Cell cycle in biology for undergrad students

CELL BIOLOGY
Dr. ESRA AYDEMİR AYAZ
Cell Cycle

The most basic function of the cell cycle is to duplicate the vast
amount of DNA in the chromosomes and then segregate the
copies into two genetically identical daughter cells. These
processes define the two major phases of the cell cycle. S and M
phases:
1) Chromosome duplication during S phase (S for DNA
synthesis) (10–12 hours)
2) Chromosome segregation and cell division in M phase (M for
mitosis), (less than an hour)
OVERVIEW OF THE CELL CYCLE
nuclear division, or mitosis
M
cytoplasmic division, or cytokinesis

The events of eukaryotic cell division as seen under a microscope
Early in mitosis at a stage called prophase: the two DNA molecules are gradually disentangled and
condensed into pairs of rigid, compact rods called sister chromatids.
Nuclear envelope disassembles later in mitosis, the sister-chromatid pairs become attached to the
mitotic spindle, a giant bipolar array of microtubules.
Sister chromatids are attached to opposite poles of the spindle and, eventually, align at the spindle
equator in a stage called metaphase. The destruction of sister-chromatid cohesion at the start of
anaphase separates the sister chromatids, which are pulled to opposite poles of the spindle. The
spindle is then disassembled, and the segregated chromosomes are packaged into separate nuclei at
telophase. Cytokinesis then cleaves the cell in two, so that each daughter cell inherits one of the two
nuclei

The Eukaryotic Cell Cycle Usually Consists of Four Phases
Most cells require much more time to grow and double their mass of proteins and organelles
than they require to duplicate their chromosomes and divide. Partly to allow time for growth,
most cell cycles have gap phases—a G1 phase between M phase and S phase and a G2 phase
between S phase and mitosis. Thus, the eukaryotic cell cycle is traditionally divided into four
sequential phases: G1, S, G2, and M. G1, S, and G2 together are called interphase
Cell growth occurs throughout the cell cycle, except
during mitosis.
The two gap phases ARE NOT only delays for the cell growth ;
provide time for the cell to monitor the internal and external
environment to ensure that conditions are suitable and
preparations are complete before the cell commits itself to the
major upheavals of S phase and mitosis

Especially important.
Length can vary greatly depending on external conditions and extracellular signals from other
cells.
If extracellular conditions are unfavorable, for example, cells delay progress through G1 and
may even enter a specialized resting state known as G0 (G zero), in which they can remain for
days, weeks, or even years before resuming proliferation.
Many cells remain permanently in G0 until they or the organism dies. If extracellular
conditions are favorable and signals to grow and divide are present, cells in early G1 or G0
progress through a commitment point near the end of G1 known as Start (in yeasts)
or the restriction point (in mammalian cells).
Start for both yeast and animal cells. After passing this
point, cells are committed to DNA replication, even if the
extracellular signals that stimulate cell growth and division are
removed.
The G1 phase

Cell-Cycle Control Is Similar in All Eukaryotes
Time required to complete certain events, vary greatly from one cell type to another,
even in the same organism.
The basic organization of the cycle, however, is essentially the same in all eukaryotic cells, and all
eukaryotes appear to use similar machinery and control mechanisms to drive and regulate cell-
cycle events.
The proteins of the cell-cycle control system have been so well conserved over the course of
evolution that many of them function perfectly when transferred from a human cell to a yeast
cell.
Several model organisms: The budding yeast Saccharomyces cerevisiae and the fission yeast
Schizosaccharomyces pombe, The early embryos frog Xenopus laevis (cell-cycle control
mechanisms), the fruit fly Drosophila melanogaster (control and coordination of cell growth
and division in multicellular organisms), and also cultured human cells

How can we tell what stage a cell has reached in the cell cycle?
To look at living cells with a microscope???
Looking at budding yeast cells
under a microscope is very
useful, because the size of the
bud provides an indication of
cell-cycle stage
By staining cells with DNA-binding
fluorescent dyes: condensation of
chromosomes in mitosis. Or with
antibodies that recognize specific
cell components such as the
microtubules: mitotic spindle).
Artificial thymidine analog
bromodeoxyuridine (BrdU)
DNA content, which
doubles during S
phase by the use of
fluorescent DNA-
binding dyes and a
flow cytometer, rapid
and automatic
analysis of large
numbers of cells

