Download cell cycle control

Overview of the Cell Cycle
The cell cycle begins when two new cells are formed by the division of a single parental cell. This division
process, called M phase, involves two overlapping events in which nucleus divides first and the cytoplasm second.
Nuclear division is called mitosis, and the division of the cytoplasm to produce two daughter cells is termed
cytokinesis. The stars of the mitotic drama are the chromosomes. The beginning of mitosis is marked by
condensation (coiling and folding) of the cell’s chromatin, which generates chromosomes that are thick enough to
be individually noticeable under the microscope.
Because DNA replication has already taken place, each chromosome actually consists of two chromosome copies
that remain attached to each other until the cell divides. As long as they remain attached, the two new
chromosomes are referred to as sister chromatids. As the chromatids become visible, the nuclear envelope breaks
into fragments. Then, guided by the microtubules of the mitotic spindle, the sister chromatids separate and—each
now a full-fledged chromosome— move to opposite ends of the cell. By this time, cytokinesis has usually begun,
and new nuclear membranes envelop the two groups of daughter chromosomes as cell division is completed. M
phase usually lasts less than an hour. Cells spend most of their time in the growth phase between divisions, called
interphase. Most cellular contents are synthesized continuously during interphase, so cell mass gradually
increases as the cell approaches division. During interphase the amount of nuclear DNA doubles, and experiments
using radioactive DNA precursors have shown that the new nuclear DNA is synthesized during a specific portion
of interphase named the S phase (S for synthesis). A time gap called G1 phase separates S phase from the
preceding M phase; a second gap, the G2 phase, separates the end of S phase from the onset of the next M phase.
FIGURE: The Eukaryotic Cell Cycle. (a) The M (mitotic) phase, the process of cell division, is the most
visually distinctive part of the cell cycle. It consists of two overlapping processes, mitosis and cytokinesis. In
mitosis, the mitotic spindle segregates the duplicated, condensed chromosomes into two daughter nuclei; in
cytokinesis, the cytoplasm divides to yield two genetically identical daughter cells. (b) Between divisions, the cell
is said to be in interphase, which is made up of the S phase (the period of nuclear DNA replication) and two “gap”
phases, called G1 and G2. The cell continues to grow throughout interphase, a time of high metabolic activity.
Although the cells of a multicellular organism divide at varying rates, most studies of the cell cycle involve cells
growing in culture, where the length of the cycle tends to be similar for different cell types. We can easily
determine the overall length of the cell cycle—the generation time— for cultured cells by counting the cells under
a microscope and determining how long it takes for the cell population to double. In cultured mammalian cells, for
example, the total cycle usually takes about 18–24 hours. Once we know the total length of the cycle, it is possible
to determine the length of specific phases.
To determine the length of the S phase, we can expose cells to a radioactively labeled DNA precursor (usually 3Hthymidine) for a short period of time and then examine the cells by autoradiography. The fraction of cells with
silver grains over their nuclei represents the fraction of cells that were somewhere in S phase when the radioactive
compound was available. When we multiply this fraction by the total length of the cell cycle, the result is an
estimate of the average length of the S phase. For mammalian cells in culture, this fraction is often around 0.33,
which indicates that S phase is about 6–8 hours in length.
Similarly, we can estimate the length of M phase by multiplying the generation time by the percentage of the cells
that are actually in mitosis at any given time. This percentage is called the mitotic index. The mitotic index for
cultured mammalian cells is often about 3–5%, which means that M phase lasts less than an hour (usually 30–45
minutes).
In contrast to the S and M phases, whose lengths tend to be similar for different mammalian cells, the length of G1
is quite variable, depending on the cell type. Although a typical G1 phase lasts 8–10 hours, some cells spend only
a few minutes or hours in G1, whereas others are delayed for long periods of time. During G1, a major “decision”
is made as to whether and when the cell is to divide again. Cells that become arrested in G1, awaiting a signal that
will trigger reentry into the cell cycle and a commitment to divide, are said to be in G0 (G zero).
Other cells exit from the cell cycle entirely and undergo terminal differentiation, which means they are destined
never to divide again; most of the nerve cells in your body are in this state. In some cells, transient arrest of the
cell cycle can also occur in G2. In general, however, G2 is shorter than G1 and more uniform in duration, usually
lasting 4–6 hours.
Cell cycle studies have been facilitated by the use of flow cytometry, a technique that permits automated analysis
of the chemical makeup of millions of individual cells almost simultaneously. In this procedure, cells are first
stained with one or more fluorescent dyes—for example, a red dye that stains DNA might be combined with a
green dye that binds specifically to a particular cell protein. The dyed cells are then passed in a tiny, liquid stream
through a beam of laser light. By analyzing fluctuations in the intensity and color of the fluorescent light emitted
by each cell as it passes through the laser beam, researchers can assess the concentration of DNA and specific
proteins in each individual cell. This information is then used to assess the chemical makeup of cells at different
points in the cycle.
MITOSIS
Mitosis Is Subdivided into Prophase, Prometaphase, Metaphase, Anaphase, and Telophase
Mitosis is subdivided into five stages based on the changing appearance and behavior of the chromosomes. These
five phases are prophase, prometaphase, metaphase, anaphase, and telophase. (An alternative term for
prometaphase is simply late prophase.) The purpose of mitosis is to ensure that each of the two daughter nuclei
receives one copy of each duplicated chromosome.
Figure: Mitotic Phases: (a)Prophase (b) Prometaphase (c)Metaphase (d) Anaphase (e) Telophase
Prophase: After completing DNA replication, cells exit from S phase and enter into G2 phase, where final
preparations are made for the onset of mitosis. Toward the end of G2, the chromosomes start to condense from the
extended, highly diffuse form of interphase chromatin fibers into the compact, extensively folded
structures that are typical of mitosis.
Chromosome condensation is an important event because interphase chromatin fibers are so long and intertwined
that in an uncompacted form. Although the transition from G2 to prophase is not sharply defined.
