and Mitosis the Cell Cycle - 1
Growth and reproduction are two of the characteristics of life. Cell division is the
process by which all the cells of a multicellular organism are formed during growth
and development. Cell division is used for replacement of cells and tissues during
one's lifetime. Asexual reproduction, a means of making more individuals for many
groups of organisms, is also accomplished by cell division. For those organisms
that have sexual reproduction in their life cycle, a special cell division, in which
chromosome number is reduced, also occurs.
Asexual Reproduction
Growth and Development
Tissue Replacement
We shall focus on the processes of cell reproduction in e ukaryotic organisms.
The process of cell division in p rokaryotic organisms, all of which are unicellular
organism, is called binary fission, and will be briefly illustrated; the single
molecule of DNA and absence of a nucleus in the prokaryotic cell account for a
number of differences in the "mechanics" of the process by which prokaryotes
divide and increase their numbers.
The collection of one's genetic information, or DNA, is known as the g enome. In
eukaryotic organisms, the genome consists of a number of c hromosomes . Each
species has a fixed chromosome number, a number that does not change from
generation to generation.
For organisms that have a sexual reproduction in their life cycles, their genome
includes two sets of genetic information, one set contributed by each parent at
fertilization. Cells having two sets of genetic information are said to be d iploid.
(Details later)
Our genetic molecule, DNA, is identical in each cell within a multicellular
organism, so that when cells divide, new cells formed must have exactly the same
DNA as the original cell. To ensure that chromosomes and DNA remain the same in
new cells (the genome remains constant) when cells divide, it is crucial to have a
mechanism that exactly d uplicates (replicates) the DNA of the original cell and
distributes , or s egregates , the copied DNA equally to the new cells.
To form new cells, we must also divide up the cytoplasm and critical organelles,
such as mitochondria and chloroplasts, of the original cell into the new cells
formed. Cells must also "know" when it's time to divide; there must be appropriate
signals to initiate the process, and checkpoints to ensure that cell division is
proceeding accurately.
Mitosis and the Cell Cycle - 2
We will look at the mechanism by which cells duplicate DNA in a later unit. At this
time we shall focus on the e ukaryotic cell cycle, which includes m itosis, the
process in eukaryotic organisms by which the duplicated chromosomes are equally
distributed to new nuclei, c ytokinesis , the distribution of the cytoplasm of the
original cell into new cells, and also look at some of the c ontrols of cell division. In
a later unit we will see how normal cell division controls are affected by cancer. We
will also address in our next section the process of m eiosis , which reduces
chromosome number by half at one point of any sexually reproducing organism's
life cycle to provide for genetic variation from generation to generation.
We will start our discussion of cell division with the vocabulary of our genetic
molecule, and in particular, the vocabulary of the c hromosome .
Chromosome Structure
DNA, as we know, is comprised of a double chain of nucleotides. It's estimated
that human DNA may consist of 140 million nucleotides per cell. Our chromosomes
average about 5 cm in length and the total DNA is about 2 meters. Chromosomes
fit into the nucleus because they are tightly coiled.
Our chromosomes are associated with a complex of proteins called h istones .
There are 5 different classes of histone proteins, all of which are positively
charged so that they are attracted to the phosphate groups of the nucleotides.
Our DNA and its associated chromosomes comprise the c hromatin material.
Prior to cell division, each 150 or so nucleotides in a chromosome coil around a
complex of 8 histone protein molecules (2 from each of 4 of the classes of histone
proteins) forming a n ucleosome . The 5th histone protein is on the exterior of the
nucleosome and may function as a clamp to hold the DNA to the histone core.
Nucleosomes further coil into "beaded" supercoils that subsequently fold into
loops, which coil even more into the, finally, visible chromosome during cell division.
Some DNA stays highly condensed in cells following division and is known as
heterochromatin . Heterochromatin may stay so tightly condensed that its DNA
cannot be read (or expressed). The DNA that can be expressed is called
euchromatin . Mutations that prevent the protein condensin from coating
chromosomes, which helps chromosomes condense during prophase, is one cause
of sterility. (How DNA gets expressed is discussed in later sections.)
Mitosis and the Cell Cycle - 3
Chromosome Terms before and after DNA Duplication (Replication)
Note: Duplication and replication mean exactly the same thing.
An unduplicated chromosome is one chromosome. A chromosome consists
of two "arms" that extend from a narrow region called the centromere. T he
centromere region is often visible in duplicated chromosomes. The two ends of
a chromosome are telomeres (see later).
When a chromosome duplicates (during the S phase of Interphase of the cell
cycle), it becomes one duplicated chromosome, and the two copies remain
attached to each other. Note it is still o ne chromosome.
