Chapter 18 Cell Cycle PDF

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Chapter 18 The Cell Cycle I.

The cell-cycle 

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II.

Cell-cycle: Process in which cell divides in two and makes a duplicate of itself. o Duration of process depends on cell type. o Neurons and skeletal muscle cells do not complete full cell cycle once fully differentiated. 4 basic phases to the cell cycle  G1: Time right after division when cell is growing.  S : Synthesis phase when DNA is duplicated.  G2: Time after S phase but before cell division.  Cell makes sure all DNA was replicated correctly and fixes any errors.  Makes sure environment is good for cell division.  M : Mitotic phase when cell divides itself into two identical daughter cells.  G0: G zero phase when cells will not move further into cell-cycle and spend their lifetime in this phase. ie neurons, skeletal muscle. a) Checkpoints are found throughout cell cycle. (a) G1 checkpoint: makes sure environment is favorable for cell division before consuming energy in DNA replication. (a) Nutrient availability (b) Proper signals in extracellular matrix signaling cell proliferation (b) G2 checkpoint: makes sure that any DNA damage is repaired before going into cell division. (i) Also ensures DNA synthesis is complete. (c) M checkpoint: ensures the replicated chromosomes are attached to mitotic spindles correctly before cell divides in to.

Cell-cycle control A. Cyclins and Cdks: Certain proteins are very important regulating the cell cycle process. a. Cyclins: proteins whose concentrations cycle during the cell cycle. i. They don’t have enzyme activity but activate kinases when binding them. b. Cyclin dependent kinase (Cdks): Kinases that are activated when cyclins bind. i. Cyclins also help bring the kinase to its target for phosphorylation. ii. Cdks don’t cycle in their concentration only in their activity. B. Activation of Cdks: The Cdk must first bind to its respective cyclin. However, this does not lead to immediate activation. a. Cdk-cyclin gets phosphorylated at 3 different sites. i. 2 are inhibitory to Cdk activity and override the first phosphorylated site that activates Cdk. b. Specific proteases remove the two phosphates from Cdk-cyclin inhibitory site. i. Now only active phosphorylated form of Cdk is present. ii.  Types of cyclins o cyclin D: Cyclin that is increased concentration during G1 o cyclin D-Cdk helps move cell through G1 phase. o cyclin E: cyclin increases during late G1 and early S phase. o cyclin E-Cdk: helps initiate S phase. o cyclin A: cyclin increases during early S phase. o cyclin A-Cdk: helps initiate S phase. o cyclin B: cyclin increases during G2 phase. o cyclin B-Cdk: helps to initiate M phase.

C. Inhibitory Cdks: 1. Removal of cyclin will turn off respective Cdk.  

Cyclin is ubiquitinated and leads to proteome degradation. This explains why Cdk is turned off very quickly along with quick loss of cyclins. o Why do you think Cdk is turned on very quickly even though there is gradual increase in the cyclin concentration? o This is due to cyclin-Cdk complex being phosphorylated and then dephosphorylated before becoming active.

2. Cdk inhibitor proteins  Proteins that prevent cyclin-Cdk assembly or prevent the cyclin-Cdk from working properly.  If Cdk inhibitor proteins are found in the G1 phase they will bind to cyclin E-Cdk or cyclin A-Cdk and prevent progression into S phase. *Serves 2 important roles 1. Prevents S phase from beginning if cell isn’t big enough…giving time for cell to grow. 2. If environment /extracellular signals are not telling the cell to divide it stops in G1.  G1: serves as the “start” site of the cell-cycle b/c once extracellular signals are present and the cell passes the G1 checkpoint it has typically committed itself to division. o Mitogen: recall these are the extracellular signals produced by other cells that stimulate cell division… often via MAP Kinase pathway. o After G1 checkpoint the rest of the cell cycle completed w/in 24 hours usually. *If cell is unable to pass G1 checkpoint for a long time it will often go into the in the G 0 phase. -This typically happens b/c many of the Cdks and cyclins are gone and Cdk inhibitor proteins inhibit those that are present. III.