THE CELL-CYCLE CONTROL SYSTEM
For many years it was not even clear whether there was a separate control system, or whether
the processes of DNA synthesis, mitosis, and cytokinesis somehow controlled themselves.
A major breakthrough (1980s): The identification of the distinct key proteins of the control
system

The Cell-Cycle Control System Triggers the Major Events of the Cell Cycle
Like a timer that triggers the events of the cell
cycle in a set sequence
If some malfunction prevents the successful completion
of DNA synthesis: signals are sent to the control system
to delay progression to M phase.
Such delays provide time for the machinery to be
repaired and also prevent the disaster that might result if
the cycle progressed prematurely to the next stage—and
segregated incompletely replicated chromosomes

Each initiates a specific cell-cycle event: increase the accuracy and reliability of cell-cycle
progression.
1) The switches are generally binary (on/off) and launch events in a complete, irreversible
fashion: if events like chromosome condensation or nuclear-envelope breakdown were
only partially initiated or started but not completed: Disaster
2) Robust and reliable: backup mechanisms and other features allow the system to operate
effectively under a variety of conditions and even if some components fail.
3) Highly adaptable and can be modified to suit specific cell types or to respond to specific
intracellular or extracellular signals.
The cell-cycle control system is based on a connected series of
biochemical switches

The first is Start (or the restriction point) in late G1, where the cell commits to cell-cycle entry
and chromosome duplication.
The second is the G2/M transition, where the control system triggers the early mitotic events
that lead to chromosome alignment on the mitotic spindle in metaphase.
The third is the metaphase-to-anaphase transition, where the control system stimulates sister-
chromatid separation, leading to the completion of mitosis and cytokinesis.
3 major regulatory transitions

It detects problems inside or outside the
cell. If the control system senses problems
in the completion of DNA replication, for
example, it will hold the cell at the G2/M
transition until those problems are solved.
If extracellular conditions are not
appropriate for cell proliferation, the control
system blocks progression through Start,
thereby preventing cell division until
conditions become favorable.
The control system blocks progression through each of these transitions if;

The Cell-Cycle Control System Depends on Cyclically Activated Cyclin-
Dependent Protein Kinases (Cdks)
Central components: a family of protein kinases known as cyclin-dependent kinases (Cdks).
The activities of these kinases rise and fall as the cell progresses through the cycle, leading to cyclical
changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of
the cell cycle.
An increase in Cdk activity at the G2/M transition, for example, increases the phosphorylation of
proteins that control chromosome condensation, nuclear-envelope breakdown, spindle assembly,
and other events that occur in early mitosis.

 The most important of these Cdk regulators are proteins known as cyclins.
 Cdks, as their name implies, are dependent on cyclins for their activity: unless they are
bound tightly to a cyclin, they have no protein kinase activity
Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other
proteins
Cyclins were originally named because they undergo a cycle of synthesis and degradation in each
cell cycle. The levels of the Cdk proteins, by contrast, are constant. Cyclical changes in cyclin
protein levels result in the cyclic assembly and activation of cyclin–Cdk complexes at specific
stages of the cell cycle

All eukaryotic cells require three (or 4) of classes of cyclins
Four classes of cyclins, each defined by the stage of the cell cycle at which they bind
Cdks and function
1. G1/S-cyclins activate Cdks in late G1 and thereby help trigger progression through Start,
resulting in a commitment to cell-cycle entry. Their levels fall in S phase.
2. S-cyclins bind Cdks soon after progression through Start and help stimulate chromosome
duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to
the control of some early mitotic events.
3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin
levels fall in mid-mitosis.
4. The G1-cyclins, helps govern the activities of the G1/S-cyclins, which control progression
through Start in late G1.

The major Cyclins and CdKs for vertebrates and Budding Yeasts

The cyclin protein does not simply activate its Cdk partner but also directs it to specific target
proteins. As a result, each cyclin–Cdk complex phosphorylates a different set of substrate
proteins.
The same cyclin–Cdk complex can also induce different effects at different times in the cycle,
probably because the accessibility of some Cdk substrates changes during the cell cycle. Certain
proteins that function in mitosis, for example, may become available for phosphorylation only
in G2.
How do different cyclin–Cdk complexes trigger different cell-cycle events?

