The Eukaryotic Cell Cycle Chapter 19

Bolded words from slides defined

Mitosis

Cell division that generates daughter cells genetically identical to their parent. The basis of growth, replacement, and regeneration.

In eukaryotic cells, the process whereby the nucleus divides, producing two genetically equivalent daughter nuclei with the diploid number of chromosomes.

Meiosis

Reductive division that halves the number of chromosomes in the resulting cells (gametes) which also recombining genetic information from maternal and paternal chromosomes to generate genetic diversity

In eukaryotes, a special type of cell division that occurs during maturation of germ cells; comprises two successive nuclear and cellular divisions with only one round of DNA replication. Results in production of four genetically nonequivalent haploid cells (gametes) from an initial diploid cell.

Sister Chromatid

The two identical DNA molecules created during DNA replication and the associated chromosomal proteins. After DNA replication, each chromosome is composed of two sister chromatids.

Sister chromatid resolution

The process of untangling of the intertwined sister chromatids during prophase.

Mitotic Cell Cycle

Orderly sequence including:

• G Phase

• S Phase

• M Phase

G Phase

Cell growth and preparation for the next stage of cell cycle

G₁: Cell growth, normal cell activities AND monitoring environment for signals to initiate division

G₂: More growth and inspection to ensure faithful chromosome replication- diploid organisms at this point have a 4n complement of chromosomes

S Phase

Faithfully copy all chromosome forming two identical sister chromatids

DNA synthesis- each parental chromosome is copied into identical sister chromatids.

M Phase

Faithfully segregate sister chromatids and other cellular material to daughter cells

following M phase and cytokinesis each daughter cell contains a 2n complement of chromosome

Cell Cycle

Ordered sequence of events in which a eukaryotic cell duplicates its chromosomes and divides into two. The cell cycle normally consists of four phases: G₁ before DNA synthesis occurs; S when DNA synthesis occurs; G₂ after DNA synthesis; and M when cell division occurs, yielding two daughter cells. Under certain conditions, cells exit the cell cycle during G₁ and remain in the G₀ state as non-dividing cells.

Tightly monitored through each of these phases. Entry/exit of each phase is tightly regulated to ensure fidelity of both replication and segregation.

Checkpoint Pathway

Surveillance mechanism that prevents initiation of each step in cell division until earlier steps on which it depends have been completed and mistakes that occurred during the process have been corrected.

monitor fidelity of each stage and prevent progression into the next until all appropriate tasks are complete.

Cell Cycle Checkpoint Pathways

Prevent initiation of the next stage of the cycle until the earlier step has been completed, and any mistakes have been corrected.

Cytokinesis

The division of the cytoplasm following mitosis to generate two daughter cells, each with a nucleus and cytoplasmic organelles.

Interphase

G1, S, and G2 are collectively called interphase.

G1 Checkpoint

Are conditions right to initiate division? Unicellular organisms, like yeast, need critical nutrients; in multicellular organisms, appropriate signals and contact to the extracellular matrix is necessary.

This checkpoint is called START in yeast and the Restriction Point in mammals. if the cell passes through the restriction point it is irreversibly committed to preparing for and completing DNA replication.

G2 Checkpoint

Have all chromosomes been replicated and have any errors been corrected?

Metaphase Checkpoint

Aka: Spindle assembly checkpoint

Are all replicated chromosomes at the metaphase plate and have all sister chromatids been attached to microtubules.

Prevents progression into anaphase unit all chromosomes are bioriented

Sensor proteins

monitor these conditions and relay information back to the proteins that drive the transition from one stage of the cell cycle to the next.

G1 cyclin-CDKs

Cyclin-CDK complexes that promote entry into the cell cycle

Entry into cell cycle. Its activity is regulated by signal transduction pathways that respond to growth factors or anti-proliferation signals. For example, they are activated downstream of the Ras/MAPK pathway.

G1/S phase cyclin CDKs

Cyclin-CDK complexes that promote entry into the cell cycle together with G1 CDKs.

Cyclin levels accumulate in late G1 and trigger the G1 to S phase transition

• the mammalian restriction point

Mitotic cyclin- CDKs

Cyclin-CDK complexes that promote entry into and progression through mitosis.