A cell is considered to be in prophase when individual chromosomes have condensed to the point of being visible
as discrete objects in the light microscope. Because the chromosomal DNA molecules have replicated during S
phase, each prophase chromosome is composed of two sister chromatids that are tightly attached to each other.
In animal cells, the nucleoli usually disperse as the chromosomes condense; plant cell nucleoli may either remain
as discrete entities, undergo partial disruption, or disappear entirely. Meanwhile, another important organelle has
sprung into action. This is the centrosome, a small zone of granular material located adjacent to the nucleus. The
centrosome functions as a microtubuleorganizing center (MTOC) where microtubules are assembled and anchored.
During each cell cycle, the centrosome is duplicated prior to mitosis, usually during S phase. At the beginning of
prophase the two centrosomes then separate from each other and move toward opposite sides of the nucleus. As
they move apart, each centrosome acts as a nucleation site for microtubule assembly and the region between the
two centrosomes begins to fill with microtubules destined to form the mitotic spindle, the structure that distributes
the chromosomes to the daughter cells later in mitosis. During this process, cytoskeletal microtubules disassemble
and their tubulin subunits are added to the growing mitotic spindle. At the same time, a dense starburst of
microtubules called an aster forms in the immediate vicinity of each centrosome.
Prometaphase: The onset of prometaphase is marked by fragmentation of the membranes of the nuclear
envelope. As the centrosomes complete their movement toward opposite sides of the nucleus (Figure b), the
break down of the nuclear envelope allows the spindle microtubules to enter the nuclear area and make contact
with the chromosomes, which still consist of paired chromatids at this stage. The spindle microtubules are destined
to attach to the chromatids in the region of the centromere, a constricted area where the two members of each
chromatid pair are held together.
Centromere protein A (CENP-A) plays a key role in recruiting additional proteins to the centromere to form the
kinetochore, which is the structure that attaches the paired chromatids to the spindle microtubules. Each
chromosome eventually acquires two kinetochores facing in opposite directions, one associated with each of the
two chromatids. During prometaphase some spindle microtubules bind to these kinetochores, thereby attaching the
chromosomes to the spindle. Forces exerted by these kinetochore microtubules then throw the chromosomes into
agitated motion and gradually move them toward the center of the cell, in a process known as congression. In
addition to kinetochore microtubules, there are two other kinds of microtubules in the spindle. Those that interact
with microtubules from the opposite pole of the cell are called polar micro tubules; the shorter ones that form the
asters (from the Greek word for “star”) at each pole are called astral microtubules. Some of the astral
microtubules appear to interact with proteins lining the plasma membrane.
Metaphase: A cell is said to be in metaphase when the fully condensed chromosomes all become aligned at the
metaphase plate, the plane equidistant between the two poles of the mitotic spindle. Agents that interfere with
spindle function, such as the drug colchicine, can be used to arrest cells at metaphase. At metaphase the
chromosomes appear to be relatively stationary, but this appearance is misleading. Actually, the two sister
chromatids of each chromosome are already being actively tugged toward opposite poles. They appear stationary
because the forces acting on them are equal in magnitude and opposite in direction.
Anaphase: Usually the shortest phase of mitosis, anaphase typically lasts only a few minutes. At the beginning of
anaphase, the two sister chromatids of each chromosome abruptly separate and begin moving toward opposite
spindle poles at a rate of about 1 mm/min. Anaphase is characterized by two kinds of movements, called anaphase
A and anaphase B. In anaphase A, the chromosomes are pulled, centromere first, toward the spindle poles as the
kinetochore microtubules get shorter and shorter. In anaphase B, the poles themselves move away from each other
as the polar microtubules lengthen. Depending on the cell type involved, anaphase A and B may take place at the
same time, or anaphase B may follow anaphase A.
Telophase: At the beginning of telophase, the daughter chromosomes have arrived at the poles of the spindle.
Next the chromosomes uncoil into the extended fibers typical of interphase chromatin, nucleoli develop at the
nucleolar organizing sites on the DNA, the spindle disassembles, and nuclear envelopes form around the two
groups of daughter chromosomes. During this period the cell usually undergoes cytokinesis, which divides the cell
into two daughter cells.
CYTOKINESIS
Cytokinesis Divides the Cytoplasm
After the two sets of chromosomes have separated during anaphase, cytokinesis divides the cytoplasm in two,
there by completing the process of cell division. Cytokinesis usually starts during late anaphase or early telophase,
as the nuclear envelope and nucleoli are re-forming and the chromosomes are decondensing. In some cases, a
significant time lag may occur between nuclear division (mitosis) and cytokinesis, indicating that the two
processes are not tightly coupled. Moreover, certain cell types can undergo many rounds of chromosome
replication and nuclear division in the absence of cytokinesis, thereby producing a large, multinucleate cell known
as a syncytium. Sometimes the multinucleate condition is permanent; in other situations, the multinucleate state is
only a temporary phase in the organism’s development.
Cytokinesis in Animal Cells. The mechanism of cytokinesis differs between animals and plants. In animal cells,
cytoplasmic division is called cleavage. The process begins as a slight indentation or puckering of the cell surface,
which deepens into a cleavage furrow that encircles the cell. The furrow continues to deepen until opposite
surfaces make contact and the cell is split into two. The cleavage furrow divides the cell along a plane that passes
through the central region of the spindle (the spindle equator), suggesting that the location of the spindle
determines where the cytoplasm will be divided.
Figure: Cytokinesis in an Animal Cell. A schematic diagram showing the position of the contractile ring during
cytokinesis, which pinches the dividing cell in two (red arrows).
Cleavage depends on a beltlike bundle of actin microfilaments called the contractile ring, which forms just
beneath the plasma membrane during early anaphase. As cleavage progresses, this ring of microfilaments tightens
around the cytoplasm, like a belt around the waist, eventually pinching the cell in two. Tightening of the
contractile ring is generated by interactions between the actin microfilaments and myosin, the motor protein whose
role in muscle contraction. The contractile ring provides a dramatic example of how rapidly actin-myosin
complexes can be assembled and disassembled in nonmuscle cells.
