BSC 1010C
Fundamentals of Biology I

Mitosis

Outline

1. Cell division functions in reproduction, growth, and repair
2. Bacteria reproduce by binary fission
3. The genome of a eukaryotic cell is organized into multiple chromosomes
4. Mitosis alternates with interphase in the cell cycle
   1. The Stages of Mitotic Cell Division
5. The mitotic spindle distributes chromosomes to daughter cells
6. Cytokinesis divides the cytoplasm
7. External and internal cues control cell division
8. Cyclical changes in regulatory proteins function as a mitotic clock
9. Cancer cells escape from the controls on cell division

The ability to reproduce distinguishes living organisms from nonliving objects- this ability has a cellular basis.

"Where a cell exists, there must have been a preexisting cell, just as the animal arises only from an animal and the plant only from a plant." "Omnis cellula e cellula" or "All cells from cells." - Rudolf Virchow (1855).

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I. Cell division functions in reproduction, growth, and repair

The perpetuation of life is based on the reproduction of cells or cell division.

• In unicellular organisms, the division of one cell to form two reproduces an entire organism (e.g. Amoeba).
• In multicellular organisms, cell division allows:

  Þ Growth and development from the fertilized egg
  Þ Replacement of damaged or dead cells.

• Precisely replicates its DNA.
• Equally distributes the DNA to opposite ends of the cell.
• Separates into two identical daughter cells

Genome = Total endowment of DNA unique to each species.

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II. Bacteria reproduce by binary fission

Prokaryotes are smaller and simpler than eukaryotes. Prokaryotes:

• Contain most genes in a single circular chromosome composed of a double-stranded DNA molecule and associated proteins.
• Contain only about 1/1000 the DNA of eukaryotes, but prokaryotic chromosomes still contain a large amount of DNA relative to the small prokaryotic cell. Consequently, bacterial chromosomes are highly folded and packed within the cell.

Prokaryotes reproduce by binary fission, a process during which bacteria replicate their chromosomes and equally distribute copies between the two daughter cells.

• The chromosome is replicated; each copy remains attached to the plasma membrane at adjacent sites.
• Between the attachment sites the membrane grows and separates the two copies of the chromosome.
• The bacterium grows to about twice its initial size, and the plasma membrane pinches inward.
• A cell wall forms across the bacterium between the two chromosomes, dividing the original cell into two daughter cells.

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III. The genome of a eukaryotic cell is organized into multiple chromosomes

Dividing eukaryotic cells replicate and distribute their tens of thousands of genes - a process that is manageable because the genes are organized into multiple chromosomes.

Chromosome = Threadlike structures in eukaryotic nuclei that are composed of DNA and protein.

• Each species has a characteristic chromosome number. (Human somatic cells have 46.)
• Gametes (sperm and ova) contain half the number of chromosomes of somatic cells. (Human gametes have 23.)

Chromatin, a DNA-protein complex, is organized into a long, thin fiber that is folded and coiled to form the chromosome. Each chromosome thus contains:

1. A long DNA molecule, with thousands of genes.
2. Various proteins that maintain chromosomal structure and help control gene activity.

Mitosis = (Mitos = thread) Nuclear division during which duplicated chromosomes are evenly distributed into two daughter nuclei; results in two daughter cells that are the genetic equivalent of the parent cell.

• Before mitosis, a cell copies its genome by duplicating every chromosome, each of which forms two identical sister chromatids joined at a specialized region called the centromere.
• During mitosis, sister chromatids are pulled apart forming two complete chromosome sets, one at each end of the cell.
• Mitosis (division of the nucleus) may be followed by cytokinesis (division of the cytoplasm).

Cytokinesis = Cytoplasmic division that forms two separate daughter cells, each containing a single nucleus.

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IV. Mitosis alternates with interphase in the cell cycle

• Includes a doubling of a cell's cytoplasm, reproduction of cellular organelles, precise duplication of DNA, mitosis and cytokinesis.
• Duration varies with cell type. Some cells divide each hour, others take more than 24 hours.
• Nerve and muscle cells never or rarely divide once they are formed.

The cell cycle alternates between M phase, or dividing phase, and Interphase, the nondividing phase:

• M phase, the shortest part of the cell cycle and the phase during which the cell divides, includes:
  1. Mitosis - division of the nucleus.
  2. Cytokinesis - division of the cytoplasm.
• Interphase, the nondividing phase, includes most of a cell's growth and metabolic activities.

