Lecture 17 Regulation of the Cell Cycle and Cell Death

The Cell Division Cycle A cell contains all the information necessary for making a copy of itself during cell division Cells reproduce by carrying out a highly ordered sequence of events called the CELL CYCLE –Typically Involves: Cell Growth DNA Replication Distribution of Duplicated Chromosomes to Daughter Cells Cell Division

Phases of the Eukaryotic Cell Cycle

Different Cell Types have Variations in Timing, Control and Progression Through Cell Cycle In the Adult Human Body -Most Cells Are Not Actively Dividing -Some Cells Are Continuously Dividing -Example: Bone Marrow Cells, Epithelial Cells Some Cells Do Not Divide After Differentiation Example: Nerve Cells Some Cells can Be Triggered to Divide to Replace Cells that have Died Example: Cells of Liver Tissue

Control of Cell Cycle Involves Monitoring Intracellular And Extracellular Conditions The Control System Needs to Regulate Progression through the Cell Cycle to Ensure: 1)Events Associated with Each Phase are Carried Out at the Appropriate Time and in the Appropriate Order 2) Each phase is complete Before Next Phase is Initiated 3) Must be Able to Respond to External Conditions Required for Cell Growth and Division. A Series of Control Points called Checkpoints Accomplish These Objectives

Cell Cycle Checkpoints Regulate Progression Through the Cell Cycle

Cell Fusion Experiments Provide Evidence for Control Molecules in the Cell Cycle. S SSMM MG1G1 G1G1 S Phase is Activated In Original G1 Nucleus M phase is Activated In Original Non-M Nucleus

The Cell Cycle Is Controlled by Cyclin Dependent Kinases (CDK) CDK forms a Complex with a Protein called a Cyclin to form a CDK Complex Different Cyclin-CDK Complexes Trigger Different Steps in the Cell Cycle 4 Major Types of Cyclin-CDK Complexes: G1-CDK G1/S-CDK S-CDK M-CDK

Regulation of the Activity of CDK Complexes 1)Proteolysis of Cyclin Component 2) Phosphorylation/Dephosphorylation 3) Binding of Inhibitory Proteins 4) Subcellular Localization

Cyclin Protein Concentration and Cyclin-CDK Activity Oscillate during the Cell Cycle

Post Transcritional Control of Cyclins: M-Cyclin is Ubiquitinated and Subsequently Degraded by the Proteasome

Cyclin Destruction Occurs at Specific Points during the Cell Cycle

Checkpoints in the Cell Cycle

Progression through the G 1 Checkpoint Cells Need to Check for : Cell Size Nutrients Mitogens and Growth Factors DNA Damage Called the Restriction Point in Mammals Commits Cell to the Process of Cell Division

G 1 checkpoint G1G1 G1G1 G0G0 Cells Can Delay Cell Division by Entering Specialized Nondividing State, G O Green Light to Proceed: Environment Favorable Red Light – Don’t Proceed Environment Unfavorable (Absence of Mitogenic Signals) Most cells in our body are In G o State:

In the Absence of Mitogenic Signals, The protein Rb Inhibits Cell Cycle Progression Rb- Retinoblastoma Protein Binds Transcription Factor E2F and Prevents Function E2F Required for: Activating Transcription Of Genes Encoding Proteins Required for G 1 /S transition G 1 /S Cyclins, S Cyclins and Components of DNA Replication Machinery

The G 1 -CDK Complex Controls the G 1 Checkpoint by Phosphorylating the Rb Protein

G 1 DNA Damage Checkpoint DNA Damage Needs to be Repaired before DNA Synthesis Occurs Activation of Transcription Factor p53 leads to synthesis of CDK inhibitor (CKI), p21 p21 binds and inhibits G1/S CDK and S-CDK Complexes Cells Arrest Until Damage is Repaired and Then Progress Or Undergo Apoptosis if Unable to Repair

p53 Mediates the G 1 DNA Damage Checkpoint Through Inactivation of G 1 /S- and S-CDK Complexes

Checkpoints in the Cell Cycle

S-CDK Complexes Are Required for DNA Replication During S-phase S-CDK Controls: 1) Initiation of Replication 2) Prevents Re-replication from a Particular Origin

Progression Through the G 2 Checkpoint to Enter Mitosis Check for: Cell Size DNA Replication Complete Passage through G 2 Checkpoint Requires Active M-CDK: Functions to Phosphorylate Proteins Involved in Early Stages of Mitosis 1)Nuclear Envelope Breakdown 2)Chromosome Condensation 3)Mitotic Spindle Formation 4)Targeted Protein Degradation

Unreplicated DNA Sensed Blocks Activating Phosphatase Unreplicated DNA Blocks Activation of M-CDK Complex T

Checkpoints in the Cell Cycle

M-CDK Controls The Spindle Assembly Checkpoint by Activating the Anaphase Promoting Complex (APC ) Check for: Proper Chromosome Attachment to Spindle Phosphorylation of APC- now Activated Sister Chromatids Can Separate If Chromosomes not Properly Attached Metaphase Arrest Will not Separate