The cyclins are synthesized during S phase but their activity is held in check until DNA synthesis is complete.

S-phase cyclin-CDKs

Cyclin-CDK complexes that promote the initiation of DNA replication

Promote DNA replication by activating DNA helicase and loading DNA polymerase onto chromosomes.

G₀

Not all cells divide

• Most terminally differentiated cells exit the cell cycle at G1 and enter an arrested state called G₀

Quiescence

Some cells are only temporarily “arrested” can be induced to become mitotically active again.

Senescence

Cell have lost the ability to receive signals (usually due to age) that would initiate cell division and actively repress genes needed for mitosis.

Cohesin

Protein complex that holds the replicated sister chromatids together.

Join sister chromatids along their lengths.

Prophase

Earliest stage in mitosis, during which the chromosomes condense, the duplicated centrosomes separate to become the spindle poles, and the mitotic spindle begins to form.

• Duplicated chromosomes condense

• Centrosomes begin migrating to opposite poles of cell, radiating microtubules to generate the mitotic spindle

• Cohesin is removed from chromosome arms, but sister chromatids remain connected by cohesion at the centromere

• Kinetochore assembles on centromere

Centromere

DNA sequence required for proper segregation of chromosomes during mitosis and meiosis; the region of mitotic chromosomes where the kinetochore forms and that appears constricted.

Kinetochore

A multilayer protein structure at or near the centromere of each mitotic chromosome form which microtubules extend toward the spindle poles of the cell; plays an active role in movement of chromosomes toward the poles during anaphase.

Kinetochore-associated tension sensing mechanism aligns sister chromatids at the metaphase plate.

Prometaphase

Second stage in mitosis, during which the nuclear envelope and nuclear lamina break down and microtubules assembled from the spindle poles “capture” chromosome pairs at specialized structures call kinetochores.

• Chromosomes continue to condense

• Nuclear envelope breaks down and retracts into ER

• microtubule (+) ends find and attach to kinetochores

Metaphase

Stage of mitosis at which condensed chromosomes are aligned equidistant between the poles of the mitotic spindle but have not yet started to segregate toward the spindle poles.

• Each sister chromatid is attached to microtubules originating from opposite poles

• Chromosomes align at central region called the metaphase plate

Anaphase

Mitotic stage during which the sister chromatids (or duplicated homologs in meiosis I) separate and move apart (segregate) toward the spindle poles.

• Microtubule shortening pulls separated chromatids to opposite poles

• Other microtubules lengthen, and aided by kinesin motor proteins, the spindle, and cell elongate

Telophase

Final mitotic stage, during which the nuclear envelope re-forms around the two sets of separated chromosomes, the chromosomes decondense, and division of the cytoplasm (cytokinesis) is completed.

• Chromosomes arrive at opposite pole-begin to decondense

• Nuclear envelope begins to reassemble

• Mitotic spindle begins to disassemble

• Actin-based contractile ring (animal cell specific) begins to assemble at midpoint.

Cytokinesis

In animal cells a cleavage furrow forms resulting from actinomyosin contraction of the contractile ring. In plant cells, the cell plate- precursor of the new cell wall forms to separate the new daughter cells.

The division of the cytoplasm following mitosis to generate two daughter cells, each with a nucleus and cytoplasmic organelles.

Mitotic spindle

A specialized temporary structure, present in eukaryotic cells during mitosis, that catures the chromosomes and then pushes and pulls them to opposite sides of the dividing cell; also called mitotic apparatus.

Anaphase A and B

Distinct mechanisms aggregate duplicated chromosomes to opposite poles and push the poles apart, lengthening the cell in preparation for division.

Centrosome cycle

During S phase, animal cells duplicate their centrosome in coordination with chromosome replication.

• paired centrioles separate- each buds a new daughter centriole

• G2- daughter centriole duplication is complete, but the two pairs of centrioles remain within a single centrosome

• At the G2 and M phase transition, the centrioles separate and migrate to opposite poles of the nucleus

Aster

Structure composed of microtubules (astral fibers) that radiate outward from a centrosome during mitosis.

aka spindle poles

Astral MTs

Project toward and engage to the cell cortex (microfilament web beneath the plasma membrane). This anchors centrosomes in place and orients the spindle to generate the axis of division.

