The Cell Cycle/Le Cycle cellulaire SMC6052/BIM6028 IRCM

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1 The Cell Cycle/Le Cycle cellulaire SMC6052/BIM6028 IRCM 1 février 2018 Benjamin H. Kwok, Ph.D. Chercheur principal, Institut de recherche en immunologie et en cancérologie Professeur sous octroi agrégé, Département de biochimie et médecine moléculaire Université de Montréal

2 Metaphase Prometaphase Anaphase The Cell Cycle Prophase Telophase Interphase (G1 S G2) Cytokinsesis

3 Main Reference : The Cell Cycle: Principles of Control By David O Morgan New Science Press Note to students: These slides are made available to the students as a courtesy. They are not meant to substitute for a presence in class and notes need to be taken following my explanations. And at courtesy of Dr. Vincent Archambault (IRIC), who prepared some of the slides.

4 1 The Cell Cycle: Introduction

5 The cell cycle. Cell reproduction begins with duplication of the cell s components, including the exact duplication of each chromosome in S phase. These components are then divided equally between two daughter cells.

6 The events of the eukaryotic cell cycle.

7 A simple cell cycle control system. In some early embryonic cell cycles, the cell cycle control system behaves like an autonomous one hand clock whose rotation is programmed to sequentially trigger cell cycle events with appropriate timing. The clock continues to operate normally even when cell cycle events fail.

8 Cyclin dependent kinase activation. The cell cycle control system is based on cyclin dependent kinases (Cdks) that are activated at specific cell cycle stages by regulatory subunits called cyclins.

9 Overview of cell cycle control. Progression through the cell cycle is governed at three major cell cycle checkpoints. In mid to late G1, Cdks are activated by G1/S and S phase cyclins, resulting in entry into the cell cycle at Start. Entry into mitosis (at the G2/M checkpoint) is triggered by activation of M phase Cdk cyclin complexes. Finally, the metaphase to anaphase transition is driven by a regulatory enzyme called the anaphasepromoting complex (APC), which triggers the destruction of cyclins and other regulators.

10 2 Model organisms to study the cell cycle

11 Variations in cell cycle structure in different cell types.

12 2a. Xenopus laevis Early embryogenesis in the frog Xenopus laevis Morgan: The Cell Cycle, Principles of Control

13 Oocyte maturation and fertilization in Xenopus Progesterone: First diploid nucleus of the zygote

14 2b. Yeast Two powerful genetic systems The budding yeast Saccharomyces cerevisiae The fission yeast Schizosaccharomyces pombe

15 The budding yeast Saccharomyces cerevisiae The fission yeast Schizosaccharomyces pombe

16 2b1. S. cerevisiae: Screens for Cell Division Cycle (CDC) genes chemical mutagenesis Look for mutants that grow at one temperature, but not at another temperature (temperature sensitive; ts mutants) Leland Hartwell Nobel prize 2001 Examine phenotype and analyze Hartwell et al (1970). Genetic control of the cell division cycle in yeast. I. Detection of mutants. PNAS 66(2):352 9.

17 Hartwell et al (1970). Genetic control of the cell division cycle in yeast. I. Detection of mutants. PNAS 66(2): Hartwell (1971). Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J Mol Biol 59(1): Culotti & Hartwell (1971). Genetic control of the cell division cycle in yeast. 3. Seven genes controlling nuclear division. Exp Cell Res 67(2): Hartwell (1971). Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp Cell Res 69(2): Hartwell et al (1974). Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics 74(2):

18 Examples of cell cycle arrests in budding yeasts cdc mutants

19 Functions of CDC genes in budding yeast

20 Some famous, conserved CDC genes CDC2: subunit of a DNA polymerase CDC3: component of the septin ring (bud neck) CDC4: F box subunit of the SCF ubiquitin ligase required for G1/S and G2/M transition CDC5: Polo like kinase with multiple functions in mitosis and cytokinesis CDC6: ATP binding protein required for DNA replication, component of the pre replicative complex CDC7: Kinase required for firing origins and replication fork progression in S phase CDC14: Protein phosphatase required for mitotic exit CDC15: Protein kinase of the Mitotic Exit Network CDC16: Subunit of the anaphase promoting complex/cyclosome (APC/C) CDC20: Cell cycle regulated activator of anaphase promoting complex/cyclosome (APC/C) CDC28: Catalytic subunit of the main cell cycle cyclin dependent kinase (CDK)

21 Some other series of genes identified in yeast MAD: Mitotic Arrest Deficient MCM: Mini Chromosome Maintenance RAD: Radiation sensitive SWI: Mating type SWItch CUP: Copper resistance ACE: Activation of CUP1 Expression BUD: Budding defective ETC

22 2b2. S. pombe: Screens for Cell Division Cycle (CDC) genes Paul Nurse Nobel Prize 2001 Schizosaccharomyces pombe (Fission yeast) (G2, cell growth) cdc genes (Cell Division Cycle) wee genes (Wee = small in Scottish)

23 2c. The fruit fly Drosophila melanogaster

24 Early divisions in Drosophila. After fertilization, rapid nuclear divisions occur without cytoplasmic division, resulting in the formation of a syncytium. After about nine nuclear divisions, nuclei begin to migrate to the surface of the embryo to form the syncytial blastoderm. Membranes grow inward from the cell surface and surround the nuclei at the end of the 13th division, resulting in the formation of the cellular blastoderm. About 15 pole cells form a separate group of cells in the posterior end of the embryo and will eventually give rise to the germ cells.

25 Drosophila Early embryonic cycles when a regulatory checkpoint is absent. Drosophila Early Embryonic Divisions In Perfect Synchrony Tubulin Rosalind V Silverman Gavrila American Society of Cell Biology Image and Video Library

26 Life cycle of Drosophila melanogaster.

27 The imaginal discs of Drosophila larva are small sheets of epithelial cells in the larva. During metamorphosis, they give rise to a variety of adult structures.

