16. Cell Cycle and Mitosis I and II_McDermott_NOTES.pdf
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Cell Cycle and Mitosis [1] Paul J. McDermott, Ph.D. Office: 792-3462 Email: [email protected] CELL CYCLE & MITOSIS A. PHASES OF CELL CYCLE 1. Mitosis 2. Interphase B. G0 (QUIESCENT STATE) C. TERMINALLY DIFFERENTIATED STATE D. CYCLINS AND CYCLIN-DEPENDENT KINASES 1. Cyclins 2. Cyclin-Dependent Kinases...
Cell Cycle and Mitosis [1] Paul J. McDermott, Ph.D. Office: 792-3462 Email: [email protected] CELL CYCLE & MITOSIS A. PHASES OF CELL CYCLE 1. Mitosis 2. Interphase B. G0 (QUIESCENT STATE) C. TERMINALLY DIFFERENTIATED STATE D. CYCLINS AND CYCLIN-DEPENDENT KINASES 1. Cyclins 2. Cyclin-Dependent Kinases (CDK) 3. Function of CDK/Cyclin Complexes 4. Maturation Promoting Factor (MPF) E. REGULATION OF CDK ACTIVITY: 3 MECHANISMS 1. Formation of a CDK/Cyclin Complexes 2. Reversible Phosphorylation of CDK/Cyclin Complexes 3. Cyclin-Dependent Kinase Inhibitors (CKI) F. CELLULAR MECHANISMS OF CYCLE REGULATION 1. G1 Restriction Point 2. Growth Factor Control 3. DNA Damage Control Checkpoints 4. Role of Tumor Suppressor Genes G. MITOSIS 1. Stages of Mitosis 2. Prophase: Key Events 3. Kinetochores 4. Structure of Metaphase Chromosomes 5. Progression to Anaphase Suggested reading: • Marks’ Basic Medical Biochemistry, 5th Ed: Ch. 13 and Ch. 18 • The Cell: A Molecular Approach, 2nd Ed., http://www.ncbi.nlm.nih.gov/books/NBK9839/ “It ain’t over ‘till it’s over” Yogi Berra Cell Cycle and Mitosis [2] OBJECTIVES 1. Describe the phases of the cell cycle. 2. Define G0 and terminal differentiation and describe how these states relate to the cell cycle. 3. Specify the functions of cyclins and cyclin-dependent kinases (CDK) in regulating different phases of the cell cycle. 4. Specify 3 mechanisms for regulating the activity of CDKs during the cell cycle. 5. Define the restriction point and its position during the cell cycle. 6. Describe the mechanism by which a growth factor promotes cell cycle progression through the restriction point. 7. Describe cell cycle checkpoints for damage control and explain how the checkpoint kinases are activated. 8. Describe how Cdc25 inhibits cell cycle progression in response to DNA damage. 9. Describe the functions of Rb, p16, and p53 in the regulation of the cell cycle. 10. Describe the key events that occur during prophase, metaphase, anaphase, telophase and cytokinesis. 11. Describe the role of Cyclin B/CDK1 in mitosis. 12. Explain the role of cohesins and condensins in mitosis. 13. Describe how the following cellular components contribute to formation of the mitotic spindle: centrosomes, centromeres, kinetochores, microtubules. 14. Define a metaphase spread and differentiate between metacentric, submetacentric and acrocentric chromosomes. 15. Describe how APC triggers the progression from metaphase to anaphase. Illustrations adapted from: • Marks' Basic Medical Biochemistry: A Clinical Approach, © 2009 Lippincott Williams & Wilkins • The Cell: A Molecular Approach, 2nd Ed. © 2007, ASM Press and Sinauer Associates, Inc. • Molecular Cell Biology, 4th Ed. © 2000, W.H. Freeman and Company Cell Cycle and Mitosis [3] A. PHASES OF THE CELL CYCLE 1. M Phase (Mitosis) G0 Prophase Metaphase Anaphase Telophase and Cytokinesis 2. Interphase G1 = Gap1: metabolically active and growing S = Synthesis: DNA synthesis (2n to 4n) G2 = Gap2: cell growth and preparation for M phase B. G0 = QUIESCENT STATE • Cells exit G1 and stop dividing. Terminal Differentiation • G0 cells remain capable of re-entering the cell cycle in response to stimuli that trigger cell proliferation. C. TERMINALLY DIFFERENTIATED STATE • Cells have exited the cell cycle permanently and can no longer divide. • Many types of highly specialized cells are terminally differentiated and therefore not