Cell CycleCell Cycle

Honors GeneticsHonors Genetics

20052005

Cell cycle and Mitosis Cell cycle and Mitosis TutorialTutorial

http://www.biology.arizona.edu/http://www.biology.arizona.edu/cell_bio/tutorials/cell_cycle/main.htmlcell_bio/tutorials/cell_cycle/main.html

http://www.cellsalive.com/http://www.cellsalive.com/cell_cycle.htmcell_cycle.htm

http://nobelprize.org/medicine/http://nobelprize.org/medicine/educational/2001/educational/2001/

Play these three animations to help you Play these three animations to help you learn about the cell cycle and controllearn about the cell cycle and control

Cell CycleCell Cycle

G1- Highest metabolic activity. G1- Highest metabolic activity. Cytoplasmic contents increase. Protein Cytoplasmic contents increase. Protein synthesis, membrane synthesissynthesis, membrane synthesisS – replication of DNA. Chromosomes S – replication of DNA. Chromosomes copied( sister chromatids form)copied( sister chromatids form)G2- Preparation for cell division – G2- Preparation for cell division – Spindle fiber formationSpindle fiber formationM – Cell divisionM – Cell divisionCytokinesis – division of the cytoplasmCytokinesis – division of the cytoplasm

Chromosome overviewChromosome overview

Chromsome structureChromsome structure

Metaphase StructureMetaphase Structure

GoGo

Many times a cell will leave the cell cycle, Many times a cell will leave the cell cycle, temporarily or permanently. It exits the cycle at temporarily or permanently. It exits the cycle at G1 and enters a stage designated G0 (G zero). A G1 and enters a stage designated G0 (G zero). A G0 cell is often called "quiescent", but that is G0 cell is often called "quiescent", but that is probably more a reflection of the interests of the probably more a reflection of the interests of the scientists studying the cell cycle than the cell scientists studying the cell cycle than the cell itself. Many G0 cells are anything but quiescent. itself. Many G0 cells are anything but quiescent. They are busy carrying out their functions in the They are busy carrying out their functions in the organism. e.g., secretion, attacking pathogens. organism. e.g., secretion, attacking pathogens. Often G0 cells are terminally differentiated: they Often G0 cells are terminally differentiated: they will never reenter the cell cycle but instead will will never reenter the cell cycle but instead will carry out their function in the organism until they carry out their function in the organism until they die. die.

Cyclins and cell cycle Cyclins and cell cycle regulationregulation

The passage of a The passage of a cell through the cell through the cell cycle is cell cycle is controlled by controlled by proteins in the proteins in the cytoplasm. cytoplasm.

Among the main Among the main players in animal players in animal cells are: cells are:

CyclinsCyclins

a a G1 cyclinG1 cyclin (cyclin (cyclin D) D)

S-phase cyclinsS-phase cyclins (cyclins E and A) (cyclins E and A)

mitotic cyclinsmitotic cyclins (cyclins B and A) (cyclins B and A)

Cyclind dependent kinasesCyclind dependent kinases

Their levels depend Their levels depend upon the stage of upon the stage of the cell cyclethe cell cycle

They add They add phosphates to phosphates to cyclins during the cyclins during the phases of the cell phases of the cell cyclecycle

Cyclin-dependent Cyclin-dependent kinaseskinases (Cdks) (Cdks)

a a G1 CdkG1 Cdk (Cdk4) (Cdk4)

an an S-phase CdkS-phase Cdk ((Cdk2) ((Cdk2)

an an M-phase CdkM-phase Cdk (Cdk1) (Cdk1)

Cyclins - eventsCyclins - events

A rising level ofA rising level of G1-cyclins G1-cyclins bind to their bind to their Cdks and signal the cell to prepare the Cdks and signal the cell to prepare the chromosomes for replication. chromosomes for replication. A rising level of A rising level of S-phase promoting S-phase promoting factorfactor ( (SPFSPF) — which includes cyclin A ) — which includes cyclin A bound to Cdk2 — enters the nucleus and bound to Cdk2 — enters the nucleus and prepares the cell to duplicate its DNA (and prepares the cell to duplicate its DNA (and its centrosomes). its centrosomes). As DNA replication continues, cyclin E is As DNA replication continues, cyclin E is destroyed, and the level of mitotic cyclins destroyed, and the level of mitotic cyclins begins to rise (in G2). begins to rise (in G2).

CheckpointsCheckpoints

DNA damageDNA damage checkpoints. These checkpoints. These sense DNA damage sense DNA damage

Before the cell enters S phase (a G1 Before the cell enters S phase (a G1 checkpoint); checkpoint);

During S phase, and During S phase, and

After DNA replication (a G2 After DNA replication (a G2 checkpoint). checkpoint).

