but not on the metaphase plate and at anaphase, that portion of the spindle between the two plates remains unaltered. The above observations pertain to the orientated part of the nucleoplasm, which alone is visible. The term mitotic apparatus as applied to the organized spindle along with centrioles, asters and chromosomes [12] is not applicable to the present material, as all of these structures are not observable. The spindle changes its shape and volume in the usual course of mitosis. The spindle is likewise alterable by many external factors which may leave the chromosomes undisturbed. A spindle which has disappeared may reappear in the same place after these external factors are removed [S]. The balance between the orientated and Experimentd
Cell Research 21
Effect of light on mitotic spindle unorientated plasm is seen to be capable of constant variations. The breakdown of spindle structure has been more often assumed than demostrated. The growing tissue is known to prefer the dark, and to avoid the light. It was Bajer’s experience that some endosperm material would divide on the microscope whereas some would not. Thus light was specifically noted to interfere with mitosis. In the case of materials less sensitive to light, the exposure had to be kept to the minimum in respect of both intensity and duration. In the course of observations on Palmae endosperm, it was possible to determine that the breakdown of mitosis was caused by light, that specifically the action was on the spindle mechanism, that the damage was proportional to duration and intensity of illumination and that within limits the process of breakdown could be reversed in the darkness. The pattern of breakdown could to a certain extent be followed, which gave some indications as to spindle structure.
MATERIAL
AND
METHODS
The fruit of two species were used, namely coconut (Cocos nucifera) and betel or areca nut (Areca cafe&u). Both showed a deterioration of spindle structure under illumination. In areca, division was immediately arrested and subsequent changes were due to light interference. Division in coconut progressed unchecked, but the illuminated spindles showed more and more the altered structure till well within I+ hours all the nuclei were showing the photoeffect. Structural changes in coconut were complicated by the alterations in spindle shape occurring as a natural consequence of mitosis (Figs. 19-22). Therefore data on the light effect were mainly taken from areca. The coconut spindle is comparatively stable at telophase and the changes due to light effect measured at this stage approximate those of areca (Table II). The method of obtaining preparations has been previously described 15, 71. Briefly, a drop of endosperm milk, taken up with a glass rod from a freshly opened fruit, was touched to a coverslip, which was then inverted over a grooved slide and at once placed under observation. Fruits before being used were stored for at least 24 hours in a sterile thermostatic chamber maintained at 26 +l”C. All glassware and apparatus were kept in this chamber in which mounting and observations were carried out. Each fruit was discarded after fresh mounts had been made, and each mounted slide was kept under observation for a maximum period of 15 minutes, before being discarded. Fresh nuclei were thus mounted straightaway in their original medium without any change in the environment, except that of the entry of light from the microscope light source, and such heat as might result from the same. The heat from the light source could not be entirely eliminated but was considerably reduced. Microscope, glassware and the material itself were kept at a constant low temperature throughout operations. Blue filters were placed in the light source. Light was used at a comparatively low intensity and for not more than 15 minutes at a time. Under these conditions and at the maximum period of illumination, the rise in temperature on the mounted slide was determined by means of a Beckmann’s differential thermometer, not to exceed 0.2”C. A Leitz ortholux microscope fitted with a built-in light source and a daylight filter, was used for observations, measurements and photomicrography. The condenser Experimental
Cell Research 21
