The Eukaryotic Cell Cycle, Part 1

Lecture 23

Cell cycle 4 phases: `

G1, S, G2, and M

cells commit to division at the

G1 START/Restriction point

cyclin-CDK complexes drive cell

cycle progression

positive and negative feedback loops drive 

CDK activity oscillations 

checkpoint pathway surveillance mechanism guarantee

each cell cycle step is completed correctly before the next is initiated

cell reproduce by the process of

cell division

cell division does not step with the formation of

mature organism but continues in certain tissues throughout life

mitosis leads to cells that are

genetically identical to their parent and serves as the basis for producing new cells

meiosis leads to the production of cells with

half of the genetic content of the parent and is basis for producing new sexually reproducing organisms

proliferating cells carry out an orderly sequence of events that lead to

faithful copying and segregation of chromosomal DNA, as well as all other cellular material required for two daughter cells to function properly

this orderly mechanism process, called the cell cycle, is tightly monitored and regulated to 

ensure efficient and faithful replication and distribution of these materials 

details of the cell cycle vary from

cell type to type and organism to organism; as does the length of the cycle, some of the molecules that drive it and the ultimate date of the daughter cells 

the cell cycle includes three main features:

1. cell growth and DNA replication

2. chromosome segregation

3. cell division

cell growth and DNA replication

duplication and division of contents

• duplication of genetic material

• duplication of organelle and cytoskeletal structures

cells, such as nerve cells, muscle cells, or red blood cells, that are

highly specialized and lack the ability to divide

• once these cells have differentiated, they remain in that state until they die 

liver cells and lymphocytes 

cells that normally do not divide but can be induced to begin DNA synthesis and divide when given an appropriate stimulus 

hematopoietic stem cells

cells that normally possess a relatively high level of mitotic activity

stem cells have

asymmetric cell division in which the daughter cells have different fates

M phase (Mitotic):

• usually the shortest

• includes segregation of duplicated chromosomes and the process of cytokinesis (separation of the cell into two daughter cells) 

interphase:

the period between M phase and the next M-phase

• includes two gap phase (G1 and G2) and 

• DNA replication (synthesis) phase known as S-phase 

Quiescent cell (Go) - are often terminally 

differentiated and have lost the ability to receive signals that would initiate cell division 

some Go cells are only only temporarily “arrested” -

not undergoing cell cycle, but can be induced to become mitotically inactive

Quiescent cell (Go) - 

• exit the cell cycle at G1 stage

• carry out their normal functional

• maintained by active repression of the genes needed for mitosis

G1 phase of cell cycle

• first phase within interpahse

• known as “gap” or “growth” phase

• runs from the end of the previous M phase until the beginning of DNa synthesis

• biosynthetic activities of cell achieve a high rate

• duration of phase is variable depending on cell type

• cell may leave the cycle and enter Go phase

S phase of cell cycle

• second phase within interphase 

• DNA synthesis and chromosome replication 

• RNA transcription and protein synthesis greatly reduced with the exception of histone proteins 

• highly mitotic cells possess an enzyme (telomerase) that extends the telomeres after DNA replication 

G2 phase of cell cycle

• third phase within interphase

• lasts until cell enters mitosis 

• marked by significant protien synthesis in preparation for mitosis 

• important checkpoints to make sure DNA replicated faithfully 

stages of mitosis - M = mitosis

duplicated chromosomes are separated into two nuclei

the mitotic spindle composed of MTs and motors separates

duplicated chromosomes into two daughter cells by a six-stage process (centrosome replicated during interphase)

mitosis - a kinetochore-associated tension-sensing mechanism aligns 

sister chromatid pairs in the spindle center at metaphase 

mitosis - distinct anaphase A and B pull

duplicated chromosomes to opposite poles and push the poles apart

mitosis - an actin-myosin0based contractile ring, positioned by the spindle, contracts 

to pinch the cell in two during cytokinesis 

in the S phase, cells duplicate their centrosome MTOC in coordination with chromosome duplication

• centrioles separate and a daughter centriole buds from each 

• G2 phase - daughter centriole growth is complete, but the two pairs of centrioles remain within a single centrosomal complex 

during mitosis, cyclin-dependent kinase (CDK) activation initiates

centrosome splitting 

• each new MTOC nucleates assembly of microtubules and is pushes to opposite sides of the nucleus to becomes a spindle pole 

interphase: 

chromosome/centrosome duplication and cohesion 

prophase:

1. chromosome condense

2. nuclear envelope breaks down by retracting into the ER, spindle poles duplicate

3. microtubules form the mitotic spindle apparatus, and 

4. kinetochore assembles 

prometaphase

spindle microtubules from each pole attach to chromosome kinetochores and center sister chromatid pairs in the spindle (coongression)

metaphase:

chromosomes align a the metaphase plate

anaphase:

spindle microtubule shortening and motor proteins pull each sister chromatid toward an opposite pole of the mitotic spindle 

three sets of microtubules (MTs), all with (-) ends at the poles 

1. astral MTs - project toward and can be linked to the cell cortex 

2. kinetochore MTs - connected to chromosomes

3. polar MTs - project toward the cell center where their (+) ends overlap (cage around sister chromatids)

chromosome capture and congressional in pro metaphase - spindle microtubule assemble at the