Cytokinesis in Plant Cells. Cytokinesis in higher plants is fundamentally different from the corresponding
process in animal cells. Because plant cells are surrounded by a rigid cell wall, they cannot form a contractile ring
at the cell surface that pinches the cell in two. In other words, rather than pinching the cytoplasm in half with a
contractile ring that moves from the outside of the cell toward the interior, the plant cell cytoplasm is divided by a
process that begins in the cell interior and works toward the periphery. Cytokinesis in plants is typically initiated
during late anaphase or early telophase, when a group of small, membranous vesicles derived from the Golgi
complex align themselves across the equatorial region of the spindle. These vesicles, which contain
polysaccharides and glycoproteins required for cell wall formation, are guided to the spindle equator by the
phragmoplast, a parallel array of microtubules derived from polar microtubules and oriented perpendicular to the
direction in which the new cell wall is being formed. After arriving at the equator, the Golgi-derived vesicles fuse
together to produce a large, flattened sac called the cell plate, which represents the cell wall in the process of
formation. The contents of the sac assemble to form the noncellulose components of the primary cell wall, which
expands outward as clusters of microtubules and vesicles form at the lateral edges of the advancing cell plate.
Eventually, the expanding cell plate makes contact with the original cell wall, separating the two daughter cells
from each other. The new cell wall is then completed by deposition of cellulose microfibrils. The plasmodesmata
that provide channels of continuity between the cytoplasms of adjacent plant cells are also present in the cell plate
and the new wall as it forms.
Figure: Cytokinesis and Cell Plate Formation in a Plant Cell. A schematic diagram showing the location of the
cellplate and the original cell wall of a dividing plant cell.
MEIOSIS
Meiosis does two things:
1) Meiosis takes a cell with two copies of every chromosome (diploid) and makes cells with a single copy of every
chromosome (haploid).
This is a good idea if you’re going to combine two cells to make a new organism. This trick is accomplished by
halving chromosome number.
In meiosis, one diploid cells produces four haploid cells.
Why do we need meiosis?
 Meiosis is necessary to halve the number of chromosomes going into the sex cells
Why halve the chromosomes in gametes?
 At fertilization the male and female sex cells will provide ½ of the chromosomes each – so the offspring has
genes from both parents
2) Meiosis scrambles the specific forms of each gene that each sex cell (egg or sperm) receives.
This makes for a lot of genetic diversity. This trick is accomplished through independent assortment and crossingover.
Genetic diversity is important for the evolution of populations and species.
 Meiosis involves two separate divisions: Two successive nuclear divisions occur, Meiosis I (Reduction) and
Meiosis II (Division).
 Meiosis I reduces the ploidy level from 2n to n (reduction) while Meiosis II divides the remaining set of
chromosomes in a mitosis-like process (division).
 In meiosis, homologous chromosomes are separated into different daughter cells
 Meiosis I and meiosis II each include prophase, metaphase, anaphase, and telophase
The First Division
Meiosis I : Separates Homologous Chromosomes
Interphase:
– Each of the chromosomes replicate
– The result is two genetically identical sister chromatids which remain attached at their centromeres
Prophase I:
Prophase I is one of the most important stages of meiosis. During this stage, many crucial events occur.
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The spindle appears.
Nuclear envelopes disappear.
The DNA of the chromosomes begin to twist and condense, making the DNA visible to the microscope.
Each chromosome actively seeks out its homologous pair (which also has a sister chromatid).
The two replicated homologous pairs find each other and form a synapse. The structure formed is referred to as
a tetrad (four chromatids).
The point at which the two non-sister chromatids intertwine is called a chiasma. Sometimes a process known
as crossing over occurs at this point.
This is where two non-sister chromatids exchange genetic material. This exchange does not become evident,
however, until the two homologous pairs separate.
Prophase I includes synapsis and crossing over
Homologous chromosomes pair and undergo synapsis
One member of a pair is the maternal homologue, the other is the paternal homologue
Synapsis is the association of four chromatids (two from each homologue)
Prophase I is divided into five stages called leptotene, zygotene, pachytene, diplotene, and diakinesis.
 The leptotene stage begins with the condensation of chromatin fibers into long, threadlike structures, similar
to what occurs at the beginning of mitosis.
 At zygotene, continued condensation makes individual chromosomes distinguishable, and homologous
chromosomes become closely paired with each other via the process of synapsis, forming bivalents.
Each bivalent has four chromatids, two derived from each chromosome. Bivalent formation is of considerable
genetic significance because the close proximity between homologous chromosomes
allows DNA segments to be exchanged by a process called crossing over. It is this physical exchange of
genetic information between corresponding regions of homologous chromosomes that accounts for genetic
recombination.
 Crossing over occurs during the pachytene stage, which is marked by a dramatic compacting process that
reduces each chromosome to less than a quarter of its previous length.
 At the diplotene stage, the homologous chromosomes of each bivalent begin to separate from each other,
particularly near the centromere. However, the two chromosomes of each homologous pair remain attached by
connections known as chiasmata (singular: chiasma). Such connections are situated in regions where
homologous chromosomes have exchanged DNA segments and hence provide visual evidence that crossing
over has occurred between two chromatids, one derived from each chromosome.
 With the onset of diakinesis, the final stage of prophase I, the chromosomes recondense to their maximally
compacted state. Now the centromeres of the homologous chromosomes separate further, and the chiasmata
eventually become the only remaining attachments between the homologues. At this stage, the nucleoli
disappear, the spindle forms, and the nuclear envelope breaks down, marking the end of prophase I.
Metaphase I:
The chromosomes line up at the equator attached by their centromeres to spindle fibers from centrioles -Still in
homologous pairs
Anaphase I:
The spindle guides the movement of the chromosomes toward the poles
 Sister chromatids remain attached
 Move as a unit towards the same pole
The homologous chromosome moves toward the opposite pole
 Contrasts mitosis – chromosomes appear as individuals instead of pairs (meiosis)
Telophase I:
 This is the end of the first meiotic cell division.