Polar MTs

Project toward the cell center. These microtubules fail to capture chromosomes. Instead, they become aligned antiparallel to polar MTs from the opposite pole. During anaphase this will drive 1) separation of poles and contribute to chromosome separation 2) and cell elongation

Kinetochore MTs

Capture sister chromatid in replicated chromosomes and will also contribute to chromosome separation during anaphase.

CENPA

special 3 histone protein which DNA is bound to

Ran in mitosis

A GEF for the nuclear import factor Ran is bound to chromosomes during mitosis

• generate high concentration of Ran-GTP around the centromere

• Ran-GTP releases a protein from importin called TPX which binds augmin and the ᵞTURK complex. This creates very shallow branches from chromosomes approaching chromosomes and enhances the number of microtubules that bind chromosomes.

Bioriented

When both kinetochores of a replicated chromosome are bound by microtubules.

Indicates that the kinetochores of sister chromatids have attached to microtubules emanating from opposite spindle poles.

Dynein-Dynactin

Complexes on both kinetochores pull each sister chromatid toward their respective poles, bioriented chromosomes experience tension.

Congression

chromosome movement toward the cell center- the metaphase plate.

Kinesin-4

Another process contributing to congression

on chromosome tips move on polar microtubules toward their (+) ends- this pulls chromosomes toward the cell center.

While this happens, a kinetochore tension sensing mechanism reads which direction the chromosomes are being pulled and

• promotes polymerization of microtubules on the short side, growing microtubules are tethered to kinetochore by kinesin-7

• While kinesin-13 on the other kinetochore promotes microtubule depolymerization

cells will not progress from metaphase to anaphase unitl all chromosomes are bioriented

Ndc80

Microtubules are captured by this outer kinetochore protein complex, its like a sleeve that a microtubule can slide into.

Aurora kinases

Serine/threonine kinases that play a crucial role in cell division by controlling chromatid segregation. Aurora B kinase destabilizes faulty microtubule-kinetochore interactions by phosphorylating microtubule-binding components within the kinetochore.

Chromosomal Passenger Complex

monitors microtubule attachment at kinetochores

The first ensures that kinetochore-microtubule attachments are weak until bi-orientation occurs, ie biorientation stabilizes kinetochore-MT attachment.

includes Ndc80 and Aurora B kinase

The second promotes tight microtubule-kinetochore association after biorientation

includes protein phosphatase 1

Aurora B Kinase

Phosphorylates Ndc80 preventing tight association with a microtubule.

Protein Phosphatase 1 (PP1)

An outer kinetochore protein, dephosphorylates Ndc80. This allows strong microtubule-kineotchore associations.

Aneuploidy

Any deviation from the normal diploid number of chromosomes in which extra copies of one or more chromosomes are present or one of the normal copies is missing.

If anaphase occurs before all chromosomes are bioriented an incorrect number of chromosomes will segregate to daughter cells.

Anaphase A

Chromosome movement toward poles is powered by microtubule shortening and sustained attachement of the microtubule tips to kinetochores

Anaphase B

Movement of poles and lengthening of cell

Model organisms for cell cycle study

budding yeast (Hartwell)

fission yeast (Nurse)

sea urchins (Hunt)

frog embryos

Maturation Promoting Factor

Also known as mitosis promoting factor

Discovered from Masui and Market injecting the cytoplasm of recently fertilized frog eggs into undertilized eggs. This induced them to enter

Cyclin Dependent Kinases (CDKs)

phosphorylate and regulate activity of stage specific proteins

positive and negative feedback first enhances CDK activity and eventually promotes inactivation leading to abrupt transitions in cell cycle stage.

Cyclins

regulatory protein that bind and activate CDKs during the appropriate stage of the cell cycle and determine their substrate specificity.