  Þ Is about 90% of the cell cycle.
  Þ Is a period of intense biochemical activity during which the cell grows and copies its chromosomes in preparation for cell division.
  Þ Consists of three periods:
  

  1. G₁ phase - first growth phase. (G stands for "gap".)
  2. S phase - synthesis phase when DNA is synthesized as chromosomes are duplicated. (S stands for "synthesis".)
  3. G₂ phase - second growth phase.

Mitosis is unique to eukaryotes and may be an evolutionary adaptation for distributing a large amount of genetic material.

• Details may vary, but overall process is similar in most eukaryotes.
• It is a reliable process with only one error per 100,000 cell divisions.

A. The Stages of Mitotic Cell Division

Mitosis and cytokinesis form a continuum, but for ease of description, mitosis is usually divided into five stages: prophase, prometaphase, metaphase, anaphase and telophase.

• G₂ of Interphase: A G₂ cell is characterized by:
  • A well-defined nucleus bounded by a nuclear envelope.
  • One or more nucleoli.
  • Two centrosomes adjacent to the nucleus (formed earlier by replication of a single centrosome).
  • In animals, a pair of centrioles in each centrosome.
  • In animals, a radial microtubular array (aster) around each pair of centrioles
  • Duplicated chromosomes that cannot be distinguished individually due to loosely packed chromatin fibers. (Chromosomes were duplicated earlier in S phase.)

• Prophase:

• In the nucleus:
  • Nucleoli disappear.
  • Chromatin fibers condense into discrete, observable chromosomes, composed of two identical sister chromatids joined at the centromere.

• In the cytoplasm:
  • Mitotic spindle forms. It is composed of microtubules between the two centrosomes or microtubule-organizing centers.
  • Centrosomes move apart, apparently propelled along the nuclear surface by lengthening of the microtubute bundles between them.

• Prometaphase:

• During prometaphase:
  • Nuclear envelo pe fragments, which allows microtubules to interact with the highly condensed chromosomes.
  • Spindle fibers (bundles of microtubules) extend from each pole toward the cell's equator.
  • Each chromatid now has a specialized structure, the kinetochore, located at the centromere region.
  • Kinetochore microtubules become attached to the kinetochores and put the chromosomes into agitated motion.
  • Nonkinetochore microtubules radiate from each centrosome toward the metaphase plate without attaching to chromosomes.
  • Nonkinetochore microtubules radiating from one pole overlap with those from the opposite pole.

• Metaphase:

• During metaphase:
  • Centrosomes are positioned at opposite poles of the cell.
  • Chromosomes move to the metaphase plate, the plane equidistant between the spindle poles.
  • Centromeres of all chromosomes are aligned on the metaphase plate.
  • The long axis of each chromosome roughly at a right angle to the spindle axis.
  • Kinetochores of sister chromatids face opposite poles, so identical chromatids are attached to kinetochore fibers radiating from opposite ends of the parent cell.
  • Entire structure formed by nonkinetochore microtubules plus kinetochore microtubules is called the spindle.

• Anaphase:

• Anaphase is characterized by movement. It begins when paired centromeres of each chromosome move apart.
  • Sister chromatids split apart into separate chromosomes and move towards opposite poles of the cell.
  • Because kinetochore fibers are attached to the centromeres, the chromosomes move centromere first in a "V" shape.
  • Kinetochore microtubules shorten at the kinetochore end as chromosomes approach the poles.
  • Simultaneously, the poles of the cell move farther apart, elongating the cell.

• At the end of anaphase, the two poles have identical collections of chromosomes.

• Telophase and Cytokinesis:

• During telophase:
  • Nonkinetochore microtubules further elongate the cell.
  • Daughter nuclei begin to form at the two poles.
  • Nuclear envelopes form around the chromosomes from fragments of the parent cell's nuclear envelope and portions of the endomembrane system.
  • Nucleoli reappear.
  • Chromatin fiber of each chromosome uncoils and the chromosomes become less distinct.

• By the end of telophase:
  • Mitosis, the equal division of one nucleus into two genetically identical nuclei, is complete.
  • Cytokinesis has begun and the appearance of two separate daughter cells occurs shortly after mitosis is completed.