The two exact copies of the duplicated chromosome, which remain attached by
the protein c ohesin , are called " sister" chromatids . They are identical to
each other. I t is essential that you remember this!
K inetochores (made of protein and DNA) form at the centromere region of
the duplicated chromosome during cell division. The three-layered kinetochores
attach to microtubules of the spindle during mitosis. The kinetochores of the
two sister chromatids face in opposite directions.
Kinetochore TEM
After the identical sister chromatids are separated during mitosis, each (called
a "daughter" chromosome now) becomes a single unduplicated chromosome
again.
R emember : "Sister" chromatids are not two chromosomes. The two sister chromatids
comprise one duplicated chromosome that consists of two identical chromatids. Once sister
chromatids are separated they are no longer chromatids!
Mitosis and the Cell Cycle - 4
To summarize - When cells divide:
We form two new cells from the original cell.
The new cells formed must have all of the genetic material for the organism,
so we need a mechanism that exactly duplicates the DNA of the original
cell's nucleus and d istributes or s egregates the copied DNA equally to two
new nuclei. Mitosis is the process by which the duplicated chromosomes are
equally distributed to new nuclei. (We will see how DNA duplication is
accomplished later.)
We must also separate the cytoplasm and critical organelles, such as
mitochondria and chloroplasts, of the original cell into the new cells formed
so that the new cells can survive, grow and function. The distribution of the
cytoplasm of the original cell into new cells is called c ytokinesis .
There must be appropriate signal controls for cell division to occur.
Mitosis and the Eukaryotic Cell Cycle.
Mitosis is one part of the cell cycle. The cell cycle includes all activities of a
cell from the time it is formed until (and if) it divides or dies.
The processes, or events, of cell division can be related to the normal lifetime of a
cell. For our convenience, these events are divided into "stages" of a cell's c ell
cycle. The cell cycle starts when a cell is formed and continues until it divides.
Some cells never divide, others divide frequently, and some only when damaged and
replacement is needed. Cell division is a brief part of the life cycle; most of the
life of a cell is spent in normal activities of growth and maintenance, a stage called
interphase . The events of interphase and cell division are:
Interphase
Gap 1 (G1 )
DNA Synthesis
Gap 2 (G2 )
Cell Division (or cell reproduction)
Mitosis
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
Eukaryotic Cell Cycle
Mitosis and the Cell Cycle - 5
Cell Cycle Details
Interphase
Growth (called G1 or First Gap in the cell cycle)
Normal growth and cell activities
The chromosomes are stretched out and grainy in appearance. They
stain well, and early on this material was called c hromatin .
DNA Duplication (called S for synthesis in the cell cycle)
DNA duplication takes place forming the duplicated chromosomes
DNA duplication is triggered in the G1 phase. Once started this process
cannot be reversed; the cell is committed to divide.
Preparation for Division ( called G2)
Condensation of chromosomes is initiated during the G2 phase of the
cell cycle, although condensed chromosomes are not visible until well
into prophase of mitosis.
Synthesis of materials, such as tubulin for the spindle microtubules
needed for mitosis and cytokinesis also takes place during G2.
Duplication of the centrosome (in organisms that have a centrosome)
and its centrioles (in organisms that have centrioles) occurs.
As G2 progresses duplicated centrosomes migrate in the cytoplasm to
either side of the nuclear envelope and the microtubule organizing
center, (centrosome) is initiated. The orientation of the centrosomes
(or microtubule organizing center) determines the "poles" of the
dividing cell.
Tubulin dimers aggregate in the centrosome regions and/or poles of
the cell in preparation for spindle formation during prophase.
In plants, a preprophase band of microtubules formed near the center
of the cell determines the plane of division.
Note:
If a cell never divides, following G1 it will be in a permanent state of
G0 (or non-dividing state).
I nterphase
Mitosis and the Cell Cycle - 6
The Stages of Mitosis: Mitosis is a continuum. Humans have decided to
separate the process into stages for the convenience of our discussions. Some
humans even separate the stages into sub-stages and intermediate stages. For
our purposes, cell division in eukaryotes involves three events:
DNA Duplication
Process of duplicating the genetic material of the nucleus (Discussed later)
This occurs during I nterphase , when growth and/or normal metabolic
activities take place.
Mitosis
Process of distributing the duplicated DNA equally to the two new nuclei.
Cytokinesis
Process of separating the cytoplasm contents
Mitosis consists of five stages (phases):
Prophase, Prometaphase, Metaphase, Anaphase,
Telophase
Prophase
Chromosome Condensation
As the cell starts prophase, the nucleoli disperse and "disappear". The
duplicated chromosomes continue condensing and thickening as mitosis
progresses and become visible as threadlike structures. Motor proteins are
involved in the condensation of chromosomes. The duplicated chromosomes
are firmly attached at their centromeres throughout this condensation and
coiling, although cohesin molecules degrade along the remainder of the
duplicated chromosome separating most of the sister chromatids.
