C mitosis

Colchicine Mitosis

1. A Presentation on Observations on C- mitosis,learning on the dynamics of spindle fiber assembly Course title: Principle of Cytogenetics (GP502) Submitted to: Dr. N.B.Patel Associate Professor Submitted by: Gami Pankajkumar B. M.Sc.(Agri.)GPB 1 st sem , 04-AGRMA-01754-2018 Dept. of Genetics and Plant Breeding, C.P.C.A, S.D.A.U, Sardarkrushinagar -385 506

2. Observations on C-mitosis, learning on the dynamics of spindle fiber assembly

3. In simple, the mitosis which affected by the colchicine is called as Colchicine mitosis or C-mitosis. Colchicine is mitotic poison. A mitotic poison are those substance which affect the cell in mitosis or prevent them to entering in it. What is C-mitosis?

4. A Brief History of Colchicine : Use of the autumn crocus or meadow saffron Colchicum autumnale for both medicinal and nefarious purposes has been known It is known since the time of ancient Greece. The plant is named after the region of Colchis, located along the Eastern tip of the Black sea. The infamous sorceress Medea may have used it in her black arts, and landowners of the region probably grew it for its toxic properties. A related species ( Colchicum parnassicum ), which contains a smaller amount of colchicine , was described as such: "The root of this is a remedy for ye toothache...But ye leaves being sodden in wine and smeared on do dissolve Oedemata (swellings) and tumors"

5. Also known as "mort a chien " (death to dogs). Earliest documented use as medicine was in the treatment of gout as early as 560 AD. Colchicine was first isolated in pure form in 1820. First structure proposed by Windaus in 1924 on the basis of degradation studies. Correct structure proposed by Dewar in 1945. In 1889, it was discovered that colchicine caused "a veritable explosion of mitosis". However, it was noticed that a majority of the dividing cells stopped at metaphase. This useful property has been used to create and study polyploidy in plants. Colchicine inhibits the formation of the microtubules necessary for chromosome transport during metaphase. It forms a 1:1 complex with tubulin , which binds to the end of the forming microtubule and inhibits further polymerization.

6. Direct evidence for this has been obtained from polarized light microscopy . This inhibition by colchicine presumably occurs either through a direct or indirect effect of the compound on the spindle. An example of indirect action would be the activation of an enzyme which attacks the spindle while a direct action might involve binding of colchicine to spindle fibers causing them to dissociate into protein subunits.

7. Colchicum autumnale Structure of Colchicine

8. BLABESLEE and AVERY (1937 ) had made the sensational discovery of the induction of chromosome doubling by colchicine treatment it became a question of prime importance to investigate the cytological mechanism causing this doubling . It has long been known that colchieine acts disturbingly on the normal course of mitosis (DIXON, 1905 ). DUSTIN (1934 ) and LITS (1934) regarded colchicine as a very active agent for inereasing the number of mitoses in a tissue ( a poison caryoclasique ). however , came to the conclusion that the increase in the number of the mitoses after colchicine treatment was due to an accumulation of arrested mitoses» rather than to a stimulation process . He attributed this effect to a failure of the mitotic spindle to form and function in the normal manner. NEBEL and RUTTLE ( 1938), who first studied the effect of colchicine on plant cells (stamen hairs of Trodesc‹intia), arrived at a similar result .

9. The material of the enperiments deal with in this topic consists of root tips of Allium fistulosum and A. c epa . Root tips of A. fistulosum were secured from bulbils that had overwintered in the field, while in the case of A. Cepa large bulbs were used. The experiments were arranged in the following way. Bulbs with rapidly growing root tips, 0.5 to 1.0 cm in length, were immersed for ex posure into colchicine solutions during periods of different length. Root tips were then fixed at certain inter vals after the transfer of the bulbs from the colchicine solutions into pure water. The material was embedded and c ut into transverse and longitudinal sections, the former being 20 µ in thickness, the latter 20-30 µ. The slides were stained in gentian violet,. The Effect of COLCHICINE on root mitosis of Allium cepa .

