Cell Cycle and Regulation Detailed Notes

G0 Phase

• Definition: A resting phase that cells can enter, technically outside the cell cycle.
• Characteristics:
  • Cells are alive but not actively growing or dividing.
• Types of G0 Cells:
  • Quiescent:
    • Reversible state.
    • Cells can re-enter the cell cycle when appropriate signals are present.
    • Example: Liver cells
  • Differentiated:
    • Irreversible state.
    • Cells are in their final form and cannot re-enter the cell cycle.
    • Examples: Neurons, differentiated blood cells

Cell Cycle Overview

• Two Main Phases:
  • Interphase (I)
  • Mitosis (M)
• Interphase Breakdown:
  • Gap 1 (G1):
    • Cell growth and preparation for DNA synthesis.
  • S Phase:
    • DNA replication.
  • Gap 2 (G2):
    • Continued cell growth and preparation for cell division.
• Mitosis:
  • Cell division.

Cell Cycle Regulation

• Checkpoints:
  • The cell cycle is heavily regulated with specific checkpoints.
  • Progression requires meeting all criteria at each checkpoint.
• Key Checkpoints:
  • G1 Checkpoint:
    • Occurs at the start of the cell cycle.
    • Determines if conditions are suitable to begin DNA synthesis.
  • G2 Checkpoint:
    • Occurs at the end of G2 phase.
    • Ensures DNA replication and repair are complete.
  • Metaphase Checkpoint:
    • Verifies proper chromosome attachment to the mitotic spindle before division proceeds.

Cell Cycle Control System

• Regulatory Proteins:
  • The cell cycle is controlled by proteins, especially protein kinases and phosphatases.
• Cyclin-Dependent Kinases (Cdks):
  • Cdks are present throughout the cell cycle but are typically inactive.
• Cyclins:
  • Cyclins are produced in cyclical waves.
  • They bind to Cdks, activating them into functional kinases.

Cyclin Levels and Cdk Activity

• Different Cdks and Cyclins:
  • Different Cdks and cyclins exist.
• M-Cyclin Example:
  • M-cyclin concentration peaks during mitosis, activating M-Cdk.
  • M-cyclin levels decrease during interphase and rise again as mitosis approaches.

Cdk and Cyclin Associations

• G1/S-Cdk:
  • Bound to G1/S-cyclin.
  • Triggers progression through the Start transition, committing the cell to complete the cell cycle.
• S-Cdk:
  • Bound to S-cyclin.
  • Triggers S phase.
• M-Cdk:
  • Bound to M-cyclin.
  • Triggers the events of M phase.
• Function:
  • Each activated Cyclin/Cdk complex phosphorylates different regulatory proteins.
  • This phosphorylation leads to differential gene expression needed for specific phases of the cell cycle.

Cyclins and Cdks of Vertebrates

• G1-Cdk:
  • Cyclin: Cyclin D
  • Cdk Partner: Cdk4, Cdk6
• G1/S-Cdk:
  • Cyclin: Cyclin E
  • Cdk Partner: Cdk2
• S-Cdk:
  • Cyclin: Cyclin A
  • Cdk Partner: Cdk2
• M-Cdk:
  • Cyclin: Cyclin B
  • Cdk Partner: Cdk1

Cyclin Cycling

• Control Factors:
  • Cyclin cyclic levels are controlled by two factors:
    • Increased gene expression (slow increase in cyclin levels)
    • Polyubiquitination and degradation by the proteosome (rapid decrease in cyclin levels)
• M-Cyclin Example:
  • M-cyclin is ubiquitinated by the anaphase-promoting complex (APC/C).
  • This leads to its degradation and inactivation of M-Cdk.

Further Cyclin Regulation

• Inhibitory Phosphorylation:
  • Cyclin-Cdk complexes are further regulated by inhibitory phosphorylation events.
  • Inhibitory kinases phosphorylate two sites on the Cdk to prevent premature activation.
  • The complex remains inactive until a phosphatase removes these phosphates.
• M-Cyclin/M-Cdk Example:
  • Wee1 is the inhibitory kinase.
  • Cdc25 is the phosphatase that removes the phosphates.

Cdk Inhibitor Proteins (CKIs)

• Function:
  • Cdk inhibitor proteins block the activity of Cyclin-Cdk complexes.
  • They bind to the complex and prevent its activity.
• Common Time:
  • Common during G1 phase.
• p27 Example:
  • During G1 phase, p27 binds to the G1/S-cyclin/Cdk complex.

Protein Phosphatases

• Function:
  • Phosphatases dephosphorylate Cdk targets to prevent them from acting at the wrong time.
• PP2A-B55 Example:
  • When active, PP2A-B55 dephosphorylates M-Cdk targets.
  • M-Cdk phosphorylates PP2A-B55 as it becomes more functional, inactivating PP2A-B55.
  • As M-cyclin degrades via APC/C and the proteosome, PP2A-B55 is reactivated.
  • Reactivated PP2A-B55 dephosphorylates the M-Cdk targets.