The kinetochores, important in chromosome movement, are formed in the
centromere regions of each duplicated chromosome at this time.
Microtubule Organization and the Mitotic Spindle
Microtubules and associated proteins form the s pindle a pparatus during
prophase. Some of the cell's cytoskeleton will disassemble to provide
spindle microtubules. The spindle also synthesizes additional microtubules
from the tubulin dimers synthesized during interphase.
Microtubules radiating from the centrosomes in animal cells are called
asters. Centrosomes migrate toward the poles by lengthening
microtubules during prophase. Plant cells also have less visible asters and
spindle formation, but lack centrosomes in the microtubule organizing
center.
Mitosis and the Cell Cycle - 7
Prophase
Prometaphase (Late Prophase)
Nuclear Envelope
The nuclear envelope degrades in prometaphase into small vesicles, used
to synthesize new nuclear membrane material in the new cells.
Spindle Apparatus
The spindle apparatus will extend from the poles of the cell through the
center of the cell to the opposite pole of the cell by the end of
prometaphase. Elongating polar microtubules go from each pole
toward the equator of the cell where they overlap with polar microtubules
from the opposite centrosome or aster forming the framework of the
mitotic spindle and stabilizing the spindle complex.
K inetochore microtubules from the opposite poles attach to
kinetochores of each chromatid of the duplicated chromosomes. The
organization of the kinetochore microtubules is such that each duplicated
chromosome has kinetochore microtubules from one pole attaching to one
of the sister chromatids, and kinetochore microtubules from the opposite
pole attaching to the other sister chromatid. If there are mistakes in
attachment, chromatids are not separated correctly during mitosis.
The spindle microtubules pull on the kinetochore regions of the sister
chromatids in a "tug-of-war" which eventually leads to a migration of the
duplicated chromosomes to the equatorial plane of the cell. Both sets of
kinetochore microtubules are pulling towards their respective "pole" that
assists the ultimate migration of the duplicated chromosomes toward the
equator. Viewed "live", chromosome motion seems random and aimless.
As the cell proceeds into metaphase aster microtubules have also
attached to the plasma membrane, anchoring the "poles".
P rometaphase
Mitosis and the Cell Cycle - 8
Metaphase
In metaphase, the spindle apparatus has moved the chromosomes to the
equator of the cell, aligning the centromeres of each duplicated chromosome
along the equator of the cell.
Centromeres of each sister chromatid are aligned with each other and each
sister chromatid is connected at its kinetochore to microtubules from its
respective pole.
Kinetochores attached to their Kinetochore Microtubules at Metaphase
This alignment of chromosomes along the equatorial plane of the cell is often
called the metaphase plate. The metaphase plate is quite distinctive when
viewed from the "top of a cell" or polar view as opposed to the view typically
shown in textbooks.
Polar View
"Side" View
Metaphase
Mitosis and the Cell Cycle - 9
Anaphase
To initiate anaphase, the remaining cohesin proteins at the centromere regions
of the sister chromatids degrade so the sister chromatids can be separated.
The hydrolytic enzyme, s eparase , catalyzes this reaction. If the chromatids
are not aligned properly, the separase precursor is inhibited, and mitosis does
not proceed. This is called the s pindle checkpoint (see later).
Separated chromosomes being moved away from the equator is the first visual
sign of anaphase. By definition, each sister chromatid is now a single
unduplicated chromosome, or "daughter" chromosome.
Anaphase
The kinetochore motor protein, cytoplasmic dynein, hydrolyzes ATP to move
the chromosomes along the kinetochore microtubules. Most cells take about
1060 minutes to complete anaphase.
In addition, kinetochore microtubules decompose at their kinetochore ends,
pulling the chromosomes, centromere first, away from each other and toward
the respective poles of the cell as the kinetochores shorten.
Polar microtubules lengthen, moving the poles of the cell further apart, and, in
animal cells elongating the cell.
Overlapping Polar Microtubules in Metaphase and Anaphase
Since sister chromatids are identical, each of the two clusters of
chromosomes being pulled to the two poles of the cell has one copy of each
original chromosome. As the chromosomes are pulled to the poles, they
begin to lengthen out.
Mitosis and the Cell Cycle - 10
Telophase
The two sets of daughter chromosomes aggregate at the poles of the cell,
uncoil and the aggregate of chromosomes becomes indistinct as chromatin.
The cell, if it is an animal cell, continues to elongate by lengthening the polar
microtubules (decreasing the overlap).