10. I . DESCRIPTION OF THE TYPICAL COURSE OF THE C-M I TOSIS . In the first series of experiments the conditions were the following : Concentrations of c olchicine: 0.125, O.25, 0.5 1.0, 2.o %. . Exposure times: 7 , 15, 30 min., 1, 2, 24, 72 hours. Fixing took place: 0, 15, 30 min., 1, 3, 6, 12, 24, 48, 72 hours after finishing the colchicine treatment.

11. The effect of colchicine on the course of mitosis is entirely specific, and the modification in mitotic’ behaviour will be abbreviated •c- mitosis*. The c-mitosis can be referred to one single moment , viz. an inactivation of the spindle apparatus connected with a delay of the division of the centromere . The effect thus produced may be expressed as a completion of the chromosome mitosis without nuclear or cellular niitosis. The prophase stages take place normally: the chromosomes divide, condense, and assume metaphase appearance. but however They are, not arranged into an - equatorial plate. Instead they are all the time scattered over the cell in a diakinesis-like manner. This condition lasts for a long time after the disappearance of the nuclear membrane.

12. The halves of each chromosome are seen to be coiled around each other in a relational spiral (Fig. 1 a—ñ). This spiral is then slowly uncoiled, and during this process the chromosomes assume a whole series of shapes exceedingly characteristic of the c-mitosis and never occurring norm a lly. At first loops are formed between the undivided centromeres and the points where, on account of the spiralisation tension, the chromatids t ouch each other. There usually occurs one such point on each of the long chromosome arms (Fig. 1 i—p ). These points of contact move slowly towards the ends, and at last the chromatids touch each other only at the still undivided centromere and at one or both ends

13. This process strikingly resembles the term- inalisation of chiasmata in diakinesis bivalents . At last the ends also slip off, and the half-chromosomes are now held together only at the undivided centromeres. Instances of such typical cross-shaped pairs (called below c-pairs) are reproduced in Figs. 2 d, n and 3 b—d. As has already been mentioned, the formation of the c-pairs is peculiar to material treated with colchieine. Their origin is evidently due to the delay of the division of the centromere. In the course of normal mitoses the centromere divides at about the same time as the chromosomes are arranged into the equatorial plate, and the orientation towards the poles is probably simply conditioned by the division of the centromeres ( » auto-orientation», DARLINozos, 1937 ). Anyhow , the later stages in the uncoiling of the relational spiral normally occur within the equatorial plate.

14. After that the centromeres separate and proceed towards the poles, pulling the chromosome arms behind. The c-pairs form the configuration most commonly found the first few hours after the colchicine treatment . This indicates that the division of the centromere is delayed for quite a considerable time. This is, partly at least, the cause of the apparent impression of mitotic stimulation, which is always found after c-treatment . . Fig. 3. The division of the centromere within the c-pairs . A. c epa : a--b, k-1; fistulosurn: c-j, m-p. - x 3500.

15. The prophases arrive at metaphase and are kept at that stage for a long period until the centromere finally divides . During this period no normal anaphases are found in the slides. If certain cells are at anaphase at the onset of colchicine action, they may form two telophase nuclei, if the anaphase is advanced far enough, and in certain cases a cell wall has been seen formed between the nuclei, but in most cases the anaphase chromosomes remain in 2 groups, which will later be included i nto one nucleus. After a few hours the division of the cen tromeres finally takes place (Fig. 3 e— I), and the two daughter chromosomes are straightened out and locate themselves parallelly like pairs of skis• (N ebel and R uttel ). The centromeres are placed opposite one another in each pair (Fig. 3 m—p). The arrangement in pairs is often maintained . A lso through the following mitoses and is especially easy to observe in the case of s1 chromosomes (Fig. 3 g).

16. Incidentally this relative stability in the chromosome arrangement through several subsequent c-mitoses is very conspicuous and results in an accumulation of the same chromo- some in one section of the nucleus . An instance of this is shown in Fig. 4 a, where at least 8 s 1 chromosomes are gathered at the same place . It is often noticed during the c-anaphase that the division of the centromeres does not take place q uite simultaneously within one cell . In Fig. 3, for instance, the still undivided pairs c and d are drawn from the same cell as the two divided ones, e and f. The division of the centromere has evidently been desyncronised as well as delayed. Diagram 1. The rhythm of the c-mitoses.