Cell Cycle Inhibition

• Environmental Monitoring:
  • The cell constantly monitors intra- and extracellular environments.
• Checkpoints and Conditions:
  • G1 Checkpoint:
    • The cell will not proceed to S phase if the environment is unfavorable.
  • G2 Checkpoint:
    • Entry into mitosis is prevented if DNA replication or repair is incomplete.
    • Cdc25 dephosphorylates target proteins.
  • Metaphase Checkpoint:
    • Mitosis completion is prevented if chromosomes are not attached to the mitotic spindle.
    • APC/C complex is inhibited.

G1 Phase Details

• Inactivation of Remaining Complexes:
  • All active Cdk-cyclin complexes remaining from mitosis are inactivated via phosphatase and cyclin degradation.
• Cellular Activities:
  • The cell continues to grow and perform its biological functions.
• Decision Point:
  • The cell decides whether to enter G0 phase, undergo quiescence, or terminal differentiation.

Mitogens

• Definition:
  • Extracellular signals that induce mitosis.
• Mechanism:
  • Mitogens bind to mitogen receptors (RTKs or GPCRs).
• G1 Phase Pause:
  • Cells pause in G1 if not exposed to mitogens.
• G0 Entry:
  • Prolonged absence of mitogens, combined with other signals, leads to quiescent G0 entry.
• Terminal Differentiation:
  • Extracellular signals induce terminal differentiation (e.g., in nerve cells).
  • This involves shutting down expression of cyclin and Cdk genes.

Mitogen Function

• Activation:
  • Mitogen signaling activates G1-Cdk and G1/S-Cdk complexes.
• Retinoblastoma (Rb) Protein Phosphorylation:
  • These complexes phosphorylate Retinoblastoma (Rb) protein.
  • Rb typically binds to transcriptional regulator regions, turning off gene expression.
• Gene Expression Release:
  • Once phosphorylated, Rb changes conformation and releases from the DNA, allowing gene expression.

DNA Damage and Cell Cycle Arrest

• p53 Regulation:
  • p53 is a transcriptional regulator that is normally translated and broken down quickly.
• Activation by DNA Damage:
  • DNA damage causes p53 to be phosphorylated, activating it.
  • Functional p53 transcribes genes involved in DNA repair.
• p21 Production:
  • p53 also promotes transcription of p21, which inhibits G1/S-Cdk and S-Cdk.
• Cancer Relevance:
  • p53 is mutated, absent, or underexpressed in approximately half of cancers.

S Phase Details

• Assembly of Replication Proteins:
  • During G1, proteins required for DNA synthesis begin to assemble on the origins of replication on the chromosomes.
• Origin Recognition Complex (ORC) Activation:
  • S-Cdk phosphorylates and activates the origin recognition complex (ORC) at the start of S phase.
• DNA Synthesis Initiation:
  • Activated ORC leads to the beginning of DNA synthesis.
• Prevention of Re-replication:
  • Once phosphorylated, these proteins cannot recognize another origin of replication until phosphatases act on them after S phase is complete.

Entry into Mitosis

• M-Cyclin Buildup:
  • During G2, M-cyclin begins to build up and form complexes with M-Cdk.
• Inhibitory Phosphorylation:
  • These complexes are initially inactivated by inhibitory phosphate groups.
• Cdc25 Activation:
  • Cdc25 is phosphorylated by another kinase and begins to dephosphorylate M-Cdk.
• Positive Feedback Loop:
  • This dephosphorylation starts a positive feedback loop, leading to rapid activation of M-Cdk.

Cohesins and Condensins

• Driven by M-Cdk:
  • This process is driven by M-Cdk.
• Cohesins:
  • Chromosomes are held together in looped structures by cohesins.
  • Specialized cohesins hold sister chromatids together after gene replication.
• Condensins:
  • During early mitosis, many cohesins are replaced by condensins, leading to chromosome condensation.

Cytoskeleton Role During Mitosis

• Cytoskeletal Filaments:
  • After chromosome condensation, two different cytoskeletal filaments begin to assemble.
• Mitotic Spindle:
  • Composed of microtubules and microtubule-associated motor proteins from the centrosomes.
  • Pulls apart the chromosomes.
• Contractile Ring:
  • Composed of actin and myosin.
  • Segregates the cell during cytokinesis.

Formation of Spindle Poles

• Centrosomes:
  • Most animal cells have a single centrosome, consisting of a pair of centrioles surrounded by a matrix of proteins.
• Centrosome Duplication:
  • Begins at the start of S phase and is complete by the end of G2.
• Centrosome Separation:
  • Initially, the two centrosomes remain together.
  • In early M phase, they separate, and each nucleates its own aster (star) of microtubules.
• Spindle Pole Formation:
  • The centrosomes move apart.
  • Microtubules that interact between the two asters elongate preferentially to form a bipolar mitotic spindle pole.

Microtubule Attachment to Kinetochores

• Kinetochores:
  • Protein assemblies that occur on the centromere regions of chromosomes.
• Microtubule Binding:
  • Microtubules bind to the kinetochore at their plus ends.
• Dynamic Interactions:
  • Protein/protein interactions do not occur exactly at the end, allowing the microtubules to shrink and grow.