 The cytoplasm divides, forming two new daughter cells.
 Each of the newly formed cells has half the number of the parent cell’s chromosomes, but each
chromosome is already replicated ready for the second meiotic cell division
Cytokinesis:
 Occurs simultaneously with telophase I
o Forms 2 daughter cells
 Plant cells – cell plate
 Animal cells – cleavage furrows
 NO FURTHER REPLICATION OF GENETIC MATERIAL PRIOR TO THE SECOND DIVISION OF
MEIOSIS
Meiosis II : Separates sister chromatids
 Proceeds similar to mitosis
 THERE IS NO INTERPHASE II !
Prophase II: Each of the daughter cells forms a spindle, and the double stranded chromosomes move toward the
equator
Metaphase II: The chromosomes are positioned on the metaphase plate in a mitosis-like fashion
Anaphase II: The centromeres of sister chromatids finally separate. The sister chromatids of each pair move
toward opposite poles (Now individual chromosomes).
Telophase II and Cytokinesis: Nuclei form at opposite poles of the cell and cytokinesis occurs. After completion
of cytokinesis there are four daughter cells. All are haploid (n).
YEAST CELL DIVISION
The cell cycle can be defined as the period between division of a mother cell and subsequent division of its
daughter progeny. The regulatory mechanisms that order and coordinate the progress of the cell cycle have been
intensely studied. Numerous proteins that have been characterized through mutations are collectively designated as
cell division cycle (Cdc) proteins.
The eukaryotic cell cycle involves both continuous events (cell growth) and periodic events (DNA synthesis and
mitosis). Commencement and progression of these events can formally been distinguished into pathways for DNA
synthesis and nuclear division, spindle formation, bud emergence and nuclear migration, and cytokinesis.
However, from a molecular viewpoint these processes are intimately coupled.
Mitotic division is initiated when cells attain a critical cell size and are stimulated by exogenous or endogenous
signals during interphase. A prerequisite is that DNA synthesis guarantees proper duplication of the genetic
material and that all substrates critical to anabolic pathways are available. In mammalian cells, the mitotic phase
can be subdivided into a number of steps that are characterized by particular states of orienting the spindle pole
bodies, sorting and movement of the duplicated chromosomes, and finally, cell separation after cytokinesis
(Figure1). While all processes pertinent to the cell cycle have been highly conserved among all eukaryotes,
ascomycetous fungi, and in particular S.cerevisiae, exhibit several peculiarities with respect to cell division and
cytokinesis
Figure1: Phases in the mitotic cycle.
Figure2: Budding yeast cell.
Budding is the most common mode of vegetative growth in yeasts and multilateral budding is a typical
reproductive characteristic of ascomycetous yeasts, including S. cerevisiae. Yeast buds are initiated when mother
cells attain a critical cell size at a time coinciding with the onset of DNA synthesis. This is followed by localized
weakening of the cell wall and this, together with tension exerted by turgor pressure, allows extrusion of
cytoplasm into an area bounded by new cell wall material. The regulation of particular cell wall synthetic enzymes
and transport of specific bud plasma membrane receptors are key steps in the emergence of a bud. Chitin forms a
ring at the junction between the mother cell and the newly emerging bud to finally result in the generation of a
daughter cell (figure 2). After cell separation, this ring will be retained at the surface of the mother cell and form
the so called bud scar and a birth scar at the surface of the daughter. The number of bud scars left on the surface of
a yeast cell is a useful determinant of cellular age.
During bud formation, only the bud but not the mother cell will grow. Once mitosis is complete and the bud
nucleus and other organelles have migrated into the bud, cytokinesis commences and a septum is formed in the
isthmus between mother and daughter. A ring of proteins, called septins, are involved in positioning cell division
in that they define the cleavage plain which bisects the spindle axis at cytokinesis. These septins encircle the neck
between mother and daughter for the duration of the cell cycle. In S. cerevisiae, cell size at division is
asymmetrical with buds being smaller than mother cells when they separate. Also cell division cycle times are
different, because daughter cells need time (in G1 phase) to attain the critical cell size before they are prepared to
bud.
CELL CYCLE CONTROL
The control system that regulates progression through the cell cycle must accomplish several tasks.
First, it must ensure that the events associated with each phase of the cell cycle are carried out at the
appropriate time and in the appropriate sequence.
Second, it must make sure that each phase of the cycle has been properly completed before the next phase is
initiated.
Finally, it must be able to respond to external conditions that indicate the need for cell proliferation (e.g., the
quantity of nutrients available or the presence of growth-signaling molecules).
The preceding objectives are accomplished by a group of molecules that act at key transition points in the cell
cycle (Figure 1).
FIGURE 1: Key Transition Points in the Cell Cycle. The red bars mark three important transition points in the
eukaryotic cell cycle where control mechanisms determine whether the cell will continue to proceed through the
cycle. That determination is based on chemical signals reflecting both the cell’s internal state and its external
environment. The two circular, dark green arrows indicate locations in late G1 and late G2 where the cell can exit
from the cycle and enter a nondividing state.
At each of these points, conditions within the cell determine whether the cell will proceed to occurs during late G1.
For example, in cultured cells the process of cell division can be stopped or slowed by allowing the cells to run out
of either nutrients or space or by adding inhibitors of vital processes such as protein synthesis. In all such cases,
the cell cycle is halted in late G1, suggesting that progression from G1 into S is a critical control point in the cell
cycle. In yeast, this control point is called Start; yeast cells must have sufficient nutrients and must reach a certain
size before they can pass through Start. In animal cells, the comparable control point is called the restriction
point. The ability to pass through the restriction point is influenced by the presence of extracellular growth factors,
which are proteins used by multicellular organisms to stimulate or inhibit cell proliferation. Cells that have
successfully passed through the restriction point are committed to S phase, whereas those that do not pass the
restriction point enter into G0 and reside there for variable periods of time, awaiting a signal that will allow them
to reenter G1 and pass through the restriction point.