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V. The mitotic spindle distributes chromosomes to daughter cells

During prophase, the mitotic spindle forms in the cytoplasm from microtubules and associated proteins.

• Microtubules of the cytoskeleton are partially disassembled during spindle formation.

  Þ Spindle microtubules are aggregates of two proteins, a - and b -tubulin.
  Þ Spindle microtubules elongate by the adding tubulin subunits at one end.
  

• Parallel microtubules form bundles called spindle fibers that are visible under a light microscope; their assembly begins in the centrosome or rnicrotubule organizing center.

• In animal cells, a pair of centrioles is in the center of the centrosome, but there is evidence that they are not essential for cell division:

  Þ If the centrioles of animal cells are destroyed with a laser microbeam, spindles still form and function during mitosis.
  Þ Plant centrosomes lack centrioles.
  

• Interphase: The centrosome replicates to form two centrosomes located just outside the nucleus.

• Prophase and Prometaphase: The two centrosomes move farther apart.
  • Spindle microtubules radiate from the centrosomes, elongating at the end away from their centrosome.

• Late Prometaphase: By the end of prometaphase, the two centrosomes are at opposite poles and the chromosomes have moved to the cell's midline.
  • Each chromatid of a replicated chromosome develops its own kinetochore, a protein structure on the centromere.

    Þ Some spindle microtubules attach to the kinetochores and are called kinetochore microtubules.
    Þ Some spindle microtubules extend from the centrosomes and overlap with those radiating from the cell's opposite pole. These are called polar or nonkinetochore microtubules.
    

  • Kinetochore microtubules interact to: (1) arrange the chromosomes so kinetochores face the poles and (2) align the chromosomes at the cell's midline.

  • The most stable arrangement occurs when sister kinetochores are attached by microtubules to opposite spindle poles.

    Þ Initially, kinetochore niicrotubules from one pole may attach to a kinetochore, moving the chromosome toward that pole. This movement is checked when microtubules from the opposite pole attach to the chromosome's other ki netochore.
    Þ The chromosome oscillates back and forth until it stabilizes and aligns at the cell's midline.
    Þ Microtubules can remain attached to a kinetochore only if there is opposing tension from the other side. It is this opposing tension that stabilizes the microtubule- kinetochore connection and allows the proper alignment and movement of chromosomes at the cell's midline.
    

• Metaphase: All the duplicated chromosomes align on the cell's midline, or metaphase plate.

• Anaphase: The chromosome's centromeres split and the sister chromatids move as separate chromosomes toward opposite ends of the cell.

• How do the kinetochore microtubtiles function in this poleward movement of chromosomes? Based on experimental evidence, the current model is that:
  • Kinetochore microtubutes shorten during anaphase by depolymerizing at their kinetochore ends; pulling the chromosomes poleward.
  • The mechanism of this interaction between kinetochores and microtubules may involve microtubule-walking proteins similar to dynein that "walk" a chromosome along the shortening microtubules.

• What is the functiotin of the nonkinetochore microtubules?
  • Nonkinetochore tubules elongate the whole cell along the polar axis during anaphase.
  • These tubules overlap at the middle of the cell and slide past each other away from the cell's equator, reducing the degree of overlap.
  • It is hypothesized that dynein cross-bridges may form between overlapping tubules to slide them past one another. Alternatively, motor molecules may link the microtubules to other cytoskeletal elements to drive the sliding.
  • ATP provides the energy for this endergonic process.

• Telophase: At the end of anaphase, the duplicate sets of chromosomes are clustered at opposite ends of the elongated parent cell.
  • Nuclei reform during telophase.
  • Cytokinesis usually divides the cell's cytoplasm during late telophase. In some exceptional cases, mitosis is not followed by cytokinests (e.g. certain slime molds form multinucleated masses called plasmodia).

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VI. Cytokinesis divides the cytoplasm

Cytokinesis, the process of cytoplasmic division, begins in telophase and is different in animal and plant cells. In animal cells, cytokinesis occurs as cleavage:

• First, a cleavage furrow forms as a shallow groove in the cell surface near the old metaphase plate.
• A contractile ring of actin microfilaments forms on the cytoplasmic side of the furrow; this ring contracts until it pinches the parent cell in two.
• Finally, the remaining mitotic spindle breaks, and the two cells become completely separate.