Exit From Mitosis Now Need to Reverse Events: Nuclear Envelope Breakdown Chromosome Condensation Mitotic Spindle Formation Destroy M-Cyclin M-CDK Activates APC APC Targets M-Cyclin for Destruction by the Proteasome

Cancer Involves Defective Cell Cycle Control Mechanisms Cancer Inducing Mutations Inherited or Introduced by Viruses Two Key Types of Mutated Genes That Can Lead to Cancer: Oncogenes - Gene whose presence can trigger development of cancer. Example: ras, bcl-2 Tumor Suppressors- Gene whose absence or inactivation can lead to cancer Example: p53, Rb

Mitogens and Growth Factors Activate the Ras Pathway Leads to Passage through the G 1 Checkpoint

p53 Can Induce Apoptosis in DNA Damaged Cells Can’t Repair DNA DAMAGE Apoptosis

Apoptosis: Regulated Cell Death Role in Killing of Unneeded, Damaged, or Potentially Deleterious Cells Occurs in Embryonic and Adult Tissues Proteins Involved are Always Present in Cells- Needs to Be Activated by Stimuli Can Result From: Developmental Cues Withdrawl of Essential Growth Factors DNA Damage Various Cell Stresses

Programmed Cell Death Cell Death Occurring at a Defined Point in Development Usually proceeds by Apoptosis Mouse Paws

Not All Cell Death is Apoptotic Apoptosis: An Active Regulated Process DNA Fragmentation Chromatin Condensation Fragmentation of Nucleus Cell Shrinks Formation of Membrane Enclosed Fragments called Apoptotic Bodies Recognition and Engulfment by Phagocytic Cells or Neighboring Cells Oncosis and Necrosis: Unregulated Cell Death Due to Injury Cell Swells (Oncosis) Nucleus Swells Disruption of Organelles and Rupture/Release of Contents Contents Released into Extracellular Space

Necrosis and Apoptosis are Morphologically Distinct Necrosis Apoptosis

The Morphological Changes of Apoptosis Are Orchestrated by Caspases Cysteine Proteases that cleave at Aspartic Acid Residues Activate Apoptosis by Cleaving Specific Substrates Present but inactive in cells Two Main Types of Caspases 1) Initiators- Need to dimerize to become active “induced proximity” 2) Executioners- Need to be proteolytically cleaved to become active - Cleavage is usually Mediated by Initiator Caspases Once Executioners are Activated their Key Targets of Proteolysis Include: 1)An Inhibitor of a DNAse- Fragmentation of DNA 2)Nuclear Lamins- Fragmentation of Nucleus 3)Other Cytoskeletal Associated Proteins- Disruption of Cytoskeleton and Cell Fragmentation 4)Activation of Additional Caspases

Caspases are Present but Inactive in Cells Zymogens: Proteins initially synthesized as inactive precursors- undergo proteolytic cleavage to become active

Caspase Activation Amplification Cascade So How Are Initiator Caspases Activated to get the Process Going??

Main Pathways Regulating Caspase Activation During Apoptosis Intrinsic Pathway- Mitochondrial Mediated Major Pathway in Mammalian Cells –Outer Mitochondrial Membrane Permeabilization (MOMP) –Release of Cytochrome C from Mitochondrial Intermembrane Space into Cytosol –Apoptosome Formation- Activation of Initiator Caspase –Effector Caspases Activated Extrinsic Pathway- Signaling through Death Receptors –Ligand Bound Death Receptors –Adaptor Protein Association –Initiator Caspase Recruitment and Activation –Effector Caspases Activated

Intrinsic Pathway of Apoptosis Activation Association of Adaptor with Procaspase allows Procaspase self cleavage MOMPs cytochrome c Release Apoptosome Formation: Adaptor (Apaf1), dATP cytochrome c and procaspase complex Active Initiator Caspase Cleaves Effector Caspases Which now Cleave Targets

Critical Regulators of Cell Death Bcl-2 Family – Regulate whether MOMPs Occurs Anti-Apoptotic Factors - Death Inhibitors A) Function to Inhibit Cytochrome C release Pro-Apoptotic Factors- Death Activators A) Bind and inhibit Death Inhibitors B) Directly cause Permeabilization of Stimulate Release of Cytochrome C ( BAX AND BAK) IAP Family (Inhibitor of Apoptosis) Bind Procaspases prevent activation Bind Caspases and inhibit Activity

Survival Factor Signaling is Required to Prevent Apoptosis

Programmed Cell Death in Neuronal Development

Survival Factors Signaling Keeps Death Inhibitor Bcl-2 Active No Survival Signal Bcl-2 Complexes with Bad Can’t prevent BAK and BAX Mediated MOMPs

Extrinsic Pathway of Apoptosis Activation: Signaling through the Death Receptors Ligand Bound Death Receptors Adaptor Protein and Procaspase Recruitment Initiator Caspase Activation Effector Caspases Activated Target cells : Viral Infected Cells or Cancer Cells Removal of Excess Lymphocytes after Infection