C mitosis

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 1st sem,
04-AGRMA-01754-2018
Dept. of Genetics and Plant
Breeding,
C.P.C.A, S.D.A.U,
Sardarkrushinagar -385 506

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

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?

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

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

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.

Colchicum autumnale Structure of Colchicine

 BLABESLEE and AVERY (1937 ) had made the sensational discovery
of the induction of chromosome doubling by colchicinetreatment 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.

 The material of the enperiments deal with in this topic consists of
root tips of Allium fistulosum and A. cepa.
 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 exposure into colchicine solutions during periods of
different length.
 Root tips were then fixed at certain intervals after the transfer of
the bulbs from the colchicine solutions into pure water.
 The material was embedded and cut 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.

I.DESCRIPTION OF THE TYPICAL
COURSE OF THE C-MITOSIS.
In the first series of experiments the conditions were the following:
Concentrations of colchicine: 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.

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.

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 normally.
At first loops are formed between the undivided centromeres and the
points where, on account of the spiralisation tension, the chromatids
touch each other. There usually occurs one such point on each of the
long chromosome arms (Fig. 1i—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

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.

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. cepa: a--b,
k-1;

 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 into one nucleus.
 After a few hours the division of the centromeres finally takes place (Fig.
3 e— I), and the two daughter chromosomes are straightened out and
locate themselves parallelly like pairs of skis• (Nebel and Ruttel ).
 The centromeres are placed opposite one another in each pair (Fig. 3
m—p). The arrangement in pairs is often maintained. Also through the
following mitoses and is especially easy to observe in the case of s1
chromosomes (Fig. 3 g).

Incidentally this relative stability in the chromosome arrangement
through several subsequent c-mitoses is very conspicuous and results
in an accumulation of the same chromosome in one section of the
nucleus.
An instance of this is shown in Fig. 4 a, where at least 8 s1
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 quite 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-

Now the chromosomes pass on into telophase and all of them are
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
chromosome number.
 The inactivation of the spindle apparatus under the influence of
colchicine 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.

 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 exposures
require a somewhat longer time for recovery than short ones.

II. CYTOLOGICAL CONSEQUENCES OF C-MITOSES:
 About 45 hours after the c-exposure was finished, the mitoses have
reassumed their normal course and persistent changes in the root cells
produced by the colchicine treatment can be observed.
 after 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 chromosome numbers are seen, 32x 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.

 Determination of greater chromosome numbers than 8x could not
always be made exactly, yet there was no difficulty in deciding whether a
chromosome plate should be classified as 8x, 16x or 32x.
 The numbers 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: there 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.

 Normal 2x cells are favoured at the expense of cells changed by
colchicine.
 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 Colchieine solution,
frequent diploid mitosis 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.

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.

 Example:
 if 5 bulbs were placed in 0.01 % colchicine 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.

IV. THE LOWER THRESHOLD VALUE OF COLCHICINE
ACTION.
 The lowest concentrations (0.0001 to 0.0005) did not produce c-
mitosis, while from 0.01% and upwards the c-mitoses were
conspicuous.
 Also on the second occasion of fixing (after 8 hours) the c-mitoses
were taking place in all the concentrations except 0.01 , 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 0.01 as in the stronger solutions.

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 chromosome number.
the colchicine action is specific. No other disturbances besides the
inactivation of the spindle apparatus are observed, at least if the c-exposure
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

Fig. : The formation of colchicine tumours. n: bulb treated with 0.1 % 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 0.1% colchicine for -8 days) Fig.
b—e) .
 This phenomenon is caused by the increase in volume of the
meristematic cells, while the formation of new cells is completely
suppressed.

An idea of the sensitivity of this macroscopic reaction may be had
from Fig. 7 b.
 This bulb has been treated with 0.5% 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.

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.

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.

Marc Kirschner and Tim MitchisonIn 1986,
proposed that microtubules use their
dynamicproperties 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.

Self-organization of molecular
componentsof possible
spatial
provides a variety
structures.This model proposes that
microtubules are nucleated
acentrosomallyspontaneous
ly bundles
an
d
near chromosomes and
assembleinto anti-
parallel adopt a spindle-
like structure

Astral microtubules.
Polar microtubules.
Kinetochore

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.

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

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.

• 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

1. Principles of anatomy and physiology by Gerard J Tortora/
Bryan Derrickson, pg-92
2. https://www.nature.com/scitable/content/types-of-
microtubules-involved-in-mitosis-14752887
3. https://en.wikipedia.org/wiki/Microtubule
4. https://en.wikipedia.org/wiki/Spindle_apparatus
5. https://en.wikipedia.org/wiki/Tubulin#Eukaryotic

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