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(n.a. 1.3) and diaphragm, were adjusted to allow of maximum illumination, the intensity of which was controlled by varying the current passing through the filament of the lamp of the microscope light source by means of a variable resistance connected to it in series. The meter incorporated in the filament circuit indicated the amount of current passing through the lamp, an Osram G\‘, 5A tungsten filament bulb. This contained the entire visible region of the spectrum. Due to the incorporation of the daylight filter in the optical path, the light beam was found to be rich in the 4500 A wavelength component. The intensities of other componcnls such as red, yellow etc. were reduced but not eliminated. Experiments were conducted by noting the time taken to complete the reaction at the different intensities of illumination. Maximum bright field was obtained at ‘4.2 A. This was reduced each time to at least 4.1 A to discern1 the material, which was sharply definable at 4.0 A and just visible at 3.3 A. l>ata were taken at 3.0 A and 4.2 A, the latter being preferred as reaction then took place at a faster rate. Immediately upon mounting, a suitable spindle figure was selected in the field and exposed to standard illumination till the completion of reaction, readings being taken at intervals. Data were taken with an achromatic / 9 micrometer eyepiece in combination with the achromatic objective / 10 (n.a. 0.25) and ,‘:42 (n.a. 0.85). Photomicrographs were taken with a Leitz attachment and Leica miniature camera on Adox KB14 film, at exposures of 5 to 14 seconds and printed on glossy Agfa Brovira paper at the magnifications indicated. Under these conditions the reaction which took place could have been due to the two causes mentioned, namely heat or light. The absence or presence of the reaction and the rate at which it occurred could be connected directly with the absence or presence of the light factor as well as of its intensity. Such a direct relationship was not found with temperature alone in the absence of light. Coconut nuclei divide at lower temperatures, but in the case of areca the limits are sharply defined. Thus at 25°C not a single division will occur, while from 26°C to 37°C many division figures will be found. In an intact fruit maintained in and opened at any temperature within this range, the spindle figures will be perfect, but they will begin to show a deterioration the moment they are exposed to light. This deterioration will be at a faster rate when exposed to the microscope light, in which case the question of a rise in temperature will be involved, but it will occur at a very slow rate if left on the laborator?, table exposed to room light, in which event there will have been no rise in temperature. IJurthermore, in material on the microscope showing the reaction at 26”C, if the temperature is increased to 2X%, the rate of reaction will slow down. These several instances illustrate that it is not the heat which brings about and enhances the reaction. Thus, if there is a rise in temperature only, in the absence of light there is no reaction, whereas a reaction occurs in light without attendant increase of temperature, which latter seems rather to retard than to enhance the reaction. The observed reaction therefore appears to demonstrate the photosensitivity of the spindle. It was necessary to isolate the factor responsible for the reaction of the spindle and to establish the optimum conditions for its action. This being completed, the pattern of reaction in the visible spindle was studied, with reference to the entire nuclear content as demarcated by the membrane, with a view to gathering evidence of structure in the living untreated spindle, under optimum conditions.
Effect of light on mitotic spindle
265
RESULTS The areca nucleus forms a perfectly shaped spindle at metaphase (Fig. 10) within the size range (36-44 X lCZ2) p, being broadest at the equator and tapering to the two poles where it may he dra\vn out at the apices. The shape at the prophase and cleavage stages is oral anti polar caps are rounded. At anaphase and telophasc the sides of that part of the spindle between the t\vo ro\vs of chromosomes are parallel, \vhile the spindle caps taper to the t\vo poles (Text-Fig. S). \\‘hen about to divide, the nucleus elongates, appearing clear and structureless and nucleoli disappear. Chromosomes are
15 Time
in minutes
“)
\ . \ \.: \'.
i /’ .I :,' ." '.
10
15
20
in minutes
2
:.,.
,‘.’ ,” : : 1 !““““,“‘,“““I’
(II:;
5 Time
1 /‘... ::* /:-
0
: s : ! )
".;.,~: .. '..:
,.--
10 mr 6 mt Omt
3 Text-Figs. 1, 2.-The mean alterations in linear length and breadth undergone by Areca spindle figures at metaphase, taking into account both the half spindles at the same time, works ont to be at the rate of a little over 0.9 p along the long axis (Text-Fig. 1) and 0.5 p across the equator (Text-Fig. 2). Text-Figs. 335 ( x 3000).-Diagrammatic representation of changes in shape of actual spindle figures of Areca nuclei, at respectively metaphase, early anaphase and late anaphase, showing the successive changes in shape undergone by each figure under continuous illumination. In Text-Figs. 3 and 4, the final form is a true sphere, but in Text-Fig. 5 due to the chromosome plates being at a distance from each other, the decrease in length of the figure is partial and the final figure is elongated. Although drastic changes are taking place in the spindle, the chromosome plates remain unaltered in dimensions, positions and relative distance from each other. Experimental
Cell Research 21
Mridula TABLE
I. Measurement Light
Record
Stage of division
tl0.
