Number of spindle fibres during Metaphase?

Number of spindle fibres during Metaphase?

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During metaphase, the chromosomes are arranged on the equatorial plate and are attached to spindle fibres. After S phase, can the cell be said to attain the configuration of 4n?

Also, during metaphase, since two spindle fibres (one from say right centriole and other from say left centriole) are attached to one tetrad, so can it be said in a well defined pattern that for a human being, 46*2=92 spindle fibres are formed in order to proceed with mitosis? Also how would the number vary in case of Meiosis I and Meiosis II?

The number of spindle fibres is actually more than total number of kinetochore pairs. The fibres attached to kinetochores are called K-fibres and the others are called polar fibres. I cant surely say that there is exactly one K-fibre per kinetochore but as per its definition and from the microscopic images you can conclude that there is one per kinetochore.

In a normal mitotic metaphase there are 2n kinetochore pairs, and as you rightly calculated, there will be 96 K-fibres.

In meiosis-I there is a tetrad which has 2 kinetochore pairs but the ones in sister chromatids behave as a single unit. So there would be 23 fibres.

The fusion of kinetochores of sister chromatids is relaxed in meiosis-II and again a separation would require 23 fibres.

Please have a look at this article. Molecular biology of Meiosis nicely explained:

More about K-fibre formation:

Centrioles are built from a cylindrical array of 9 microtubules, each of which has attached to it 2 partial microtubules. Figure (PageIndex<1>) is an electron micrograph showing a cross section of a centriole with its array of nine triplets of microtubules.

Figure (PageIndex<1>): Cross Section of a Centriole (courtesy of E. deHarven). The magnification is approximately 305,000.

When a cell enters the cell cycle and passes through S phase, each centriole is duplicated. A "daughter" centriole grows out of the side of each parent ("mother") centriole. Thus centriole replication &mdash like DNA replication (which is occurring at the same time) &mdash is semiconservative.

  • Functional microtubules grow out only from the "mother".
  • When stem cells divide, one daughter cell remains a stem cell the other goes on to differentiate. In two animal systems that have been examined (mouse glial cells and Drosophila male germline cells), the cell that receives the old ("mother") centriole remains a stem cell while the one that receives what had been the original "daughter" centriole goes on to differentiate. (You can read about these findings in Wang, X., et. al., Nature, 15 October 2009.)

Centrioles are a key feature of eukaryotic cells and presumably arose with the first eukaryotes. A few groups have since lost their centrioles including most fungi (but not the primitive chytrids), "higher" plants (but not the more primitive mosses, ferns, and cycads with their motile sperm) and animal eggs lose their centriole during meiosis and must have it restored by the sperm that fertilizes it

In nondividing cells, the mother centriole can attach to the inner side of the plasma membrane forming a basal body. In almost all types of cell, the basal body forms a nonmotile primary cilium. In cells with a flagellum, e.g. sperm, the flagellum develops from a single basal body. (While sperm cells have a basal body, eggs have none. So the sperm's basal body is absolutely essential for forming a centrosome which will form a spindle enabling the first division of the zygote to take place.)

In ciliated cells such as the columnar epithelial cells of the lungs and ciliated protozoans like the paramecium, many basal bodies form, each producing a beating cilium. Most of their centrioles are produced by repeated duplication of the daughter centriole of centrosome and are temporarily assembled in a special organelle called the deuterosome (not to be confused with deuterostome). Centrioles organize the centrosome in which they are embedded.

The Plant, the Cell and its Molecular Components

P.M. Dey , . J.B. Harborne , in Plant Biochemistry , 1997

(c) Prometaphase

At prometaphase (late prophase) the chromosomes condense inside the nuclear envelope and asters of fibers appear on the outside of the chromosomes. When the nuclear envelope has disappeared, a spindle forms in prometaphase. The spindle fibers comprise bundles of microtubules radiating from the opposite ends and referred to as poles of the cell. The chromosomes then migrate to the equatorial plane where they attach to one of the spindle fibers. In animal cells, spindle formation occurs by centrosomes, which are composed of twin centrioles at right angles surrounded by amorphous material. The centrosome is the major microtubule-organizing center during interphase. The centrosome replicates in late G 1 and S phases and the pair can be observed just outside the nuclear envelope. However, higher plants have no characterized centrosomes although the nucleation and dynamics of their microtubules suggest that plants possess cell cycle-dependent microtubule organizing center (MTOC) activities. Initiation of microtubule polymerization within a cell usually occurs at specific nucleating sites referred to as MTOCs. In most higher plants, initiation of mitosis is characterized by two successive events involving different microtubule populations. These are the production of the preprophase band and the development of the bipolar spindle. Cytoplasmic microtubules of higher plants radiate from the surface of the nucleus towards the cell cortex. This is one of the particular aspects of the plant cytoskeleton in comparison with other cell types. The plant nuclear surface may comprise an MTOC activity and this may represent one of the important factors in the control of the initiation of mitosis. There is experimental evidence to support the role of the plant nuclear surface as a microtubule nucleation site. When mitosis begins and chromosomes undergo condensation, tubulin incorporation has been shown to increase on the nuclear surface of Haemanthus endosperm cells in prophase, potentially a cell-cycle-dependent control of nucleation. The formation of the bipolar spindle ( Fig. 1.22 ) around the nucleus is achieved through a transient convergence of microtubules forming aster-like centers, which subsequently produce spindle poles. This increased microtubule interaction is believed to be mediated by specific microtubule-associated proteins (MAPs). Calmodulin is found at higher concentration at the centriolar polar regions of animal cells. It is also found in these prophase microtubule centers, suggesting that there is a calmodulin-regulated mechanism in higher plants.