A second important transition point occurs at the G2-M boundary, where the commitment is made to enter into
mitosis. In certain cell types, the cell cycle can be indefinitely arrested at the end of G2 if cell division is not
necessary; under such conditions, the cells enter a nondividing state analogous to G0. This occurs in a few cases,
such as the division of fertilized frog eggs or in some skin cells, G2 arrest is more important.
A third key transition point occurs during M phase at the junction between metaphase and anaphase, where
the commitment is made to move the two sets of chromosomes into the newly forming daughter cells. Before cells
can pass through this transition point and begin anaphase, it is important to have all the chromosomes properly
attached to the spindle. If the two chromatids that make up each chromosome are not properly attached to opposite
spindle poles, the cell cycle is temporarily arrested to allow spindle attachment to occur.
1. Progression Through the Cell Cycle Is Controlled by Cyclin-Dependent Kinases (Cdks)
The phosphorylation of target proteins by protein kinases, and their dephosphorylation by enzymes called protein
phosphatases, is a common mechanism for regulating protein activity that turns out to be widely used in
controlling the cell cycle.
Progression through the cell cycle is driven by a series of protein kinases—including the protein kinase produced
by the cdc2 gene—that exhibit enzymatic activity only when they are bound to a special type of activator protein
called a cyclin. Such protein kinases are therefore referred to as cyclin-dependent kinases or simply Cdks. The
eukaryotic cell cycle is controlled by several different Cdks that bind to different cyclins, thereby creating a variety
of Cdk-cyclin complexes.
Cyclins required for the G2-M transition and the early events of mitosis are called mitotic cyclins, and the Cdks to
which they bind are known as mitotic Cdks. Likewise, cyclins required for passage through the G1 restriction
point (or Start) are called G1 cyclins, and the Cdks to which they bind are G1 Cdks. Yet another group of cyclins,
called S cyclins, are required for events associated with DNA replication during S phase.
2. Mitotic Cdk-Cyclin Drives Progression Through the G2-M Transition by Phosphorylating Key
Proteins Involved in the Early Stages of Mitosis
Biochemical studies of these mitosis-inducing molecules revealed that they consist of two subunits: a Cdk and a
cyclin. In other words, MPF (Maturation Promoting Factor) is a mitotic Cdk-cyclin complex. Mitotic Cdk is active
as a protein kinase when it is bound to mitotic cyclin, and mitotic cyclin is not always present in adequate
amounts. Instead, the concentration of mitotic cyclin gradually increases during G1, S, and G2; eventually it
reaches a critical threshold at the end of G2 that permits it to activate mitotic Cdk and thereby trigger the onset of
mitosis. Halfway through mitosis, the mitotic cyclin molecules are abruptly destroyed. The resulting decline in
mitotic Cdk activity prevents another mitosis from occurring until the mitotic cyclin concentration builds up again
during the next cell cycle. In addition to requiring mitotic cyclin, the activation of mitotic Cdk involves
phosphorylation and dephosphorylation of the Cdk molecule itself.
FIGURE 2: Regulation of Mitotic Cdk-Cyclin by Phosphorylation and Dephosphorylation.
As shown in Figure 2, the binding of mitotic cyclin to mitotic Cdk yields a Cdk-cyclin complex that is initially
inactive (step 1). To trigger mitosis, the complex requires the addition of an activating phosphate group to a
particular amino acid in the Cdk molecule. Before this phosphate is added, however, inhibiting kinases
phosphorylate the Cdk molecule at two other locations, causing the active site to be blocked (step2 ). The
activating phosphate group, highlighted with yellow in step 3 , is then added by a specific activating kinase. The
last step in the activation sequence is the removal of the inhibiting phosphates by a specific phosphatase enzyme
(step 4). Once the phosphatase begins removing the inhibiting phosphates, a positive feedback loop is set up: The
activated mitotic Cdk generated by this reaction stimulates the phosphatase, thereby causing the activation process
to proceed more rapidly. After mitotic Cdk-cyclin has been activated through the preceding steps, its protein
kinase activity triggers the onset of mitosis (Figure 19-37).
FIGURE 3: The Mitotic Cdk Cycle.
3. The Anaphase-Promoting Complex Coordinates Key Mitotic Events by Targeting Specific Proteins for
Destruction
Besides triggering the onset of mitosis, mitotic Cdk-cyclin also plays an important role later in mitosis when the
decision is made to separate the sister chromatids during anaphase. Mitotic Cdk-cyclin exerts its influence on this
event by phosphorylating and thereby contributing to the activation of the anaphase-promoting complex, a
multiprotein complex that coordinates mitotic events by promoting the destruction of several key proteins at
specific points during mitosis. The anaphase-promoting complex functions as a ubiquitin ligase, a type of
enzyme that targets specific proteins for degradation by joining them to the small protein ubiquitin.
FIGURE 4: The Anaphase-Promoting Complex and the Mitotic Spindle Checkpoint.
As shown in Figure 4, sister chromatids are held together prior to anaphase by adhesive proteins called cohesins,
which become bound to newly replicated chromosomal DNA in S phase following the movement of the replication
forks. Securin maintains this sister chromatid attachment by inhibiting a protease called separase, which would
otherwise degrade the cohesins.
At the beginning of anaphase, however, the anaphase-promoting complex attaches to securin and thereby triggers
its destruction, releasing separase from inhibition. The activated separase then cleaves cohesin, which frees sister
chromatids to separate from each other and begin their anaphase movements toward the spindle poles. Besides
initiating anaphase by causing cohesins to be destroyed, the anaphase-promoting complex induces events
associated with the end of mitosis by targeting another crucial protein for destruction, namely mitotic cyclin.