Time
of light intensity Length of spindle in p
Datta effect in Areca catechu. at 4.2 A. Breadth of spindle in p
Diameter of chromosome plate in p
Distance between chromosome plates in ,u
2.55 p.m. 3.00 p.m. 3.05 p.m.
42 32 28
22 26 27
Metaphase
12.45 p.m. 12.49 p.m. 12.53 p.m.
44 36 29
18 30 29
18 18 18
3
Rletaphase
6.30 p.m. 6.33 p.m. 6.37 p.m.
38 32 27
20 24 25
20 20 20
4
Metaphase
1.20 p.m. 1.25 p.m.
26 19
16 18
16 16
5
Metaphase
3.20 p.m. 3.25 p.m. 3.30 p.m.
40 34 30
24 26 29
24 24 24
6
Metaphase
2.50 p.m. 2.55 p.m. 3.00 p.m.
28 26 21
16 18 20
16 16 16
7
Anaphase
2.00 2.10 2.20 2.25
p.m. p.m. p.m. p.m.
44 37 33 30
1x 20 22 22
IX 18 plates bending plates bending
16 16 16 16
8
Anaphase
3.30 p.m. 3.35 p.m. 3.40 p.m.
34 30 28
20 22 26
20 20 plates bending
12 12 12
9
Cleavage
5.40 p.m. 5.45 p.m. 5.48 p.m.
34 30 29
25 28 29
20 20 20
10
Cleavage
11.35 a.m. 11.40 a.m. 11.50 a.m.
34 22 21
16 18 21
14 14 14
1
Late prophase
2
Experimental
Cell Research 21
22 26 chromosomes disappear
Effect of light on mitotic spindle hydrated prior to metaphase, but from this stage to telophase, condense and are clearly visible. Whatever the stage of mitosis, the elongated spindle commences to alter its shape instantaneously upon exposure to light. At metaphase a well-defined sequence of changes is witnessed (Table I). The acute polar tips retract and TABLE
II. Measurements of light Light
Record no.
Stage of division
Time
1
Metaphase
12.07 p.m. 12.17 p.m. 12.32 p.m.
2
Xnaphase
3
intensity
Spindle length in ,u
effect in Cocos nucifera. at 4.2 A.
Spindle breadth in p
Diameter of chromosome plate in p
Distance between chromosome plates in p
42 36 32.5
22 24 27
22 23 24
4.00 p.m. 4.10 p.m.
36 40
30 24
20 plates bending
10 18
Anaphase
3.40 3.50 4.00 4.10
p.m. p.m. p.m. p.m.
36 31 31 31
22 23 25 26
22 22 -
10 16 18 -
4
Cleavage
4.55 p.m. 5.00 p.m. 5.10 p.m.
52 44 42
20 26 30
20 20 20
5
Cleavage
3.15 p.m. 3.25 p.m. 3.37 p.m.