The important chromosomal event of prometaphase is the attachment of the chromosomes to the spindle and their movement towards the center of the spindle. Attachment of the chromosome to the spindle occurs at the kinetochore, which contains proteins for chromatid attachment. The breakdown of the nuclear envelope permits the kinetochores to attach to the spindle microtubules.

Notes on Microtubule and Spindle

During the process of cell division the cytoplasmic network of microtubules disappears and the microtubules are reassembled in the form of spindle in the dividing cell (in both mitotic and meiotic cell division). Though the controlling pattern of this disappearance and recognized is not very clear, but it has been realised that there are several regions which act as micro­tubule organising centres (MOCs or MTQCs).

The following are the examples of different microtubule organising centres involve in cell division:

They form the mitotic spindle and also the centriole satellites that form other centrioles.

These are the regions on the centromeres of chromosomes which are the attachment sites for microtubules of mitotic spindle.

(iii) Pericentriolar Cloud:

It has also been shown that the microtubules are assem­bled from the pericentriolar cloud and not from the centrioles.

In plant cells as they do not have the centrioles, so other centres play the role for organization of micro­tubule. Assembly of microtubules occurs in three steps. In the first step, free α-β tubulin dimers associate longitudinally to form short unstable protofi lament. Next the short protofilaments associate laterally into more stable curved sheet.

In the final step, thirteen such protofilaments join laterally to form the cylinder (Fig. 5.7). Micro­tubule then grows by the addition of the subunits to the ends of protofilaments.

The process of microtubule assembly requires tubulin monomers bound to GTP, Mg 2+ and ions. Though GTP binding is necessary for microtubule assembly but GTP hydrolysis does not provide the energy to drive the pro­cess. GTP remains bound to the tubulin in microfilaments.

When GTP is hydrolysed to GDP, the monomers become less stable and disaggregate. Polymerisation and disaggre­gation of microtubules may occur at either end, and may proceed independently. Microtubules have plus and minus ends. In the plus end microtubule assembly or disassembly occurs faster than in the minus end.

Colchicine, vinblastine, vincristine and podophyllotoxin inhibit microtubule assembly whereas taxol promotes and stabilizes the micro­tubule formation.

Spindle Apparatus:

The term ‘mitotic apparatus’ or ‘spindle apparatus’ has been applied to the asters that surround the centrioles together with the mitotic spindle.

The spindle apparatus has the chromo­some fibres, joining the chromosomes to the poles the continuous fibres, extending pole to pole the inter-zonal fibres observed between the daughter chromosomes and nuclei in anaphase and telophase all of which are composed of microtubules (Fig. 5.8A).

The EM and polarization microscopic stu­dies have revealed that in plant cells, which are devoid of centrioles and asters, the first spindle fibres appear at prophase in a clear zone sur­rounding the nucleus. Birefringence is strongest near the kinetochores but becomes weaker towards the poles. During anaphase, the chro­mosomes are led by intensely birefringent chro­mosomal spindle fibres (Fig. 5.8B).

The continu­ous fibres, in which birefringence is low in early anaphase, become more conspicuous in late anaphase and telophase. During anaphase in a plant cell, it is possible to differentiate the micro­tubules attached to the kinetochores of the chro­mosomes from those forming the continuous and inter-zonal fibres.

A study on the number of microtubules has shown that there may be as few as single micro­tubule per chromosome in the spindle of yeast cell and as many as 5000 in the spindle of a higher plant cell. The chromosomal fibres are also called kinetochore tubules.

Among the so- called continuous microtubules which point towards the poles, all of them are not long enough to reach the pole, only a few micro­tubules may be so long as to span between the poles are called as polar tubules and the rests are called free tubules (Fig. 5.9).

In vitro studies have revealed that the assembly of microtubules is controlled by the poles and also by the kinetochores. The lateral interaction between the spindle microtubules may also be involved. When a cell enters prophase, the cytoplasmic microtubules become depolymerized and replaced by the mitotic spindle.

At metaphase, only the spindle microtubules are present at anaphase with the movement -of the chromosomes, the spindle becomes de-polymerised and at telophase the daughter cells are held by the mid-body, and the cytoplasmic microtubules reappear.

The Ca ++ ions and the Ca ++ binding protein, calmodulin, appear to have a controlling role in the assem­bly and disassembly of spindle microtubules. The microtubules have distinct polarity with a fast growing or plus end and a slow growing or minus end (Fig. 5.10).


The major event of Anaphase is the sister chromatids moving to opposite poles of the cells, due to the action of the condensing spindle fibres. The chromatids only start separating when the pressure is sufficient to split the centromere. At this point, each chromatid effectively becomes a chromosome. The moving sister chromatids form a V shape as they move through the cytoplasm. This is because the centromeres are pulled by the spindle fibres, and lead the rest of the chromatid.