In the absence of cyclin, the
active site in the Cdk protein is
partly
obscured by a protein loop, like a
stone blocking the entrance to a
cave
Cyclin binding causes the loop to
move away from the active site,
resulting in partial activation of the
Cdk enzyme
A separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the
entrance of the Cdk active site; a small conformational change increases the activity of the
Cdk, kinase phosphorylates its target proteins effectively and thereby induce specific cell-
cycle events

Cdk Activity Can Be Suppressed By Inhibitory Phosphorylation and Cdk Inhibitor
Proteins (CKIs)
Phosphorylation at a pair of amino acids in the roof
of the kinase active site inhibits the activity of a
cyclin–Cdk complex. Phosphorylation of these sites
by a protein kinase known as Wee1 inhibits Cdk
activity, while dephosphorylation of these sites by a
phosphatase known as Cdc25 increases Cdk
activity
Phosphatases remove phosphate groups from molecules

Binding of Cdk inhibitor proteins (CKIs) inactivates cyclin–Cdk complexes. CKI binding
stimulates a large rearrangement in the structure of the Cdk active site, rendering it
inactive. Cells use CKIs primarily to help govern the activities of G1/S- and S-Cdks
early in the cell cycle.

Regulated Proteolysis Triggers the Metaphase-to-Anaphase Transition
Progression through the metaphase-to-anaphase transition is triggered not by protein
phosphorylation but by protein destruction, leading to the final stages of cell division
The key regulator: anaphase promoting complex, or cyclosome (APC/C), a member of the
ubiquitin ligase family of enzymes
These enzymes are used in numerous cell processes to stimulate the proteolytic destruction of
specific regulatory proteins. They polyubiquitylate specific target proteins, resulting in their
destruction in proteasomes. Other ubiquitin ligases mark proteins for purposes other than
destruction

Cell-Cycle Control Also Depends on Transcriptional Regulation
Cell-cycle control depends exclusively on post-transcriptional mechanisms that involve
the regulation of Cdks and ubiquitin ligases and their target proteins.
In the more complex cell cycles of most cell types, however, transcriptional control
provides an important additional level of regulation. Changes in cyclin gene
transcription, for example, help control cyclin levels in most cells.

The Cell-Cycle Control System Functions as a Network of Biochemical Switches
These proteins are
functionally linked to form
a robust network, which
operates essentially
autonomously to activate a
series of biochemical
switches, each of which
triggers a specific cell-cycle
event
Major Cell Cycle Proteins

When conditions for cell proliferation are right, various external and internal signals stimulate
the activation of G1-Cdk, which in turn stimulates the expression of genes encoding G1/S- and
S-cyclins
The resulting activation of G1/S-
Cdk then drives progression
through the Start transition.
G1/S-Cdks unleash a wave of S-
Cdk activity, which initiates
chromosome duplication in S
phase and also contributes to
some early events of
mitosis.

M-Cdk activation then triggers progression through the G2/M transition and the events of
early mitosis, leading to the alignment of sister-chromatid pairs at the equator of the
mitotic spindle.
Finally, the APC/C, together with its activator Cdc20, triggers the destruction of securin and
cyclins, thereby unleashing sister-chromatid separation and segregation and the
completion of mitosis.
When mitosis is complete, multiple mechanisms collaborate to suppress Cdk activity,
resulting in a stable G1 period.

S PHASE
The central event of chromosome duplication—DNA replication—poses two problems for
the cell.
1) Replication must occur with extreme accuracy to minimize the risk of mutations in the
next cell generation.
2) Every nucleotide in the genome must be copied once, and only once, to prevent the
damaging effects of gene amplification.