42 36 29
22 25 27
14 14 14
become rounded. From then on the figure continues to decrease in length and at the same time increase in girth. This gradual modification continues till the figure turns into a sphere, when no further changes are witnessed. Measurements show that in no case is the full girth attained before the decrease in length is completed. The alteration along the two directions is synchronous (TextFigs. 3, 4; Figs. 3, 4, 5; Table I). The two halves of the spindle alter at an equal rate. Variations, such as the bending of the metaphase plate occur rarely, but are corrected if the light is cut off. The decrease in length takes place at the average rate of 0.9 ,U per minute (Text-Fig. 1) and the increase in breadth at 0.5 ,U per minute (Text-Fig. 2). Experimental
Cell Research 21
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A similar sequence of changes is followed at early anaphase and cleavage stages (Text-Fig. 4; Figs. 14, 15, 16). At late anaphase and telophase the initial reaction is the same. The final shape, however, is not that of a sphere, for the spindle remains more or less elongated, according to the stage of mitosis attained at the moment of exposure to light (Text-Fig. 5). The chromosome plate appears to he inert to the action of light; as seen from side vie\\-, it alters neither position nor dimensions. At mctaphase, the chromosome plate remains unchanged, in the centre of a mitotic figure that is undergoing a drastic alteration, although the spindle membrane moves away from the plate. The cleavage plate behaves in the same \vay as the metaphase plate. At anaphase not only do the t\\-o plates remain unaltered as to relative positions, hut the portion of spindle between them remains apparently unaltcrctl as well. l’rior to metaphase the plate is less rigid and may extend hreadthwise as the figure increases in girth (Fig. 0). Often under strong illumination, the \vcakl\ defined chromosomes of late prophase may disappear altogether (Fig. 2). The reaction is considered to be complete when the figure turns into a sphere. The spherical shape is comparatively stable. This is evident in hoth the resting nuclcolated nuclei and in the altered ones, which have been found to remain unaffected by subsequent illumination for a period of at least one hour. Figs. I-li.-The nuclei of Areca undergoing mitosis in endospermal sap are generally free of cytoplasm. Division is arrested in the illuminted figure which within the permanent nuclear membrane begins at once to undergo a simultaneous decrease in length and increase in girth till finally it turns spherical. Late Prophase.-Figs. l-2 ( x 665). The reaction is completed in 2 minutes while the scattered chromosomes, barely visible at first (l), finally disappear completely (2). Figs. 3-5 ( y 460). In this already rounded figure (3) after 4 minutes of light treatment (4) and again 3 minutes later (5), the chromosome plate is not rigid but extends along with the waxing figure. Fig. 9 ( x 475) depicts an almost completed sphere in which chromosomes stretch right along the equator. Metaphase.-Figs. 6-S (X 523). The spindle almost rounds out within 12 minutes, but the chromosome plate does not extend. It bends slightly (8). Fig. 10 ( x 582). The sharply defined spindle at 1.30 p.m. progressively shows the reaction in Figs. 11, 12 ( x 650), at respectively 1.40 p.m. and 1.43 p.m. Fig. 13 ( x 600). An almost spherical spindle in which the unchanged plate has been left clear in the centrc. Chromosomes are placed linearly along the long axis of the spindle. Lnle l’elophuse and Cleavage.--Pigs. 14-16. These are different nuclei placed at the successive stages of photoreaction. Fig. 14 ( x 600). The untreated cleavage figure with the plate touching the membrane. At later stages the figure reduces in length (Fig. 15, x 560) ancl the membrane moves away from the plate. In Fig. I6 ( x 620) some cytoplasmic gel may bc noticed. Resting nucleus.-Fig. 17 ( x 600). The resting nucleus is round in shape and nucleoli are visible. Figs. 18-22 ( x 200).-Coconut nuclei undergoing mitosis in endospermal sap, in the illuminated mount. Fig. 18. A perfectly shaped untreated metaphase figure. Figs. 1%22.-The nucleus undergoing mitosis is partly rounded out in a mount illuminated for 40 minutes. Fig. 19, Metaphase, 1.40 p.m.; Fig. 20, Anaphase, 1.57 p.m. IJig. 21, Telophase, 2.7 p.m.; Fig. 22, Cleavage, 2.25 p.m. Apart from the light effect the achromatic figure undergoes alterations in shape and volume in the course of mitosis. Experimental
Cell Research 21
Effect of light on mitotic spindle
3
6
269
4
7
5
8
10
lb 15
18
19
20
Experimental
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270
Mridula
Datta