Telophase sees the nuclear envelope reform around the chromosomes at opposite poles of the cell

Cell Cycle and Cell Division Model Question Papers

1. Which of the following options gives the correct sequence of events during mitosis?
A. Condensation – nuclear membrane – disassembly crossing over segregation – telophase
B. Condensation – nuclear membrane disassembly – arrangement at equator centromere division – segregation telophase
C. condensation – crossing over nuclear – membrane disassembly – segregation telophase
D. condensation – arrangement at equator centromere division – segregation telophase

2. Anaphase Promoting Complex (APC) is a protein degradation machinery necessary for proper mitosis of animal cells. If APC is defective in a human cell, which of the following is expected to occur?
A. Chromosomes will not condense
B. Chromosomes will be fragmented
C. Chromosomes will not segregate
D. Recombination of Chromosomes arm will occur

3. During cell growth, DNA synthesis takes place in-
A. G1 Phase
B. S-Phase
C. M Phase
D. G2-Phase

4. When cell has stalled DNA replication fork, which checkpoint should be predominantly ctivated?
A. G2/M
B. G1/S
C. Both G2/M and M
D. M

5. In meiosis crossing over is initiated at
A. Zygotene
B. Diplotene
C. Pachytene
D. Leptotene

6. Which of the following is not a characteristic feature during mitosis in somatic cells?
A. Chromosome movement
B. Synapsis
C. Spindle fibres
D. Disappearance of nucleolu

7. Which of these is not a key feature of meiosis?
A. Meiosis involves two sequential cycles of nuclear and cell division
B. Meiosis involves pairing of homologous chromosomes
C. Two cycles of DNA replication occur during meiosis
D. There is recombination between the paired homologous chromosomes

8. Which of the following phases correspond to the interval between mitosis and initiation of DNA replication?
A. S phase
B. G phase
C. G2 phase
D. M phase

9. The checkpoint in cell cycle plays important role in-
A. repair DNA damage
B. apoptosis initiation
C. assess DNA damage
D. inhibit cell damage

10. Arrange the following events of meiosis in correct sequence-
(i) Crossing over
(ii) Synapsis
(iii) Terminalisation of chiasmata
(iv) Disappearance of nucleolus

A. (i), (ii), (iii), (iv)
B. (ii), (iii), (iv), (i)
C. (ii), (i), (iv), (iii)
D. (ii), (i), (iii), (iv)

11. Which one of the following is wrong for meiosis?
A. It leads to formation of sister chromatids
B. It occurs in diploid cell
C. It occurs in haploid cell
D. It occurs by splitting of centromeres and separation of sister chromatids

12. Which of the following does not occur in the interphase of eukaryotic cell division?
A. Increase of ATP synthesis
B. Increase of DNA synthesis
C. Increase of RNA synthesis
D. d Reduction in cell size

13. Which one of the following is the significance of mitosis?
A. Restricted to haploid cells
B. Cell repair
C. Increases the genetic variability
D. Recombination of chromosomes

14. Find out the correct statement-
A. During mitosis endoplasmic reticulum and nucleolus disappear completely at early prophase
B. Chromosomes are arranged along the equator during prophase of mitosis
C. Chromosome is made up of two sister chromatids at anaphase of mitosis
D. Small disc shaped structures at the surface of the centromeres that appear during metaphase are kinetochores

15. In a typical eukaryotic cell cycle, Gap 1, Synthesis and Gap 2 are the three phases included in the-
A. Prophase
B. metaphase
C. anaphase
D. interphase

16. An example of mitogen is
A. cytokinin
B. glucose
C. glycerol
D. fructose

17. Crossing over occurs in the ________ stage of meiosis.
A. zygotene
B. leptotene
C. pachytene
D. diplotene

18. The stage between two meiotic divisions is called as ________
A. Diakinesis
B. Interkinesis
C. diplotene
D. interphase

19. Chromosomes start pairing in which stage of meiosis?
A. Leptotene
B. Zygotene
C. Pachytene
D. Diplotene

20. During meiosis I, the number of chromosomes is-
A. Halved
B. Tripled
C. Doubled
D. Quadrupled

21. Some cells in adult animals do not divide. They exit G, phase and enter an inactive stage which is called as
A. G2 phase
B. G0 phase
C. S phase
D. M phase

22. Identify the correct combination regarding anaphase, anaphase I and anaphase II
A. Anaphase – centromere splits, Anaphase I – centromere splits, Anaphase II – centromere splits
B. Anaphase – chromatids move to opposite poles, Anaphase I – homologous chromosomes separate, Anaphase II – centromere splits
C. Anaphase – chromosomes cluster at opposite poles, Anaphase I – homologous chromosomes separate, Anaphase II – centromere splits
D. Anaphase – chromosomes move to one pole, Anaphase I – homologous chromosomes separate, Anaphase II –centromere splits.

23. Assertion (A) (A): Events in pachytene play a key role in evolutionary changes in organisms
Reason (R): Exchange of genetic material takes place between sister chromatids or homologous chromosomes
A. A and R are true, R is correct explanation of A
B. Both A and R are true, R is not the correct explanation of A
C. A is true, R is false
D. A is false, R is true.

24. Which is the longest phase of the cell cycle?
A. M-phase
B. Leptotene
C. Interphase
D. S-phase

25. During which phase(s) of cell cycle, amount or DNA In a cell remains at 4C level if the initial amount is denoted as 2C?
A. G1 and S
B. G2 and M
C. G0 and G1
D. Only G2

26. In’S phase of the cell cycle
A. amount of DNA doubles in each cell
B. amount of DNA remains same in each cell
C. chromosome number is increased
D. amount of DNA is reduced to halt in each cell

27. The enzyme recombinase is required at which stage of meiosis?
A. Pachytene
B. Diplotene
C. Zygotene
D. Diakinesis

28. Select the correct statement related to mitosis –
A. Amount of DNA in the parent cell is first doubled and then distributed into four daughter cells
B. Amount of DNA in the parent cell is first halved and then distributed into four daughter cells
C. Amount of DNA in the parent cell is first doubled and then distributed into two daughter cells
D. Amount of DNA in the parent cell is first halved and then distributed into two daughter cells

29. Statement A For a particular character in an individual, each gamete gets only one allele.
Statement B: Chromatids of a chromosome split (separate) and move towards opposite poles during anaphase of mitosis.
A. statement A is correct and statement B is wrong
B. Both the statements are correct and B is the reason for A
C. Statement B is correct and statement A is wrong
D. Both the statements are correct and B is not the reason for A.