capable of cell division. Examples: neurons, cardiac muscle cells, skeletal myofibers, circulating leukocytes (neutrophils, eosinophils and basophils). D. CYCLINS AND CYCLIN-DEPENDENT KINASES (CDK) 1. Cyclins [A, B, D, E] • Family of proteins that controls progression through the cell cycle. • Cyclins bind specifically to enzymes called Cyclin-dependent Kinases (CDK) to form active complexes. • Level of each cyclin type fluctuates during different phases of the cell cycle to regulate activity of a specific CDK. 2. Cyclin-Dependent Kinases (CDK) • Family of protein kinases that phosphorylates a broad range of protein substrates (effectors) required to complete the cell cycle. • Levels of CDK remain relatively stable during the cell cycle. • Activation of CDK is dependent upon binding to its specific cyclin(s). Cyclin Cyclin + CDK CDK Inactive Active complex D. CYCLINS AND CYCLIN-DEPENDENT KINASES (CDK Cell Cycle and Mitosis [4] 3. Function of CDK / Cyclin Complexes Cell Cycle Cyclin/CDK Complex G1 Phase CDK4, CDK6/Cyclin D G1 to S Transition CDK2/Cyclin E S Phase CDK2/Cyclin A S to G2 Transition CDK1/Cyclin A G2 to M Transition CDK1/Cyclin B (MPF) 4. Maturation Promoting Factor (MPF) MPF is another name for the CDK1/Cyclin B complex. MPF functions as the master regulator of the G2 to M transition by phosphorylating proteins involved in mitotic processes including chromatin condensation, breakdown of the nuclear envelope and formation of the mitotic spindle. E. REGULATION OF CDK ACTIVITY: 3 MECHANISMS 1. Formation of CDK/Cyclin Complexes Cyclin levels fluctuate during different phases of the cell cycle. The amount of each cyclin available for formation of active CDK/cyclin complexes is regulated during the cell cycle by changes in its synthesis and degradation. Synthesis Cyclin Degradation (proteolysis) 2. Reversible Phosphorylation of CDK/Cyclin Complexes a) CDK-activating kinase (CAK): Phosphorylates CDKs to fully activate CDK/cyclin complex. b) CDK inhibitory kinases: Phosphorylate CDKs on other amino acids to inhibit the activity of the CDK/cyclin complex. c) Cdc25 family of phosphatases: Activate CDK/cyclin complexes by dephosphorylation of CDKs. c) Cdc25 b) Inhibitory kinases Pi Pi Inhibited a) CAK Activated Pi Fully Activated Cell Cycle and Mitosis [5] E. REGULATION OF CDK ACTIVITY: 3 MECHANISMS 3. Cyclin-Dependent Kinase Inhibitors (CKI) Family of proteins that binds to CDK/cyclin complexes and inhibit activity. Growth factors regulate the levels of CKIs by multiple mechanisms that control their synthesis and degradation. a) INK4 Family of CKIs [p15, p16, p18, p19] Specific inhibitors of CDK4,CDK6/Cyclin D - Blocks G1 phase b) Cip/Kip Family of CKIs [p21, p27, p57] Broader range of inhibition Inhibits CDK2/Cyclin E - Blocks G1 to S Inhibits CDK2/Cyclin A - Blocks S Inhibits CDK1/Cyclin A - Blocks S to G2 Inhibits CDK1/Cyclin B - Blocks G2 to M F. CELLULAR MECHANISMS OF CELL CYCLE REGULATION 1. G1 Restriction Point Decision point that occurs late in the G1 phase. If a cell passes through this point, then it becomes committed to S phase entry and completion of the cell cycle. a) Presence of growth factor(s): Cells stimulated to progress through G1 restriction point to S phase. b) Absence of growth factor(s): Cells stop at the the restriction point and can enter G0. Restriction Point S G0 G1 G2 M Cell Cycle and Mitosis [6] F. CELL CYCLE REGULATION 2. Growth Factor Control • Growth factors activate numerous intracellular signaling pathways that regulate the cell cycle by facilitating the G1 to S transition through the restriction point. • This example shows activation of the EGF receptor, which is a receptor tyrosine kinase that initiates the Ras-MEK-MAP kinase pathway. • The protein product of oncogenes are often gain of function mutations that result in loss of cell