S-Cdk Initiates DNA Replication Once Per Cycle
DNA replication begins at origins of replication, which are scattered at numerous locations in
every chromosome. During S phase, DNA replication is initiated at these origins when a DNA
helicase unwinds the double helix and DNA replication enzymes are loaded onto the two
single-stranded templates. This leads to the elongation phase of replication, when the
replication machinery moves outward from the origin at two replication forks

To ensure that chromosome duplication
occurs only once per cell cycle, the
initiation phase of DNA replication is
divided into two distinct steps that occur
at different times in the cell cycle

1) Occurs in late mitosis and early G1, when a pair of inactive DNA
helicases is loaded onto the replication origin, forming a large complex
called the prereplicative complex or preRC. This step is sometimes
called licensing of replication origins because initiation of DNA
synthesis is permitted only at origins containing a preRC.
2) Occurs in S phase, when the DNA helicases are activated, resulting in
DNA unwinding and the initiation of DNA synthesis. Once a replication
origin has been fired in this way, the two helicases move out from the
origin with the replication forks, and that origin cannot be reused until
a new preRC is assembled there at the end of mitosis. As a result,
origins can be activated only once per cell cycle.

Molecular details underlying the control of the two steps in
the initiation of DNA replication.
A key player: Large multiprotein complex called the origin
recognition complex (ORC), which binds to replication
origins throughout the cell cycle. In late mitosis and early
G1, the proteins Cdc6 and Cdt1 collaborate with the ORC
to load the inactive DNA helicases around the DNA next to
the origin. The resulting large complex is the preRC, and
the origin is now licensed for replication.

At the onset of S phase, S-Cdk triggers origin activation by phosphorylating specific initiator
proteins, which then nucleate the assembly of a large protein complex that activates the DNA
helicase and recruits the DNA synthesis machinery.
Another protein kinase called DDK is also activated in S phase and helps drive origin
activation by phosphorylating specific subunits of the DNA helicase.
As S-Cdk initiates DNA replication, several mechanisms prevent assembly of new preRCs.
S-Cdk phosphorylates and thereby inhibits the ORC and Cdc6 proteins. Inactivation of the APC/C in
late G1 also helps turn off preRC assembly.
In late mitosis and early G1, the APC/C triggers the destruction of a Cdt1 inhibitor called geminin,
thereby allowing Cdt1 to be active.
When the APC/C is turned off in late G1, geminin accumulates and inhibits the Cdt1 that is not
associated with DNA.
Also, the association of Cdt1 with a protein at active replication forks stimulates Cdt1 destruction. In
these various ways, preRC formation is prevented from S phase to mitosis, thereby ensuring that
each origin is fired only once per cell cycle

At the end of mitosis, APC/C activation leads to the inactivation of Cdks and the destruction
of geminin. ORC and Cdc6 are dephosphorylated and Cdt1 is activated, allowing preRC
assembly to prepare the cell for the next S phase.

Chromosome Duplication Requires Duplication of Chromatin Structure
Duplication of a chromosome is not simply a matter of replicating the DNA at its core but also
requires the duplication of these chromatin proteins and their proper assembly on the DNA.
The production of chromatin proteins increases during S phase to provide the raw materials
needed to package the newly synthesized DNA.
S-Cdks stimulate a large increase in the synthesis of the four histone subunits that form the
histone octamers at the core of each nucleosome. These subunits are assembled into
nucleosomes on the DNA by nucleosome assembly factors, which typically associate with the
replication fork and distribute nucleosomes on both strands of the DNA as they emerge from the
DNA synthesis machinery.

Chromatin packaging helps to control gene expression. In some parts of the chromosome, the
chromatin is highly condensed and is called heterochromatin, whereas in other regions it has a more
open structure and is called euchromatin
These differences in chromatin structure depend on a variety of mechanisms
1) Modification of histone tails
2) Presence of non-histone proteins.
These differences are important in gene regulation and crucial that chromatin structure, like the DNA
within, is reproduced accurately during S phase.
How chromatin structure is reproduced is not well understood, however. During DNA synthesis, histone-
modifying enzymes and various non-histone proteins are probably deposited onto the two new DNA
strands as they emerge from the replication fork, and these proteins are thought to reproduce the local
chromatin structure of the parent chromosome

Cohesins Hold Sister Chromatids Together
At the end of S phase, each replicated chromosome consists of a pair of identical sister
chromatids glued together along their length.
This sister-chromatid cohesion sets the stage for a successful mitosis because it greatly
facilitates the attachment of the two sister chromatids to opposite poles of the mitotic spindle.
It would be very difficult to achieve this bipolar attachment if sister chromatids were allowed to
drift apart after S phase. Indeed, defects in sister-chromatid cohesion—in yeast mutants, for
example—lead inevitably to major errors in chromosome segregation.