30. The centrosome duplicates during the-
A. G2 phase of cell cycle
B. S-phase of cell cycle
C. Prophase of cell cycle
D. G1 phase of cell cycle

31. Cell cycle includes the sequence
A. S, G1, G2, M
B. S, M, G1, G2
C. G1, S, G2, M
D. M, G1, G2, S

32. What are spindle fibres that connect the centromere to respective poles called-
A. Astral rays
B. Interphase fibres
C. Chromosomal fibres
D. Interchromosomal fibres

33. The complex formed by a pair of synapsed homologous chromosomes is called
A. Bivalent
B. equatorial plate
C. Kinetocnore
D. axoneme

34. Assertion: Meiosis ll is Similar to mitosis.
Reason : Meiosis I cannot occur in haploid cells.
A. If both are true with reason being correct explanation
B. Both true but reason is not correct explanation
C. Assertion true but reason is wrong
D. both are wrong

35. Which of the following events takes place during anaphase stage of mitosis
I. Spindle fibre attach to Kinetochores of chromosomes
II. Centromeres split and chromatids separate
III. Chromatids move to opposite poles
IV. Nucleolous, Golgi complex and E.R. reform
A. I and II only
B. III and IV only
C. II and III only
D. L and IV only

36. During meiosis I, the chromosomes start pairing at
A. Zygotene
B. Diplotene
C. Pachytene
D. leptotene

37. During the metaphase stage of mitosis, spindle fibres attach to chromosomes at
A. Kinetochore
B. both centromere and kinetochore
C. centromere, kinetochore and areas adjoining centromere
D. centromere

38. A bivalent of meiosis I consists of
A. four chromatids and two centromeres
B. two chromatids and one centromere
C. two chromatids and two centromeres
D. four chromatids and four centromeres.

39. The homologous genes are separated at
A. Anaphase
B. Diplotene
C. Pachytene
D. Anaphase II

40. Metaphase chromosome appears to be longitudinally divided into two identical parts known as
A. sister chromosomes
B. satellites
C. daughter chromosomes
D. sister chromatids

41. Microtubule depolymerising drug such as colchicine is expected to
A. include formation of multiple contractile rings
B. allow mitosis beyond metaphase
C. inhibit cytokinesis
D. inhibit spindle formation during mitosis

42. Which of the following statement is incorrect about G0 Phase?
A. Mitosis occurs after G0 phase
B. Biocatalysts can be used to exit G0 phase
C. Cell volume keeps on increasing during this phase
D. Cell metabolism occurs continuously in G0 phase

43. Identify the meiotic stage in which the homologous chromosomes separate while the sister chromatids remain associated at their centromeres
A. Metaphase I
B. Anaphase
C. Metaphase I
D. Anaphase

44. During gamete formation, the enzyme recombinase participates during-
A. metaphase-I
B. prophase- II
C. anaphase-II
D. prophase-I

45. Find the correctly matched pairs and choose the correct option
A. Leptotene – The chromosomes become invisible
B. Zygotene-Pairing of homogenous chromosomes
C. Pachytene-Dissolution of the synaptonemal complex takes place
D. Diplotene-Bivalent chromosomes appear as tetrads
E. Diakinesis – Terminalisation of chiasmata take place
A. A and B are correct
B. B and D are correct
C. B and E are correct
D. B and Care correct

46. Which of the following events are not characteristic features of telophase?
A. Chromosome material condenses to form compact mitotic chromosomes
B. Nucleolus, Golgi complex and ER reform
C. Nuclear envelope assembles around the chromosome clusters
D. Centromeres split and chromatids separate
E. Chromosomes cluster at opposite, spindle poles and their identity as discrete elements is lost
A. A, B and D only
B. B and C only
C. A and D only
D. C, D and E only

47. Visible expression of the genetic phenomenon of crossing over is called-
A. Recombination
B. Condensation
C. Chiasmata
D. spiralisation

48. The chromosomes become gradually visible with compaction of chromatin during the meiotic stage
A. diplotene
B. leptotene
C. zygotene
D. pachytene

49. Yeast cell can progress through the cell cycle in about-
A. 30 minutes
B. 90 minutes
C. 60 minutes
D. 120 minutes

50. In onion root tip during metaphase stage of mitosis the number of kinetochores will be
A. 4
B. 8
C. 32
D. 16


We have identified structural connections between metaphase KMTs and regions at or near the pole-proximal ends of non-KMTs in spindles from two species, suggesting that mechanical interactions within the spindle’s body may play an important part in the mechanical stability of these spindles. In small spindles with well-structured poles, like those in yeasts, different classes of MTs connect directly with the pole, allowing the polar plate to link the constrictive and extensive actions of different MTs, helping the spindle to a stable mechanical equilibrium. When poles are absent, as in higher plants, algae like Chlamydomonas, and some meiotic spindles (Redemann et al., 2018), mechanical links between MT classes must be made elsewhere bridges between KMTs and mcMTs can do this job. Even in the complex spindles found in mammalian spindles, where some MTs are linked mechanically with the poles, the observed connections between KMTs, and mcMTs may play an important role in metaphase spindle stability.

