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Lecture 2, part 2 of 3
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III. A MOLECULAR OVERVIEW OF MITOSIS

Mitosis - nuclear division leading to  equal sets of chromosomes  in the daughter nuclei. Characteristic of somatic cells.

Meiosis - nuclear division leading to a  reduction in chromosome number of the parent cell to produce hapliod gametes.

In a nutshell: As we mentioned above, the extended, loosely-coiled structure of chromosomes that is required for gene expression is incompatible with an orderly division of chromosomes between daughter cells.  Therefore, the chromosomes coil into dense rods that tend to retain stains, which makes them visible in light microscopy. When we can see the chromosomes, we call it prophase. The nuclear envelope is also disassembled during prophase. In metaphase, the chromosomes, attached to the spindle fibers, are pulled to the center of the cell in a kind of 'tug of war' between centrosomes on opposite poles of the cell. During anaphase, chromosomes are pulled along the spindle fibers toward the poles. Finally, in telophase, the nuclear envelope reassembles, the chromosomes decondense, and the nucleus reorganizes, allowing normal cellular functions to resume.
 

http://biog-101-104.bio.cornell.edu/BioG101_104/tutorials/cell_division/wf_review.html

Whitefish Mitosis -- Review


Interphase
Prophase
Metaphase
Anaphase
Telophase
Interphase

A. Interphase

It is important to remember that interphase is defined by what we can see under the microscope with conventional staining techniques.  While it may look like nothing is happening, we have already seen that the term "Interphase" includes G 1, S and G 2 phases of the cell cycle.



Onion root tip cells in interphase, stained with aceto carmine. Nuclei stain red, nucleoli appear white.
Image from Yaping Wang and Brian Fristensky, University of Manitoba.


Organization of spindle fibers prior to mitosis


Centrosomes are the site of spindle fiber organization, and thus provide a polarity to the dividing cell.  They  also serve as "basal bodies" in cells with flagellae. In animal cells, discrete centrioles can be seen in centrosomes . Centrioles are not visible in plant cells.
From https://en.wikipedia.org/wiki/Centrosome
Spindle fibers are microtubules which originate at the centrosomes. Microtubules are tubes formed from polymers of alternating α- and β-tubulin heterodimers. As illustrated above, bundles of microtubules also form the centrioles. Centrosomes are also referred to as "microtubule organizing centers" (MTOC).

Displayed from http://www.cryst.bbk.ac.uk/pps97/course/section11/mtube3.gif

The role of centrosomes in the cell cycle are illustrated in the  figure. (from http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.figgrp.2472 ) Prior to the visible onset of prophase, the following steps occur:

1. Centrosome replication preceeds the onset of DNA synthesis

Centrosomes roughly double in size. A substantial amount of the proteins in centrosomes appear to be tubulins.

2. DNA synthesis occurs. This is the S phase of the cell cycle

Note that the significance of this point is that by the time you can see the chromosomes at prophase, the DNA has already replicated. (ie. S phase of cell cycle) DNA has already replicated (2n -> 4n)

3. Centrosomes divide and begin migration to the poles.

As the centrosomes migrate to opposite poles, they play out polar spindle fibers, such that fibers from one pole are interdigitated with fibers from the opposite pole. These polar fibers will be used in anaphase B to push the poles of daughter cells in opposite directions.

To view a GIF animation file (137K)  illustrating the centrosomes in the cell cycle click here. Centrosomes are shown in black, chromosomes in brown and orange, and spindle fibers in green. [taken from http://www.stanford.edu/group/Urchin/mitosis.html]

B. Prophase 

1. The initiation of chromatin condensation coincides with the disassembly of the nuclear membrane.  

 
Prophase in onion root tips with aceto-carmine staining. A - C show the progression of chromosome condensation. Note that as chromosomes condense, they become thicker and less diffuse.

Images from Yaping Wang and Brian Fristensky, University of Manitoba.
A

B

C


 

2.  Switch in microtubule stability accompanies formation of spindle fibers.

Spindle fibers extend randomly in all directions. The length of the fibers varies due to  polymerization and depolymerization of the microtubule from tubulin dimers. Polymerization occurs at both ends, but is relatively slow at the (-) end, which is anchored at the centrosome. The (+) end grows more rapidly, as it extends into the cytoplasm. However, when a microtubule encounters the kinetochore at the centromere of a chromosome, it's (+) end is  stabilized by the kinetochore. The kinetochore is a protein matrix, consisting of proteins that attach to centromeric DNA sequences (inner layer) and proteins that attach to the spindle fibers (outer layer).

The kinetochore is a protein/DNA complex which forms at the centromere. It is the site of attachment of kinetochore microtubules to the mitotic chromosome.

Fluroescently-tagged spindle figers are shown in green. DNA in chromosomes is counterstained in blue flurorscence.

http://en.wikipedia.org/wiki/Image:Mitotic_spindle_color_micrograph.gif

3. By end of prophase, sister chromatids have separated but remain joined at centromeres.

Despite the fact that DNA replication occurs prior to prophase, the two sister chromatids cannot be distinguished microscopically during early prophase. This is referred to as relational coiling.
As the chromosomes condense during proplase, the relational coils are unwound, and each chromatid coils tightly with itself.  Relational coiling disappears as prophase continues and the chromatids disengage to lie side by side.




Displayed from
http://www.lbl.gov/Publications/Currents/Archive/view-assets/Feb-04-2005/kinetochore.jpg


 

C) Metaphase


In metaphase the chromosomes are fully condensed and arranged in a plane equidistant from the poles of the spindle. This is the highest level of coiling, and the chromosomes are shorter and thicker than any other stage and therefore ideal for cytogenetic study. There is no longer much relational coiling present, and the chromatids lie side by side, not twisted around each other.
Metaphase in onion root tips.
Image from Yaping Wang and Brian Fristensky, University of Manitoba.
 

Experimental evidence indicates that centrosomes appear to pull the spindle fibers taut, in a kind of "tug of war". Thus, the chromosomes migrate toward equator of the cell and become aligned.
 
DEMO: YOU can simulate migration of chromosomes to the metaphase plate, with only twine, bolts and rubber bands!

VIDEO (12.7 Mb) PLNT3140-CongressionOfChromosomes.webm


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Lecture 2, part 2 of 3
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