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Lecture 2, part 3 of 3
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D) Anaphase

Anaphase is the shortest phase of mitosis. The beginning of anaphase is observed microscopically when the synapsed sister-chromatids separate simultaneously, and begin migrating to the opposite cellular poles. During anaphase, longer chromosomes often appear V-shaped or J-shaped, as if the chromosomes were being dragged by their centromeres, with the arms lagging behind.

Anaphase results in two compact bundles of chromosomes at opposite poles of the parent cell.

 
Early anaphase


Late Anaphase


Images from Yaping Wang and Brian Fristensky, University of Manitoba.
  
kinetochore fibers
- spindles attached to kinetochore
.

polar fibers - spindles interdigitated with spindles from opposite poles.


1. Poleward movement of chromosomes (anaphase A) is powered by microtubule depolymerization.

Begins with a splitting of the kinetochore, releasing the tension and allowing chromosomes to "dangle" at the ends of their respective spindles.  Depolymerazition of microtubules at the kinetochores causes sister chromatids separate into independent chromosomes.  Depolymerization provides energy for chromosome movement.

DEMONSTRATION THAT CHROMOSOMES MOVE POLEWARD ALONG STATIONARY KINETOCHORE MICROTUBULES [
Adapted from G. J. Gorbsky, P. J. Sammak, and G. Borisy, 1987, J. Cell Biol. 104:9; and G. J. Gorbsky, P. J. Sammak, and G. Borisy, 1988, J. Cell Biol. 106:1185.]
The experiment shown above verifies this model. At top, cells have been labeled with fluorescently-tagged tubulin proteins, so that microtubules (eg. spindle fibers) fluoresce red in UV microscopy. A laser is used to bleach part of the spindle in metaphase cells, so that the bleached spot will not fluoresce.  During anaphase, the distance from the chromosomes to the bleached spots decreases, while the distance from the centrioles to the bleached spots remains constant. This suggests that the kinetochore spindle fibers are shortening due to depolymerization at the kinetochore.


 

2. Separation of the poles (anaphase B) involves sliding of adjacent microtubules, requiring ATP

A second set of spindle fibers, called "polar fibers", are not attached to the kinetochore. Rather they are interdigitated with each other. That is, one polar fiber from one centrosome polymerizes until it overlaps a fiber from the opposite pole.


At the end of anaphase, the spindle fibres converge at the pole and the chromosomes are jammed together. Polymerization of + ends of polar fibers push the poles apart, bring the chromosomes with them.



E) Telophase

Telophase starts when chromosomes reach opposite poles and form a dense chromatid boll. It is the termination of mitosis and is the most difficult stage to study cytogenetically because the chromosomes are so jammed together that it is difficult to see the details.

During telophase the nuclear envelope is reconstituted from vesicles leftover from the parent nucleus, and chromosomes begin to decondense.


Image from Yaping Wang and Brian Fristensky, University of Manitoba



Displayed from http://upload.wikimedia.org/wikipedia/commons/8/8a/Nuclear_envelope_breakdown_and_reassembly_in_mitosis.jpg

Telophase restores the daughter cells to the interphase state. The nuclear envelope reforms using vesicles left over from the parent nucleus. Envelope vesicles first enclose each individual chromosome, after which vesicles fuse to form a single genetically complete nucleus at each pole. Nucleoli and the nuclear membrane reforms and the spindle fibres disappear. Chromatin decondenses, and the nuclear matrix re-assembles.

F) Cytokinesis

The division of the cytoplasm and its organelles between daughter cells is called cytokinesis, which begins during late telophase portion of the cell at the equatorial plate. In plants, cytokinesis takes place by the formation of the cell plate. In animals, cytokinesis works by formation of a cleavage furrow, pinching off the cell much like a drawstring.

Cytokinesis divides the cytoplasm- ribosomes, organelles, enzymes into sufficiently adequate portions for each daughter cell to function. The result of no cytokinesis following nuclear division is a bi-nucleate cell. Asymmetrical division gives rise to cells of different sizes and shapes, with different numbers of organelles with the result that the daughter cells are physiologically different.

 

G) Chromosome orientation at interphase and prophase

After mitotic anaphase, chromosomes remain localised during interphase and eventually reappear in the same position during the next prophase. The telomeres lie at one side of the nucleus, visible as the chromocenter or dark area in the nucleus. The kinetechore structures are found opposite to the chromocenters at a position that was the spindle pole position from the last division. Chromosome arms extend from the kinetechore region to the chromocenters which represents their telomeric ends.