Mechanical solutions employed by even larger spindles

EM of serial sections cut from spindles in Haemanthus endosperm has revealed well-developed K-fibers that can include >100 MTs/kinetochore (Jensen, 1982). These spindles also contain large bundles of non-KMTs that run approximately parallel to the spindle axis, passing by the chromosomes during all stages of mitosis. During metaphase and anaphase there is a clear commingling of KMTs with non-KMTs in the region just poleward from each kinetochore, opening the possibility of mechanically significant interactions between them, though neither structural nor morphometric evidence for connections between these MT classes is yet available. Given the situation we describe in Chlamydomonas, it seems likely that analogous interactions provide the mechanical support necessary to withstand pole-directed tension at kinetochores in higher plants as well as an algae (Figure 4E).

Spindles in blastomeres from Caenorhabditis elegans provide a complement to the cases described above. Their metaphase spindles have been reconstructed in their entirety by ET (Redemann et al., 2017). These 3D studies demonstrate that most KMTs are not long enough to reach the spindle poles they terminate in a network of non-KMTs that emanates from the spindle poles, suggesting that force-bearing linkages to the pole are indirect. Moreover, almost all non-KMTs in this metaphase spindle are too short to reach the spindle equator, so there is little evidence for mcMTs at metaphase. These spindles appear to lack a framework of mcMTs to support tension at the centromeres, although such a structure is visible by fluorescence microscopy in early anaphase (Saunders et al., 2007). Here, as in PtK cells, the MT bundles found between the separating chromosome slow, rather than drive, anaphase spindle elongation. Anaphase B appears to be effected by pulling forces that act through astral MTs, which connect each spindle pole with cortex-associated dynein (Grill et al., 2001). Cortical forces are also probably acting with sufficient strength during metaphase to support the tension that acts on kinetochores and would otherwise pull the poles toward the spindle equator.

The spindles formed in extracts from Xenopus eggs are an even more extreme example of supporting centromeric tension by indirect linkages. These spindles are 40–50 µm long and contain tens of thousands of MTs that are short relative to the spindle’s length (Brugués et al., 2012). During prometaphase, MTs form in the neighborhood of the chromosomes, then reorient and reposition to form the spindle (Karsenti and Vernos, 2001). Although kinetochores have not yet been identified by EM, these spindles contain many bundles of MTs, some of which are probably kinetochore associated (Tranfield et al., 2014 Weber et al., 2014). Clever use of laser-mediated cutting of spindle MTs that have been labeled with small amounts of fluorescent mammalian tubulin, followed by detailed study of the space and time dependence of fluorescence redistributions, has shown that these spindles are formed by assemblies of MTs that point in opposite directions. Average MT lengths vary from ∼3 µm near the poles to ∼13 µm far from the pole (Brugués et al., 2012). Time-dependent visualization of speckles induced in these spindles by sparse labeling of the MTs with fluorescent tubulin shows that the polymers are in slow but continuous flux toward one spindle pole or the other (Mitchison et al., 2004), making a two-way conveyer belt. There are no well-defined structures that could serve as spindle poles, so the support for kinetochore tension comes from relatively short non-KMTs that are linked together to form the necessary framework.

Why are direct connections between kinetochores and poles few or absent in big spindles?

The design of small spindles seems efficient and effective for forming a mechanically stable metaphase. Why is this design not used in the bigger spindles described here? A direct polar connection is of course impossible in cells with no structured pole, so linkages between MTs become necessary. Mammalian spindles possess structured poles, but many spindle MTs, both KMTs and non-KMTs, terminate before reaching the poles. In spindles that are bigger still, MTs long enough to extend from pole to kinetochore, or beyond to the spindle midplane, are very rare, defining a need for bridge-­mediated connections to make a stable metaphase structure.

MTs in nonspindle systems can be almost arbitrarily long, probably as a result of the right MT-associated proteins. For example, flagellar MTs in sperm of Drosophila bifurca extend ≥5 mm (Pitnick et al., 1995). Why, then, do not big spindles retain the efficient design of small spindles but instead resort to coupling short MTs to make a framework that can support kinetochore tension? One factor may be that spindle MTs are necessarily dynamic. They form for the occasion of division but disappear before the following interphase. Moreover, dynamic instability is important for the likelihood that MTs will encounter an appropriate load, for example, a kinetochore (Kirschner and Mitchison, 1986 Magidson et al., 2011) or an mcMT from the opposite pole. Labile MTs displaying dynamic instability show a distribution of lengths that is well described by a negative exponential function (Verde et al., 1992 Redemann et al., 2017), so the most numerous MTs are short. This situation may be exacerbated by MT-severing enzymes that cut spindle components in some systems, making the average MT length even shorter (Srayko et al., 2006). To get a significant number of long, dynamic MTs, one must therefore make a very large number of short ones. When the distance from one pole to the far side of the spindle midplane is large, pole-initiated MTs long enough to cross the spindle midplane are expensive, given the tubulin needed to form the many short MTs characteristic of an exponential distribution of lengths. Thus, even in mammalian spindles, whose half-spindles are commonly ∼5 µm, only a very few pole-initiated MTs extend far enough to cross the midplane. To build a robust interpolar structure in a big spindle, the augmin complex can function to initiate MTs along the way from the pole to the midplane (Kamasaki et al., 2013). Many of the resulting MTs can then interdigitate with their counterparts from the opposite side of the spindle, yet none of them needs be too long. Indeed, when augmin levels are reduced by RNAi, the structure and function of a mammalian spindle are seriously compromised (Kamasaki et al., 2013).