Deposited at many locations along the length of each sister chromatid as the DNA is
replicated in S phase.
Two of the subunits of cohesin are members of a large family of proteins called SMC
proteins (for Structural Maintenance of Chromosomes).
Cohesin forms giant ringlike structures, and it has been proposed that these surround the
two sister chromatids
Sister-chromatid cohesion depends on a large protein complex: Cohesin

Sister-chromatid cohesion also results, at least in part, from DNA catenation, the intertwining of
sister DNA molecules that occurs when two replication forks meet during DNA synthesis.
The enzyme topoisomerase II gradually disentangles the catenated sister DNAs between S phase
and early mitosis by cutting one DNA molecule, passing the other through the break, and then
resealing the cut DNA
Once the catenation has been removed, sister-chromatid cohesion depends primarily on cohesin
complexes. The sudden and synchronous loss of sister cohesion at the metaphase-to-anaphase
transition therefore depends primarily on disruption of these complexes.

MITOSIS
Following the completion of S phase and transition through G2, the cell undergoes the
dramatic rise of M phase.
This begins with mitosis: the sister chromatids are separated and distributed (segregated) to
a pair of identical daughter nuclei, each with its own copy of the genome.
Mitosis is traditionally divided into five stages—prophase, prometaphase, metaphase,
anaphase, and telophase—defined primarily on the basis of chromosome behavior as seen
in a
microscope.
As mitosis is completed, the second major event of M phase—cytokinesis—divides the cell
into two halves, each with an identical nucleus

Each governed by distinct components of the cell-cycle control system.
1) An abrupt increase in M-Cdk activity at the G2/M transition triggers the events of early mitosis
(prophase, prometaphase, and metaphase).
M-Cdk and several other mitotic protein kinases phosphorylate a variety of proteins, leading to
the assembly of the mitotic spindle and its attachment to the sister-chromatid pairs.
2) The metaphase to-anaphase transition, when the APC/C triggers the destruction of securin,
liberating a protease that cleaves cohesin and thereby initiates separation of the sister
chromatids.
The APC/C also promotes the destruction of cyclins, which leads to Cdk inactivation and the
dephosphorylation of Cdk targets, which is required for all events of late M phase, including the
completion of anaphase, the disassembly of the mitotic spindle, and the division of the cell by
cytokinesis
Mitosis can be divided into two major parts

M-Cdk Drives Entry Into Mitosis
M-Cdk must induce the assembly of the mitotic spindle and ensure that each sister chromatid
in a pair is attached to the opposite pole of the spindle.
It also triggers chromosome condensation, the large-scale reorganization of the intertwined
sister chromatids into compact, rodlike structures.
In animal cells, M-Cdk also promotes the breakdown of the nuclear envelope and
rearrangements of the actin cytoskeleton and the Golgi apparatus.

To phosphorylate key proteins involved in early mitosis.
Two additional families of protein kinases, the Polo-like kinases and the aurora kinases, also
make important contributions to the control of early mitotic events.
The Polo-like kinase Plk, for example, is required for the normal assembly of a bipolar mitotic
spindle, in part because it phosphorylates proteins involved in separation of the spindle
poles early in mitosis.
The Aurora kinase Aurora-A also helps control proteins that govern the assembly and
stability of the spindle, whereas Aurora-B controls attachment of sister chromatids to the
spindle
M-Cdk does not act alone

Dephosphorylation Activates M-Cdk at the Onset of Mitosis
M-Cdk activation begins with the accumulation of M-cyclin or (cyclin B)
In embryonic cell cycles, the synthesis of
M-cyclin is constant throughout the cell
cycle, and M-cyclin accumulation results
from the high stability of the protein in
interphase. In most cell types, however,
M-cyclin synthesis increases during G2
and M, owing primarily to an increase in
M-cyclin gene
transcription

The increase in M-cyclin protein leads to a corresponding accumulation of M-Cdk (the complex of Cdk1
and M-cyclin) as the cell approaches mitosis. Although the Cdk in these complexes is phosphorylated at
an activating site by the Cdk-activating kinase (CAK), the protein kinase Wee1 holds it in an inactive state
by inhibitory phosphorylation at two neighboring sites
Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that
is primed and ready to act but is suppressed by phosphates that block the active site of the
kinase.