OncogenesOncogenes

All the checkpoints examined require All the checkpoints examined require the services of a complex of proteins. the services of a complex of proteins. Mutations in the genes encoding Mutations in the genes encoding some of these have been associated some of these have been associated with cancer; that is, they are with cancer; that is, they are oncogenesoncogenes. This should not be . This should not be surprising since checkpoint failures surprising since checkpoint failures allow the cell to continue dividing allow the cell to continue dividing despite damage to its integrity. despite damage to its integrity.

Cyclins and cell cycle Cyclins and cell cycle regulationsregulations

Spindle fibers have three Spindle fibers have three destinations:destinations:

Some attach to one Some attach to one kinetochorekinetochore of a of a dyaddyad with those growing from the opposite with those growing from the opposite centrosome binding to the other centrosome binding to the other kinetochore of that dyad. kinetochore of that dyad.

Some bind to the arms of the Some bind to the arms of the chromosomes. chromosomes.

Still others continue growing from the two Still others continue growing from the two centrosomes until they extend between centrosomes until they extend between each other in a region of overlap. each other in a region of overlap.

How Spindle Fibers WorkHow Spindle Fibers Work

MicrotubulesMicrotubules

Grow at each end Grow at each end by the by the polymerization of polymerization of tubulin dimers tubulin dimers (powered by the (powered by the hydrolysis of GTP), hydrolysis of GTP), and and

Shrink at each end Shrink at each end by the release of by the release of tubulin dimers tubulin dimers (depolymerization) (depolymerization)

All types of spindle fibers All types of spindle fibers participate inparticipate in

the assembly of the chromosomes at the the assembly of the chromosomes at the metaphase plate at metaphase plate at metaphasemetaphase. Proposed . Proposed mechanism (the diagram shows only 1 and 2): mechanism (the diagram shows only 1 and 2): Microtubules attached to opposite sides of the Microtubules attached to opposite sides of the dyad shrink or grow until they are of equal length. dyad shrink or grow until they are of equal length. Microtubules motors attached to the kinetochores Microtubules motors attached to the kinetochores move them move them – toward the toward the minusminus end of end of shrinkingshrinking microtubules (a microtubules (a

dyneindynein); ); – toward the toward the plusplus end of end of lengtheninglengthening microtubules (a microtubules (a

kinesinkinesin).).

The chromosome The chromosome armsarms use a different use a different kinesinkinesin to to move to the metaphase plate.move to the metaphase plate.

Microtubule MotorsMicrotubule Motors

There are two major groups of There are two major groups of microtubule motors: microtubule motors:

kinesinskinesins (most of these move (most of these move toward the plus end of the toward the plus end of the microtubules) and microtubules) and

dyneinsdyneins (which move toward the (which move toward the minus end).minus end).

Kinesins and dyneinsKinesins and dyneins

The sister kinetochores separate and, The sister kinetochores separate and, carrying their attached chromatid, carrying their attached chromatid, move along the microtubules powered by move along the microtubules powered by minus-end motors, minus-end motors, dyneinsdyneins, while the , while the microtubules themselves shorten microtubules themselves shorten (probably at both ends). (probably at both ends). The overlapping spindle fibers move past The overlapping spindle fibers move past each other (pushing the poles farther each other (pushing the poles farther apart) powered by plus-end motors, the apart) powered by plus-end motors, the "bipolar" "bipolar" kinesinskinesins. . In this way the sister chromatids end up at In this way the sister chromatids end up at opposite poles.opposite poles.

Mitosis/Meiosis promoting Mitosis/Meiosis promoting factorfactor

M-phase promoting factorM-phase promoting factor (the complex of (the complex of mitotic cyclins with the M-phase Cdk) initiates mitotic cyclins with the M-phase Cdk) initiates – assembly of the mitotic spindle assembly of the mitotic spindle – breakdown of the nuclear envelope breakdown of the nuclear envelope – condensation of the chromosomes condensation of the chromosomes

These events take the cell to These events take the cell to metaphasemetaphase of mitosis. of mitosis. At this point, the M-phase promoting factor At this point, the M-phase promoting factor activates the activates the anaphase-promoting complex anaphase-promoting complex ((APC/CAPC/C) which ) which Allows the sister chromatids at the metaphase Allows the sister chromatids at the metaphase plate to separate and move to the poles (= plate to separate and move to the poles (= anaphase), completing mitosis; anaphase), completing mitosis; Destroys Destroys cyclin Bcyclin B. It does this by attaching it to . It does this by attaching it to the protein the protein ubiquitinubiquitin which targets it for which targets it for destruction by destruction by proteasomesproteasomes. .