4. G1 Cdk-Cyclin Regulates Progression Through the Restriction Point by Phosphorylating the Rb Protein
This is another type of Cdk-cyclin regulates entry into S phase. As mentioned earlier, the restriction point (Start in
yeast) is a control mechanism located in late G1 that determines whether a cell will enter S phase, proceed through
the rest of the cell cycle, and divide. Because passing through the restriction point is the main step that commits a
cell to the cell division cycle, it is subject to control by a variety of factors such as cell size, the availability of
nutrients, and the presence of growth factors that signal the need for cell proliferation. Such signals exert their
effects by activating G1 Cdkcyclin, whose protein kinase activity triggers progression through the restriction point
by phosphorylating several target proteins. A key target is the Rb protein, a molecule that controls the expression
of genes whose products are needed for moving through the restriction point and into S phase.
FIGURE 5: Role of the Rb Protein in Cell Cycle Control.
In its dephosphorylated state, the Rb protein binds to the E2F transcription factor. This binding prevents E2F from
activating the transcription of genes coding for proteins required for DNA replication, which are needed before the
cell can pass through the restriction point into S phase. In cells stimulated by growth factors, the Ras pathway is
activated, which leads to the production and activation of a G1 Cdk-cyclin complex that phosphorylates the Rb
protein. Phosphorylated Rb can no longer bind to E2F, thereby allowing E2F to activate gene transcription and
trigger the onset of S phase. During the subsequent M phase (not shown), the Rb protein is dephosphorylated so
that it can once again inhibit E2F.
CELL CYCLE CHECKPOINTS
There are 3 check points in the cell cycle. They are
1. Monitor for chromosome to spindle attachment
2. Completion of DNA replication
3. DNA damage
It would obviously create problems if cells proceeded from one phase of the cell cycle to the next before the
preceding phase had been properly completed. For example, if chromosomes start moving toward the spindle poles
before they have all been properly attached to the spindle, the newly forming daughter cells might receive extra
copies of some chromosomes and no copies of others, a situation known as aneuploidy (an = “not,” eu = “good,”
and “ploidy” refers to chromosome number). Similarly, it would be potentially hazardous for a cell to begin
mitosis before all of its chromosomal DNA had been replicated.
To minimize the possibility of such errors, cells utilize a series of checkpoint mechanisms that monitor conditions
within the cell and transiently halt the cell cycle if conditions are not suitable for continuing.
First check point:
 The checkpoint pathway that prevents anaphase chromosome movements from beginning before the
chromosomes are all attached to the spindle is called the mitotic spindle checkpoint.
 It works through a mechanism in which chromosomes whose kinetochores remain unattached to spindle
microtubules produce a “wait” signal that inhibits the anaphase-promoting complex.
 As long as the anaphase-promoting complex is inhibited, it cannot trigger destruction of the cohesins that hold
sister chromatids together.
 The wait signal responsible for inhibiting the anaphase promoting complex is transmitted by proteins that are
members of the Mad and Bub protein families. Mad and Bub proteins accumulate at unattached chromosomal
kinetochores, where they are converted into a multiprotein complex that inhibits the anaphase promoting
complex by blocking the action of one of its essential activators, the Cdc20 protein.
 After all the chromosomes have become attached to the spindle, the Mad and Bub proteins are no longer
converted into this inhibitory complex, thereby freeing the anaphase promoting complex to initiate the onset of
anaphase.
Figure 1: The mitotic spindle checkpoint prevents anaphase from starting until all chromosomes are attached to
the spindle. Unattached chromosomes keep the “checkpoint on” by organizing Mad and Bub proteins into a
complex that prevents Cdc20 from activating the anaphase-promoting complex. After all chromosomes are
attached, the Mad-Bub complex is not formed (“checkpoint off”) and the anaphase-promoting complex is free to
initiate anaphase.
Second check point:
A second checkpoint mechanism, called the DNA replication checkpoint, monitors the state of DNA replication
to help ensure that DNA synthesis is completed before the cell exits from G2 and begins mitosis.
The existence of this checkpoint has been demonstrated by treating cells with inhibitors that prevent DNA
replication from being finished. Under such conditions, the phosphatase that catalyzes the final dephosphorylation step involved in the activation of mitotic Cdk-cyclin is inhibited through a series of events triggered by
proteins associated with replicating DNA. The resulting lack of mitotic Cdk-cyclin activity halts the cell cycle at
the end of G2 until all DNA replication is completed.
Figure 2: Regulation of Mitotic Cdk-Cyclin by Phosphorylation and Dephosphorylation.
Third check point:
A third type of checkpoint mechanism is involved in preventing cells with damaged DNA from proceeding
through the cell cycle unless the DNA damage is first repaired.
In this case, a multiple series of DNA damage checkpoints exist that monitor for DNA damage and halt the cell
cycle at various points—including late G1, S, and late G2—by inhibiting different Cdk-cyclin complexes.
A protein called p53, sometimes referred to as the “guardian of the genome,” plays a central role in these
checkpoint pathways.
when cells encounter agents that cause extensive double-stranded breaks in DNA, the altered DNA triggers the
activation of an enzyme called ATM protein kinase (catalyzes the phosphorylation of kinases known as checkpoint
kinases), which in turn phosphorylate p53 (and several other target proteins).
Phosphorylation of p53 prevents it from interacting with a protein, Mdm2.
ATM-catalyzed phosphorylation of p53 therefore protects it from degradation and leads to a buildup of p53 in the
presence of damaged DNA.
The accumulating p53 in turn activates two types of events: 1) cell cycle arrest and 2) cell death.
Both responses are based on the ability of p53 to bind to DNA and act as a transcription factor that stimulates the
transcription of specific genes.
One of the crucial genes activated by p53 is the gene coding for p21, a protein that halts progression through the
cell cycle at multiple points by inhibiting the activity of several different Cdk-cyclins.
Phosphorylated p53 also stimulates the production of enzymes involved in DNA repair. But if the damage cannot
be successfully repaired, p53 then activates a group of genes coding for proteins involved in triggering cell death
by apoptosis. A key protein in this pathway, called Puma (p53 upregulated modulator of apoptosis), promotes
apoptosis by binding to and inactivating a normally occurring inhibitor of apoptosis known as Bcl-2.