cycle control because of unregulated progression through the G1 restriction point. EGF Receptor Activation of MAP kinase (ERK) ↑ Cyclin D synthesis Activated CDK complexes Rb = Retinoblastoma gene product E2F = Family of Transcription Factors Phosphorylation of Rb by Cyclin D/CDK4, CDK6 causes its dissociation from E2F transcription factors Non-phosphorylated Rb binds to and inhibits E2F transcription factors Nucleus Target genes E2F activates transcription of target genes Ex: Cyclin E, Cyclin A Cell cycle progression to S phase a) Binding of growth factor to receptor: Induces expression of Cyclin D through the Ras/Raf/MEK/ERK signaling pathway. b) Binding of Cyclin D to CDK4 and CDK6: Causes formation of active complexes. c) Phosphorylation of Rb protein by Cyclin D/CDK4,CDK6 complexes: Causes the dissociation of Rb from E2F family of transcription factors. d) Function of E2F: Moves to nucleus and activates transcription of target genes required for cell cycle progression through the restriction point to the S phase. Examples include Cyclin E, Cyclin A and regulatory proteins of DNA Polymerases. Cell Cycle and Mitosis [7] F. CELL CYCLE REGULATION 3. DNA Damage Control Checkpoints Damage control checkpoints function as a mechanism to arrest cycle cell progression in response to either DNA damage or unreplicated DNA. The damage control checkpoint in G1 shown here is not the same as the restriction point. S G1 a) Detection of DNA Damage: 2 related protein kinases function part of DNA repair machinery by detecting DNA damage. • ATR: Kinase activated by single-strand breaks, UV damage, or by unreplicated DNA. • ATM: Kinase activated by double-strand breaks in DNA. G2 M ATM = Ataxia-Telangiectasia Mutated ; ATR = ATM- and Rad3-related b) Cell Cycle Arrest by Checkpoint Kinases • ATR: Activates Checkpoint Kinase 1 (CHK1) by phosphorylation. • ATM: Activates Checkpoint Kinase 2 (CHK2) by phosphorylation. UV irradiation, single-strand breaks or unreplicated DNA ATR Activation Double-strand breaks in DNA ATR ATM Activation ATM ATP ATP Pi Pi CHK1 CHK2 ATP Pi Cdc25 CDK1 CDK2 • CHK1 and CHK2 phosphorylate Cdc25 • Cdc25 phosphatase activity inhibited Inhibitory kinases Pi Pi Inhibited • CDK1 inhibition: G2 Arrest • CDK2 inhibition: G1 and S Arrest Active c) Ataxia Telangiectasia : Mutations in ATM gene cause inability to detect double-strand breaks in DNA. Consequently, CHK2 is not activated to trigger cell cycle arrest. • Severe neurological disorders (wheelchair by age 10) • Carriers have increased sensitivity to X-rays and radiation-induced breast cancer Cell Cycle and Mitosis [8] F. CELL CYCLE REGULATION 4. Role of Tumor Suppressor Genes Tumor suppressor genes encode proteins that function normally by regulating the cell cycle in response to stimuli such as DNA damage. Loss of function mutations mutations to both copies of these genes can cause cellular transformation and and lead to tumor development. Tumor Suppressor Gene Effect on Cell Cycle Protein Rb Binds to E2F transcription factors to inhibit transcriptional activation of target genes G1 arrest p16 INK4 family of CKI- inhibits CDK4 & CDK6 G1 arrest p53 Transcription factor induced by DNA damage activates transcription of p21 other genes G1 to S arrest a) Function of Rb • When Rb is non-phosphorylated, it binds to E2F transcription factors and inhibits activation of target genes required for the G1 to S transition. • When Cyclin D levels increase, Cyclin D/CDK4 and Cyclin D/CDK 6 complexes are activated and phosphorylate Rb, which causes it to dissociate from E2F transcription factors. ↑ Cyclin D synthesis Activated CDK complexes Phosphorylation of Rb by Cyclin D/CDK4, CDK6 causes its dissociation from E2F transcription factors Nucleus Non-phosphorylated Rb binds to and inhibits E2F transcription factors Target genes