At each pole membrane vesicles and membrane fragments form new nuclear
envelopes around each group of chromosomes resulting in two distinct nuclei
in the cell.
The spindle microtubules disperse and the spindle apparatus disappears.
New nucleoli form, concluding mitosis.
Telophase
Mitosis in Blood Lily
Mitosis and the Cell Cycle - 11
Cytokinesis:
Separation of the Cytoplasmic Contents
Speaking precisely, mitosis describes events of chromosomes and nuclei. Most
cells accompany mitosis with cytokinesis, the separation of the cytoplasm of the
original cell into two new cells. This is not always the case. Some organisms
(including many fungi and algae) are "multinucleate"; they just have one cell body
with many nuclei. Some animal tissues are also multinucleate.
Cytokinesis typically coincides with the events of late anaphase and telophase, so
that at the completion of mitosis, the original cell is separated into two cells, each
with a nucleus and DNA identical to that of the original cell. Although the end
result of cytokinesis is always two new cells, the mechanism of separation is
different in plants and animals, so we shall discuss them separately.
Cytokinesis in Animal Cells
The cells of animals lack cell walls. Cytokinesis in animal cells is started with the
formation of a cleavage furrow, a depression or pinching in of the plasma
membrane.
This is caused by a ring of microfilaments, the c ontractile ring, composed of the
protein, actin, associated with myosin, which forms across the middle of the cell
after the chromosomes are separated in anaphase. This ring contracts, pinching
or drawing in the plasma membrane toward the center of the cell, which eventually
pinches the cell in two. The additional membrane surface needed is supplied by
membrane material synthesized during Interphase. Cytokinesis, hence cell division,
can be disrupted by mutations that affect microfilament function.
Cytokinesis in Animal Cells
Mitosis and the Cell Cycle - 12
Cytokinesis in Plant Cells
Each cell of a plant is surrounded by a rigid cell wall. Plant cells can not form a
cleavage furrow. Instead, plant cells are separated by the cell plate formation.
Cell plate formation involves making a cross wall at the equator of the original cell
and plasma membrane. Kinesin motor proteins move Golgi vesicles containing wall
material along microtubules to the equator of the cell. The vesicles contents
contribute to a disk-like structure that is called the p hragmoplast . As cellulose
and other fibers are deposited, the cell plate results creating a boundary and new
cell wall between the two new cells. Membrane material from the original vesicles
fuses to each side of the cell plate forming new cell membranes on the dividing
sides of the original cell.
Cytokinesis in Plant Cells
In both plants and animals, organelles within the cytosol are distributed into the
two new cells formed by cytokinesis.
Variations in Mitosis in Eukaryotes
The process of mitosis in most eukaryotes is remarkably the same. However,
some protists, such as dinoflagellates and diatoms, and some yeasts do not
degrade the nuclear membrane during mitosis. Spindles may not form at all (some
protists), may form within the nucleus (yeasts and diatoms), or may form outside
of the nucleus and pass through nuclear pores (dinoflagellates).
Dinoflagellates
Yeast Fungi
Diatoms
Mitosis and the Cell Cycle - 13
Binary Fission in Prokaryotes
The process of cell division in prokaryotes, called b inary fission, parallels that of
eukaryotes. Bacteria have just one long continuous, or circular molecule of DNA
with some associated proteins, although the DNA is routinely folded and compacted
to fit into the cell. That, and the absence of a nucleus in the prokaryotic cell,
account for a number of differences in the "mechanics" of the process. Polar
proteins attached to the DNA molecule facilitate the folding needed for the DNA to
fit within the cytoplasm of the cell. The rate of cell division in prokaryotes is
related to environmental conditions, dividing more rapidly when conditions are good,
and less rapidly in poor environments. Binary fission is used to increase population
numbers.
The single DNA molecule attaches to the plasma membrane prior to
duplication and cell division at a site on the chromosome identified as the
origin, or ori. (The termination region of replication is the "ter" and signals
the completion of DNA duplication.)
As the DNA is duplicated using a complex of enzymes (very similar to that of
eukaryotes), the duplicated molecule's ori region is attached to the plasma
membrane.
The cell elongates by synthesizing new membrane and wall material between
the two ori regions, separating the two DNA molecules.
After a period of elongation, in which the original cell about doubles its
length, plasma membrane is pulled inward (much as occurs with eukaryotic
animal cells), pinching off the two halves of the original cell. New cell wall
material is also synthesized.