17. Now the chromosomes pass on into telophase and all of them a re included in one nucleus, which will consequently contain the double somatic chromosome number. After the short exposures, 7 min to 1 hour, there usually occurs only one c-mitosis, but after long exposures several c-mitoses may follow each other in the same cell, and each of them doubles the chro mosome number. The inactivation of the spindle apparatus under the influence of col c hicine is reversible; after a period of 12-24 hours in pure water the spindle begins to regenerate. The regeneration takes place very characteristically and in the course of the transition to normal spindle all kinds of abnormities are seen like multipolar spindles , asymmetrically or not at all compact spindles, etc. After 36-45 hours the mitoses again run their normal course.

18. In Diagr . 1 is given a survey of the rhythm of the c-mitoses, calculated from the experiments now dealt with. From this diagram is seen that after short exposures an interval of 30 minutes passes before the typical c-mitoses appear. This interval is necessary for the full development of the c-pairs, while the inactivation of the spindle probably occurs much earlier. Diagr . 1 also indicates that long ex- posures require a somewhat longer time for recovery than short ones.

19. II. CYTOLOGICAL CONSEQUENCES OF C-MITOSES: About 45 hours after the c-exposure was finished, the mitoses have r eassumed their normal course and persistent changes in the root cells produced by the colchicine treatment can be observed. a fter the short exposures (7-30 min.) such changes are rarely met with, nevertheless a few cells with 4x chromosomes may be found. The majority of cells show the normal diploid number. After an exposure of 1-2 hours a great percentage of 4x cells are found together with occasional 8x cells, and after the longest exposures (72 hours) cells with still greater chro- mosome numbers are seen , 32 x being the upper limit in these series . In order to get an idea of the distribution of different chromosome numbers in the roots after different c-exposures , examined that the cross- sections of the whole meristematic region of a few roots and plotted on diagrams all the chromosome numbers which could be determined .

20. Determination of greater chromosome numbers than 8x could not alwa ys be made exactly, yet there was no difficulty in deciding whether a chromosome plate should be classified as 8x, 16x or 32x. The num bers were not strictly euploid , which is easy to understand in view of the frequently occurring anaphase disturbances. The plate reproduced in Fig. 4 d shows, for instance, 135 chromosomes, and though this number may not be exact it is certainly greater than 128, which corresponds to l6x. The root diagrams were grouped into 5 or 6 regions from each root and the chromosome numbers from these regions were tabulated in 'Table 1, the treatment of the roots being specified in Table 2. A study of Table 1 will at once reveal one very important fact: t here is a decided correlation between the chromosome number of a cell and the location of the cell in the root. A greater percentage of changed cells occurs in older parts of the root, while close to the root tip among the new meristematic cells normal diploid numbers are there.

21. Normal 2x cells are favoured at the expense of cells changed by colchi c ine . The primary cause of this is probably the division rate, which seems to be more rapid in diploid than in polyploid cells . Moreover , normal unchanged cells start their first division after the c-treatment more readily than do changed cells . At a certain moment after the transfer from the C olchieine solution, frequent diploid mitos i s are seen, while highly polyploid giant nuclei still linger in prophase stages. As regards the roots recorded in Table 1, the diploid cells have in no case totally disappeared. Even after an exposure of 72 hours there occur numerous diploid cells in the sections close to the tip. When colchicine is used for the production of polyploid forms, this fact must be kept in mind. Throughout a considerably prolongated colchicine treatment single cells of the meristeme can keep themselves in a condition resistant to colchicine .