Augmin-initiated MTs have the additional advantage in that they commonly occur along the walls of existing MTs (Kamasaki et al., 2013). This behavior may endow them with the ability to form functionally significant mcMT bundles, like the ones described here and by the Tolic´ lab. Indeed, some of the links between KMTs and the ends of non-KMTs described here may be the augmin complex bound to a KMT wall. The same logic could apply in any big spindle, although current evidence from genome sequences has not identified augmin-like molecules in nematodes. Other molecules, like Tangled1 from plants, are known to promote MT-MT binding, particularly the association of an MT end with an MT wall (Martinez et al., 2019), similar to the connections seen here. There are probably additional molecular players with similar properties that are yet to be identified. Whatever the molecular mechanisms, it seems that when cells need a large and labile spindle, they abandon the strategy that works in small spindles and make mechanically equivalent structures from shorter MTs, connected to make a framework that can support kinetochore tension.


The process that produces haploid gametes is meiosis. Meiosis is a type of cell division in which the number of chromosomes is reduced by half. It occurs only in certain special cells of the organisms. During meiosis, homologous chromosomes separate, and haploid cells form that have only one chromosome from each pair. Two cell divisions occur during meiosis, and a total of four haploid cells are produced. The two cell divisions are called meiosis I and meiosis II. The overall process of meiosis is summarized in Figure below. You can watch an animation of meiosis at this link:

Overview of Meiosis. During meiosis, homologous chromosomes separate and go to different daughter cells. This diagram shows just the nuclei of the cells. Notice the exchange of genetic material that occurs prior to the first cell division.

Phases of Meiosis

Meiosis I begins after DNA replicates during interphase of the cell cycle. In both meiosis I and meiosis II, cells go through the same four phases as mitosis - prophase, metaphase, anaphase and telophase. However, there are important differences between meiosis I and mitosis. The flowchart in Figure below shows what happens in both meiosis I and II.

Phases of Meiosis. This flowchart of meiosis shows meiosis I in greater detail than meiosis II. Meiosis I&mdashbut not meiosis II&mdashdiffers somewhat from mitosis. Compare meiosis I in this flowchart with the earlier figure featuring mitosis. How does meiosis I differ from mitosis?

Compare meiosis I in this flowchart with the figure from the Mitosis and Cytokinesis concept. How does meiosis I differ from mitosis? Notice at the beginning of meiosis (prophase I), homologous chromosomes exchange segments of DNA. This is known as crossing-over, and is unique to this phase of meiosis.

7. Dissecting the Chromosome-Based Signal for Spindle Assembly

7.1. Ran-GTP

30 micrometers apart. Subsequent studies employed different sensors and fluorescence lifetime imaging (FLIM) rather than the measurement of donor/acceptor signal ratios alone, which can be sensitive to fluorophore concentration and bleed-through of fluorescence signal [146,147]. These measurements were consistent with a chromosome-centered Ran-GTP-dependent signal, which can release spindle assembly factors from importin-β, covering distances that extend all the way across the spindle [146,147]. A possible explanation for how this gradient could induce asymmetry in microtubule aster organization came from modeling and experimental data that indicate that the Ran-gradient may be combined with the activities of a Ran-regulated kinase (CDK11) and phosphatases [148]. FRET-based sensors also revealed the presence of a Ran-GTP gradient in somatic cells. This spatial gradient was much steeper, and extended across a shorter distance (3–4 μm) compared to what was detected in spindles assembled in Xenopus egg extracts [147]. A more recent study reported an even more localized spatial gradient, extending only

2 μm in dividing somatic cells [149].

7.2. Chromosomal Passenger Complex (CPC)

3 min) disrupted bipolar spindles assembled in egg extracts, reducing microtubule density and overall spindle size. In egg extracts depleted of Op18, the formation of microtubules around chromatinized DNA-beads was accelerated. Other studies suggest that phosphorylation reduces Op18’s binding to tubulin [153]. Together, these data are consistent with phosphorylation suppressing Op18’s inhibitory effect on microtubule formation, and support a model in which chromosomes control the activity of microtubule assembly factors.

7.3. Interplay between Ran-GTP and the CPC


Spermatocyte Culture

Larvae in the fourth instar were selected from a laboratory colony. Testes were isolated in tricine insect buffer (Begg and Ellis, 1979) and submerged under a droplet of Voltalef 10s oil (Ugine Kuhlmann, Paris, France) on a coverslip, where spermatocytes released upon rupturing of the testicular sheath were smeared as a monolayer at the oil-coverslip interface. A ring of Vaseline placed around the oil droplet and four drops of VALAP (a molten mixture of one part each of Vaseline, lanolin, and paraffin) placed at the corners of the coverslip served as spacers upon mounting the coverslip on a glass microscope slide. Spermatocytes survived in such oil preparations for a few hours, sufficient for observation of both meiotic divisions in an individual living spermatocyte.

Cold Treatments That Induced Chromosome Malorientation

For cold treatments to induce chromosome malorientation, selected larvae were transferred from the moist tissue paper mulch that serves as their culture medium in the lab to fresh mulch contained in a 90 × 50-mm crystallizing dish, which was subsequently put on ice in a refrigerator for the duration of cold exposure, typically 24–36 h. Those conditions maintained the temperature of the mulch, and the larvae, at 0–1°C. For microscopy during cold recovery, larvae were removed from the cold, transferred to fresh mulch at room temperature (∼24°C), and then after ∼10 min of recovery, testes were isolated and oil preps were made as with control, untreated material. Typically, 20–30 min of recovery time were spent on specimen preparation before cold-recovering cells were actually located and imaged with the polarizing microscope. This sacrifice of recovery time in specimen preparation was necessitated by the room temperature environment of the microscope. As a consequence of using this approach of preparing cells for observation after recovery onset, complete historical records of the induction of malorientation, which were obtained in earlier studies (Janicke and LaFountain, 1986), were not obtained here.