(+) end and search for the duplicated chromosomes

chromosome capture and congressional in pro metaphase - multiple microtubules will connect 

to the sister chromatids at the kinetochore 

chromosome capture and congressional in pro metaphase - attachment of microtubules from both poles, a phenomenon known as 

chromosome biorientation, allows the chromosomes to be positioned in the center of cell 

kinetochores:

• assemble on the centromere of each sister chromatid 

• mediate attachment between chromosomes and kinetochore MTs (+) ends, which connect to the outer layer 

• animal cell kinetochores consist of centromeric DNA and inner and outer kinetochore layers

the chromosomal passenger complex regulates

microtubule attachment at kinetochores 

two mechanisms ensure all chromosomes are correctly bi-oriented before anaphase begins (first mechanism)

ensures that all kinetochore-microtubule interactions are weak (no tension) until bi-orientation occurs and forces equalize 

two mechanisms ensure all chromosomes are correctly bi-oriented before anaphase begins (second mechanism)

spindle assembly checkpoint pathway, a signaling pathways stops the mitosis progression into anaphase until tension is present at all kinetochores 

chromosome capture and congressional in pro metaphase - after attachment to the kinetochore, the chromosome is drawn towards

the spindle pole by dynein-dynactin that is associated with one of the kinetochores walking towards the kMT (-) end 

chromosome capture and congressional in pro metaphase - a kMT from the opposite pole becomes attach to the 

free kinetochore to bi-orient from the chromosome 

chromosome capture and congressional in pro metaphase - bi-oritented chromosomes (with several emts attached to each kinetochore in animal cells) move

to a central point between the spindle poles (congression)

congression -

bi-directional oscillations of chromosome position (tug-of-war), with one set of kMTs shortening while the others are elongating 

Anaphase A

chromosome movement: kinetochore microtubules shorten and the attached chromosome move poleward 

tubular dimers are lost from both ends of kinetochore microtubules

• (A1) MT-shortening kinesin-13 proteins at the kinetochore

• (A2) MT-shortening kinesin-13 proteins at the spindle pole

Anaphase B

movement of poles and associated chromosomes away from each other:

• (B1) sliding of antiparallel polar microtubules powered by a kinesin-5 (+) end-directed motor

• (B2) pulling on astral microtubules by dynein-dynactin located at cell cortex

telophase

chromosome decondense, and each presumptive daughter cell reassembles a nuclear membrane around its chromosomes

cytokinesis

1. cell division into two separate daughter cells

2. contractile ring (actin and myosin) forms the cleavage furrow to split the cell 

3. usually follows mitosis, but not required 

different cyclin present only in

the cell cycle stage they promote activate CDKs at different cell cycle stages

the ubiquitin-proteasome systems limits 

presence of a cyclin to the appropriate cell cycle stage

activating and inhibitory phosphorlyation of the CDK subunit

regulates CDK activity

CDK inhibitors (CKIs) inhibit CDK activity by

binding directly to the cyclin-CDK complex

CDKs initiate every aspect of each cycle stage by

phosphorylating many different target proteins

during the two gap phases (G1 and G2) cells grow and monitor internal as well as external cues in order to determine: 

1. if conditions are appropriate for division

2. if the cell has completes all necessary events required to divide

at specific points within the cycle, called checkpoints, the cell makes

decisions whether to enter the next phase or if it should pause for more time to prepare 

during all of interphase, cells generally

continue gene transcription and preforming cellular activities - this includes (in addition to DNA replication) 

• creasing cell mass, 

• creating more membrane replication organelles in preparation for M  phase - these are the principle cellular processes that are checkpoints monitor 

interphase cells receive signals from the 

environment that stimulates either division and progression through the cycle, or halt to the cycle 

internally, at the various checkpoints, cell monitor the fidelity of the various processes required for cell division:

1. G1 to S

2. G2 ro M 

3. M

G1 to S checkpoint

is the environment favorable - signals, nutrients, appropriate substrate

G2 to M checkpoint

is all DNA replicated and was replication faithful

M checkpoint

are all chromosomes attached to the mitotic spindle

these monitoring points allow the cell to give

go-ahead signals or pause the cycle and are collectively refereed to as cell-cycle checkpoints 

• at each checkpoint, specific protiens monitor cycle progress and signal whether to continue or halt 

the major checkpoints occurs at the:

1. G1 checkpoint (StART/restriction point): G1 to S transition

2. G2 checkpoint: G2 to M transition

3. mitotic checkpoint (spindle checkpoint): mediated the completion of mitosis 

this system is deeply conserved in eukaryotes - meaning

the same checkpoint proteins are utilized in virtually all eukaryotic cells

in fact, the most powerful model organisms for studying the cell cycle have remained the

single-celled yeasts, Saccharomyces and cerevisiae and Schizosaccharomyces pombe

progression into each phase of the cell cycle and the activities that occur within each phase are regulated by specific proteins: 

• cyclin dependent proteins kinases (CDKs)

• protien phosphatases

• CDK inhibitors (allosteric regulators)

• ubiquitin-protein ligases 

the cell cycle control system depends on

cyclically activated proteins kinasease CDKs 

CDKs in the control system 

• Each Cdk phosphorylates distinct target proteins in order toregulate their activities (either positively or negatively).