In plant cells, cytokinesis occurs by cell plate formation across the parent cell's midline (old metaphase plate).

• Golgi-derived vesicles move along microtubules to the cell's center, where they fuse into a disc-like cell plate.
• Additional vesicles fuse around the edge of the plate, expanding it laterally until its membranes touch and fuse with the existing parent cell's plasma membrane.
• A new cell wall forms as cellulose is deposited between the two membranes of the cell plate.

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VII. External and internal cues control cell division

Normal growth, development and maintenance depend on the timing and rate of mitosis. Various cell types differ in their pattern of cell division; for example:

• Human skin cells divide frequently.
• Liver cells only divide in appropriate situations, such as wound repair.
• Nerve, muscle and other specialized cells do not divide in mature humans.

Using tissue culture, researchers have identified some factors that influence cell division:

1. Contents of the culture medium.
   • If essential nutrients are left out of the culture medium, cells will not divide.
   • Specific regulatory substances called growth factors are necessary for some cultured mammalian cells to divide. For example:

     Þ Binding of platelet-derived growth factor (PDGF) to cell membrane receptors, stimulates cell division in fibroblasts. This regulation probably occurs not only in cell culture, but in the animal's body as well - a response that helps heal wounds.
     Þ Other cell types may have membrane receptors for different growth factors or for different combinations of several growth factors.
     

2. Cell density.
   • Crowding inhibits cell division in a phenomenon called density-dependent inhibition. Cultured cells stop dividing when they form a single layer on the container's inner surface. If some cells are removed, those bordering the open space divi de again until the vacancy is filled.
   • Density-dependent inhibition is apparently a consequence of the fact that:

     Þ Quantities of nutrients and growth regulators may be insufficient to support cell division, if cell density is too high.
     Þ For normal cell division, cells must adhere to a substratum - such as the surface of a culture dish. Cells will stop dividing if they become detached.
     

   • Density-dependent inhibition probably occurs in the body's tissues as well as in cell culture. Cancer cells are abnormal and do not exhibit density-dependent inhibition.

3. G₁ phase of the cell cycle.
   • Whether a cell is destined to divide is determined at the restriction point which occurs late in the G₁ phase of the cell cycle.

     Þ If a cell is destined to divide, it progresses beyond the restriction point into the S phase when DNA synthesis begins, and then proceeds through cell division.
     Þ If the cell is not destined to divide, it may exit from the cell cycle at the restriction point and switch to a nondividing state called the G₀ phase.
     

   • Most human body cells are in the G₀ phase. Some most specialized cells, such as nerve and muscle, stay in G₀ and never divide. (Recent experimental evidence suggests that it may be possible to reinitiate cell division in some n erve cells. This holds out the possibility that some day spinal cord injuries may be repairable.). Other cells, such as liver cells, can be induced by environmental cues, such as injury, to continue through the cell cycle and divide.

4. Cell size.
   • For actively dividing cells, the ratio of cytoplasmic volume to genome size is the most important indicator of whether a cell will pass the restriction point.
   • Once a cell's volume-to-genome ratio reaches a critical threshold value, it may pass the restriction point and copy its DNA. Thus, a cell must add enough cytoplasm to attain a certain size, before DNA synthesis can begin. This prevents daughter ce lls from becoming progressively smaller with each cell cycle.

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VIII. Cyclical changes in regulatory proteins function as a mitotic clock

Once the cell passes the restriction point, it is destined to divide.

• The onset of S phase commits the cell to continue through G₂ and M phases and divide.
• Biologists are only beginning to work out the switches that control the precise sequence of events in cell division. We do know that this sequence probably depends on the completion of each task before the cell can progress to the next stage.

The ordered sequence of cell cycle events is synchronized by rhythmic changes in the activity of regulatory proteins, some of which are protein kinases.

• Protein kinases are enzymes that catalyze the transfer of a phosphate group from ATP to a target protein.
• Phosphorylation, in turn, induces a conformational change that either activates or inactivates the target protein.

Cyclical changes in kinase activity are controlled by another class of regulatory proteins called cyclins.

• These regulatory proteins are named cyclins, because their concentrations change cyclically during the cell cycle.
• Protein kinases that regulate cell cycles are cyclin-dependent kinases (or Cdks); they are active only when attached to a particular cyclin.
• Even though Cdk concentration stays the same throughout the cell cycle, its activity changes in response to the changes in cyclin concentration.