Prokaryote Cell Cycle
E coli dividing
Pseudomonas dividing
Binary Fission
Mitosis and the Cell Cycle - 14
Regulating the Cell Cycle
The control of cell division is one of the most active areas of biological research, in
part because cancers are diseases that involve cells that have lost their cell
division controls. In humans, some cells routinely divide: bud taste cells and cells
lining the digestive tract divide about every three days, skin cells monthly and red
blood cells are produced by the millions each day. Other cells in mature humans
never divide. Nerve cells and muscle cells are two examples (although one can
increase one's muscle mass by making muscles work). Much of our human brain
development occurs in the first two years. Liver cells divide only if damage
occurs. (Animal growth and development is discussed in Biology 212).
Plants have an open pattern of growth. They are making new cells "constantly", not
for replacement, but for continued growth. Plants have a tissue, m eristem, t hat
is specialized for cell division. However, there are numerous exceptions in plants,
including some that are a part of normal development. In addition, many plant cells
have a remarkable ability to d edifferentiate and become "embryonic", something
that rarely happens in animals. Many plant cells die at maturity, but continue to do
their function (Plant growth is discussed in Biology 213).
But what tells a cell to divide - or not to divide? In cell fusion studies during the
1970's, researchers learned that there are definite c ell-cycle controls that
direct and coordinate the events of the cell cycle. The cell cycle is subject to both
internal and external chemical control mechanisms.
Cell-cycle Control Checkpoints
As is common, most of what we know comes from research on animal processes.
For cell division, we also have research on yeast cells, and the processes are
remarkably similar. The animal cell cycle has at least three "checkpoints" (in G1, G2
and mid-mitosis) where the cell cycle remains in that stage until over-ridden by
chemical signals, which we shall discuss. Cells have both external and internal
signal transduction pathways controlling cell division.
Mitosis and the Cell Cycle - 15
There are three well-studied checkpoints for cell division that function during G1,
G2 and in Mitosis (M).
T he first checkpoint, called the G 1 /S checkpoint, or sometimes restriction
(R) point, is in G1 and determines whether DNA replication should proceed.
R
Cells that never leave G1 are said to be in a non-dividing cycle called G0. Cells
will stay in G1 until they receive a signal to proceed with DNA duplication.
The second checkpoint, G2 /M , is in G2 just prior to mitosis and determines if
mitosis will begin.
The third checkpoint, which is in metaphase of mitosis (M ), is the spindle or
M
APC (anaphase-promoting complex) checkpoint.
The checkpoint signals involve protein kinases, called c yclin-dependent kinases
(Cdk) , which are activated by proteins called c yclins , whose concentration is
cyclical (hence cyclin). Cyclin is a cyclin-dependent kinase allosteric regulator
molecule. {Kinases are enzymes with serine, threonine or tyrosine amino acids that that
function in cell signal relay pathways when phosphorylated by ATP. Kinase-relay pathways
are discussed with cell communication and signaling.}
Cell Cycle Cyclin-dependent Kinase Controls
Cyclin Activation of Cdk
Mitosis and the Cell Cycle - 16
Cyclin concentration rises and falls during the cell cycle. When levels are high,
cyclins combine with the cyclin-dependent kinases (Cdks) forming a complex
(cyclin-Cdk) .
When activated by their regulator cyclin, the cyclin-dependent kinases
phosphorylate proteins needed for DNA synthesis and for mitosis. The
phosphorylated protein changes shape to initiate important steps in the cell cycle.
m
The first cyclin-Cdk complex discovered was called M PF for "maturation ( or
mitosis) promoting factor", a cyclin-Cdk complex that activates the start of
mitosis in G2. Its activity coincides with peak levels of cyclins. MPF activates
kinase transduction relay pathways that promote a number of mitosis activities,
including degradation of the nuclear membrane, spindle formation and chromosome
condensation.
Kinase relay pathway
There are a variety of different Cdk and cyclin molecules that function in the cell
cycle. For example:
The cyclin/Cdk at the spindle (M) checkpoint activates the Anaphase
promoting complex, APC.
The cyclin/Cdk at G1/S phosphorylates the protein, RB to start a
transduction cascade for DNA duplication. Until phosphorylated, RB blocks
DNA duplication.
The Cdk-cyclin complexes involved at the cell division checkpoints promote their
own degradation by activating proteolytic enzymes that destroy cyclin at each
checkpoint. Enzymes that dephosphorylate are p hosphatases. This fluctuation in
the level of cyclins regulates the cell cycle.
Mitosis and the Cell Cycle - 17
Cell Control Checkpoint Details
G 1 /S (or R { restriction}) checkpoint
)
Nutritional status and cell size signals at the G1/S checkpoint ensure that the cell
has sufficient resources and volume to divide. External signals include a number of
growth factors that promote cell division (see below). Such signals promote the
synthesis of G1 cyclin molecules to complex with Cdk (the Cdk/G1 cyclin complex),
which activates the synthesis of proteins needed for DNA duplication. DNA
integrity is also monitored at G1. (p21, a protein that blocks DNA duplication by binding
to the G1/S cyclin/Cdk is activated when DNA is damaged. Mutations in p21 are involved in
some cancers.)