22. III . THE RE ITERATION OF THE C-MITOSES . The preceding chapter leads to the question: How far can the cells be changed by colchicine, where is the limit of the capability of the cell to respond to an extremely prolongated c-exposure with new c-mitoses? Even the strongest dose of colchicine used in the above experiments (2 %' colchicine for 72 hours) did not entirely kill the cells . After their transfer into water the cells might revert again to the normal mitosis . Judging from the chromosome numbers then observed there had taken place at least 4 generations of c-mitoses in the course of the exposure. It is clear, however, that in this experiment the lethality limit cannot have been far off . After the conclusion of the treatment these cells may constitute a very trifling minority, but thanks to their favoured situation in the competition with polyploid cells they will pre- dominate again after some time. The tissue thus shows a tendency to return to its original cytological state.

23. Example: if 5 bulbs were placed in 0.01 % col - chicine and 6 in 0.1% They were transferred daily into new-prepared colchicine solutions in order to diminish the risk of putrefaction. 6, 8, 10, 12, and 14 days after the beginning of the exposure one bulb from each series was transferred into pure water, and root tips were fixed several times from each bulb. A poisoning effect of colchicine could actually be observed in these experiments. The bulbs immersed in 0.1% solution manifested a much slower growth of the leaf shoots than the bulbs in 0.01 % . On the seventh day the former showed leaf shoots about 8 cm in length, the latter on an average 15 cm, while an untreated control bulb had shoots 34 cm in length. In many of these bulbs the limit of cell lethality had been passed. C-mitoses were, it is true, going on in most of them at the first fixation time, but as a rule they were incapable of reverting into normal mitoses.

24. IV. THE LOWER THRESHOLD VALUE OF COLCHICINE ACTION. The lowest concentrations ( 0.0001 to 0.0005 ) did not produce c- mitosis, while from .01% and upwards the c-mitoses were con spicuous. Also on the second occasion of fixing (after 8 hours) the c-mitoses were taking place in all the concentrations except .01 , where the new meristematic cells showed normal mitosis . The recovery of the spindle is evidently more rapid after exposures in low concentrations. At first, however, the c-mitoses were as conspicuous in .01 as in the stronger solutions.

25. THE PRACTICAL SIGNIFICANCE OF COLCHICINE . . A many facts go to prove that colchicine will constitute the agent long which , without detrimental secondary effects and with full certainty, induces polyploidy. From a practical point of view colchicine has many advantages over the various methods for increasing the chro- mosome number . the colchicine action is specific. No other disturbances besides the in activation of the spindle apparatus are observed, at least if the c-ex posure is not prolonged too much and the concentrations employed are not too strong. The completeness of the action of colchicine is remark- able. All the mitoses taking place within the exposed tissue turn into c-mitoses and bring about chromosome doubling. importance is also the reversibility of the inactivation process of the spindle After the conclusion of the exposure the spindle apparatus recovers and is able to function in the normal manner. Cells with increased chromosome number are thus brought back to normal mitoses after their period of rest.

26. Fig . : The formatio n o f colchicin e tumours . n: bul b treate d wit h . 1 % colchieine for 8 days; the calyptra is clearly visible; b: eolchicine trealment alternating with periods in pure water; the tumours take the shape of strings of pearls. The possibility of. macroscopically diagnosing colchicine-induced polyploidy may be of some practical use. As is seen from Figs. 6 and 7, tumours are formed by the root meristeme, while the length growth ceases altogether [compare control Fig. 6 a with c-treated bulbs ( with .1% colchicine for - 8 days) Fig . b—e) . T his phenomenon is caused by the increase in volume of the meristematic cells, whil e th e formatio n o f new cell s i s completel y suppressed.

27. An idea of the sensitivity of this macroscopic reaction may be had from Fig . 7 b . This bulb has been treated with .5% colchicine solution alternating with periods in pure water. The colchicine tumour then takes the shape of a string of pearls, each period in water being distinguishable as a constriction of the tumour . Considering all the obvious advantages of colchicine for producing varieties with increased chromosome number, it seems very likely that colchicine, on account of its specific and infallible action, will be of great practical use and possibly take the place of the more favourable acting agents for the production of polyploids .

