Key Events in the Cell Cycle

Proper Chromosome Attachment to Spindle Corrected Slide from Lecture 17- New Title The Spindle Assembly Checkpoint Blocks Activation of 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

Cell Division Lecture 18 We will revisit and complete Apoptosis Lecture on Monday

Cell Cycle Overview

Preparation for the Onset of M-phase Duplication of Chromosomes Member of pair referred to as Sister Chromatid Held together by Proteins Complexes called Cohesions Duplication of Centrosomes Centriole Pair and other Components Duplicated Remain Together as a Single Complex in Nucleus

Kinetochore microtubule M phase in Animal Cells Depends upon Chromosome and Centrosome Duplication in the Preceding Interphase G2 PROPHASE PROMETAPHASE Centrosomes (with centriole pairs) Chromatin (duplicated) Aster Centromere Fragments of nuclear envelope Kinetochore Nucleolus Nuclear envelope Plasma membrane Kinetochore microtubule Nonkinetochore microtubules MT IF

Replicated Chromosomes Are Composed of Two Sister Chromatids Held Together by Cohesion Protein Complexes Centromeres DNA Replication Sister Chromatids

M-phase Requires Formation of a New Apparatus Called the Mitotic Spindle A Symmetrical, Bipolar Structure composed of Microtubules- with associated Motors and Other Proteins Each pole has a Centrosome Functions in M-phase: Responsible for Separating the Replicated Chromosomes into Daughter Nuclei during Division of the Nucleus Directs the Process of Dividing the Cytoplasm

Centrosome Duplication is Required for the Formation of a Bipolar Mitotic Spindle

M-CDK Activity is Required for Mitosis Key Ways to Regulate CDK Complex Activity: Proteolysis of Cyclin Subunit Phosphorylation/ Dephosphorylation Binding by Inhibitors Subcellular Localization

Mitosis Requires Active M-CDK During G2- Cyclin Levels are Increasing Form M-CDK Complexes But Held Inactive due to Inhibitory Phosphate

Active M-CDK Indirectly Activates More M-CDK Leads to Sudden Explosive Increase in Active M-CDK Levels

M–CDK Activity is Required for Triggering the Mitotic Machinery 1) Phosphorylation and Activation of Various Kinases 2) Direct Phosphorylation of Targets Chromosome Condensation Assembly of Mitotic Spindle Nuclear Envelope Breakdown

Stages of Mitosis: Prophase Individual Replicated Chromosomes Condense Nucleoli Disperse (in Animal Cells) Two Centrosomes Separate From Each Other Assembly of the Mitotic Spindle Outside of the Nucleus

Chromosome Condensation During Prophase: M-CDK Phosphorylates Condensin Proteins Cohesions Keep Sister Chromatids Together Condensins bind DNA Phosphorylated by M-CDK Results in Chromosome Condensation

G2 to Prophase Transition PROMETAPHASE Centrosomes (with centriole pairs) Chromatin (duplicated) Aster Early mitotic spindle Kinetochore Centromere Nonkinetochore microtubules Nucleolus Plasma membrane Chromosome, consisting of two sister chromatids Kinetochore microtubule Nuclear envelope

Prometaphase Marked by: 1) The Breakdown of Nuclear Envelope 2) Kinetochores Assembled on Chromosomes can now be attached to Spindle Microtubules 3) Chromosomes Attach to Mitotic Spindle Begin to move around

Nuclear Envelope Breakdown Marks Transition into Prometaphase by M-CDK

A Kinetochore Assembles on the Centromere Region of Each Sister Chromatid One Kinetochore Per Chromatid Sister Chromatids

Prophase to Prometaphase Transition G2 OF INTERPHASE PROPHASE PROMETAPHASE Centrosomes (with centriole pairs) Chromatin (duplicated) Early mitotic spindle Aster Fragments of nuclear envelope Kinetochore Nucleolus Nuclear envelope Plasma membrane Chromosome, consisting of two sister chromatids Kinetochore microtubules Interpolar Microtubules Now MT Have Access To Chromosomes!