The crucial event is the activation of the protein phosphatase Cdc25, which removes the inhibitory
phosphates that restrain M-Cdk
What, then, triggers the activation of the M-Cdk stockpile?
At the same time, the inhibitory activity of the kinase Wee1 is suppressed, further ensuring that M-
Cdk activity increases. The mechanisms that unleash Cdc25 activity in early mitosis are not well
understood. One possibility is that the S-Cdks that are active in G2 and early prophase stimulate
Cdc25.
The ability of M-Cdk to
activate its own activator
(Cdc25) and inhibit its own
inhibitor (Wee1) suggests
that M-Cdk activation in
mitosis involves positive
feedback loops

Condensin Helps Configure Duplicated Chromosomes for Separation
At the end of S phase, the immensely long DNA molecules
of the sister chromatids are tangled in a mass of partially
catenated DNA and proteins. Any attempt to pull the sisters
apart in this state would undoubtedly lead to breaks in the
chromosomes.
To avoid this disaster, the cell devotes a great deal of energy
in early mitosis to gradually reorganizing the sister
chromatids into relatively short, distinct structures that can
be pulled apart more easily in anaphase.
These chromosomal changes involve two processes:
chromosome condensation, in which the chromatids are
dramatically compacted; and sister-chromatid resolution,
whereby the two sisters are resolved into distinct, separable
units

Decatenation of the sister DNAs, accompanied by the partial removal of cohesin molecules along
the chromosome arms. As a result, when the cell reaches metaphase, the sister chromatids
appear in the microscope as compact, rodlike structures that are joined tightly at their
centromeric regions and
only loosely along their arms
Resolution results from
The condensation and resolution of
sister chromatids depend, at least in
part, on a five-subunit protein complex
called condensin. Condensin structure
is related to that of the cohesin complex
that holds sister chromatids together

It is not clear how condensin catalyzes the
restructuring and compaction of chromosome DNA,
but it may form a ring structure that encircles loops of
DNA within each sister chromatid

The Mitotic Spindle Is a Microtubule-Based Machine
The central event of mitosis—chromosome segregation—depends in all
eukaryotes on a complex and beautiful machine called the mitotic spindle
The spindle is a bipolar array of microtubules, which
pulls sister chromatids apart in anaphase, thereby
segregating the two sets of chromosomes to opposite
ends of the cell, where they are packaged into
daughter nuclei
M-Cdk triggers the assembly of the spindle early in mitosis, in
parallel with the chromosome restructuring

The core of the mitotic spindle is
a bipolar array of microtubules,
the minus ends of which are
focused at the two spindle
poles, and the plus ends of
which radiate outward from the
poles
The plus ends of some microtubules—called the interpolar microtubules—overlap with the plus
ends of microtubules from the other pole, resulting in an antiparallel array in the spindle
midzone. The plus ends of other microtubules—the kinetochore microtubules—are attached to
sister-chromatid pairs at large protein structures called kinetochores, which are located at the
centromere of each sister chromatid. Finally, many spindles also contain astral microtubules that
radiate outward from the poles and contact the cell cortex, helping to position the spindle in the
cell

In most somatic animal cells, each spindle pole is focused at a protein organelle called the
centrosome. Each centrosome consists of a cloud of amorphous material (called the
pericentriolar matrix) that surrounds a pair of centrioles

The pericentriolar matrix nucleates a radial array of microtubules, with their fast-
growing plus ends projecting outward and their minus ends associated with the
centrosome. The matrix contains a variety of proteins, including microtubule-
dependent motor proteins, coiled-coil proteins that link the motors to the centrosome,
structural proteins, and components of the cell-cycle control system. Most important, it
contains γ-tubulin ring complexes, which are the components mainly responsible for
nucleating microtubules

