Advanced Cell ch. 18 Cell Cycle: an orderly sequence of events in which a cell duplicates its contents and then divides in two. Answer three questions: How do cells duplicate their contents-including the chromosomes, which carry the genetic info? Ch. 6 Ch. 7, 11, 15, and 17 How do they partition the duplicated contents and split in two? How do they coordinate all the steps and machinery required for these two processes? Overview of the Cell Cycle: Eukaryotic Cell Cycle Includes Four Phases: M phase: when the nucleus divides (mitosis) cell splits in two (cytokinesis) in mammalian cell, this phase takes about an hour Interphase: The period between one M phase and another Includes three phases S phase: synthesis phase The cell replicates its DNA G1 and G2: gap phases Flank either side of the s phase The cell continues to grow The cell monitors both its internal state and external environment Ensures conditions are suitable for reproduction Checks to make sure preparations are complete before cell starts S phase (follows G1) and Mitosis (follows G2) Has checkpoints where the cell pauses to decide to proceed or pause to allow more time to prepare. During all of Interphase a cell continues to: Transcribe genes Synthesize proteins Grow in mass In first stages of frog embryo: G1 and G2 phases are drastically shortened This allows the embryo to divide into as many cells as possible in a quick time Result is smaller cells with each division because no time to grow in mass Cell-Cycle Control System: Made of regulatory proteins Makes sure cell cycle occur in a set sequence and that each process has been completed before the next one begins The control system is regulated in itself at certain critical points of the cycle by feedback from the process currently being performed If there was no feedback, an interruption or delay would be disastrous How? Uses molecular breaks, or checkpoints Pauses the cycle at certain transition points Control system does not trigger the next step in cycle unless the cell is properly prepared. Three main transition points: 1. From G1 to S phase: Confirms that the environment is favorable for proliferation before committing to DNA replication Proliferation requires both sufficient nutrients and specific signal molecules in the extracellular environment. Progress will be stalled in G1 if not favorable Can enter a G0 phase= resting state2. From G2 to M phase: Confirms that the DNA is undamaged and fully replicated Ensures that the cell does not enter mitosis unless the DNA is intact3. During Mitosis: Ensures that the duplicated chromosomes are properly attached to a cytoskeletal machine called the mitotic spindle Does this before the spindles pulls the chromosomes apart and segregates them into two daughter cells If the control system malfunctions: Cell division is excessive Cancer can result The Cell-Cycle Control System: Two types of machinery present: One manufactures the new components of the growing cell Another hauls the components into their correct places and partitions appropriately when the cell divides in two. Control system switches all of the machinery on and off at the correct times Coordinates the various steps of the cycle this way The cell-cycle control system depends on cyclically activated protein kinases called Cdks: Control system governs the cell-cycle machinery by cyclically activating and then inactivating the key proteins and protein complexes that initiate or regulate DNA replication, mitosis, and cytokinesis. Regulation is carried out through phosphorylation and dephosphorylation of proteins. Cyclin-dependent protein kinases (Cdks): The kinases of the cell-cycle control system They are only enzymatic when bound to a protein called Cyclin. Cyclin is not enzymatic in itself Activated only at the correct times of the cell-cycle This makes them seem cyclic. The cyclical concentration of Cyclin help drive the cyclic assembly and activation of the cyclin-Cdk complexes. Once activated, the cyclin-Cdk complexes help drive the cell-cycle into M phase or S phase, etc. Different cyclin-Cdk complexes trigger different steps in the cell cycle: There are several types of cyclins and several types of Cdks involved in cell-cycle control. Different cyclin-Cdk complexes trigger different steps of the cell cycle. Major cyclins and cdks of vertebrates: Cyclin-Cdk ComplexCyclinCdk Partner

G1-CdkCyclin D1, D2, D3Cdk4, Cdk6

G1/S-CdkCyclin ECdk2

S-CdkCyclin ACdk2

M-CdkCyclin BCdk1