28. When designing experimental procedures , we must remember that microtubules not only play an important part in separating chromosomes, but also are involved in other cellular activities. Disassembly of microtubules by colchicine does not only terminate cell division, but also affects the structure, intracellular organization, and macromolecule transport of the cells. We must determine what features in a dividing cell population might tell us that colchicine treatment has successfully inhibited spindle formation selectively, and what features might have simply stopped mitosis because it killed the cell. For example, if we see many empty cells, or cells with a densely stained nuclear zone but no evidence of mitosis in treated onion root tips, it would probably mean that the colchicine has simply killed the cells. However, an observation that most cells are in prophase and metaphase, but very few are in anaphase or telophase in treated onion root tips would more clearly indicate that colchicine has arrested cell division of most cells by inhibiting the formation of a spindle apparatus. If the guidance and mechanical force of the apparatus are not impaired (as in the control onion root tips), we should see cells in all stages of mitosis represented equally.

29. Spindle fibre assembly

30. Cell Centrosomes Microtubule

31. The spindle apparatus (or mitotic spindle ) refers to the cytoskeletal structure of eukaryotic cells that forms during cell division to separate sister chromatids between daughter cells. It is referred to as the mitotic spindle during mitosis, a process that produces genetically identical daughter cells, or the meiotic spindle during meiosis, a process that produces gametes with half the number of chromosomes of the parent cell.

32. M ar c K i r s ch ne r a nd T i m M i t chi s on In 1986, prop o s e d t hat m i c ro t ubu l es u se t h e i r d y n am i c properties of growth and shrinkage at their plus ends to probe the three dimensional space of the cell. Plus ends that encounter kinetochores or sites of polarity become captured and no longer display growth or shrinkage. In contrast to normal dynamic microtubules, which have a half-life of 5–10 minutes, the captured microtubules can last for hours.

33. Self-organization of molecular components of possible spatial provides a variety structures . This model p r o p oses t h at microtubules are nucleated acentrosomally s p ont a ne o usly b u ndl e s and near chromosomes and as se m bl e i nto ant i - p aral l el adopt a spindle-like structure

34. Astral microtubules. Polar microtubules. Kinetochore microtubules.

35. Astral microtubules develop in the actin skeleton and interact with the cell cortex to aid in spindle orientation . They are organized into radial arrays around the centrosomes. The turn-over rate of this population of microtubules is higher than any other population. The role of astral microtubules is assisted by dyneins specific to this role. These dyneins have their light chains (static portion) attached to the cell membrane , and their globular parts (dynamic portions) attached to the microtubules . The globular chains attempt to move towards the centrosome, but as they are bound to the cell membrane, this results in pulling the centrosomes towards the membrane , thus assisting cytokinesis.

36. Polar microtubules interdigitate at the spindle midzone and push the spindle poles apart via motor proteins.

37. Kinetochore microtubules directly connect to the kinetochores. Each chromosome has two chromatids, and each chromatid has a kinetochore. The two kinetochores associated with a region of the chromosome called the centromere.

38. Spindle assembly is largely regulated by phosphorylation events catalyzed by mitotic kinases . Cyclin dependent kinase complexes (CDKs) are activated by mitotic cyclins, whose translation increases during mitosis. CDK1 (also called CDC2 ) is considered the main mitotic kinase in mammalian cells and is activated by Cyclin B1 . Aurora kinases are required for proper spindle assembly and separation. Aurora A associates with centrosomes and is believed to regulate mitotic entry . Aurora B is a member of the chromosomal passenger complex and mediates chromosome- microtubule attachment and sister chromatid cohesion . Polo-like kinase, also known as PLK , especially PLK1 has important roles in the spindle maintenance by regulating microtubule dynamics.

39. Pr inci p l e s o f ana t o m y a nd physiol o gy by Ger a rd J T or t ora/ Bryan Derrickson, pg-92 https:// www.nature.com/scitable/content/types-of- microtubules-involved-in-mitosis-14752887 https://en.wikipedia.org/wiki/Microtubule https://en.wikipedia.org/wiki/Spindle_apparatus https://en.wikipedia.org/wiki/Tubulin#Eukaryotic