E2F activates transcription of target genes Ex: Cyclin E, Cyclin A Cell cycle progression to S phase b) Function of p16 • p16 is a member of INK4 family of cyclin-dependent kinase inhibitors (CKI). • p16 inhibits Cyclin D/CDK4 and Cyclin D/CDK6 - Blocks G1 phase • Loss of p16 function allows Cyclin D/CDK4 and Cyclin D/CDK6 complexes to remain active. Cyclin D Cyclin D p16 Cyclin D p16 p16 CDK4 CDK6 Active Cyclin D CDK4 CDK6 Inactive G1 Arrest Cell Cycle and Mitosis [9] F. CELL CYCLE REGULATION 4. Role of Tumor Suppressor Genes c) Function of p53 DNA Double Strand Breaks ATM Activation Nota Bene: p53 is referred to as the “Guardian of the Genome”. More than 50% of all human tumors contain p53 mutations. ATP ATM Pi CHK2 ATP p53 p21 Gene Pi Active p53 stabilized by phosphorylation, p53 levels increase Pi Cyclin E p53 p21 p21 G1 to S blocked CDK2 Increased expression of p21 (CKI) Stimulate transcription of the p21 gene Inhibition of Cyclin E/CDK2 Molecular Structure & Function of p53: p53 is a specific transcription factor that activates target genes involved in regulating the cell cycle and apoptosis. N TAD PRD DBD 4D CTD C Transactivation Domain (TAD) - Activation of RNA Polymerase II holoenzyme Proline Rich Domain (PRD) - Binding site for other regulatory proteins DNA Binding Domain (DBD) - Binds to specific sequences of target genes Tetramerization Domain (4D) - p53 binds to DNA as a tetramer Cytoplasmic Tail Domain (CTD) - Ubiquitination site that controls stability of p53 Cell Cycle and Mitosis [10] G. MITOSIS Mitosis is the M phase of the cell cycle during which the chromosomes are divided equally into 2 distinct nuclei. It is followed by cytokinesis which generates 2 daughter cells. Note that DNA replication and cell growth occur during interphase, before the G2 to M transition. Interphase G1 S G2 DNA = 2n M G1 DNA = 4n DNA = 2n DNA Replication Mitosis Cytokinesis DNA = 2n Chromosome pair Sister chromatids 1. Stages of Mitosis Chromosome condensation Alignment on metaphase plate Separation of sister chromatids • Prophase • Metaphase • Anaphase • Telophase & Cytokinesis • Transitional stages- Prometaphase, Early Anaphase and Late Anaphase Interphase Metaphase Early Prophase Anaphase Late Prophase Telophase Stages of mitosis visualized by fluorescent tags for chromatin (blue), microtubules (green) and intermediate filaments (red). Cell Cycle and Mitosis [11] 1. Stages of Mitosis Interphase (G1 → S → G2) • Chromosomes are replicated by DNA synthesis, thus, each chromosome consists of 2 sister chromatids. • Centrosome (2 centrioles) is replicated. Mitosis (M) Prophase • Chromosomes condense; sister chromatids are held together at the centromere. Proteins bind to centromere and form the kinetochore. • Centrosomes move to opposite poles and microtubules form the mitotic spindle. • Nuclear envelope breaks down and eventually disappears. Microtubules that form the mitotic spindle can attach to kinetochores. Metaphase • Chromosomes align along equator (metaphase plate) of the mitotic spindle. • Kinetochore microtubules complete attachment of sister chromatids to opposite poles of the mitotic spindle. Anaphase • Sister chromatids separate and move to opposite poles of the mitotic spindle. • Kinetochore microtubules shorten and spindle poles move away from each other as sister chromatids separate. Telophase • Daughter chromosomes decondense. • Nuclear envelope reassembles. • Cytoplasm is divided as contractile ring forms. Contractile ring Cytokinesis • Cytoplasm is completely divided by contractile ring of microfilaments composed of actin and myosin. Cell Cycle and Mitosis [12] G. MITOSIS 2. Prophase: Key Events The transition from G2 of interphase to prophase is triggered by activation of Cyclin B/CDK1. It phosphorylates multiple target proteins that are required for