28 Mammalian cells growing in culture (Red: actin; Blue: DNA) Interphase Telophase and cytokinesis

29 The cellular DNA content can be analyzed by flow cytometry. Synchronous progression through the cell cycle

30 3 The S phase: Chromosome Duplication

31 Overview of chromosome duplication in the cell cycle

32 Mechanismsthatlimitpre replicative complex formation to G1

33 4 Mitosis: Chromosome Segregation

34 Stages of mitosis in a vertebrate cell

35 Mitosis is mainly controlled by cyclical Cyclin B Cdk1 activity. Activity Cyclin B Cdk1 APC Mitotic entry Mitotic exit time Interphase Mitosis Nuclear envelope breakdown Chromosome condensation Spindle assembly etc Interphase

36 Overview of mitotic control mechanisms. In early mitosis, mitotic cyclin Cdk complexes trigger the events leading to metaphase. Mitotic Cdks promote their own activation, resulting in positive feedback. M Cdks also activate the ubiquitinprotein ligase APC Cdc20, resulting in the destruction of two proteins: securin, a protein that inhibits sister chromatid separation, and mitotic cyclins. The destruction of securin results in sister chromatid separation. Cyclin destruction causes Cdk inactivation, which leads to the completion of mitosis and cytokinesis. Progression of mitosis can be blocked at the G2/M checkpoint. In all eukaryotes, defects in spindle assembly inhibit APC Cdc20, thereby preventing progression through the metaphase to anaphase transition.

37 5 The cell cycle control system

38 Approaches to study the cell cycle: Genetics versus Biochemistry Science: Trying to understand how things work Genetics versus Biochemistry: A geneticist view: These components as well as others are essential to make the car run. A biochemist view: This multi component complex can generate force when an energy source is provided.

39 A biochemical approach to study the cell cycle: A brief History Yoshio Masui Clement Markert Biochemistry: Extraction, perturbation & monitoring Masui & Markert, J Exp Zoo 1971

40 The early days: MPF and CSF Maturation (Maturation Promoting Factor / MPF) Tunquist & Maller, Genes & Dev 2003

41 1. Chasing MPF with Biochemistry: Fundamental Principle: Purify, Purify, Purify! From crude to purity! Biochemistry: Extraction, perturbation & monitoring Lohka et al., PNAS 1988

42 A brief overview of the cell cycle control system.

43 Cyclin dependent kinase activation. The cell cycle control system is based on cyclin dependent kinases (Cdks) that are activated at specific cell cycle stages by regulatory subunits called cyclins.

44 The Cyclin Cdk system is conserved among species.

45 Two steps in Cdk activation. Cyclin binding alone causes partial activation of Cdks, but complete activation also requires activating phosphorylation by CAK.

46 Control of Cdk activity by inhibitory phosphorylation. The fully active cyclin Cdk complex (center) can be inhibited by further phosphorylation at one or two sites in the active site of the enzyme. Phosphorylation of Tyr15 by Wee1, or phosphorylation of both Thr14 and Tyr15 by Myt1, inactivates the cyclin Cdk complex. Dephosphorylation by the phosphatase Cdc25 leads to reactivation.

47 Cyclins are destroyed by the ubiquitin proteasome system. Steps in protein ubiquitination: The SCF complex (E2 E3, active mostly in G1/S, targets G1 and S cyclins and not M cyclins) The APC complex (E2 E3, active mostly in M and M/G1, targets M cyclins) Cdc20 or Cdh1

48 Mitotic cyclin levels in a somatic mammalian cell. Cyclin A is primarily a nuclear protein whose levels and associated Cdk2 activity rise in early S phase and drop in prometaphase (pale purple line). Cyclin B1 (dark purple) begins to accumulate in G2 and reaches peak levels just before its destruction in metaphase. The protein kinase activity of cyclin B1 Cdk1 complexes (red) remains low in G2, owing to inhibitory phosphorylation of Cdk1. Removal of these inhibitory phosphorylations in prophase results in the sudden, all or none activation of Cdk1. In late prophase, cyclin B1 Cdk1 complexes are translocated from the cytoplasm to the nucleus. The nuclear envelope breaks down several minutes later, defining the onset of prometaphase.

49 Nuclear accumulation of cyclin B1 Cdk1 and Cdc25C in late prophase

50 Multiple feedback loops governing Cdk1 activation in early mitosis

51 The spindle assembly checkpoint (SAC) during mitosis Musacchio & Salmon, 2007, Nat Rev Mol Cell Biol Peters, 2006, Nat Rev Mol Cell Biol

52 Control of late mitotic events by the APC. M Cdk activity promotes the events of early mitosis, resulting in the metaphase alignment of sister chromatids on the spindle. M Cdk activity also promotes the activation of APC Cdc20, which triggers anaphase and mitotic exit by stimulating the destruction of regulatory proteins, such as securin and cyclins, that govern these events. By promoting cyclin destruction and thus Cdk inactivation, APC Cdc20 also triggers activation of APC Cdh1, thereby ensuring continued APC activity in G1.

53 Kinase in control: Assembling a Cdk oscillator. Embryonic cell cycle (no G1) Cell cycle in most cells (with G1)

54 Major kinases in the mammalian cell cycle Malumbres, M. (2011), Physiological Reviews.

55 The Polo kinase is required for multiple steps of cell division. Discovered in Drosophila as polo Sunkel & Glover, Petronczki et al, 2008, Dev Cell

56 Different strategies targeting mitosis in anti cancer therapies Manchado et al, (2012). Cell Death & Differentiation