S phase * Recall that DNA synthesis occurs at origins of replication found throughout the chromosome. A. Origin Recognition Complex (ORC): is a protein complex that binds to the Ori and remains bound throughout cell cycle.  Cdc6: important regulatory protein who increases in concentration during G1. o Cdc6 binds to ORC and makes the Ori ready for replication. It is poised to start but still needs a trigger.  S cyclin-Cdk (i.e. cyclin A) phosphorylates Cdc6 which leads to its degradation and allows DNA synthesis to occur. o Also prevents re-replication of DNA at that Ori. o This is due to Cdc6 being degraded and loss of the trigger while which ensures the DNA is not re-replicated again throughout the rest of the cell cycle.  Cohesins: protein complex that holds the 2 copies of the replicated chromosomes (sister chromatids) together.

B. Damaged DNA is not replicated 1. If DNA is damaged it will not pass the G1 checkpoint.  p53: protein that serves as transcription activator of the Cdk inhibitor protein p21.  Mutated p53 gene is found in ~50% of cancer cases.  p21: Cdk inhibitor protein that binds G1/S-cdk (cyclin E) and S-cdk (cyclin A). This prevents S phase from initiating.

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If DNA is damaged during replication or G2 phase or not completely replicated it will not enter M phase.  Damaged / incomplete DNA replication leads to cascade of events that inhibits phophatase of Mcdk (cyclin B).  Recall that cyclin-cdks are phosphorylated 3 times and a phosphatase must remove phosphate before the cyclin-cdk is activated.  w/o phosphatase the M-cdk complex is inactive and progression into mitosis doesn’t occur. M phase A. Initiation of M phase.  M-cdk: is phosphorylated by inhibiting and activating kinases and accumulation begins during G2 phase. i. Currently inactive during accumulation process.  Cdc25: phosphatase that removes inhibitory phosphates and leads to activation of M-cdk. i. Once M-cdk is active it phosphorylates Cdc25 in a positive feedback loop that activates Cdc25 which in turn activates more M-cdk.  This leads to a sudden activation of M-cyclin-cdk and the cell quickly goes from G2 to M phase. B. Quick overview of the 5 phases of Mitosis 1. Prophase: replicated chromosomes condense; mitotic spindles begin to assemble which are made of microtubules. 2. Prometaphase: Nuclear envelope breaks down; spindle fibers attach to chromosomes.  Kinetochore: protein complex on chromosome where microtubules attach. 3. Metaphase: chromosomes align in the middle of cell. 4. Anaphase: sister chromatids separate with each being pulled to opposite polls by mitotic spindles; cleavage furrow begins to separate the cell in two. 5. Telophase: The separated sister chromatids arrive at opposite ends of the cell; nuclear envelope reforms; contractile ring continues to divide cell (cytokinesis). C. Condensation of chromosome (Prophase):  Condensins: proteins complexes that are used to condense chromosomes. o Activated by M-cdk phophorylation  

Cohesins work with chromosome to keep sister chromatids together. Condensins work with each chromatid to coil the DNA into a condensed chromosome.

D. Centrosome duplication and mitotic spindle formation (Prophase): 1. G1/S-cdek and S-Cdk: trigger duplication of centrosome at the start of the S phase.  Centrosome duplication and replication of DNA occur at same time.  Aster: The 2 centrosomes separate and move to opposite poles during prophase and prometaphase. i. During this the microtubules form out of centrosome and look like a star… hence the name aster. 2. M-Cdk: Phosphorylates microtubules (MT) associated proteins which increases the dynamic instability of the microtubule filaments.  Spindle fibers 1st form during prophase.  As the spindle fibers form they search the interior of cell.  Interpolar microtubule: Some spindle fibers from one centrosome interact with spindle fibers from another centrosome.

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Nuclear envelope breakdown and attaching chromosome to spindle fiber (Prometaphase): 1. Nuclear envelope breakdown: intermediate filament proteins and nuclear pore proteins of nuclear lamina are phosphorylated.  Causes lamina to destabilize and envelope to breakdown into small membrane vesicles. 2. Once nuclear envelope is gone the mitotic spindles can interact with condensed chromosomes.  Kinetochore: protein complex where spindle fibers attach to chromosome. i. Centromere: more condensed region of chromosome where kinetochore is formed and microtubule attach to sister chromatids. ii. One kinetochore is attached to each sister chromatid and faces in opposite directions.  When each sister chromatid kinetochore is attached to spindle fibers they are pulled in opposite directions creating tension. o This is a control mechanism… if tension is present the spindle fibers are attached correctly; if no tension than cell won’t continue through mitosis. *At this point there are 3 types of mitotic spindles 1. Aster microtubules: constantly undergoing dynamic instability 2. Kinetochore microtubule: Microtubule that have attached centrosome to the chromosome. a. Multiple microtubules (20-40) attach per kinetocohore. 3. Interpolar microtubule: Microtubule from each aster that interact with one another and are held together by motor proteins. F.