Anaphase promoting Anaphase promoting complexcomplex

The The anaphase-promoting anaphase-promoting complex (APC)complex (APC). (The APC is also . (The APC is also called the called the cyclosomecyclosome, and the , and the complex is often designated as the complex is often designated as the APC/CAPC/C.) The APC/C .) The APC/C Triggers the events leading to Triggers the events leading to destruction of the destruction of the cohesinscohesins thus thus allowing the sister chromatids to allowing the sister chromatids to separate; separate; Degrades the mitotic cyclin B. Degrades the mitotic cyclin B.

Proteosome – Core ParticleProteosome – Core Particle

The core particle is The core particle is made of 2 copies of made of 2 copies of each of 14 different each of 14 different proteins. proteins. These are These are assembled in assembled in groups of 7 forming groups of 7 forming a ring. a ring. The 4 rings are The 4 rings are stacked on each stacked on each other (like 4 donuts) other (like 4 donuts)

Proteosome – Regulatory Proteosome – Regulatory ParticleParticle

There are two identical There are two identical RPs, one at each end of RPs, one at each end of the core particle. the core particle. Each is made of 14 Each is made of 14 different proteins (none different proteins (none of them the same as of them the same as those in the CP). those in the CP). 6 of these are 6 of these are ATPasesATPases. . Some of the subunits Some of the subunits have sites that have sites that recognize the small recognize the small protein protein ubiquitinubiquitin. .

UbiquitinUbiquitin

A small protein (76 amino acids) A small protein (76 amino acids)

Conserved throughout all the Conserved throughout all the kingdoms of life; that is, virtually kingdoms of life; that is, virtually identical in sequence whether in identical in sequence whether in bacteria, yeast, or mammals. bacteria, yeast, or mammals.

Used by all these creatures to target Used by all these creatures to target proteins for destruction. proteins for destruction.

Proteosome and proteinsProteosome and proteins

Are conjugated to a molecule of ubiquitin Are conjugated to a molecule of ubiquitin which binds to the terminal amino group of which binds to the terminal amino group of a a lysinelysine residue. residue. Additional molecules of ubiquitin bind to Additional molecules of ubiquitin bind to the first forming a chain. the first forming a chain. The complex binds to ubiquitin-recognizing The complex binds to ubiquitin-recognizing site(s) on the site(s) on the regulatory particleregulatory particle. . The protein is unfolded by the ATPases The protein is unfolded by the ATPases using the energy of ATP using the energy of ATP The unfolded protein is translocated into The unfolded protein is translocated into the central cavity of the core particle. the central cavity of the core particle.

Proteosomes and Proteosomes and Particles(2)Particles(2)

Several active sites on the inner surface of the two Several active sites on the inner surface of the two middle "donuts" break various specific peptide middle "donuts" break various specific peptide bonds of the chain. bonds of the chain. This produces a set of peptides averaging about 8 This produces a set of peptides averaging about 8 amino acids long. amino acids long. These leave the core particle by an unknown route These leave the core particle by an unknown route where where they may be further broken down into individual they may be further broken down into individual amino acids by peptidases in the cytosol or amino acids by peptidases in the cytosol or in mammals, they may be incorporated in a class I in mammals, they may be incorporated in a class I histocompatibility molecule to be presented to the histocompatibility molecule to be presented to the immune system as a potential immune system as a potential antigenantigen [ [see belowsee below]. ]. The regulatory particle releases the ubiquitins for The regulatory particle releases the ubiquitins for reuse. reuse.

Spindle CheckpointsSpindle Checkpoints

Spindle checkpoints. Some of Spindle checkpoints. Some of these that have been discovered these that have been discovered

Detect any failure of spindle Detect any failure of spindle fibers to attach to kinetochores fibers to attach to kinetochores and arrest the cell in metaphase and arrest the cell in metaphase (M checkpoint — (M checkpoint — exampleexample); );

Detect improper alignment of Detect improper alignment of the spindle itself and block the spindle itself and block cytokinesiscytokinesis; ;

Mitosis occurs in somatic Mitosis occurs in somatic cellscells

http://www.johnkyrk.com/http://www.johnkyrk.com/mitosis.htmlmitosis.html

Check this animationCheck this animation

Mitosis - ProphaseMitosis - Prophase

The two The two centrosomescentrosomes of the cell, each with of the cell, each with its pair of centrioles, move to opposite its pair of centrioles, move to opposite "poles" of the cell. "poles" of the cell. The The mitotic spindlemitotic spindle forms. This is an forms. This is an array of spindle fibers, each containing array of spindle fibers, each containing ~20 ~20 microtubulesmicrotubules. Microtubules are . Microtubules are synthesized from tubulin monomers in the synthesized from tubulin monomers in the cytoplasm and grow out from each cytoplasm and grow out from each centrosome. centrosome. The chromosomes become shorter and The chromosomes become shorter and more compact. more compact.

