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Lecture 10

BIO1140 Lecture 10: Topic 10 readings

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University of Ottawa
Caroline Petit- Turcotte

OVERVIEW OF THE CELL CYCLE AND ITS CONTROL Expression The Cell Cycle Is an Ordered Series of Events Leading to Replication of Cells • S (synthesis) phase – chromosomes are replicated • M phase – mitosis and cytokinesis o Prophase – chromosomes condense by tightly folding loops of chromatin fiber attached to the chromosome scaffold o Metaphase – sister chromatids remain attached at the centromere become aligned in the center of the cell o Anaphase – sister chromatids separate and move to opposite spindle poles, turning them into daughter cells o Telophase – nuclear envelope that broke down into small vesicles earlier in mitosis will reform around the segregated chromosomes as they deconden se o Cytokinesis - the mitotic spindle forms within the nuclear envelope, which then pinches off, forming two nuclei • Post-mitotic cells in multicellular organism s can “exit” the cell cycle, enter G a0d remain there without proliferating further o Some cells stay in G temporarily and re-enter into the S phase that is regulated 0 Regulated Protein Phosphorylation and Degradation Control Passage through the Cell Cycle • Cell cycle is regulated by small number of heterodimeric protein kinases called cyclins • Cyclins increase/decrease in phase with the cell cycle • Catalytic subunits called cyclin -dependent kinases (Cdks) because they don’t have kinase activity unless associated with cyclin o their activity is regulated by phosphorylation at specific inhibitory and activating sites in the catalytic subunit o they regulate progress of a cell through the cell cycle o Large protein complexes also mark specific inhibitors of cell - cycle events for proteolytic degradation by proteasomes. • Each Cdk associate with different cyclins, and the associated cyclin determines which proteins are phosphory lated by the Cdk- cyclin complex • G 1dk complexes are expressed first when cells are stimulated to replicate o prepare the cell for S phase by activating transcription factors that cause expression of enzymes required for DNA synthesis and the genes encoding S -phase Cdk complexes • S-phase Cdk complex o activity of this is initially held in check by specific inhibitor o in late G1, G1 Cdk complexes induce the degradation of the S -phase inhibitor, releasi ng the activity of the S-phase Cdk complexes, which s timulate entry into the S phase o once activate, the complex phosphorylates regulatory sites in proteins that form DNA pre -replication complexes which assemble on replication origins during G1 , and also prevents re-assembly of new pre- replication complexes o Because of this inhibition, each chromosome is replicated just once during passage through the cell cycle, ensuring that the proper chromosome number is maintained in the daughter cells • Mitotic Cdk comple x o synthesized during S phase and G2, but not activated until after DNA synthesis o when activated, they induce chromosome condensation, breakdown of the nuclear envelope, assembly of the mitotic spindle apparatus, and alignment of condensed chromosomes at the metaphase plate o after proper association of all chromosomes with spindle microtubules, the complex activates anaphase- promoting complex (APC) . • APC directs the ubiquitin-mediated proteolysis of anaphase inhibitors, leading to inactivation of the protei n complexes that connect sister chromatids at metaphase o APC also directs proteolytic degradation of the mitotic cyclins, r esulting in decrease in mitotic Cdk activity permits the now separated chromosomes to decondense, the nuclear envelope to re -form around daughter-cell nuclei during telophase, and the cytoplasm to divide at cytokinesis, yielding the two daughter cells • Phosphatases dephosphorylate the proteins that form pre -replication complexes during early G1 of next cell cycle o these complexes can assemble at replication origins in pr eparation for the next S phase • G1 Cdk complexes o inactivates APC by phosphorylation, allowing the subsequent accumulation of mitotic cyclins during the S phase and G2 of the following cycle o Extracellular growth factors, c alled mitogens, induce the synthesis of G1 Cdk complexes o Once mitogens have acted for a sufficient period, the cell cycle continues through mitosis even when they are removed o The point in late G1 where passage through the cell cycle