This evidence supports the hypothesis that chromosomes retain a relatively fixed position in the nucleus.

Figure A shows wheat root tissue which has been hybridized with fluorescent probes specific for centromeres (green) and telomeres (red). As illustrated in B, it appears that in these nuclei the centromeres for all chromosomes are on one side of each nucleus, and the telomeres are all on the opposite side.

Franklin, A.E. & Cannde, W.Z. (1999) Nuclear organization and chromosome segregation. Plant Cell 11:523-534.

Copyright American Society of Plant Biologists (permission statement)

centtelo.jpg
http://www.plantcell.org/content/vol11/issue4/images/large/PC980434.f4.jpeg

What is it about the process of mitosis that would cause this uniform orientation to occur? During anaphase, sister chromatids separate and are pulled along the spindle fibers towards the centrosomes at opposite poles of the cell. Kinetochores, found at the centromeres, are pulled through depolymerization of spindle fibers, and chromosome arms drag behind, leaving the telomeres oriented away from the centromeres. In daughter cells, centromeres for all chromosomes will be clumped at one end of the newly-forming nucleus, and telomeres at the other end. Therefore, the telomeres in two adjacent daughter cells will be adjacent, and the centromeres will be on opposite poles of the two cells.


Uniform alignments of centromeres and telomeres are more likely to occur in plants than in animals, because in plants, cell divisions often occur along a plane.

TAKE HOME LESSON: Chromosomes stay pretty much where they were at the end of anaphase.


H) DURATION OF MITOSIS.

The duration of mitosis varies with plant species, tissues, temperature and environment.The duration is similar between diploid and their polyploid counterparts in some species eg. Triticum sp. and different in others. The range is from 9 to 29 hours. In general the interphase takes the longest and the period from prophase to telophase is shorter than the remainder of the cycle.

Mitotic cycle time (h) in several plant species
Species  2n Ploidy 

level

Mitotic

cycle (hr.)

Reference
Haplopappus gracilis 4 2x 10.50 Sparvoli et al., 1966
Crepis capiliaris 6 2x 10.75 Van't Hof, 1965
Trillium erectum 10 2x 29.00 Van't Hof and Sparrow, 1963
Tradescantia paludosa 12 2x 20.00 Wimber, 1960
Vicia faba 12 2x 13.00 Van't Hof and Sparrow, 1963
Impatiens balsamina 14 2x 8.80 Van't Hof, 1965
Lathyrus angulatus 14 2x 12.25 Evans and Rees, 1971
Lathyrus articularis 14 2x 14.25 Evans and Rees, 1971
Lathyrus hirsutus 14 2x 18.00 Evans and Rees, 1971
Avena strigosa 14 2x  9.80 Yang and Dodson, 1970
Secale cereale 14 2x 12.75 Ayonoadu and Rees, 1968
Alliam cepa 16 2x 17.40 Van't Hof, 1965
Allium fistulosum 16 2x 18.80 Van't Hof, 1965
Hyacinthus orientalis 16 2x 24.00 Evans and Rees, 1971
Zea mays 20 2x 10.50 Evans and Rees, 1971
Melandrium album 22 2x 15.50 Choudhun, 1969
Lycopersicon esculentum 24 2x 10.60 Van't Hof, 1965
Tulipa kaufmanniana 24 2x 23.00 Van't Hof and Sparrow, 1963
Avena strigosa 28 4x 9.90 Yang and Dodson, 1970
Pisum sativum 28 4x 12.00 Van't Hof et al., 1960
Triticum durum 28 4x 14.00 Avanzi and Deri, 1969
Allium tuberosum 32 4x 20.60 Van't Hof, 1965
Helianthus annuus 34 2x 9.00 Van't Hof and Sparrow, 1963
Triticum aestivum 42 6x 10.50 Bennett, 1971
Table 3.1 From Singh, R.J. (1993) Plant Cytogenetics CRC Press

I. SUMMARY

Time lapse movies of mitosis can be found at : http://www.bio.unc.edu/faculty/salmon/lab/mitosis/mitosismovies.html

DIC microscopy of cell division in a newt lung cell
     by Vicki Skeen, Robert Skibbens, and E.D. Salmon

Stereo image of a mitotic spindle in mammalian cells
     by Julie C. Canman and Elise Shumsky
Get out your red/green 3-D glasses


In class exercise: 2005 Midterm, Question 1



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previous page PLNT3140 Introductory Cytogenetics
Lecture 2, part 3 of 3
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