Polarization Microscopy

Images of birefringent spindle fibers were obtained with a polarizing microscope that was equipped with a liquid crystal universal compensator (LC-PolScope, Cambridge Research and Instrumentation, Woburn, MA) and was operated as described by Oldenbourg and Mei (1995) and by Oldenbourg et al. (1998). The optical set-up included a 60×/1.4 NA plan apochromat oil immersion objective and apochromat oil immersion condenser. Images were stored as TIFF files and imported into NIH image for analysis (NIH image is public-domain software for image analysis available online from NIH Image For the present study, we made extensive use of a stepper motor to make Z-focus series images of cells, in which important data regarding their numerous spindle fibers were in different focal planes. Two Z-focus series (cells 6 and 14) were made with steps of 0.5-μm stage traverse, but all subsequent trials were made with steps of 0.3 μm in order to maintain high spatial resolution.

The metaphase positions of bivalents in LC-PolScope images were visualized by image overlays. In each plane of a Z-focus series, the image of the bivalent in that plane was overlaid by solid paint. The maloriented bivalent was identified using a different pixel value than the properly oriented bivalents in the same cell. In addition, the positions of kinetochores and basal bodies of the polar flagella were also identified in their respective focal planes and overlaid with a small dot of a different pixel value. Pixel values of the rest of the image were set to zero. Then, a stack of overlays was created for only the structures of interest (i.e., bivalents, kinetochores and basal bodies). Each stack was projected into a single plane using a maximum pixel value algorithm. Projections were merged to produce a final projection of the positions of all structures of interest relative to one another. The spindle equator was included as the midline between basal bodies. Maloriented and properly oriented bivalents appear in the projections with different gray values (see Results).

For the quantitative analysis of birefringence retardation (also called retardance), we measured the magnitude of retardance within selected areas of images of individual kinetochore fibers. With our system, unlike with traditional polarized light microscopes, the gray scale (brightness) level is directly proportional to the retardance within the area of interest of a polarized light image. Furthermore, the LC-PolScope measures the retardance independent of the orientation of the birefringence axis in the selected area. For the purpose of comparing retardance values of different microtubule bundles, we used an algorithm that computed “retardance area” within the domain of each kinetochore fiber that was selected. As shown by Oldenbourg et al. (l998), the retardance area is directly proportional to the number of microtubules in a fiber, with each microtubule contributing ∼7.5 nm 2 to the retardance area of a fiber. In addition, the measured retardance area is independent of the exact focus position, as long as the fiber boundaries can be discerned. The fiber boundary is best discerned when the focal plane extends through the center of the fiber. In that focus position, the fiber boundary is distinct and retardance contributed by out of focus parts of the kinetochore fiber is inside the boundary, assuming an approximate cylindrical shape of the fiber.

For measuring the retardance area of a kinetochore fiber, we made a line scan perpendicular to the axis of the fiber being analyzed at a distance of ∼0.5 μm from its associated kinetochore. The line had the shape of an elongated rectangle 4 pixels wide by 3–4 μm long. The line scan included the retardance measured across the fiber and surrounding background. The following regimen was used for determining the boundary limits of a birefringent fiber: 1) the image to be analyzed was rotated to put the long axis of the to-beanalyzed fiber parallel to the Y-axis, and 2) the X-Y pixel coordinates of the fiber's left and right boundaries along the line scan were determined. Using that information, the algorithm then computed the retardance area of the birefringent fiber.

The retardance area is defined as the integral (or area) under the curve of measured retardance in the line scan. Hence, the retardance area has the unit: length square (L 2 ), because both retardance and the width of the fiber under analysis have linear units of measurement. The retardance area of a kinetochore fiber was measured as the difference of the fiber retardance and the background retardance (see Results). The differential value of retardance area, then, provides an estimate of the number of microtubules within a fiber over background, and thus, it provides the desired measure for making comparisons among different fibers within the same cell.

The uncertainty in measuring the differential retardance area is largely determined by the uncertainty in estimating the background retardance. The background retardance originates from the birefringence of other spindle microtubules that have the same average orientation as the kinetochore fibers and are located in Z-sections either above or below the fiber. For estimating the background that contributes to the retardance measured in the image of a kinetochore fiber we measured the spindle retardance on either side of the fiber and linearly interpolated between the two values (see Figures 2 and 3). For estimating the uncertainty in this background subtraction procedure we considered the typical variation of spindle retardance over a distance equivalent to the fiber thickness. We estimate the typical variation of the spindle retardance beyond the linear interpolation to be around ±0.02 nm over a distance of 2 μm, leading to an uncertainty in the retardance area of 0.02 × 2000 = 40 nm 2 , which equals nearly 6 microtubules. Hence, we estimate the uncertainty in determining the number of microtubules in a given kinetochore fiber to be about ±6 microtubules. This uncertainty should increase for fibers that are thicker than 2 μm and decrease if they are thinner. On the basis of this uncertainty we also estimate that the thinnest fiber that can reliably be identified should contain around 10 microtubules.