Figure: Role of the p53 Protein in Responding to DNA Damage
The ability of p53 to trigger cell cycle arrest and cell death allows it to function as a molecular stoplight that
protects cells with damaged DNA from proliferating and passing the damage to daughter cells.
CELL DIFFERENTIATION
Complex unicellular organisms represent one evolutionary pathway. In the evolution of multicellular organisms
different activities are conducted by different types of specialized cells. A single organism consists of a complex
mixture of specialized or differentiated cell types—for example, nerve, muscle, bone, blood, cartilage, and fat—
brought together in various combinations to form tissues and organs. Differentiated cells are distinguished from
each other based on differences in their microscopic appearances and in the products they manufacture. For
example, red blood cells synthesize hemoglobin, nerve cells produce neurotransmitters, and lymphocytes make
antibodies. Such differences indicate that selectively controlling the expression of a wide variety of different
genes must play a central role in the mechanism responsible for creating differentiated cells. Differentiated
cells are produced from populations of immature, non specialized cells by a process known as cell
differentiation. The classic example occurs in embryos, in which cells of the early embryo produce all the cell
types that make up the organism. A fertilized human egg, for example, will progress through a course of
embryonic development that leads to the formation of approximately 250 distinct types of differentiated cells.
Some cells become part of a particular digestive gland, others part of a large skeletal muscle, others part of a bone,
and so forth (Figure 1.17). The pathway of differentiation followed by each embryonic cell depends primarily on
the signals it receives from the surrounding environment; these signals in turn depend on the position of that cell
within the embryo.
FIGURE: Pathways of cell differentiation. A few of the types of differentiated cells present in a human fetus.
As a result of differentiation, different types of cells acquire a distinctive appearance and contain unique materials.
Skeletal muscle cells contain a network of precisely aligned filaments composed of unique contractile proteins;
cartilage cells become surrounded by a characteristic matrix containing polysaccharides and the protein collagen,
which together provide mechanical support; red blood cells become disk-shaped sacks filled with a single protein,
hemoglobin, which transports oxygen; and so forth. Despite their many differences, the various cells of a
multicellular plant or animal are composed of similar organelles. Mitochondria, for example, are found in
essentially all types of cells. In one type, however, they may have a rounded shape, whereas in another they may
be highly elongated and thread-like. In each case, the number, appearance, and location of the various organelles
can be correlated with the activities of the particular cell type. An analogy might be made to a variety of orchestral
pieces: all are composed of the same notes, but varying arrangement gives each its unique character and beauty.
CHARACTERISTICS OF CELL DIFFERENTIATION
There are four characteristics of cell differentiation. They are
1. It is stable phenomenon
2. It is induced by specific stimuli
3. It precede morphological difference
4. It is controlled by genetic factors
1. It is stable phenomenon
One of the principle characteristics of cell differentiation in higher cells is that once established, the differentiated
state is very stable and can persist throughout many cell generations. For example, a neuron will persist as such
throughout the lifetime of an individual.
2. It is induced by specific stimuli
Another important characteristic of cell differentiation is that it is induced in the organism by various stimuli but
once it has been established, it can persist even in the absence of the initial stimulus. For example, differentiated
cloned cell lines, such as steroid-secreting cell lines are able to grow indefinitely in vitro. The differentiated state
is maintained over many cell generations.
3. It precede morphological difference
In many cases before morphological difference appears, the cell is committed to a particular change due to cell
differentiation process. The best example of determination is provided by the imaginal discs of Drosophila. When
the discs were transplanted in Drosophila to different parts, they give rise to its original morphology irrespective of
its transplantation.
4. It is controlled by genetic factors
Genes (genetic factors) enacted crucial role in controlling and executing cell differentiation process. This was
clearly shown by mutational studies. Certain genes are called as homeotic genes, which when inactivated change
one segment of the fly into another e.g. haltere into wings or an antenna into a leg. The fruit fly, Drosophila
melanogaster development may be conceived as a series of binary divisions in which selector genes are switched
on or off, giving rise to the different segments of the fly and then to smaller compartments within each
segment. Each selector gene modifies the activity of entire sets of other genes. A conserved protein-encoding
segment of 180 nucleotides, the homeobox, has been found in several Drosophila homeotic genes. These genes
code for DNA-binding proteins, which are involved in controlling the early differentiation of the
embryo. Homeobox-containing genes have been isolated from vertebrates and this may provide key to
understanding development and differentiation.
TYPES OF DIFFERENTIATED CELLS
The cells of adult animals can be grouped into three general categories with respect to cell proliferation and
replacement.
 A few types of differentiated cells, such as cardiac muscle cells in humans, are no longer capable of cell
division and cannot be replaced if they are lost due to injury or cell death. These cells are produced during
embryonic development, differentiate and are then retained throughout the life of the animal.
 Second types of differentiated cells retain their ability to proliferate. These cells enter the G0 phase of the
cell cycle but resume proliferation as needed to replace cells that have been injured or have died. Cells of
this type include liver cells, skin fibroblasts, smooth muscle cells and the endothelial cells of blood vessels
etc.,
 In third group cells, most fully differentiated cells, however, are no longer capable of cell division but can
be replaced by the proliferation of less differentiated cells, called stem cells, that are present in the tissues
of adult animals. Because they retain the capacity to proliferate and replace differentiated cells throughout
the lifetime of an animal, stem cells play a critical role in the maintenance of adult tissues.
DETERMINANT OF CELL DIFFERENTIATION
Mainly there are two determinants namely cytoplasmic and nucleocytoplasmic determinants.
Cytoplasmic determinants:
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•
•
An egg’s cytoplasm contains RNA, proteins, and other substances that are distributed unevenly in the
unfertilized egg
Cytoplasmic determinants are maternal substances in the egg that influence early development
As the zygote divides by mitosis, cells contain different cytoplasmic determinants, which lead to different
gene expression
Two sources of information “tell” a cell, like a myoblast or even the zygote, which genes to express at any given
time. Once source of information is both the RNA and protein molecules, encoded by the mother’s DNA, in the
cytoplasm of the unfertilized egg cell. Messenger RNA, proteins, other substances, and organelles are distributed
unevenly in the unfertilized egg. This impacts embryonic development in many species. These maternal
substances, cytoplasmic determinants, regulate the expression of genes that affect the developmental fate of the
cell.