Mitosis - PrometaphaseMitosis - Prometaphase

The The nuclear envelopenuclear envelope disintegrates because of disintegrates because of the dissolution of the the dissolution of the laminslamins that stabilize its that stabilize its inner membrane. inner membrane. A protein structure, the A protein structure, the kinetochorekinetochore, appears at , appears at the the centromerecentromere of each chromatid. of each chromatid. With the breakdown of the nuclear envelope, With the breakdown of the nuclear envelope, spindle fibers attach to the kinetochores as well spindle fibers attach to the kinetochores as well as to the arms of the chromosomes. as to the arms of the chromosomes. For each dyad, one of the kinetochores is For each dyad, one of the kinetochores is attached to one pole, the second (or sister) attached to one pole, the second (or sister) chromatid to the opposite pole. Failure of a chromatid to the opposite pole. Failure of a kinetochore to become attached to a spindle fiber kinetochore to become attached to a spindle fiber interrupts the process. interrupts the process.

MetaphaseMetaphase

At metaphase all At metaphase all the dyads have the dyads have reached an reached an equilibrium equilibrium position midway position midway between the poles between the poles called the called the metaphase platemetaphase plate. . The chromosomes The chromosomes are at their most are at their most compact at this compact at this time. time.

AnaphaseAnaphase

The sister The sister kinetochores kinetochores suddenly separate suddenly separate and each moves to and each moves to its respective pole its respective pole dragging its dragging its attached chromatid attached chromatid (chromosome) (chromosome) behind it. behind it.

TelophaseTelophase

The chromosomes The chromosomes reach the polesreach the poles

A nuclear envelope A nuclear envelope reforms around reforms around each cluster of each cluster of

These return to These return to their more their more extended form. extended form.

CytokinesesCytokinesesIn In animal cellsanimal cells, a belt of , a belt of actin filamentsactin filaments forms around forms around the perimeter of the cell, the perimeter of the cell, midway between the poles. midway between the poles. The interaction of actin and a The interaction of actin and a myosinmyosin (not the one found in (not the one found in skeletal muscle) tightens the skeletal muscle) tightens the belt, and the cell is pinched belt, and the cell is pinched into two daughter cells.into two daughter cells.In In plant cellsplant cells, a membrane-, a membrane-bounded bounded cell platecell plate forms forms where the metaphase plate where the metaphase plate had been. The cell plate, had been. The cell plate, which is synthesized by the which is synthesized by the Golgi apparatus, supplies the Golgi apparatus, supplies the plasma membrane that will plasma membrane that will separate the two daughter separate the two daughter cells. Synthesis of a new cells. Synthesis of a new cell cell wallwall between the daughter between the daughter cells also occurs at the cell cells also occurs at the cell plate.plate.

Telomeres and agingTelomeres and aging

http://runews.rockefeller.edu/http://runews.rockefeller.edu/index.php?page=engine&id=138index.php?page=engine&id=138

TelomeresTelomeres

....TTGGGGTTGGGGTTGGG....TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGGTTGGGGTTGGGGTTGGGGTTGGGGTT 3'TTGGGGTT 3'....AACCCCAACCCCAACCCC....AACCCCAACCCCAACCCC 5' 5'

The extra G-T rich The extra G-T rich sequences are not present sequences are not present in telomeres as single in telomeres as single stranded DNA. stranded DNA. G-T rich strands can, in the G-T rich strands can, in the test tube, form an unusual test tube, form an unusual structure in which 4 G's structure in which 4 G's are paired with one are paired with one another. A metal ion (M+) another. A metal ion (M+) assists the formation of assists the formation of these G quartets. They these G quartets. They stack on top of one stack on top of one another. The T-stretches another. The T-stretches connect the sides of the connect the sides of the quartet. quartet. Intermolecular G quartets Intermolecular G quartets can form. can form.

Telomere repeatsTelomere repeats

Telomeric factsTelomeric factsAt their ends, telomeres can assume special structures, the At their ends, telomeres can assume special structures, the G quartets. G quartets. G quartets can account for the stability properties of G quartets can account for the stability properties of chromosome ends. chromosome ends. The formation of intermolecular G quartets can account for The formation of intermolecular G quartets can account for the apparent interaction of telomeres (bouqet formation). the apparent interaction of telomeres (bouqet formation). The studies using TRF2 suggest that the single strand The studies using TRF2 suggest that the single strand telomeric end is not arranged as a doubled-back G-quartet, telomeric end is not arranged as a doubled-back G-quartet, but rather invades duplex DNA near the junction of but rather invades duplex DNA near the junction of telomeric and non-telomeric DNA. telomeric and non-telomeric DNA. The analysis of psoralen-treated nuclei suggests that the The analysis of psoralen-treated nuclei suggests that the TRF2-facilitated structures probably exist in actual cells. TRF2-facilitated structures probably exist in actual cells. There is no analogous evidence favoring a biological There is no analogous evidence favoring a biological relevance of G-quartet structures. relevance of G-quartet structures.