The Cell Cycle Control PDF

The Cell Cycle Reading: 1- Chapter 17. Mol.Cell.Biol. 6th edition. 2- Review article provided Cell Cycle 1- Duplication and 2-division, known as the cell cycle. It is sequence of events with order. Cell division: 1- Growth and 2- replacement. Many millions...

The Cell Cycle Reading: 1- Chapter 17. Mol.Cell.Biol. 6th edition. 2- Review article provided Cell Cycle 1- Duplication and 2-division, known as the cell cycle. It is sequence of events with order. Cell division: 1- Growth and 2- replacement. Many millions of cells produced every second simply to compensate for loss. Certain events are universal between organisms. Proteins performing and controlling the events are so well conserved over the course of evolution that many of them function perfectly when transferred from a human cell to a yeast cell. Model systems like yeast, drosophila, Xenopus laevis, contribute well to our understanding of eukaryotic systems  :Cell Cycle in Eukaryotes phases. 2 phases with 2 Gaps 4 Gaps are not simple time S-phase: 10- delay and rest. It is for 12 hr.quality control G0-Days, weeks or years. or permanent before dying G1 may be extended depending on internal and external environment :M phase Much less than an hour Cell-Cycle Progression Can Be Studied in Various Ways Microscopic-e.g. In culture rounded up cells are mitotic. Cytokinesis can be seen. Budding in fungi under microscope. Buds vary in size according to the cell-cycle stage. Unbudded cells are in G1 BrDU incorporation  S-phase cells : Supplying cells with visualizable molecules that are incorporated into newly synthesized DNA, such as the artifcial thymidine analog bromodeoxyuridine (BrdU); and then using anti- BrdU antibodies and check under the microscope BrdU is also present in fluorescent form Fluorescent anti-BrdU antibodies Labeling S-phase cells. An immunofluorescence micrograph of BrdU-labeled epithelial cells of the zebrafish gut. The fish was exposed to BrdU, after which the tissue was fixed and prepared for labeling with fluorescent anti- BrdU antibodies (green). MTT Assay Cell viability and proliferation MTT assay principle Measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan. MTT assay The insoluble formazan crystals are dissolved using a solubilization solution. The resulting colored solution is quantified by measuring absorbance at 500-600 nanometers using spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells Other ways. Flow Cytometry Measuring cells DNA content, which doubles during S phase. This is facilitated by using fluorescent DNA- binding dyes and a flow cytometer. We can use flow cytometry to determine the lengths( Proportion of cell population) of G1, S, and G2 + M phases, by measuring DNA content in a synchronized cell population as it progresses through the cell cycle. fluorescence-activated cell sorter ( FACS)----Flow Cytometry Analysis of DNA content with a flow cytometer. (A flow cytometer, also called a fluorescence-activated cell sorter, or FACS, The cells were stained with a dye that becomes fluorescent when it binds to DNA, so that the amount of fluorescence is directly proportional to the amount of DNA in each cell. The cells fall into 3 categories:  unreplicated complement of DNA are in G1,  Fully replicated complement of DNA (twice the G1 DNA content) and are in G2 or M phase,  an intermediate amount of DNA and are in S phase.  The distribution of cells indicates that there are greater numbers of cells in G1 than in G2 + M phase, showing that G1 is longer than G2 Principle Overview of the control mechanism of cell cycle progression Control of the cell cycle progression from G1 to S phase. The Retinoblastoma pathway Senescence The cell cycle control system work much like the washing machine control system The cell-cycle control system operates much like the control system of an automatic clothes-washing machine. The washing machine functions in a series of stages: it takes in water, mixes it with detergent, washes the clothes, rinses them, and spins them dry. The Cell cycle control system  A connected series of biochemical switches, each of which initiates a specific cell-cycle event.  First switches are generally binary (on/of) and launch events in a complete, irreversible fashion.  (Imagine the disastrous consequences if chromosome condensation or nuclear-envelope breakdown were only partially initiated or started but not completed.)  Imagine the cell enter M phase before completing S phase.  Second, the cell-cycle control system is remarkably robust and reliable, partly because backup mechanisms and other features allow the system to operate effectively under a variety of conditions  Finally, the control system is highly adaptable and respond to specific intracellular or extracellular signals. Cell cycle checkpoints Control system: Three major regulatory transitions ( Checkpoints) Start (or the restriction point) in late G1, where the cell commits to cell- cycle entry and DNA duplication. G2/M transition, where the control system triggers the early mitotic events that lead to chromosome alignment on the mitotic spindle in metaphase. Metaphase-to-anaphase transition, where the control system stimulates sister-chromatid separation, leading to the completion of mitosis and cytokinesis.  