G 2 /M Checkpoint
The MPF cyclin-dependent kinase that operates at the G2/M checkpoint triggers
cell activities that check the integrity of the duplicated DNA molecules and
commits the cell to mitosis.
At this checkpoint, a phosphorylated Cdk (called c dc2) must be dephosphorylated
to activate mitosis by appropriate signals. Once cdc2 is dephosphorylated, the
MPF activates its own phosphatase enzyme. The checkpoint works by assessing
the balance of kinases that add phosphates (maintaining inhibition) with the
phosphatases that remove phosphates. When DNA damage is detected, the
balance favors MPF phosphorylation activity, and mitosis does not take place. (One
DNA damage-sensing protein, p53, when mutated and non-functional, is associated with
nearly half of human cancers.)
T he Spindle Checkpoint the Anaphase-Promoting Complex (APC)
The spindle checkpoint is regulated by the kinetochores of the sister chromatids.
They delay anaphase until the kinetochores of all the duplicated chromosomes are
attached to kinetochore microtubules and oriented correctly to their poles with
the appropriate tension between the poles. Proteins associated with the
kinetochores have a signal pathway that blocks the enzyme that hydrolyzes
securin , the s eparase inhibitor . Separase cannot hydrolyze the remaining
cohesin holding sister chromatids together until its inhibitor is removed. Once
cohesin is degraded, anaphase commences. This checkpoint ensures that each cell
formed will have the correct complement of chromosomes; no stray chromosome
can avoid the metaphase plate and impact mitosis.
Mitosis and the Cell Cycle - 18
External Signals and Cell Division Control
Cell division, like many other cell activities, cannot occur if essential nutrients are
not available. As mentioned in the G1 checkpoint discussion, G rowth factors are
a class of proteins involved with signal transduction pathways that can promote or
arrest cell division, so that cells divide when needed and do not divide
inappropriately (except when cancers happen).
It takes a certain concentration of growth factor(s) for cell division to proceed. It
also takes sufficient appropriate nutrients to divide so that growth factors and
nutrient supply are both important in regulating the rate of cell division. Absence
of appropriate growth factors is one means of keeping a cell in G0.
In plant tissue culture, a cell will remain undivided until and unless an appropriate
mixture of nutrients and growth regulators (plant hormones) is provided. Early
plant tissue culture succeeded when researchers added coconut endosperm
(coconut milk) to the culture medium. The endosperm is rich in both plant
hormones and nutrients because its normal job is providing nutrients to the
developing embryo.
External Factors and Growth
Density-dependent Inhibition
Normal growth is also regulated by external environmental conditions. In animal cell
cultures, cell division is halted when the cell population gets too dense and exceeds
the nutrient and growth factor concentration needed to continue to divide.
Crowded cells stop dividing. As in population biology and ecology, such regulation of
cell division is known as a d ensity-dependent activity or in this case a
density-dependent inhibition . It's important to note that density-dependent
inhibition is chemical. Physical contact is less important in stopping cell division.
Mitosis and the Cell Cycle - 19
Anchorage Dependence
Animal cell division in cultures is also arrested when cells do not have an
appropriate substrate or an extracellular matrix of cells to attach to during
division. Normally, membrane proteins and components of the cytoskeleton trigger
anchoring signal pathways that have a role in regulating cell division. Such cell
division control is known as a nchorage dependence .
Normal Cell Anchorage Dependence
Cancers and Cell Controls
In cancers, the normal controls of the cell cycle are disrupted. Cancer cells seem
to lack cell density and cell anchoring controls. Abnormal cell cycle patterns are
also present in cancers, as are significant changes in chromosomes. Although
most cells cease division after 20 30 cycles, cancer cells may divide indefinitely.
Tumor cells removed from a woman named Henrietta Lacks in 1951 are still
dividing. Cancer cells also continue to divide when growth factors are missing. It is
likely that for some cancers, signal pathways activated normally by a growth
factor remain active, or that cancer cells synthesize their own growth factors.
(Cancer and gene regulation will be discussed later in Biology 211.)
Cell Death and the Cell Cycle
Death of cells is a normal part of multicellular organisms. Cells have two common
ways of dying. If a cell is damaged by injury (mechanical damage) or toxins, or if
the cell lacks sufficient nutrients to function, it will generally swell and burst. This
is called n ecrosis . Our immune system responds to necrosis with inflammation
and other appropriate responses to "clean up" the damaged area.