The described changes are dependent on the intensity of light, being completed at 4.2 A illumination in 5-10 minutes, at 4.0 A in 15-20 minutes and at 3.3 A or under, not only taking a longer time but often not being completed at all. Data were taken at 26”C, at 4.2 amp intensity of illumination. Any other spindle figures that happened to be in the treated mount, were found also to have undergone photoreaction. The degree of reaction was related to the duration of illumination. In the metaphase figure of areca, if the light vvas cut off for 10 minutes just as the polar tips were being drawn in, they could be reformed within this period of darkness. If the figure was illuminated until it became oval and then placed in darkness for 10 minutes, the process of rounding up continued in the dark until the figure turned into a sphere. Coconut nuclei are similarly affected but at a slower rate. Division The reaction will not be completed in progresses despite the photoreaction. the division cycle of one nucleus, but in illuminated material, the nuclei which enter mitosis at later stages will be progressively more altered in shape. This process will continue till within l+ hours all the mitotic figures will have been completely rounded and no more divisions will occur. Apparently spindles that yet deviate from spherical form may enter division, but once the figure has turned spherical, the spindle mechanism can no longer function. DISCUSSION
Mazia [lo], Swann [17] and InouC [8] demonstrated the reality of the highly orientated continuous fibres connecting the mitotic centres and stretching at least from pole to equator. While conceding that these elements are parallel, Wada [ 191 and Sato [15] tend to believe that they are discontinuous. It is accepted generally that the sub-microscopic structure of the achromatic figure is composed for the most part of protein micelles which are extended and linked to form long chains, and such fibrils may be integrated into hbres. If these links are caused to be broken by some agency, the figure will revert to the amorphous gel state and will no longer be capable of its mechanical function. The disturbing factor being removed, the orientation may be reimposed upon the gel and the bipolar spindle figure re-emerge [8, 11, 191. The spindle can retain its structural integrity despite a considerable amount of distortion, in material which has been centrifuged or otherwise subjected to mechanical pressure. Mitosis was seen to progress normally in spindles flattened gradually by bajer [l] to almost chromosome thickness. Experimental
Cell Research 21
Effect of light on mitotic spindle It can be noticed that in these cases the polar caps remained undamaged. Rajer could destroy the spindle by sudden pressure. In this case, the bipolarity of the figure was probably destroyed. The functional spindle must necessarily have an elongated form and intact polar extremities. In the present work nuclei are observed in almost their natural state, except for one factor, namely light, the action of which differs according to the material. On the whole, animal material appears to be more resistant [9] as ill effects of illuminution are mentioned but rarely. Even under illumination the nuclear membrane remains as a comparatively indestructible elastic structure, its outer shape reflecting the orientation of the nuclear micelles within it, elongating when they are longitudinally extended and assuming the static spherical form when the latter lose their linear orientation. An aspect of mitotic structure that becomes strikingly apparent as such a dissociation of micelles takes place due to the effect of light, is the firm bonding established between kinetochore and spindle fibre at metaphase, which is known to be a critical phase [ 131 with respect to spindle structure. Spindle dissolution stops short of the chromosome plate, resulting in undamaged preservation at metaphase of the chromosome plate, and at anaphase of the two plates as well as of the spindle area bounded by them (Text-Fig. 5). This behavior may be explained in two ways; either the spindle dissolution proceeds in the direction of pole to equator or else the passage of kinetochores at the anaphase movement alters spindle structure. The bonding agents that diffuse from the kinetochores at metaphase permeate that area, with the result that the so-called plate behaves as a unit in being structurally differentiated from the rest of the spindle. Prior to metaphase the plate is not rigid and the chromosomes are sensitive to the action of light. As the chromosome plate remains unaltered from metaphase onwards the site of spindle dissolution may possibly be situated at the poles, the plasm which retracts from the poles increasing the girth of the figure. This process has to be considered as a reversal of the work carried out by the mitotic ccntres at prophase. In the altering spindle the polar orientation is possibly maintained, as borne out by Inoub’s diagrams and the preliminary observations with coumarin. This accounts for the continuation of mitosis in the partially rounded nucleus of coconut. Inoue explained the decrease in length of the spindle figure during colchirine treatment as due to the lengthwise contraction of spindle fibres, although it occurred to him that this would not be a satisfactory explanation when spindle size prior to disappearance approached zero; therefore some Experimental