The control system blocks progression through each of these transitions if it detects problems inside or outside the cell ( Subject of feed back loops). Feedback loops simplified version The Cell Cycle Control Machinery Positive and negative regulators  DNA Cell cycle replication arrest ve+ ve- regulator regulator s s  ve+ ve- regulator regulator s s - - - v v + e v + e v + e v e v e e Grow Seru th m facto Prolifer starv Anti r ative ation Prolifer signal ative  - Negative v Positive + e regulators v e regulators p15 INK4 Cyclins-D P21 CIP1 Cyclin P16 INK4 dependent P27 KIP1 kinase4/6 P18 INK4 Cyclin P57 KIP2 activating  In cancer Tumor Proto- suppressor oncogenes Genes - Negative v Positive+ e regulators v e Mutated/ regulators p15downregulated INK4 Cyclins-D P21 CIP1 Cyclin P16 INK4 dependent P27 KIP1 kinase4/6 P18 INK4 Cyclin P57 KIP2 activating Positive regulators of the cell cycle transition Cyclins 1- G1/S-cyclins activate Cdks in late G1 and thereby help trigger progression through Start, Their levels fall in S phase. 2. S-cyclins bind Cdks soon after progression through checkpoint and help stimulate DNA duplication. S- cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events. 3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid- mitosis. The concentrations of the three major cyclin types oscillate during the cell cycle, while the concentrations of Cdks (not shown) do not change and exceed cyclin amounts. A separate regulatory protein complex, the APC/C, initiates the metaphase-to anaphase transition The structural basis of Cdk activation. These drawings are based on three-dimensional structures of human Cdk2 and cyclin A, (A)In the inactive state, without cyclin bound, the active site is blocked by a region of the protein called the T-loop (red) like a stone blocking the entrance to a cave. (B) The binding of cyclin causes the T-loop to move out of the active site, resulting in partial activation of the Cdk2. (C) Phosphorylation of Cdk2 (by CAK) at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop, improving the ability of the enzyme to bind its protein  The cell cycle engines Simplified version P substrate CDK cyclin product ADP + ATP CAK Cyclin H-CAK Prognostic value of Cyclins Negative regulators of cell cycle. G1/S phase transision Inhibitors of CDK4 (hence their CIP/KIP proteins are capable of name INhibitors of CDK4), and of inhibiting all CDKs. CDK6. p27 null mice spontaneously Tumor suppressors and loss-of- develop tumors in the pituitary function mutations lead to gland and are more susceptible to carcinogenesis chemical carcinogens or irradiation. INK4 proteins are highly similar in P57 is an imprinted gene. terms of structure and function, Maternally expressed. By Dimopoulos K, Gimsing P, Grønbæk K. - The role of epigenetics in the biology of multiple myeloma.Dimopoulos K1, Gimsing P1, Grønbæk K1., CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php? Proliferative and anti-proliferative signals effect Cyclins Negative regulators  Switching between Inhibitor and substrate functions P p27kip1 P Switch Cell cycle CDK CDK cyclin cyclin p27kip1 progression p27kip1 Inhibited Active Phosphorylation does not mean always activation Phosphorylation at a pair of amino acids in the roof of the kinase active site by a kinase known as Wee1 inhibits the activity of a cyclin–Cdk complex. while dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity The Retinoblastoma pathway G1/S transition control system Retinoblastoma is among the first tumor suppressor genes identified  Cell-cycle dependent phosphorylation of Rb Phosphorylation of Rb Hyperphosphorylated Rb allows cells to transit p p p p the restriction point Rb Rb and enter S phase p p p p Restriction point p S phase Rb p G1 phase G0 G2 phase Quiescent cells M p p phase Rb p p p Hypophosphorylated Rb Rb p p p Rb http://www3.kumc.edu/jcalvet/PowerPoint/23 p p The E2F family is generally split by function into two groups: transcription activators and repressors. Activators such as E2F1, E2F2, E2F3a promote and help carryout the cell cycle, while repressors inhibit.the cell cycle  E2F transcribe PCNA ( Sliding Clamp) A link between the control system and replication machinery  Gate keeper M model G2 G1 Cdk4/6 S Cyclin D Rb R E2F released S-phase genes expressed Positive feedback loop Two waves of phosphorylation  DNA synthesis DNA synthesis not allowed allowed E2F1 P P E2F1 Р Р pRB P P Р P P Р P P Р P P pRB Р P PР P PР Cyclin D/CDK4 Cyclin Е/СDK2 Cyclin complexes Cyclin complexes are not active are active pRB less phosphorylated pRB more phosphorylated E2F1 bound to pocket E2F1 is free to promote cell cycle  Viral oncoproteins can interact with RB1 and occupate its pocket ,Adenoviral E1A ,Large T antigene of SV40 E1A, SV40-TAg, HPV-E7 E7 protein of HPV type 16 or18 Viral proteins Р bind to pocket domain of RB1 pRB !Set E2F factors free P PР P PР Cell is going to proliferate despite low phosphorylation of RB1 E2F1  Epigenetic repression of E2F E2F gene Binding with DP1,2 provides a second DNA binding site, increasing E2F [.binding stability ?E2F1 inhibits RB via H19 ?Does E2F induces miR-675 E2F1 inhibits RB via H19? Does E2F induces miR-675?  