Alternatively cells may be are programmed to die when they are no longer needed
or when they are damaged genetically beyond repair. This set of genetic events is
called apoptosis. Apoptosis is common and normal during development,
particularly of the nervous system and for morphological changes in development,
such as the formation of digits or reabsorption of parts used by the embryo, but
not the adult. Apoptosis is also part of the normal functioning of the immune
system and in the normal sloughing off of epithelial tissues, such as the linings of
the digestive tract.
Human Day 41
Day 56
Mitosis and the Cell Cycle - 20
During apoptosis, the cell's DNA is destroyed, organelles are destroyed and the cell
shrinks and becomes lobed in appearance, a process called b lebbing. The cell
components are packaged into vesicles to be engulfed by the cells of the immune
system.
Normal WBC
WBC in Apoptosis
Apoptosis is important in cells in which there has been genetic damage and cell
division would perpetuate the damage, possibly leading to abnormal growth and
cancers. Apoptosis is common in all animals and the regulatory genes are so
similar that an apoptosis gene from one organism spliced into a second will
function in the second organism. Apoptosis genes are activated when defective
DNA is discovered. Apoptosis may have a role in both Alzheimer's and Parkinson's
diseases, and failure of apoptosis is involved in some cancers. In some plants,
apoptosis is used in the hypersensitive response in response to some pathogens.
(Plant defense responses are discussed in Biology 213.) Plant apoptosis does not
involve blebbing. The cell contents are digested within the central plant vacuole,
and the digested molecules released from the cell.
Gene Control of Apoptosis
By studying a small nematode worm, Caenorhabditis elegans, researchers were
able to identify genes involved in apoptosis called ced genes. (ced = cell death).
The ced genes are coded, but the proteins in the cytosol are inactive until called
into action for apoptosis during normal development or when cells are damaged.
The activated ced proteins hydrolyze nucleic acids and proteins of the cell. In C
elegans, the ced-4 and ced-3 proteins are involved in apoptosis. A third protein,
ced-9, located on the mitochondrial membrane, inhibits ced-3 and ced-4. When
ced-9 is inhibited, ced-4 initiates apoptosis.
Humans have a set of enzymes, called capases, which function in the same
manner. The protein, Bcl-2 inhibits apoptosis similarly to ced-9 in C elegans.
Apaf1 activates apoptosis.
Apoptosis can be triggered by signals when either DNA is too damaged or proteins
are not being assembled correctly in the ER. Capases are then released from the
mitochondria through holes formed in the mitochondrial membranes. Cytochrome
c, an electron carrier for cell respiration promotes apoptosis by facilitating the
movement through the mitochondrial membrane.
Mitosis and the Cell Cycle - 21
Ced 9 Control of Apoptosis
Capase Activation in Apoptosis
Comparing Apoptosis in C elegans and Human neuron
Mitosis and the Cell Cycle - 22
When and Where Does Mitosis Occur?
Growth
All growth (increase in numbers of cells) in individual organisms takes place by
mitosis, from the fertilized egg (zygote) to death.
Repair and Replacement
Mitosis is used for replacement of damaged cells or tissues, as well as for the
routine replacement of cells that is a part of normal growth, development and
maintenance. When cells die, they are replaced by mitosis. The rate of cell
replacement varies with tissue type. Some human cells, such as those that line
the digestive tract, are replaced every 1 3 days. Our red blood cells last about
four months. Some, such as nerve cells, are never replaced.
Non-Sexual (Asexual) Reproduction
Mitosis is used for all asexual reproduction or p ropagation . This is especially
common in plants, fungi and protists. Animals less commonly reproduce asexually.
There are many claims for the world's largest organism based on the ability to
make more. Asexual reproduction produces offspring genetically identical to the
original parent, as would be expected of any mitosis. Cloning is a variant of
asexual reproduction.
Asexual Reproduction in Aspen, Fungi, Protist and Hydra (an Animal)
Mitosis and the Cell Cycle - 23
Cloning A Variant of Asexual Reproduction
The zygote of any organism has total genetic competence. It is said to be
totipotent because its DNA, or genome, has all of the instructions for the
organism that is going to develop. In multicellular organisms, as growth and
development take place (all by mitosis), cells and tissues differentiate for their
specialized functions and their DNA becomes determined; different genes are
activated or repressed in different tissues, so we have differential or selective
gene expression depending on the cell and tissue type, although each cell retains
the total complement of DNA found in the zygote.
Animal Cell Lines and Differentiated Cell Types for Tissue Development
The idea of cloning is to produce a genetically identical organism using a single cell
from a "mature" multicellular organism. For cloning to succeed, that single cell
must be able not only to divide, but also to differentiate its cells into the tissues
and organs of the "adult". One compelling reason for cloning research today is not
to have more "copies" of any one individual, but for the production of stem cells,
which have the ability to generate many kinds of tissues for treatment of diseases
and injuries (Stem cell research and applications discussed in our section on Genetic Technology).