Cell Research 21
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Datta
micelles should have to be excluded. The areca records show this expulsion actually taking place and that the characteristic pattern of spindle reduction, leading to a turnover of nuclear gel to the unorientated state, as far as can be followed, is similar to that observed for colchicine in sea urchin egg and for coumarin in coconut. This entire pattern of changes cannot be followed with the polarizing microscope. There is this difl’erencc in the degrees of reaction observed that whereas the light intensities employed are not sufficient to break the bonding at the metaphase plate, the colchicine solntion used is of sufficient strength to break it as a result of lvhich the chromosomes scatter. ostergren [l-r] listed a number of diverse substances having the ell’ect of colchicine on mitosis and was led to deduce that spindle destruction appeared to be a non-specific reaction to any ill treatment. Present observations indicate that the arrest of mitosis is related to the breakdolvn of the spindle mechanism and moreover that the breakdown pattern is similar in the cases of such unrelated causative factors as colchicine and roumarin on the one hand and light on the other, which latter is incidentally a natural agent in the environment of the plant. SUMMARY
In coconut and arecanut endosperm, it is possible to observe the mitotic spindle in its natural state; the only and unavoidable disturbing factor being the light from the microscope light source. The nuclear membrane is permanent, carrying within it, at all stages of division and reduction, the total content of nuclear matter. The bipolar highly orientated state of the achromatic figure is changeable by visible light, the whole mitotic figure assuming a spherical shape at a certain intensity and duration of the illumination. The visible light has probably the effect of dissociating the linked chains of extended protein micelles which compose the bulk of spindle substructure, resulting in their return to the amorphous gel. The bonding of chromosomes at their kinetochores with the spindle fibres has the effect of altering spindle structure both in the areas occupied by them and that over which they have passed. As a result, at metaphase and anaphase the chromosome plates and that area of the spindle bounded by them remain inert to the action of light. The metaphase spindle remaining unaltered at the equator, the dissociating plasm is released at the polar ends. The spindle retains its bipolar orientation while it is undergoing reduction and can remain functional as long as it has the elongated shape. Experimenful
Cell Research
21
Effect of light on mitotic spindle
273
This pattern of breakdown under illumination compares favourably in under colchicine points of similarity with the observed pattern of breakdown and coumarin treatment, and may well take place in all cases of arrested mitosis destruction has had to be deduced.
reflect the sequence of events which in which, usually, the fact of spindle
It is a pleasure to acknowledge my indebtedness to Dr. D. M. Bose, Director, Bose Institute, for kindly extending laboratory facilities, to Sri Jyoti Dutt of the Chemistry Department, Bose Institute, for checking the temperature on the mounted slide, as well as to Dr. T. C. Bhadra of the Physics Department, Bose Institute, for determining the spectral properties of the light source. This work has been aided in part by the grant of an I.C.I. fellowship by the National Institute of Sciences, India. REFERENCES 1.
A., Ezperientiu 2, 221 (1955). Expff. Cell Research 13, 493 (1957). ibid. 14, 245 (1958). BAJER, A. and MoLF: BAJER, J., Chromosoma 7, 558 (1956). D.zw.\, M., I’runs. Llose Inst. 19, 117 (1952). --Ann. Rep. Bose Inst. 47 (1957). DIXT, iU., lVntwe 171, 799 (1953). Isouk, S., Ezptf. Cell Research, Suppf. 2, 305 (1952). K.~wanfun.~, K., Cyfofogiu 22, 347 (1957). iWiz~.i, D., Symposi” Sot. &pi/. Rid. 9, 335 (1955). -Advances in Hiof. Med. Phys. 4, 69 (1956). hJER,
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Sat!. kd Sci. 38, 826 (1952). A, Ezpff. Cd Research 15, 138 (1958). 30, 429 (1911). SiTO, S., Cytoloyia 23, 383 (1958). SWINX, M. M., Intern Reu. Cyfof. 1, 195 (1952). --Symposia Sot. E&pff. Riof. 6, 89 (1952). TARI.WI, S. and T.AKAHISIIA, O., Ezpff. Cell Research 11, 346 (1956). \Vma, B., Cyfoloyia 15, 88 (1929). PTOC.
13. hlhm, D. and %mERmNN 14. ~TISRGREX, G., Heredifas 15. 16. 17. 18. 19.
20. --~~
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7, 389 (1955).
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