Multiple Mechanisms Collaborate in the Assembly of a Bipolar Mitotic
Spindle
The mitotic spindle must have two poles if it is to
pull the two sets of sister chromatids to opposite
ends of the cell in anaphase. In most animal cells,
several mechanisms ensure the bipolarity of the
spindle. One depends on centrosomes. A typical
animal cell enters mitosis with a pair of
centrosomes, each of which nucleates a radial
array of microtubules. The two centrosomes
provide prefabricated spindle poles that greatly
facilitate bipolar spindle assembly. The other
mechanisms depend on the ability of mitotic
chromosomes to nucleate and stabilize
microtubules and on the ability of motor proteins
to organize microtubules into a bipolar array.
These “self-organization” mechanisms can
produce a bipolar spindle even in cells lacking
centrosomes.

Centrosome Duplication Occurs Early in the Cell Cycle
Centrosome duplication begins at about
the same time as the cell enters S phase.
The G1/S-Cdk (a complex of cyclin E and
Cdk2 in animal cells) that triggers cell-
cycle entry also helps initiate centrosome
duplication. The two centrioles in the
centrosome separate, and each
nucleates the formation of a single new
centriole, resulting in two centriole pairs
within an enlarged pericentriolar matrix
Most animal cells contain a single centrosome that nucleates most of the cell’s cytoplasmic
microtubules. The centrosome duplicates when the cell enters the cell cycle, so that by the time
the cell reaches mitosis there are two centrosomes.
Centrosomes, like chromosomes, must replicate once and only once per cell cycle, to ensure that the cell enters
mitosis with only two copies: an incorrect number of centrosomes could lead to defects in spindle assembly and
thus errors in chromosome segregation

M-Cdk Initiates Spindle Assembly in Prophase
Spindle assembly begins in early mitosis, when the two centrosomes move apart along the nuclear
envelope, pulled by dynein motor proteins that link astral microtubules to the cell cortex
Early mitosis, the number of
γ-tubulin ring complexes in
each centrosome increases
greatly, increasing the ability
of the centrosomes to
nucleate new microtubules, a
process called centrosome
maturation

The balance of opposing forces generated by different types of motor proteins determines
the final length of the spindle. Dynein and kinesin-5 motors generally promote centrosome
separation and increase spindle length. Kinesin-14 proteins do the opposite: they tend to pull
the poles together
We don’t know how the cell
regulates the balance of opposing
forces to generate the appropriate
spindle length.

The Completion of Spindle Assembly in Animal Cells Requires Nuclear-Envelope
Breakdown
The attachment of sister-chromatid pairs to the spindle requires the removal of this barrier.
In addition, many of the motor proteins and microtubule regulators that promote spindle
assembly are associated with the chromosomes inside the nucleus, and they require nuclear
envelope breakdown to carry out their functions also phosphorylates components of the nuclear
lamina, the structural framework beneath the envelope.
The phosphorylation of these lamina components and of several inner-nuclear-envelope
proteins leads to disassembly of the nuclear lamina and the breakdown of the envelope
membranes into small vesicles.

Mitotic Chromosomes Promote Bipolar Spindle Assembly
By creating a local environment that favors both microtubule nucleation and microtubule
stabilization, they play an active part in spindle formation. The influence of the chromosomes
can be demonstrated by using a fine glass needle to reposition them after the spindle has
formed.
This property of the chromosomes seems to depend, at least in
part, on a guanine nucleotide exchange factor (GEF) that is bound
to chromatin; the GEF stimulates a small GTPase in the cytosol
called Ran to bind GTP in place of GDP.
The activated Ran-GTP, which is also involved in nuclear transport,
releases microtubule-stabilizing proteins from protein complexes
in the cytosol, thereby stimulating the local nucleation and
stabilization of microtubules around chromosomes

The ability of chromosomes to stabilize and organize microtubules enables cells to form
bipolar spindles in the absence of centrosomes.
Acentrosomal spindle assembly is thought to begin with the formation of microtubules
around the chromosomes. Various motor proteins then organize the microtubules into a
bipolar spindle,

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