Types of Microtubules in the Mitotic Spindle

• Kinetochore Microtubules:
  • Attach duplicated chromosomes to the spindle poles.
• Non-kinetochore Microtubules:
  • Form a network that does not connect to the chromosomes or to the centrosome.
  • Microtubule-associated proteins (including motor proteins and nucleation factors) cross-link these microtubules, producing a dense, dynamic meshwork.
• Astral Microtubules:
  • Reach outward toward the cell cortex and help position the spindle.

Cell State at the End of G2

• Cell Growth:
  • The cell has grown in size.
• Genome Duplication:
  • The genome has been duplicated.
• Organelle Duplication:
  • Organelles have duplicated.

Stages of Mitosis

• Interphase (I): Covered previously.
• Prophase (P)
• Prometaphase (P)
• Metaphase (M)
• Anaphase (A)
• Telophase (T)
• Cytokinesis (C)

Prophase

• Chromosome Condensation:
  • Chromosomes condense.
• Mitotic Spindle Assembly:
  • The mitotic spindle begins to assemble, associated with the two centrosomes.
• Centrosome Movement:
  • The two centrosomes begin to move apart.

Prometaphase

• Nuclear Envelope Breakdown:
  • The nuclear envelope breaks down.
• Spindle Pole Formation:
  • The spindle poles form.
• Chromosome Attachment:
  • Chromosomes begin to attach to the mitotic spindle's microtubules at the kinetochores.
• Chromosome Movement:
  • Movement of chromosomes begins.

Metaphase

• Chromosome Alignment:
  • Chromosomes are aligned in the middle of the cell at the metaphase plate.
• Kinetochore Microtubule Attachment:
  • Kinetochore microtubules from opposing spindle poles are attached to sister chromatids.

Anaphase

• Cohesion Breakdown:
  • Starts when the remaining cohesion is broken down by proteases.
• Sister Chromatid Separation:
  • Sister chromatids separate and are pulled to the opposing spindle poles.
• Mechanism:
  • Kinetochore microtubules get shorter while the spindle poles move further apart.

Separation of Sister Chromatids

• Separase Activation:
  • Separase exists in an inactive state bound to securin until anaphase.
• Securin Destruction:
  • During anaphase, APC/C ubiquinates and causes the destruction of securin.
• Cohesin Cleavage:
  • Separase cleaves the cohesin complexes, allowing the mitotic spindle to pull the sister chromatids apart.
• APC/C Role:
  • APC/C was originally called the anaphase-promoting complex because of its central role in this process.

Chromosome Separation Details

• Anaphase A:
  • Sister chromatids are pulled toward opposite poles as the kinetochore microtubules depolymerize.
  • The force driving this movement is generated mainly at the kinetochore.
• Anaphase B:
  • The two spindle poles move apart due to two separate forces:
    • Elongation and sliding of non-kinetochore microtubules past one another pushes the two poles apart (mediated by myosin II).
    • Forces exerted on the outward-pointing astral microtubules at each spindle pole pull the poles away from each other, toward the cell cortex.
  • Both forces depend on the action of motor proteins associated with the microtubules.

Telophase

• Chromosome Arrival:
  • The two chromosomes arrive at the spindle poles.
• Spindle Disassembly:
  • Mitotic spindle disassembles.
• Nuclear Envelope Reformation:
  • New nuclear envelopes form around the chromosomes.
• Contractile Ring Assembly:
  • The contractile ring begins to assemble in the cytoplasm.

Nuclear Envelope Reformation Details

• Dephosphorylation:
  • During prometaphase, the nuclear lamina and nuclear pore proteins are phosphorylated.
  • These are then dephosphorylated, allowing for their reassembly during telophase.
• Vesicle Fusion:
  • Vesicles form around the chromosomes and begin fusing to reform the nuclear envelope.

Cytokinesis

• Definition:
  • The process by which the cell is segregated into two.
• Cleavage Furrow:
  • Separation begins along the cleavage furrow, which forms between the two sets of segregated chromosomes.
• Chromosome Number:
  • Each daughter cell winds up with the same number of chromosomes.

Direction of Cleavage Furrow

• Formation Mechanism:
  • The exact mechanism of cleavage furrow formation is not completely understood.
  • Non-kinetochore microtubules recruit proteins that generate a signal.
• RhoA Activation:
  • This signal activates a protein called RhoA (GTPase).
• Contractile Ring Control:
  • RhoA controls the assembly and contraction of the contractile ring midway between the spindle poles.

Contractile Ring Details

• Composition:
  • Actin filaments and myosin II work together to form the contractile ring.
• Attachment:
  • Attached to the inner leaflet of the plasma membrane.
• Force Generation:
  • The force separating the cells comes from the movement of myosin.
• Ring Dynamics:
  • The ring shrinks in size as cytokinesis progresses and eventually disappears after the two cells have been separated.