Figure 2. Birefringent kinetochore fibers in a control (untreated) spermatocyte (cell 29) imaged with the LC-PolScope. (A and B) Two sections from a series of optical sections made through the cell at focus steps of 0.26 μm (0.3 μm of stage travel). Lower right: slice number/total slices in the focus series. In viewing these polarized light images, brightness represents the magnitude of birefringence retardation (black = 0 nm and white = 2.5 nm retardance), irrespective of the orientation of the brifringence axis. Bar, 5 μm. (C) A duplicate image of B, including the line from which retardance area data were obtained. The shaded area in the plot is the retardance area (461 nm 2 ) of the fiber, which was evaluated for the number of kinetochore microtubules (62). (D) The metaphase positions of the three bivalent chromosomes in this cell upon projection of all images within the Z-focus series to make a 2-D profile. Two dots indicate the positions of the flagellar basal bodies within the centrosomes at the two spindle poles. The numbers on each kinetochore fiber indicate the number of kinetochore microtubules in that fiber, based on retardance area analysis. Estimated inclination angles of kinetochore fibers ranged between 3 and 10° and were used to correct retardance values (see Materials and Methods).

Figure 3. Birefringent kinetochore fibers of a bivalent exhibiting amphisyntelic orientation during cold recovery. Cold treatment: 29 h at 0.2°C. (A–D) Four slices from the Z-series made through cell 47 at focus steps of 0.26 μm. Time elapsed after onset of cold recovery: 61 min. (A–C) Bivalent 2 is on the left bivalent 3 is on the right. Bivalent 3 is maloriented. (D) Bivalent 1 is on the left one of the sex univalents is on the right. Lower right: slice number/total slices in the Z-series. Bar, 5 μm. (A) White arrowhead locates one of the amphitelic kinetochores of bivalent 3 and its kinetochore fiber extends to the upper pole. (B) White arrowhead is positioned at the same X,Y pixel coordinates as in A black arrowhead locates the other amphitelic sister kinetochore and its kinetochore fiber extends to the lower pole. (C) White and black arrowheads are positioned at the same X,Y coordinates as in A and B, respectively. In focus above the white arrowhead are the two syntelic sister kinetochores of the partner homologue and its kinetochore fiber extends to the upper pole. (D) White arrowheads locate the positions of the two basal bodies at the two spindle poles of cell 47. (AA) A duplicate image of A including the line from which retardance area data were obtained. The shaded area in the plot is the retardance area of the selected fiber. The inclination angle of the fiber was estimated to be 11° and retardance data were corrected accordingly. (BB) A duplicate of B with the plot of retardance area data obtained from it (inclination angle 13°). (CC) A duplicate of C with the plot of retardance area data obtained from it (inclination angle 17°). DIC: cell 47 during anaphase imaged with differential interference contrast microscopy showing the anaphase laggard that derived from the amphitelically oriented homolgue depicted in A and B. Time elapsed after initiation of cold recovery: 86 min.

The conversion factor that we used (i.e., 7.5 nm 2 per microtubule) was obtained by Oldenbourg et al. (l998) using in vitro preparations of microtubules containing negligible microtubule-associated proteins and other solutes in the surrounding medium. Inside a living cell, however, the surrounding medium contains many proteins, which tend to increase the refractive index of the cytoplasm (based on our own interferometric measurements, the average refractive index of the cytoplasm varies around 1.36). Also, spindle microtubules are known to have other proteins associated with them, leading to a higher mass per unit length of a kinetochore fiber microtubule compared with a microtubule prepared from purified tubulin. Although the higher medium refractive index decreases the effective retardance area of a kinetochore fiber microtubule, its higher mass per unit length increases its effective retardance area. Hence, the two effects might cancel each other, and the effective retardance area of a kinetochore fiber microtubule might be close to the one measured in vitro. Nevertheless, the value of 7.5 nm 2 used in this study might lead to absolute numbers of microtubules per kinetochore fiber that are somewhat higher or lower than the actual values. However, a change in the effective retardance area per microtubule would affect all measurements equally and in no way changes the disparities listed and conclusions drawn from our measurements.

In all cells analyzed, the spindle was oriented nearly parallel to the focal plane. Nevertheless, the inclination of the spindle axis and especially of the kinetochore fibers has a systematic effect on the retardance measurements. The retardance measured in a given fiber decreases with increasing inclination angle. To correct for this effect, we estimated the inclination angle by first measuring the X-Y-Z coordinates of individual kinetochores and of the polar basal bodies at the poles to which kinetochore fibers were directed. The coordinate values were gleaned from Z-focus series and then imported into a spread sheet program for computing the inclination angles, based on calibration of pixels and Z-focus steps (see below). Inclination angle of the spindle axis was measured as the angle between the x-y plane and the line between the two basal bodies. Inclination angles of kinetochore fibers were estimated by calculating the angle between the x-y plane and the line connecting the basal body and kinetochore locations. The average inclination angle of all spindle axes was 3° (range 0–10°) the average inclination angle of all kinetochore fibers was 8° (range 0–19°).

Inclination angles of individual kinetochore fibers were used to correct the retardance value of the fibers. The measured retardance value was multiplied by the factor 1/cos 2 (α), where α is the inclination angle (see Born and Wolf, 1980).

For our statistical analysis of kinetochore fiber microtubules reported in Tables 1 and 2, we used the common expressions for the average and standard deviation (StdDev) of measured numbers of microtubules. The percent disparity of kinetochore fiber microtubules that attach the same bivalent to opposite poles was calculated using the same expressions for the StdDev and average. For a given bivalent that is attached to one pole by n1 microtubules and to the other pole by n2 microtubules, the percent disparity is calculated as the ratio of the StdDev and the Average:

Table 1. Summary of retardance area analysis on birefringent kinetochore fibers in control (untreated) spermatocytes.

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