Mitosis and the Cell Cycle - 24
However, our initial start in this subject dates from the 1950's when FC Steward
at Cornell University successfully cultured carrots from single cells taken from
the root of the carrot. This was the first laboratory "clone". Although the plant
cells used were differentiated root tissue cells, they were totipotent no genetic
material had been permanently lost or "turned off". That plants naturally have
cells that dedifferentiate to form new meristem regions was a good clue to this.
Most cells of plants retain the ability to "dedifferentiate" and become embryoniclike. (Plant growth and development is discussed in Biology 213.)
Cloning many kinds of plants turned out to be fairly easy provide the totipotent
cell with the right mix of hormones, nutrients, chemical signals and a growth
medium, such as agar, and genetically identical plants grow. In the 1970's one
could buy test-tube plants to share with one's friends as a novelty it was so
successful. This method, known as tissue culture, is a common way of cloning
plants. Cloning is used in agriculture, forestry and orchid cultivation today, and
augments the myriad ways plants have for "natural" asexual propagation.
Plant Cloning from Root Cell
Animal Cloning
Cloning animals has not been so easy, although experiments have been ongoing for
decades. In contrast to plant cells, nuclei in animal tissues do undergo
"permanent" changes during development so that taking one cell and treating it
with hormones and nutrients and other chemical signals does not result in the cell
developing into a new organism.
Mitosis and the Cell Cycle - 25
Early animal cloning research used nuclear transplantation a process that takes a
nucleus from an embryo and transplants it into an egg cell whose nucleus has been
destroyed, typically by UV light. Thomas King and Robert Briggs did extensive
nuclear transplant experiments on frog embryos in the 1950s while doing research
on genetic determination in embryo cell lines, and also by John Gurdon in the
1970s. How and if the embryo developed helped researchers identify how long an
embryonic cell line remained totipotent. Only early embryo nuclei were successful
in the transplant studies. Cells transplanted from tadpoles typically did not
develop. The totipotency of very early human embryos provides for some genetic
screening that requires cell DNA observation.
Nuclear Transplant Study in Frog Embryos
Similar studies with mammalian embryo nuclear transplants were also successful,
but efforts with adult or fully differentiated cells did not succeed until 1997.
All cloning in animals still involves nuclear transplantation into an egg cell. The
cytoplasm of the egg cell is essential, and plays significant roles in early
development. In animal cloning, the new organism has a nucleus from the "parent",
but the nucleus is injected into an egg cell from which the egg cell nucleus has been
removed. This is called cell fusion. The fused cell, or "clone", is then implanted
into a surrogate mother for development.
In 1996, Ian Wilmut cloned a sheep using the nucleus from an altered mammary cell
of an adult sheep and an enucleated ovum from a second donor. They induced the
nuclei of the mammary cells to dedifferentiate by growing isolated mammary cells
in a nutrient-poor medium that forced the cells into a stalled G1 growth phase prior
to implantation into the egg cell (ovum). At that time, they used mitosisstimulating chemicals to initiate division. Once the diploid egg cell divided and
started developing, the embryos were transplanted into a surrogate "mother" and
one survived, the now famous Dolly, who aged prematurely and is no longer alive.
Since Dolly, many mammals have been "cloned" using this technique. It was
announced in 2001 that cloned human embryos had been formed, but not implanted
in surrogate mothers. Researchers in South Korea in 2004 developed human
embryonic clones through the blastocyst stage. Many such announcements have
occurred during the past decade.
Mitosis and the Cell Cycle - 26
Mammal Cloning
Parent and Cloned Offspring
Apart from ethical concerns with cloning, the nuclear transplantation method of
animal cloning has a very low success rate, and many, if not most, embryos fail to
develop and many that do have abnormalities, as did Dolly.
Both donor cell and donor nucleus undergo trauma, and implantation has additional
risks. In addition, during normal development, the chromatin within cells is altered
by DNA methylation and histone modifications to deactivate many genes. The DNA
of donor cells for cloning has been affected by these generally non-reversible
changes. Although donor nuclei are treated to "dedifferentiate" prior to
transplanting into egg cells, the reversal appears to be incomplete in cases
studied. Some newer techniques cause less trauma to the donor nucleus and using
a mixture of chemicals from rapidly dividing cells promotes more stable DNA in the
donor nucleus. This technique is called c hromatin transfer. None the less,
cloning is ever increasing, and used for many purposes, including increasing the
populations of endangered species, ensuring that organisms whose genome humans
value for "economic" reasons increase their numbers, and even replicating the
genome of desired pets. However, clones are never "identical" despite the common
DNA, showing the role of specialization within cell lines during development and the
role of the environment.