After fertilization, the cell nuclei resulting from mitotic division of the zygote are exposed to different cytoplasmic
environments.
Cytoplasmic determinants include mRNA, proteins, chemicals, and organelles and how they are distributed in the
egg. They are distributed unevenly - and this can set up gradients that says ‘head’ side, ‘tail’ side , etc.
The best example of cytoplasmic determinants is provided by the granules present in germ cells. When they are
centrifuged or transplanted into different positions, they will induce the formation of germ cells in a different
position. In nematode embryos these granules can be followed with monoclonal antibodies and have been
observed to move to one side of the cytoplasm during prophase, so that only one of the daughter cells inherits the
cytoplasmic determinants.
Nucleocytoplasmic determinants
Experiments in which a nucleus is placed in a foreign cytoplasm have provided insights on nucleocytoplasmic
determinants for cell differentiation. Immortality character provided by fusion of B lymphocytes with myeloma
cell lines during monoclonal antibodies production is an example for the influence of nucleocytoplasmic
determinants. Frog oocyte cytoplasm is able to reprogram the expression of genes in transplanted nuclei. This
reprogramming ability was found when kidney nuclei of Xenopus were injected into oocytes of the salamander
Pleurodeles. The work with somatic cell fusion and with transplantation of nuclei into oocytes suggests that the
cytoplasm can indeed reprogram the activity of nuclear genes.
STEM CELLS AND ITS BIOLOGICAL IMPORTANCE
Definition: A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s)
of cells/tissues.
Stem Cell Characteristics:
 Blank cells’ (unspecialized)
 Capable of dividing and renewing themselves for long periods of time (proliferation and renewal)
 Have the potential to give rise to specialized cell types (differentiation)
 Self renewable: a cell that has the ability to continuously divide
 Pluripotent: ability to develop into several different kinds of cells/tissues
 Repair: ability to return function to damaged cells in the living organism
Kinds of Stem Cells:
Stem cell type
Totipotent
Description
Each cell can develop into a new
individual
Pluripotent
Cells can form any (over 200) cell types
Multipotent
Cells differentiated, but can form a
number of other tissues
Stem Cell Differentiation:
Examples
Cells from early (1-3 days)
embryos
Some cells of blastocyst (5 to
14 days)
Fetal tissue, cord blood, and
adult stem cells
Kinds of Stem Cells:
Embryonic stem cells: come from a five to six-day-old embryo. They have the ability to form virtually any type
of cell found in the human body.
Embryonic germ cells: are derived from the part of a human embryo or foetus that will ultimately produce eggs
or sperm (gametes).
Adult stem cells: are undifferentiated cells found among specialised or differentiated cells in a tissue or organ
after birth. Based on current research they appear to have a more restricted ability to produce different cell types
and to self-renew.
Sexual Reproduction:
Embryonic Stem Cells (ESC): received from:
 Embryos created in vitro fertilization
 Aborted embryos
Source of ESC:
1. Blastocyst
 “ball of cells”
 3-5 day old embryo
 Stem cells give rise to multiple specialized cell types that make up the heart, lung, skin, and other tissues
2. Human ESC were only studied since 1998
 It took scientists 20 years to learn how to grow human ESC following studies with mouse ESC
How are embryonic stem cells harvested?
 Human ES cells are derived from 4-5 day old blastocyst
 Blastocyst structures include:
 Trophoblast: outer layer of cells that surrounds the blastocyst & forms the placenta
 Blastocoel: (“blastoseel”) the hollow cavity inside the blastocyst that will form body cavity
 Inner cell mass: a group of approx. 30 cells at one end of the blastocoel:
o
Forms 3 germ layers that form all embryonic tissues (endoderm, mesoderm, ectoderm)
Blastocyst Diagram:
Stages of Embryogenesis:
Stages of Embryogenesis
Day 1
Fertilized egg
4/16/2014
Day 2
2-cell embryo
Day 11-14
Dr. Hariom Yadav
Tissue Differentiation
Day 3-4
Multi-cell embryo
Day 5-6
Blastocyst
Adult stem cells:
Adult Stem Cells (ASC): can be received from:
 Limited tissues (bone marrow, muscle, brain)
 Discrete populations of adult stem cells generate replacements for cells that are lost through
normal wear and tear, injury or disease
 Placental cord
 Baby teeth
Adult or somatic stem cells have unknown origin in mature tissues
 Unlike embryonic stem cells, which are defined by their origin (inner cell mass of the blastocyst)
Adult stem cells typically generate the cell types of the tissue in which they reside
 Stem cells that reside in bone marrow give rise to RBC, WBC and platelets
 Recent experiments have raised the possibility that stem cells from one tissue can give rise to other
cell types
 This is known as PLASTICITY
Ex: Blood cells becoming neurons, Liver cells stimulated to produce insulin, Hematopoietic
(blood cell producing) stem cells that become heart cells
Applications:
Basic research – clarification of complex events that occur during human development & understanding molecular
basis of cancer
 Molecular mechanisms for gene control
 Role of signals in gene expression & differentiation of the stem cell
 Stem cell theory of cancer
Biotechnology(drug discovery & development) – stem cells can provide specific cell types to test new drugs
 Safety testing of new drugs on differentiated cell lines
 Screening of potential drugs
 Cancer cell lines are already being used to screen potential anti-tumor drugs
 Availability of pluripotent stem cells would allow drug testing in a wider range of cell types & to
reduce animal testing
Cell based therapies:
 Regenerative therapy to treat Parkinson’s, Alzheimer’s, ALS, spinal cord injury, stroke, severe burns, heart
disease, diabetes, osteoarthritis, and rheumatoid arthritis
 Stem cells in gene therapy
 Stem cells as vehicles after they have been genetically manipulated
 Stem cells in therapeutic cloning
 Stem cells in cancer