Movement of attached chromosomes to equator of cell (metaphase).  Metaphase plate: the pulling back and forth of chromosomes between opposite spindle poles leads to all chromosomes lining up at the middle of the cell.  Tension between chromosomes in metaphase plate is constant.

G. Breaking of sister chromatids and separation to opposite poles (anaphase) 1. Anaphase Promoting Complex(APC): promotes degradation of M-cyclin and securing.  Breakdown of M-cyclin leads toward exit of mitosis. 2. Securin: inhibitory protein that prevents the protease separase from degrading cohesion.  APC promotes degradation of securing which frees up separase to proteolytic breakdown cohesion.  Loss of cohesion means sister chromatids are no longer held together.  Once sister chromatids are unbound the constant tension / pulling of mitotic spindles leads to separation of replicated DNA. 3. Two processes lead to movement of sister chromatid to opposite poles.  Anaphase A: Kinetochore microtubules shorten and pull apart sister chromatids and more toward spindle pole / centrosome. i. ATP hydrolysis helps remove tubulin from kinetochore microtubules.  Anaphase B: The spindle poles / centrosomes are pushed and pulled further apart. o Pushed further apart by interpole microtubules which use motor proteins to slide the interacting microtubules apart from each other. o Pulled further apart by Aster microtubules which bind to cell cortex just beneath plasma membrane and pull spindle pole toward plasma membrane. 4. Spindle Assembly Checkpoint: If there are any unattached chromosomes an inhibitor blocks APC activation.

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This presents sister chromatids from separating until all chromosomes are attached to kinetochore microtubules and aligned at the metaphase plate.

H. Re-forming of nuclear envelope (telophase) 

Phosphatases remove phosphate placed on nuclear pore proteins and lamins that were placed on during prometaphase.

The dephosphorylated nuclear pore and lamin protein aggregate together around individual chromosomes at the poles.  These aggregates then fuse together and reform the nuclear envelope. *Once nuclear envelope is reformed the chromosomes decondense and transcription resumes. 

V. Cytokinesis *Cytokinesis: is the dividing of the cytoplasm in two. o Begins during anaphase. o Completes at end of telophase and marks end of mitosis. o Use Actin filaments and myosin motor proteins. A. Placement of cleavage furrow determined in Anaphase.  Cleavage furrow: 1st sign of where cytokinesis will cut the cytoplasm. o During Anaphase the pushing and pulling of kinetochore and interpolar microtubules make the cleavage furrow form perpendicular to the microtubules.  This ensures cell is cut in ½ in such a way that the DNA is properly split in two. o The cleavage furrow is typically formed so that cytokinesis ends with equal portions of cytoplasm in the two daughter cells.  During development this may be manipulated so that the contents are not shared evenly and helps lead to differentiation of cells. B. Contractile Ring  Composed of actin and myosin filaments  Forms during anaphase 

*Actin and myosin filaments attach to cell cortex just beneath plasma membrane.  Formation occurs perpendicular to the interpolar microtubules.  The sliding of actin over myosin filaments, powered by ATP, leads to a force that pinch the cytoplasm in two.

*While a cell divides it often undergoes significant change in shape and contact with other cells.  Due to contractile ring pinching cell in half.  Also due to cell not adhering to surface as tightly during mitosis. o Integrins: Transmembrane proteins that bind cell to surface. (recall cell crawling)  Are phosphorylated which cause them to not bind as well and allow cell to change shape during cytokinesis. C. Dividing of organelles 1. Mitochondria or chloroplasts : double in number during cell cycle and the splitting of the cytoplasm evenly results in even split of these organelles. 2. ER: is extension of nuclear envelope.  When nuclear envelope breaks down the ER stays intact.  During cytokinesis the ER is split in ½. 3. Golgi Apparatus: Disassembles during mitosis into small vesicles.  These fragments bind to spindle fibers via motor proteins and travel with chromosomes to spindle poles during anaphase. 