Classification of Spindle Microtubules Kinetochore Microtubules- Attach to Kinetochores Polar Microtubules- Interact with Microtubules from Opposite Pole of the Cell Astral Microtubules- Nucleate from Centrosome Some Interact with Motor Proteins Associated with at Cell Cortex

Organization of the Mitotic Spindle

TELOPHASE AND CYTOKINESIS Metaphase Centrosome at one spindle pole Daughter chromosomes METAPHASE ANAPHASE TELOPHASE AND CYTOKINESIS Bipolar Spindle Metaphase plate Nucleolus forming Cleavage furrow Nuclear envelope reforming Maximal Chromosome Condensation Aligned at Metaphase Plate -Not actually still- jerky motions due to constant tension Forces Acting on Chromosomes to Align them At Metaphase Plate

Anaphase Abrupt Separation of Sister Chromatids Movement to Chromosomes to Opposite Pole Cell Elongates For Anaphase to Occur: 1) Activation of the Anaphase Promoting Complex (APC) Mediated Indirectly through M-CDK 2) Must Pass : SPINDLE ATTACHMENT CHECKPOINT All Chromosomes are properly attached to the spindle before Sister Chromatid Separation Occurs

Activation of the Anaphase Promoting Complex (APC), a Highly Regulated Ubiquitin Ligase APC Functions to Bring About: Cleavage of Cohesion Complex to Separate Sister Chromatids (through proteolytic degradation of inhibitory protein) Proteolytic Cleavage and Inactivation of M-phase Cyclin thereby inactivating M-CDK

Proteasome Degradation Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Protein to be degraded Ubiquinated protein Proteasome and ubiquitin to be recycled Protein fragments (peptides) Protein entering a proteasome E2 E3 Degradation Signal (APC) Key Targets: Securin And Cyclin of M-CDK

The Mechanism of Separation of Sister Chromatids Requires APC Activity Securin Separase Spindle Attachment Checkpoint: If A Kinetochore is Not Attached to The Spindle It Is Sensed The APC is Kept Inactive T APC-Anaphase Promoting Complex

Chromosome Separation During ANAPHASE Centrosome at one spindle pole Daughter chromosomes METAPHASE ANAPHASE TELOPHASE AND CYTOKINESIS Metaphase plate Nucleolus forming Cleavage furrow Nuclear envelope reforming Anaphase A- Sister Chromatids are Pulled to Opposite Poles Centromeres First Toward the Spindle Poles as Kinetochore MT Depolymerize Anaphase B- The Spindle Poles Move Away from Each Other as Polar MT Lengthen Anaphase A and B May Occur Simultaneously or Shortly After One Another Movements Rely on Motor Proteins that Associate with the Various MT

Movement Involved in Daughter Chromosome Separation During Anaphase A Anaphase A Movement Plus End of Kinetochore Microtubules Dissassembles Minus End Directed Motor (Dynein) Proteins Pull Toward Spindle Pole Pac Man Mechanism MT Depolymerize Dynein Walks Toward Minus End (-) Taxol Inhibits Grows to 2X the Metaphase Length

Movement Involved in Daughter Chromosome Separation During Anaphase B Polar MT Assembly Occurs at Plus End Plus End Directed Motor Proteins of Antiparallel Polar MT Slide Filaments 2.Astral MT Motor Proteins- Cytoplasmic Dynein Attach to Cell Cortex Moving toward Centrosome -Exert Outward pull on the spindle- help separate spindle poles 1. 2. Taxol Inhibits

Cytokinesis Divides the Cytoplasm Usually starts in late Anaphase and continues through Telophase (once sister chromatids are separated) Occurs by a Process called Cleavage in Animal Cells Requires Formation of a Contractile Ring Contractile Ring: A Dynamic Assembly of Actin, Bipolar Myosin II Filaments, and many structural and regulatory proteins Forms Under Surface of Plasma Membrane and Contracts to Pinch Two Daughter Cells Apart Cytokinesis Cannot Occur Until: 1) M-CDK is Inactivated- M-cyclin Targeted by APC for destruction by proteasome 2) Sister Chromatids have migrated to opposite poles of the cell – Remaining MT form Midbody - determines where Contractile Ring Formation will occur

Cleavage Requires The Contractile Ring Remaining Polar MT form Midbody Determines where Contractile Ring Formation will occur

Cleavage Furrow Formation in Animal Cells 1)Slight Indentation or Puckering on Cell Surface 2) Rapidly Deepens to Form Cleavage Furrow 3) Continues Until Opposite Surfaces Make Contact and Cell is Split in Two.

Telophase 1)Sister Chromatids have arrived at poles of the spindle 2) Now M-cyclin Degraded by APC Reversal of M-CDK Effects Including: -Nuclear Envelope Reforms around two groups of daughter chromosomes to form Two Interphase Nuclei -Chromosomes Decondense -Disassembly of Mitotic Spindle

Nuclear Envelope and Portions of Endomembrane System Reform During Telophase

Completion of Mitotic Phase of Cell Cycle Centrosome at one spindle pole Daughter chromosomes METAPHASE ANAPHASE TELOPHASE AND CYTOKINESIS Metaphase plate Nucleolus forming Cleavage furrow Nuclear envelope reforming