a) condensation of chromosomes, b) breakdown of the nuclear envelope, and c) formation of the mitotic spindle. Condensins & Histones H3 and H1 Cyclin B CDK1 Centrosomes & Microtubuleassociated Proteins (MAPs) Condensation of chromosomes Formation of mitotic spindle Lamins, Nuclear pore complexes & Inner nuclear membrane proteins Breakdown of nuclear envelope a) Condensation of Chromosomes G2 Prophase Cohesins Condensins Cohesins • Cohesins are a family of proteins that bind to DNA during the S phase and hold replicated chromosomes (sister chromatids) Metaphase together by creating interstrand crosslinkages. • Upon entry into prophase, Condensins are phosphorylated by Cyclin B/CDK1 and bind to DNA. Condensins produce intrastrand cross-links to condense chromatin. Histone phosphorylation is involved also in chromosome condensation. Cohesins are replaced except in the centromere regions where sister chromatids remain attached. Sister chromatids b) Breakdown of the Nuclear Envelope • Phosphorylation of lamins by Cyclin B/CDK1 causes depolymerization of the filaments that constitute the nuclear lamina, leading to breakdown of the nuclear envelope. • Phosphorylation of proteins in the nuclear pore causes disruption of nuclear pore complexes, while phosphorylation of proteins in the inner nuclear membrane causes its detachment from B type lamins in the nuclear lamina. • Lamins are dephosphorylated during telophase, which enables repolymerization of lamins to form intermediate filaments of the nuclear matrix. Late Prophase Prometaphase Nota Bene: Breakdown of the nuclear envelope occurs in late prophase. The transitional phase referred to as prometaphase starts when the nuclear envelope disappears. Cell Cycle and Mitosis [13] G. MITOSIS 2. Prophase: Key Events c) Formation of Mitotic Spindle • Cyclin B/CDK1 activation triggers migration of the centrosomes to opposite poles. • Microtubules are anchored at the minus ends in the pericentriolar material. • The mitotic spindle forms as microtubules grow rapidly at the plus ends by polymerization of tubulin. Pericentriolar material Centriole TEM of centrosome showing MTs radiating from the pericentriolar material 3. Kinetochores Kinetochore MTs of mitotic spindle • The kinetochore consists of a complex of proteins bound to DNA of the centromere. It serves as the attachment site for microtubules of the mitotic spindle. • When the sister chromatids separate during anaphase, kinetochore proteins function as a molecular motor to facilitate movement of the daughter chromosome along microtubules in the minus end direction, that is, toward the centrosome. Cohesins 4. Structure of Metaphase Chromosomes Homologous Chromosomes Sister Sister Chromatids Chromatids Telomere Centromere Telomere Metacentric short arm “p” long arm “q” Submetacentric Acrocentric Cell Cycle and Mitosis [14] G. MITOSIS 4. Structure of Metaphase Chromosomes Metaphase spread: karyotyping Cells arrested in metaphase are used for isolation of chromosomes in order to analyze their number and structure by staining or fluorescence in situ hybridization (FISH). Giemsa stained metaphase spread: 46,XY Chromosome “painting” by FISH: 46,XX 5. Progression to Anaphase a) Spindle Assembly Checkpoint: Metaphase does not progress to anaphase until kinetochore microtubules are attached and all chromosomes are aligned properly along the metaphase plate. b) Anaphase Promoting Complex (APC) • Progression from Metaphase to anaphase is dependent upon activation of APC. • APC activity is inhibited until all chromosomes are attached to mitotic spindle and aligned. • Activation of APC triggers the degradation of 2 main targets: Cohesins and Cyclin B. Cohesin degradation Metaphase Separation of sister chromatids APC activation Anaphase Cyclin B Cyclin B degradation CDK1 CDK1 Inactivation of CDK1