VI Apoptosis

Apoptosis: is a form of programmed cell death in which the cell kills itself off from the inside out. A. Important during development for sculpting the body.  Our hands and fingers for instance; loss of tadpole tail. B. Important in adults to maintain proper organ size and cell count.  Liver is kept at correct size via cell proliferation and apoptosis working together. Cell necrosis: cell death due to acute injury.  Results in splitting of cell and release of intracellular contents to environment. o

This induces inflammation response and can have damaging effects on neighboring cells.

*Apoptosis kills the cell off neatly prevents the release of intracellular contents.  Cytoskeleton and nuclear envelope breakdown; DNA fragments and plasma membrane signals for macrophages to breakdown cell by phagocytosis. A. Caspases: family of protein proteases that through cleavage of specific protein induce apoptosis. 

Procaspases: are the inactive precursor of caspases. i. Are always present in the cell waiting to be signaled to be turned on. ii. When proteolytically cleaved they are turned into caspases.

*Once a procaspase is cleaved and turned into a caspase it will cleave other procaspases and cause a very fast activation of apoptosis. B. Bcl2 family: family of proteins that regulate activation of procaspases.  Some activate procaspases to caspases.  Others inhibit activation of procaspases. 1) Bax and Bak: proteins that activate procaspases indirectly.  Relase cytochorme C from mitochondria.  Cytochrome C binds with special proteins that in turn bind procaspase-9 *Apoptosome: protein complex formed by Cytochrome C / protein and Procaspase-9 2) Bcl2: the protein Bcl2 of the Bcl2 family serves to inhibit activation of procaspases  Turned on by PKB (Akt) phosphorylating the inhibitor of Bcl2 and inactivating it. VI Cell growth *Cells must grow in size before they divide. Requires 2 things: 1) Nutrients 2) Extracellular signal informing cell it needs to go ahead and grow. Three general categories for promoting cell survival, cell division, cell growth. 1) Survival factors: primarily suppress apoptosis. 2) Mitogens: stimulate cell division by providing signal through checkpoints. 3) Growth factors: stimulate cell growth by promoting synthesis of microtubules.

A. Survival factors:

*Recall from our discussion on cell signaling that if no signals are present the cell will die via apoptosis. o Survival factors case a cascade of events that blocks apoptosis from occurring within that cell. o Important in development to ensure only the cells needed for organism to function properly survive. ie. Embryo o Embryonic neurons are significantly overproduced. o To determine which neurons are eliminated the target cell will secrete survival factors. o The neuron that binds the survival factor while those that don’t undergo apoptosis. *Survival factor binds receptor and causes cascade that may activate transcription of Bcl2 which would prevent apoptosis from occurring. B. Mitogens  Induce cell division and cell proliferation.  Are extracellular signals that bind to cell surface receptors.  Induce the MAP-kinase Pathway. Retinoblastoma protein (Rb): is inactivated by mitogens. *Serves as a transcription repressor by binding transcription activator needed for progression into S phase. 1) Mitogen binds receptor and induces signaling cascade. 2) cyclin E & A / Cdk are activated. 3) The active G1/Cdk and G1S/Cdk phosphorylates Rb and inactivates it. 4) With Rb inactive it no longer binds transcription activator. 5) Transcription activator induces transcription of genes needed for movement into S phase. *Rb is a tumor suppressor gene and is mutated in numerous cancer cells…not just in the eye. C. Growth Factors  Cell growth of single celled organisms only require nutrients.  Cell growth of multicellular organisms rely on nutrients and communication with neighboring cells.  Growth factors are part of extracellular matrix and bind to receptors on plasma membrane. i. Leads to increase in macromolecule synthesis ii. Can also lead to decrease in macromolecule breakdown. D. Growth inhibitors *Inhibitors are used to maintain proper homeostasis of growth factors. Myostatin: protein that inhibits growth and proliferation of myoblasts that make up skeletal muscle. o If gene is defective organism has abnormally large muscles and extra muscle cells. o In 2004 a baby boy was born in Germany with a documented double allele mutation of myostatin.  Born with very defined muscles in legs and arms for a baby....