becomes independent of mitogens is called the restriction point • Passage through G1 → S phase, metaphase → anaphase, and anaphase → telophase and cytokinesis, is irreversible because they’re triggered by the regulated degradation of proteins o cells are forced to traverse the cell cycle in one direction only • Control of the cell cycle is achieved by regulating the synthesis and activity of G1 Cdk complexes Diverse Experimental Systems Have Been Used to Identify and Isolate Cell-Cycle Control Proteins • Diffusible factors in mitotic cells, now known to be mitotic Cdk complexes, cause chromosome condensation and vesiculation of the nuclear envelope in G1 and G2 cells when they are fused to mitotic cells. Similarly, S - phase Cdk complexes stimulate DNA replication in the nuclei of G1 cel ls when they are fused to S-phase cells. • Diffusible factors (S-phase Cdk complexes) in an S-phase cell can enter the nucleus of a G1 cell and stimulate DNA synthesis, but these factors cannot induce DNA synthesis in a G2 nucleus • The isolation of yeast cell -division cycle (cdc) mutants led to the identification of genes that regulate the cell cycle CELL-CYCLE CONTROL IN MAMMALIAN CELLS Introduction • Control of cell cycle during development and growth is critical for determining the size and shape of each tissue • Cell replication controlled by a network of signaling pathways that integrate extracellular signals about the identity and numbers of neighboring cells and intracellular cues about cell size and developmental program • Most cells leave cycle during G1 and enter G0 to differentiate o some can be stimulated to re-enter and replicate (i.e. fibroblasts and lymphocytes) o cells that don’t re-enter = postmitotic cells Mammalian Restriction Point is Analogous to start in Yeast Cells • Most studies are done with mammalian cultured cells that need certain polypeptide growth factors (mitogens) to stimulate cell proliferation • Binding mitogens to recep tor proteins on plasma membrane initiates signal transduction, that influence transcription and cell cycle control • Mammalian cells without mitogens are arrested with diploid chromosomes in G0 o If mitogens are added, these quiescent cells (cell that has exited the cell cycle) pass through the restriction point and enter the S phase and complete the rest of the cell cycle o restriction point = cells become committed to entering the S phase and completing the cell cycle o If the cells are moved from medium with mitogens, to one without before passing restriction point, they don’t enter the S phase Multiple Cdks and Cyclins Regulate Passage of Mammalian Cells through the Cell Cycle • Mammalian cells use a small family of related Cdks to regulate progression through the cell cycle • The principle Cdks active in most mammalian cells have been named Cdk1, 2, 4, and 6 • Cdk 1 o ability of a cDNA clone encoding it to complement S. pombe cdc2− mutants • Cdk 2 o ability to complement S. cerevisiae cdc28 mutants • Cdk 3 and Cdk 5 o the encoded proteins are not expressed at significant levels in most mammalian cells, and they function in processes other than cell cycle control • Mammalian cells express multiple cyclins • Cyclins A and B o function in S phase, G2, and early mitosis • Cyclins D and E o ability to complement S. cerevisiae cells mutant in all three CLN genes encoding G1 cyclins o expressed during G1 and are essential for passage through the restriction point • Different Cdk-cyclin complexes act in G0-arrested mammalian cells stimulate to divide by addition of growth factors o The absence of these proteins critical for stimulating the many molecular processes required for cell -cycle progression explains why G0 cells do not replicate Regulated Expression of Two Classes of Genes Returns G0 Mammalian Cells to the Cell Cycle • Addition of growth factors to G0 -arrested mammalian cells induces transcription of multiple genes, most of which fall into one of two classes — early-response or delayed-response genes — depending on how soon their encoded mRNAs appear • Early response genes o their transcription is induced quickly after addition of growth factors by a signal transduction cascade that leads to activation of specific transcription factors o their induction isn’t blocked by inhibitors of protein sy nthesis because the required transcription factors are present in G0 cells and are activated by post -translational modifications (i.e. phosphorylation) o they encode transcription factors that stimulate transcription of