• Each Cdk is always present in the cell, but is only activated through association with its partner cyclin protein.

the anaphase-promoting complex (APC/C cyclostome) ubiquitin-protein ligase:

•Ubiquitinylates proteins, including cyclins, targeting them for degradation by proteosomes

•Catalyzes two key cell cycle transitions: anaphase and exit from mitosis

Mechanisms Involved in the Regulation of Cdk’s

1.Cyclin Binding

2.Cdk Phosphorylation state (can be activated or inhibited by different phosphorylation states).

3.Cdk Inhibitors

4.Controlled Proteolysis

5.Subcellular localization

cells harbor different types of CDKs that initiate

different events of the cell cycle

cyclins are not always presents → instead, their transcription/translation

oscillates through the cell cycle

each checkpoint activate a unique cyclin/CDK pair that propels the cell into the next phase and regulates cellular activities specific to that phase: 

• G1/S phase CDKs are most active at the G1-S phase transition to trigger entry into the cell cycle 

• S phase CDKs are most active during S phase

• Mitotic CDKs are most active during mitosis 

cyclin-dependent kinases: Activity is cell-cycle-stage-specific 

• small serine/theorine kinases

• require a regulatory cyclin subunit for activity 

• are regulated by activating and inhibitory phosphorylations of a flexible T loop-activation loop 

• activity and substrate specificity of any given CDKs is defined by the particular cyclin to which it is bound 

cyclin: present only during the cell cycle stage that they trigger

• synthesis is regulated by transcriptional control 

• degradation is regulated by ubiquitin-mediated, proteasome-dependent degradation

• synthesis-degradation cyclin control ensures cell cycle progresses in one direction 

inactive CDK2 not bound to cyclin - 

the T loop blocks access of protein substrates to the y phosphate of the bound ATP

nonphosphorylated, low activity cyclin A-CDK2 complex - 

Cyclin A-induced conformational changes cause the T loop to pull away from the active site so substrate proteins can bind. (The CDK2 α1 helix interacts extensively with cyclin A and moves several angstroms into the catalytic cleft, repositioning key catalytic side chains required for the phosphotransfer reaction.)

phosphorylated, high-activity cyclin A-CDK2 complex -

Phosphorylation of Thr-160 induces conformational changes that alter the substrate-binding surface, greatly increasing the active site affinity for protein substrates

CDK phosphorylation/dephosphorylation

• CDC-activating kinase (CAK) phosphorylates both a threonine and a tyrosine on the CDK subunit

• the doubly phosphorylated CDC-cyclin is inactive

• a phosphatase (cdc25) removes the phosphates

• the CDK becomes active, driving the cell to mitosis 

• CAK and cdc25 are activated by other kinases and phosphatases 

CDK inhibitors (CKIs): a protein Sic1 controls 

cell cycle progression by allosterically inhibiting the S phase CDK complex preventing the cell entering the S phase 

controlled proteolysis 

• occurs via the ubiquitin-proteasome pathway

• mutlisubunit complexes function as ubiquitin ligases

destruction of the mitotic cyclins allows a 

cell to exist mitosis and enter a new cell cycle 

SCF ubiquitin ligase enables

the entry into S phase by promoting degradation of the S-phase cyclin-dependent kinase inhibitor Sic1

sub cellular localization

• movement of cyclin between the cytoplasm and the nucleus is another point of control 

• cyclin B1 (tagged with green fluorescent protein): serine phosphorylation of NES sequence allows it to enter nucleus 

• if nuclear accumulation of cyclin is blocked, cells fail to initiate mitosis 

extracellular signals - nutritional state (in yeast) and presence of mitogens and anti-mitogens (in vertebrates) -

regulate cell cycle entry

molecular event promoting entry into the cell cycle are

conserved across sites

G1/S CDKs trigger

chromosome duplication at DNA origin of replication sites

cohesions linked to 

replicated DNA molecules to ensure accurate segregation during mitosis 

the G1-S transition is a cell cycle checkpoint

where DNA integrity is assessed

G1-S phase transition is the point at which cells

become irreversible committee to division by transition to the S phase

G₁/S phase CDK phosphorylation of a single activating Sic1 site would cause a

sluggish G₁/S phase transition. G₁/S phase CDKs accumulation during G₁ would cause slow progressive degradation of Sic1 and initiation of S phase

Six suboptimal phosphorylation sites ensure that

Sic1 is fully phosphorylated and targeted for destruction by SCF only when G₁/S phase CDKs have reached high levels. This ensures that Sic1 degradation and DNA synthesis initiation occurs rapidly, when G₁/S phase CDKs have accomplished all their other G₁ tasks

Metazoan entry into S phase is regulated by a mechanism similar to that in

budding yeast but involving the CKI p27 instead of Sic1