Regulation of cell cycle transitions

A) activity of stage specific CDKs at different points in the cell cycle- though all CDKs are always present, they are only active when bound by partner cyclins- this panel also shows the activity of a key regulator of entry into anaphase and exit from the cell cycle called APC

B) the expression of the various cyclins at different points in the cell cycle

C) the kinase activity of the various cyclin/CDK complexes through the cell cycle

Anaphase Promoting Complex (APC)

A ubiquitin ligase that targets securing, mitotic cyclins, and other proteins for proteasomal degradation from the onset of anaphase until entry into the subsequent cell cycle.

Not a cyclin-CDK complex, but is a ubiquitin ligase complex that triggers exit from mitosis (as well as promotes the metaphase to anaphase transition)

Promotes the degradation of:

A) Anaphase inhibitory proteins to promote separation of sister chromatids at the anaphase to metaphase transition

B) As well as S phase and mitotic cyclins to promote exit from M phase and reentry into Interphase.

SCF

Ubiquitin-protein ligase that ubiquitinylates inhibitors of S-phase CDKs and many other proteins, marking them for degradation by proteasomes.

• Functions to degrade inhibitors of S phase promoting proteins. It therefore helps trigger the G1-S phase transition

• Active SCF promotes degradation of G1 and G1/S phase cyclins and CDK inhibitory proteins

CDK activating kinase (CAK)

After being bound by a cyclin, each CDK is activated via phosphorylation by CAK

Phosphorylates CDKs on a threonine residue near the active site. This phosphorylation is essential for CDK activity.

Inhibitory Phosphorylation

Most CDKs also have residues that are phosphorylated by inhibitory kinases. This pauses the CDK after CAK phosphorylation- allowing rapid activation at the appropriate time via the action of phosphatase.

CDK Inhibitors (CKIs)

Prevent premature activation of S and M phase CDKs

These are allosteric regulators prevent cells with damaged DA from passing the restriction point, and in doing serve as major roadblocks to tumor formation.

Mitogen

Any extracellular molecule, such as a growth factor, that promotes cell proliferation.

E2F

A TF family that will promote entry into S phase

G1 cyclin-CDKs indirectly activate this. During G1 E2Fs are bound to their target genes but are inhibited from promoting transcription by a negative cell cycle regulator Retinoblastoma (Rb)

Retinoblastoma (Rb)

negative cell cycle regulator

a transcriptional repressor that must be removed in order for the cell to progress into S phase.

recruits deacetylases and methyltransferases, which make the DNA inaccessible to transcription by promoting heterochromatin.

Rb is inactive in many cancer cells (either by mutation or hyperphosphorylation due to mutation in other genes.) This allows proliferation even in the absence of growth factors.

G1/S phase GDKs prepare DNA for replication

• Turning off APC- which otherwise promotes S phase cyclin degradation

The two functions, and stage specific targets, of this ubiquitin ligase are mediated by distinct allosteric activators

During late anaphase the protein Cdh1 binds APC giving it specificity for S and M phase cyclins- this promotes exit from mitosis and reentry into interphase

When G1/S phase cyclins accumulate and activate their CDKs, they phosphorylate Cdh1 which dissociate from APC

APC can no longer target S phase cyclins for degradation, and they rise to threshold levels to promote S phase entry.

• Inducing degradation of a CKI that inhibits S phase CDKs

As S phase cyclin-CDks accumulate they are bound and inactivated by a CKI this CKI was first identified in yeast and named Sic1.

When G1/S-cyclins reach a threshold level they phosphorylate Sic1 (targeting it for poly-ubiquitination by SCF). S-phase cyclin-CDKs can now activate proteins necessary for DNA replication

Degradation of an S phase CDK Inhibitor Triggers DNA Replication

The mammalian homolog of Sic1 named p27 which inhibits bothe S-phase and G1/S CDKs

Two mechanisms of p27 inactivation

1. Like Sic1, p27 is also phosphorylated by high G1/S and S phase CDKs

2. MAPK- activated via RTK transduction by growth factors- phosphorylates and activates p27

Both events promote poly-ubiquitination proteosomal degradation of p27

Origin of recognition complexes

Initiates DNA replication that occurs throughout S phase

No ORC initiates replication more than once

MCM helicase

During early G1, when S phase cyclin-CDK levels are low, ORC recruits MCM helicase to form pre-replication complex, but remains inactive.