the delayed -response genes o Most of the immediate early proteins are unstable and, consequently, fall in concentration as the level of their mRNAs, and hence their rate of synthesis, falls. This drop in transcription is blocked by inhibitors of protein synthesis, indicating that it depends on production of one or more of the early-response proteins • Delayed response genes o not expressed when mitogens are added to Go -arrested cells in the presence of an inhibitor of protein synthesis because their expression requires the synthesis of early-response proteins o they encode additional transcription factors (i.e. E2Fs), D-type cyclins, cyclin E, Cdk2, Cdk4, and Cdk6. Cdk4, Cdk6 o If growth factors are withdrawn before passage through the restriction point, transcription of these G1-phase Cdks and cyclins ceases o these proteins and the mRNAs encoding them are unstable, their concentrations fall precipitously = the cells do not pass the restriction point and do not replicate. Passage through the Restriction Point Depends on Activation of E2F Transcription Factors • Mammalian genes encoding many of the proteins involved in DNA and deoxyribonucleotide synthesis are induced as cells pass through the G1 → S transition • A small family of E2F transcription factors is required for transcription of several of these ge nes and those encoding Cdk2 and cyclins A and E o E2Fs autostimulate transcription o f the genes encoding themselves • The transcription-activating ability of E2Fs is inhibited by their binding of Rb protein and two related proteins, p107 and p130 • Rb Proteins o binding of Rb to E2Fs converts them from transcriptional activators to repressors because Rb interacts with histone deacetylase complexes o they were initially identified as the product of the prototype tumor - suppressor gene Rb o Phosphorylation of Rb protein inhibits its repressing function, permitting activation of the genes required for entry into the S phase by E2Fs o phosphorylation of Rb protein is initiated by Cdk4 – cyclin D and Cdk6 – cyclin D in mid G1; once expression of Cdk2 and cyclin E is stimulate d, Cdk2 – cyclin E further phosphorylates Rb in late G1 o maintained in the phosphorylated state throughout the S, G2, and M phases by Ckd2 -and Cdk1-cyclin complexes o After cells complete mitosis and enter early G1 or G0, the fall in Cdk -cyclin levels leads to dephosphorylation of Rb protein by the a ction of unopposed phosphatases, à, hypophosphorylated Rb protein is available to inhibit E2F activity duri ng early G1 of the next cycle • Since E2Fs also stimulate their own expression, these processes form positive feedback loops for phosphorylation of Rb protein • Initial phosphorylation of Rb leads to generation of Cdk2 – cyclin E, which accelerates further phosphorylation of Rb o At this point, passage through the cell cycle is independent of Cdk4/6 – cyclin D activity, so that progression occurs even when mitogens are withdrawn and cyclin D le vels fall • The positive cross-regulation of E2F and Cdk2 – cyclin E produces a rapid rise of both activities as cells approach the G1 → S transition point o Events in addition to Rb phosphorylation also contribute to control of passage through the restrictio n point Cyclin A Is Required for DNA Synthesis and Cdk1 for Entry into Mitosis • Cyclin A o Synthesis begins as cells approach the G1 → S transition, and the protein is immediately transported into the nucleus o Disruption of cyclin A function inhibits DNA syn thesis in mammalian cells o Cdk2 – cyclin A may trigger initiation of DNA synthesis by pre -replication complexes assembled in early G1 and inhibit the assembly of new pre -replication complexes • Cdk 1 o is the principle mammalian Cdk in G2 o associates with cyclins A and B, and mRNAs encoding either of these mammalian cyclins can promote oocyte maturation when injected into Xenopus oocytes o the MPF activity of Cdk1 – cyclin A and Cdk1 – cyclin B, which induces entry into mitosis, appears to be regulated by proteins analogous to S. pombe Wee1, CAK, and Cdc25 • Cyclin B o is first synthesized during the S phase and increases in concentration as cells proceed through G2, peaking in early mitosis and dropping after anaphase o it first accumulates in the cytoplasm and then enters he nucleus just before the nuclear envelope breaks down early in mitosis o MPF activity may be controlled not only by phosphorylation/ dephosphorylation but also by regulation of the nuclear trans
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