The cyclin that acts on G2 to trigger entry into M phase is called M Cyclin The active complex it forms with its Cdk is called M-Cdk. S cyclins and G1/S cyclins bind to distinct Cdk protein late in the G1 to form S-Cdk and G1/S-Cdk Help launch the S phase G1 cyclins act earlier in G1 and bind to other Cdk proteins to form G1-Cdks Help drive the cell through G1 towards the S phase. Each cyclin-Cdk complex phosphorylates a different set of target proteins in the cell. i.e. G1-Cdks phosphorylate regulatory that activate transcription of genes required for DNA replication. Cyclin concentrations are regulated by transcription and by Proteolysis: The concentration of a given protein in the cell is determined by the rate at which the protein is synthesized and the rate at which it is degraded. Over the course of the cell cycle: Cyclin concentrations rise gradually Due to the increase in transcription of cyclin genes Then they fall abruptly Due to a full scale targeted degradation of the protein M and S cyclins are abruptly degraded part way through the M phase Depends on a large enzyme complex called anaphase-promoting complex (APC) Complex tags the cyclins with a chain of ubiquitin Proteins marked this way are directed to proteasomes where they are rapidly degraded. Ubiquitylation and degradation of the cyclin returns its Cdk to an inactive state. Cyclin destruction can help drive the transition from one phase of the cell-cycle to the next. i.e. inactivation of M-cdk leads to the molecular events that take the cell out of mitosis. The activity of cyclin-Cdk complexes depends on Phosphorylation and Dephosphorylation: Cyclin-Cdk complexes tend to switch on right at the correct time How? Cyclin-Cdk complex contains inhibitory phosphates To become active, cyclin-Cdk must be dephosphorylated Done by a specific protein phosphatase (i.e. Cdc25) Protein kinases (i.e. Wee1) and phosphatases regulate the activity of specific cyclin-Cdk complexes and help control progression through the cell cycle. Cdk activity can be blocked by Cdk inhibitor proteins: The activity of cyclin-Cdk complexes can also be modulated by the binding of Cdk inhibitor proteins System uses these to block the assembly or activity of the cyclin-Cdk complexes Some, like p27, help maintain the Cdks in an inactive state during the G1 phase of the cycle Delays the progression into the S phase Allows the cell more time to grow or until conditions are favorable. The cell-cycle control system can pause the cycle in various ways: G1 to S transition: It uses Cdk inhibitors to keep cells from entering S phase and replicating their DNA. G2 to M transition: It suppresses the activation of M-Cdk by inhibiting the phosphatase required to activate the Cdk Exit from Mitosis: Can delay this by inhibiting the activation of APC, thus preventing the degradation of M cyclin. G1 Phase: Based on intracellular signals on cell size and extracellular signals on environmental conditions, cell-cycle control machinery can: Hold the cell transiently in G1 (or G0) Allow it to prepare for entry into the S phase Once past this checkpoint, the cell usually continues through the full cell-cycle. Cdks are stably inactivated in G1: During M phase, when cells are activily dividing, the cell is full of active cyclin-Cdk complexes. Cell has to disable these if it is to proceed through G1 at the normal speed If goes through too fast, makes smaller and smaller cells with each cycle Used in some early stage embryos. To inactivate all of the S-Cdk and M-Cdk Blocks the synthesis of new ones Deploys Cdk inhibitor proteins to muffle the activity of any remaining complexes. Uses many different mechanisms to ensure complete shut down. Mitogens promote the production of the cyclins that stimulate cell division: Mammalian cells will multiply only if they are stimulated by extracellular signals called Mitogens (produced by other cells) If no signal, cell arrests in G1 and can enter G0 for any amount of time Escape from cell-cycle arrest: Requires the accumulation of cyclins Mitogens turn on cell signaling pathways that stimulate the synthesis of G1 cyclins, G1/S cyclins and other proteins involved in DNA synthesis and chromosome duplication Buildup of cyclins triggers a wave of G1/S-Cdk activity Relieves the negative controls that block progression from G1 to S phase Example of negative control: Retinoblastoma (Rb) protein binds to transcription regulators Prevents them from turning on the genes required for cell proliferation In childhood eye cancer, this protein is missing Rb is inactivated by G1-Cdks and G1/S-Cdks They phosphorylate the protein which releases it from the transcription factor Cell proliferation genes are transcribed DNA damage can temporarily halt progression through G1: G1-S transition: Prevents the cell from replicating damaged DNA DNA damage in G1 cause and increase in concentration and activity of a protein called p53 Transcription regulator that activates the transcription of the gene encoding a Cdk inhibitor protein called p21 P21 binds to G1/S-Cdk and S-Cdk Prevents them from driving the cell into S-phase Arrest of the cell-cycle in G1 allows: Time to repair DNA before replicating it If damage is too severe, p53 can induce a form of apoptosis If p53 is missing or defective Unrestrained replication of damaged DNA leads to increase in mutations Cells tend to become cancerous Mutation of p53 gene found in about half of all human cancers Cells can delay division for prolonged periods by entering