Once S phase cyclin-CDK reach their peak, they phosphorylate ORC cofactor proteins and MCM helicase

this releases ORC and recruits several MCM helicase activators as well as the DNA polymerases

Entry in Mitosis

When S phase is complete cells must:

• Radiate the mitotic spindle

• Breakdown the nuclear envelope

• Restructure or modify almost all organelles

• Capture chromosomes on kinetochore microtubules

• Segregate sister chromatids to opposite poles of the spindle

All of these events are triggered and controlled by the mitotic cyclin-CDK complexes.

CDK-activating Kinase (CAK)

Phosphorylates CDKs on a threonine residue near the active site. This phosphorylation is essential for CDK activity

Wee1

protein-tyrosine kinase; phosphorylates CDKs on threonine 14 and tyrosine 15 to inhibit CDK activity

an inhibitory kinase which also phosphorylates CDK1 on conserved tyrosine and threonine residues- this pauses the complex and prevents it from phosphorylating target proteins until those phosphates are removed

Is a dual-specificity kinase (it can phosphorylate both tyrosines as well as serine/threonines).

Promotes early division by skipping G2, therefore smaller cells.

Cdc 14 phosphatase

A dual-specificity protein phosphates that triggers mitotic CDK inactivation at the end of mitosis.

Cdc25B phosphatase

One of a pair of dual-specificity phosphatases, along with Cdc25C, that dephosphorylates CDKs on threonine 14 and tyrosine 15, thereby activating CDKs

Cdc25C phosphatase

One of a pair of dual-specificity phosphatases, along with Cdc25B, that dephosphorylates CDKs on threonine 14 and tyrosine 15, thereby activating CDKs.

Cdc25 (aka String)

dual specificity phosphatase which removes the inhibitory phosphorylation on CDK1.

positive and negative feedback regulation:

• Active mitotic CDK1 phosphorylates and stimulates Cdc25, further promoting its own activation

• Additionally, CDK1 phosphorylates Wee1 leading to its ubiquitin mediated degradation by the SCF complex

Prevents division, but allows the cell to continue growing in an extended G2 phase. They are essentially trapped in G2 and therefore grow into long (string-like) cells.

Lamins

A group of intermediate filament proteins that form a fibrous network, the nuclear lamina, on the inner surface of the nuclear envelope.

Nucleoporin

Large group of protein that make up the nuclear pore complex. One class (FG-nucleoporins) participates in nuclear import and export.

Mitotic CDKs Promote Nuclear Envelope Breakdown

Active mitotic CDKs phosphorylate specific serine residues in all three nuclear lamins causing depolymerization of lamin intermediate filaments and disintegration of the lamina promoting disassembly of the nuclear envelope .

Mitotic CDKs also phosphorylate specific nucleoporins, causing nuclear pore complexes to dissociate into subcomplexes during prophase.

Phosphorylation of integral membrane proteins of the inner nuclear membrane is thought to decrease their affinity for chromatin and further contributes to the disassembly of the nuclear envelope and its collapse into the ER.

Aurora Kinases

Serine/Threonine kinases that play a crucial role in cell division by controlling chromatid segragation. Aurora B kinase destabilizes faulty microtubule-kinetochore interactions by phosphorylating microtubule-binding components within the kinetochore.

Compaction

Condensation of chromosomes and untangling of sister chromatids (aka sister chromatid resolution) Reduces chromosome length up to 10,000x

without compaction DNA would become entangled and break as the chromosomes segregate during anaphase

Condensins

Protein complex that promotes chromosome condensation

loops each individual chromatid arm into the dense, recognizable M-phase chromosomes, still joined at the centromeres by cohesin.

Topoisomerase II

As condensins work to compact the chromosomes entanglements (knots) between sister chromatids are resolved by topoisomerase II enzymes

Cleaves one sister chromatid, passes the other sister chromatid through this break and then re-ligates the cut ends back together