Entry into Mitosis The G2/M Checkpoints CyclinB-cdc2 activity is specific to the G2/M checkpoint. Cdc2 activity is further regulated by phosphorylation/dephosphorylation of its corresponding activators and inhibitors. Through a positive feedback loop, CyclinB-Cdc2 activates the phosphatase Cdc25 which in turn deactivates the CyclinB- Cdc2 inhibitors, Wee1 and Myt1. Cdc25 activates the complex through the removal of phosphates from the active site while Wee1 inactivates the complex through the phosphorylation of tyrosine residues,  Other Scenario's of regulation Regulated protein destruction by ubiquitination. Metaphase-to-Anaphase Transition Metaphase-Anaphase regulation Regulated Proteolysis Triggers the Metaphase-to-Anaphase Transition Progression through the metaphase-to-anaphase transition is triggered not by protein phosphorylation but by protein destruction. The key regulator is the anaphase promoting complex, or cyclosome (APC/C), APC/C is a member of the ubiquitin ligase family of enzymes. They polyubiquitylate specific target proteins, resulting in their destruction in proteasomes APC/C activate separase Separase degrade cohesins APC/C targets  The APC/C catalyzes the ubiquitylation and destruction of two major types of proteins. 1- Securin, which protects the protein linkages that hold sister-chromatid pairs together in early mitosis ( cohesin ). Destruction of securin in metaphase activates a protease ( Seperase) that separates the sisters and unleashes anaphase. 2- S- and M-cyclins are the second major targets of the APC/C. Destroying these cyclins inactivates most Cdks in the cell. This allows the cell to exit from mitosis. Activation of APC/C by Cdc20 (A)The APC/C is activated in mitosis by association with Cdc20. (B)This complex recognizes specific amino acid sequences on M-cyclin and other target proteins. (C)With the help of two additional proteins called E1 and E2, the APC/C assembles polyubiquitin chains on the target protein. (D)The polyubiquitylated target is then Ubiquitination is not confined to metaphase-telophase only The cell-cycle control system also uses another ubiquitin ligase called SCF Ubiquitylate certain Cyclin dependent kinase inhibitors proteins in late G1. Responsible for the destruction of G1/S-cyclins in early S phase Control by SCF Interplay b/w phosphorylation and ubiquitination  The activity of the ubiquitin ligase SCF depends on substrate- binding subunits called F-box proteins, of which there are many different types.  The phosphorylation of a target protein, such as the CKI shown, allows the target to be recognized by a specific F-box subunit An overview of the cell cycle control system.  The core of the cell cycle control system consists of a series of cyclin–Cdk complexes (yellow).  The activity of each complex is also influenced by various inhibitory mechanisms, which provide information about the extracellular environment, cell damage, and incomplete cell-cycle events (top).  These inhibitory mechanisms are not present in all cell types; many are missing in early embryonic cell cycles, for example. The S-Phase DNA replication phase S-Cdk Initiates DNA Replication Once Per Cycle DNA replication begins at the origin of replication scattered at numerous locations in each chromosome. DNA replication is initiated at these origins when a DNA helicase unwinds the double helix and DNA replication enzymes are loaded. This leads to the elongation phase of replication, when the replication machinery moves outward from the origin bidirectionally. Duplication occurs only once per cell cycle. How controlled? Initiation phase : 2 distinct steps that occur at different times in the cell cycle Step 1: in late mitosis and early G1, a pair of inactive DNA helicases is loaded onto the replication origin, forming a large complex called the pre- replicative complex or preRC “licensing”. The proteins Cdc6 and Cdt1 collaborate with the ORC to load the inactive DNA helicases around the DNA next to the origin. The second step occurs in S phase, when the DNA helicases are activated, by a protein kinase called DDK resulting in DNA unwinding and the initiation of DNA synthesis. Once a replication origin has been fired the two helicases move out from the origin, and that origin cannot be reused until a new preRC is assembled there at the end of mitosis. As a result, origins can be activated only once per cell cycle.