An example of a cyclin-dependent kinase is MPF (maturation promoting factor), which controls the cell's progress from late interphase (G₂) to mitosis.

• Active MPF phosphorylates chromatin proteins, causing chromosomes to condense during prophase.
• During prometaphase, the nuclear envelope disperses when some of its membrane proteins are phosphorylated.

Cyclin's rhythmic changes in concentration regulate MPF activity, and thus act as a mitotic clock that regulates the sequential changes in a dividing cell.

• Cyclin is produced at a uniform rate throughout the cell cycle, and it accumulates during interphase.
• Cyclin combines with Cdk to form active MPF, so as cyclin concentration rises and falls, the amount of active MPF changes in a similar way.
• Active MPF activates proteins that participate in mitosis and initiates prophase.
• Near the end of mitosis, cyclin is destroyed by an enzyme that is activated by MPF.
• The destruction of cyclin causes the decline in active MPF at the end of mitosis. Since MPF is activated by cyclin, it is indirectly responsible for its own decline.
• Continuing cyclin synthesis raises the concentration again during interphase. This newly synthesized cyclin binds to Cdk to form active MPF, and mitosis begins again.

Rhythmic changes in different cyclin-Cdk complexes regulate other cell cycle stages.

To summarize the control of the cell cycle:

• The G₁ phase of the cycle is the most variable phase in duration and in the variety of external and internal controls over cell division.
• Nutritional status, growth factors, cell density, and developmental state of the cell affect the length of G₁ and whether the cell will pass the restriction point and divide.
• A cell that does not pass the restriction point will enter the G₀ phase as a nondividing cell.
• If conditions favor cell division, the cell will proceed through the restriction point. When the cell acquires enough cytoplasm to reach the threshold volume-to-genome ratio, the cell will be irreversibly committed to divide.
• Rhythmic changes in cyclin concentration activate protein kinases at key regulatory points in the cell cycle, controlling the sequence of events during cell division.

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IX. Cancer cells escape from the controls on cell division

Cancer cells do not respond normally to controls on cell division. They divide excessively, invade other tissues and, if unchecked, can kill the whole organism.

• Cancer cells in culture do not stop growing in response to cell density (density-dependent inhibition); they continue to grow until nutrients in the medium are exhausted.
• Cancer cells that stop dividing do so at random points in the cycle instead of at the restriction point in G₁.
• Cancer cells in culture are immortal in that they continue to divide indefinitely, as long as nutrients are available. Normal mammalian cells in culture divide only about 20 to 50 times before they stop.

Cells in tissue culture that have lost the normal controls on growth are said to be transformed.

Transformation = The conversion of eukaryotic cells in tissue culture to a condition of unregulated growth. The immune system normally destroys abnormal cells that have converted from normal to cancer cells.

• If abnormal cells evade destruction, they may proliferate to form a tumor, an unregulated growing mass of cells within otherwise normal tissue.
• If the cells remain at this original site, the mass is benign and can be completely removed by surgery.
• A tumor is malignant if the cells have the ability to spread to other parts of the body. Only a malignant tumor is said to be cancer.

Besides having anomalous cell cycles, malignant cells are abnormal in other ways. For example, they may have:

• Unusual numbers of chromosomes.
• Aberrant metabolism.
• Lost attachments to neighboring cells and substratum - usually a consequence of abnormal cell surface changes.

Detached cancer cells may spread into other tissues surrounding the original tumor and may even enter the blood and lymph vessels of the circulatory system.

• These circulating cells can invade other parts of the body and proliferate to form more tumors.
• This spread of cancer cells beyond their original sites is called metastasis.
• If a tumor metastasizes, it is usually treated with radiation and chemotherapy, which is especially harmful to actively dividing cells.

Researchers are beginning to understand how a normal cell is transformed into a cancerous one. In most cases, it is probably caused by an alteration of genes that control cell division.

• Michael Bishop and Harold Varmus won the Nobel Prize for demonstrating that genetic alterations can cause cancer.
• Knowledge of how changes in the genome lead to the heritable abnormalities of cancer cells is still rudimentary and depends upon our increased understanding of how normal cells work.

Course Pages maintained by
Dr. Graeme Lindbeck .