specialized nondiving states: Can withdraw permanently or for a short period of time into G0 phase Become terminally differentiated S phase: S-Cdk initiates DNA replication and Blocks Re-Replication: Prep: Begins early in G1- DNA is made replication ready by the recruitment of proteins to the sites along each chromosome Where replication will begin Nucleotide sequences are called origins of replication Serve as landing pads for the proteins and protein complexes that control and carry out DNA synthesis One complex, the origin recognition complex (ORC) remains perched atop the replication origins throughout the cell cycle First step of replication initiation: The ORC recruits a protein called Cdc6 Its concentration rises early in G1 Together these proteins load the DNA helicases that will open up the double helix and ready the origin for replication Once this prereplicative complex is in place, the replication origin is loaded and ready to go. Replication: The signal to commence replication comes from S-Cdk The cyclin-Cdk complex that triggers S phase S-Cdk is assembled and activated at the end of G1. During S phase, it activates the DNA helicases in the prereplicative complex and promotes the assembly of the rest of the proteins that form the replication fork S-Cdk essentially pulls the trigger that initiates DNA replication S-Cdk also helps prevent re-replication It phosphorylates Cdc6 which marks the protein for degradation Incomplete Replication can arrest the cell cycle in G2 Mechanism to delay entry into M phase Activity of M-Cdk is inhibited by phosphorylation at particular sites to progress into Mitosis, inhibitory phosphates must be removed by an activating protein phosphatase called Cdc25 When DNA is damaged or incompletely replicated, Cdc25 is itself inhibited Prevents the removal of inhibitory phosphates M-Cdk remains inactive and M phase is delayed until DNA replication is complete and any DNA damage is repaired. M Phase: M-Cdk drives entry into M phase and Mitosis: M-Cdk Helps prepare the duplicated chromosomes for segregation Induces the assembly of the mitotic spindle M-Cdk accumulates throughout G2 Not activated until the end of G2 Activating phosphatase Cdc25 removes the inhibitory phosphatases holding M-Cdk activity in check Activity is self-reinforcing when activated Each active M-Cdk can indirectly activate additional M-Cdk complexes by phosphorylating and activating more Cdc25 Once activated: shuts down the inhibitory kinase Wee1 further promotes the production of activated M-Cdk causes cell to go from G2 to M abruptly Cohesins and Condensins help configure duplicated chromosomes for separation: Chromosomes become dense with the help of protein complexes called condensins Help carry out this chromosome condensation Reduces mitotic chromosomes to compact bodies that can be easily segregated Help double helices to coil up into a more compact form Assembly of condensing complexes onto the DNA is triggered by the phosphorylation of condensins by M-Cdk. Immediately after a chromosome is replicated in S phase Two copies remain tightly bound together The identical copies, sister chromatids, each contain a single, double-stranded molecule of DNA Also associated proteins Sisters held together with protein complexes called cohesins Assemble along the length of each chromatid as the DNA is replicated Broken only in late Mitosis Both protein complexes form ring structures Different cytoskeletal assemblies carry out Mitosis and cytokinesis: Mitotic Spindle: Nuclear division (Mitosis) Composed of microtubules and various proteins that interact with them Microtubule-associated motor proteins Responsible for separating the duplicated chromosomes Responsible for allocating one copy of each chromosome to each daughter cell Contractile Ring: Carries out cytoplasmic division Consists mainly of actin filaments and myosin filaments They are arranged in a ring around the equator of the cell Starts assembly just under the plasma membrane toward end of Mitosis As it contracts, pulls the membrane inward Divides the cell in two M Phase occurs in Stages: Stages of Mitosis: Prophase, prometaphase, metaphase, anaphase, telophase, cytokinesis. Mitosis: Centrosomes duplicate to help for the two poles of the Mitotic Spindle: Before M phase begins: DNA must be fully replicated Centrosome must be duplicated Centrosome- the principle microtubule-organizing center in animal cells Duplicates to form two poles of the mitotic spindle Makes sure each daughter cell receives its own centrosome Duplication begins at the same time as DNA replication Triggered by the same Cdks that initiate DNA replication: G1/S-Cdk and S-Cdk Originally, after duplication, centrosomes remain together as one complex on one side of the nucleus As mitosis begin they start to separate Each nucleates a radial array of microtubules called aster The two asters move to opposite sides of the nucleus to form the poles of the spindle This whole process called Centrosome cycle. The Mitotic Spindle starts to Assemble in Prophase: Microtubules continuously polymerize and depolymerize the addition and loss of their tubulin subunits Called dynamic instability Start of Mitosis: M-Cdk phosphorylates microtubule-associated proteins Influences the stability of the microtubules Result is rapidly growing and shrinking microtubules extending in all directions from the two centrosomes Microtubules from one centrosome interact with the microtubules from the other centrosome Provides stability Keeps from depolymerizing Forms basic framework of Mitotic Spindle Centrosomes are now called spindle poles Interacting microtubules are called interpolar microtubules Chromosomes attach to the Mitotic Spindle at Prometaphase: Prometaphase starts abruptly with the disassembly of the nuclear envelope Breaks into small membrane vesicles Triggered by the phosphorylation and disassembly of nuclear pore proteins and the intermediate filament proteins of the nuclear lamina Spindle microtubules gain access to chromosomes and connect Connect at kinetochores Protein complexes that assemble on the centromere of each condensed chromosome during late prophase Recognize the special DNA sequence present on the centromeres Kinetochore links the chromosome to one pole The kinetochores on each sister chromatid face in different directions This allows them to be sorted to different poles Before pulled apart: Chromatids are briefly attached to both poles: bi-orientation Tension builds, signaling to sister kinetochores that they are attached correctly Chromosomes assist in the assemble of the Mitotic Spindle: The chromosomes can do the work of the centrosome and arrange a bi-polar spindle They control the microtubules and motor proteins put them into place Some plants and animal cells do not have centrosomes Chromosomes line up at the spindle equator at metaphase: Chromosomes align at center Make metaphase plate This is the beginning of Metaphase Chromatids still under tremendous tension Proteolysis triggers sister-chromatid separation at anaphase: Anaphase begins abruptly with the breakage of the cohesion linkages that hold sister chromatids together Chromatids now become chromosomes Chromosomes pulled to their poles Cohesin linkage is destroyed by a protease called separase Before anaphase this protease is inactive due to inhibitory protein called securin at beginning of anaphase securin is targeted for destruction by APC Chromosomes segregate during anaphase: Anaphase A: The kinetochore microtubules shorten and the attached chromosomes move poleward. Due to loss of tubulin subunits on microtubules Anaphase B: Spindle poles themselves move apart, further segregating the two sets of chromosomes Due to two sets of motor proteins Members of kinesin and dynein families An unattached chromosome will prevent sister-chromatid separaton: If two chromatids did not separate: Spindle assembly checkpoint Activation of APC is blocked, causing the sisters to remain glued together So none of the chromosomes can be pulled apart until every chromosome has been positioned correctly on the spindle Nuclear envelope reforms at Telophase: Mitotic spindle disassembles Nuclear envelopes form: Vesicles of nuclear membrane cluster around individual chromosomes Then fuse to reform the nuclear envelope Nuclear pore proteins and nuclear lamina are dephosphorylated Allows them to reassemble Once formed pores pump in nuclear proteins and the nucleus swells Chromosomes decondense Cytokinesis: Process by which the cytoplasm is cleaved in two Completes M phase The mitotic spindle determines the plane of cytoplasmic cleavage Puckering during anaphase that is perpendicular to spindle Ensures the cleavage furrow is in the correct spot During anaphase: The overlapping microtubules of spindle, central spindle, recruit and activate proteins that signal to the cell cortex to initiate the assembly of the contractile ring at a halfway position Cytokinesis in plant cells involves the formation of a new cell wall Daughter cells are separated by the construction of a new wall New wall starts to assemble at the start of telophase Assembly process is guided by a structure called the phragmoplast Is formed by the remains of the interpolar microtubules at the equator of the old mitotic spindle Small membrane enclosed vesicles from the golgi apparatus are filled with polysaccharides and glycoproteins Required for cell wall matrix Fuse to form a disc like, membrane enclosed structure Reaches both plasma membranes Cellulose microfibrils are laid down to complete contruction Control of cell numbers and cell size: Apoptosis Mediated by an intracellular proteolytic cascade Involves a family of proteases called caspases Two types: Initiator Caspases: Cleave and activate the downstream executioner caspases. Executioner caspases activate additional executioners Causes amplified cascade Others dismember other key proteins in the cell like lamin proteins Cell division Cell growth