Cell Cycle Regulation Quiz

Cell Cycle Overview

• The cell cycle is a series of molecular and morphological events that accompany the replication of somatic eukaryotic cells.
• The cycle conceptually has two main components: Interphase and Mitosis.
• Interphase is the period where cells double their constituents (proteins, lipids, and DNA).
• Mitosis is where cells divide their contents into two daughter cells.

Cell Cycle Lectures - Objectives

• Students should be able to describe the phases and steps of the cell cycle.
• Students should be able to list critical regulatory steps in the cell cycle transitions.
• Students should be able to explain how cyclin/CDK complexes regulate G1/S and G2/M transitions.
• Students should be able to explain how APC promotes chromosome segregation and resets the cell cycle.
• Students should be able to describe how bistable switches work and why they are crucial to the cell cycle.
• Students should be able to explain Geminin's role in loading and firing replication origins.

Different Modes of Somatic Cell Growth

• Cellular hypertrophy: increase in cell size without affecting the number of cells. (e.g., cardiac muscle cells, neurons).
• Cell division: increase in cell number through cell division. (e.g., fibroblasts, endothelial cells, keratinocytes).

Historical Considerations

• Early observations (1800s) showed cell growth through increasing volume (interphase) followed by division (mitosis) with continuous oscillation between mitosis and interphase.
• Experiments in the 1950s using radioisotopes revealed DNA replication during interphase, subdividing interphase into G1, S, and G2 phases.
• The 1970s saw the discovery of tightly regulated cell cycle progression, primarily at the G1/S and G2/M transitions.
• The 1980s identified cyclins and cyclin-dependent kinases (CDKs) as crucial regulators of these transitions.

Cell Cycle Phases (Graphic)

• G1: Cells grow and expand in volume.
• S: Cells synthesize DNA and replicate their genome.
• G2: Cells prepare for division.
• M: Cells divide to produce two daughter cells.

DNA Content Fluctuations

• DNA content increases during the S phase as DNA is replicated.
• DNA content is doubled at the end of S phase.
• The S phase follows G1.
• The G2 phase follows the S phase, which prepares the cell for the M phase.
• The M phase follows the G2 phase and involves cell division.

Flow Cytometry

• A technique utilizing fluorescence to measure DNA content per cell, distinguishing cell cycle stages (e.g., G1, S, G2/M).

Excluding Doublets

• DNA profiles are further refined by excluding doublets (two cells together) to prevent inaccurate analysis, because doubled cells take longer to pass through the laser beam, producing a wider pulse in flow cytometry.

Restriction Point

• A point in G1 where cells become committed to proceeding through the entire cell cycle, independent of growth factors.
• Cells must cross the restriction point to enter S phase.
• Cell cycle progression beyond this point is largely independent of extracellular growth factors.

G1/S Transition & Restriction Point

• Signals from growth factor receptors increase cyclin D expression for CDK4/6 activation, which phosphorylates RB and activates E2F.
• This release of E2F results in Cyclin E production, activating CDK2 and complete phosphorylation/inactivation of RB, triggering S-phase genes (including Cyclin A) transcription.
• Cyclin A then replaces Cyclin E for continued CDK2 activity.
• CDK2 phosphorylates pre-replication complexes to initiate DNA synthesis at origins of replication.

DNA Replication (S Phase)

• Origins of replication (ORCs) are licensed at the M-to-G1 transition and become active only during S phase.
• Separation of DNA strands involves DNA helicase, single-stranded DNA-binding proteins, topoisomerases, and helicases.
• Replication begins with the synthesis of RNA primers by Primase, laying the groundwork for DNA polymerase to extend leading strands.
• Lagging strands proceed in short fragments, known as Okazaki fragments, later joined together by DNA ligase.

Replication of Telomeres

• Telomeres are composed of DNA repeats(in humans, ds-TTAGGG)and proteins, capping the ends of chromosomes.
• This protein-DNA complex protects chromosome ends from breakdown, fusions, or recognition as double-stranded DNA breaks.
• Telomerase activity is crucial in maintaining telomeric DNA repeats.

The "End Replication Problem"

• The end replication problem results from the inability of DNA polymerases to replicate the 5' end of the lagging strand to the very end, leading to a gradual shortening of telomeres.
• Lagging strand synthesis involves internally primed Okazaki fragments, distinct from the leading strand synthesis process.

Measuring Telomere Size

• Telomere shortening is studied by measuring telomere length changes during cell division via G-banding(chromosome staining)and flow cytometry.

Biochemical Properties of Telomerase

• A ribonucleoprotein enzyme that adds telomere repeats to the 3' end of DNA strands, compensating for the end replication problem.
• Telomerase comprises components like hTR, HTERT, and other proteins.

S Phase and Restriction Point

• Crossing the restriction point commits a cell to proceed completely through the cell cycle.
• S phase marks DNA replication.
• Entering S phase signifies passage of the restriction point.

Mitosis (M Phase)

• This phase encompasses stages like prophase, metaphase, anaphase, and telophase, culminating in cytokinesis, the actual division of the cytoplasm.
• Prophase initiates chromosome condensation.
• Mitosis promotes chromosome segregation.
• Metaphase positions chromosomes at the metaphase plate, facilitated by spindle microtubules, centrosomes, and kinetochores.
• Anaphase involves sister chromatid separation and chromosome migration to opposite poles.
• Telophase marks chromosome decondensation and nuclear envelope reformation, leading to two distinct nuclei.
• Cytokinesis completes the process by partitioning cytoplasm and cellular components to the two newly formed daughter cells.

Mitosis-Promoting Factor (MPF)

• MPF is a kinase complex formed by the association of B-type cyclins with the CDC2 kinase; it drives cell entry into mitosis and inhibits anaphase.
• MPF is crucial in the various steps of mitosis.
• It is responsible for chromosome condensation, nuclear envelope breakdown, and mitotic spindle assembly.
• This role is crucial for successful mitosis.

Phosphorylation of Lamins

• Nuclear lamina is a meshwork composed of lamin proteins (A, B, C) in the nuclear envelope.
• Lamins B is anchored to the nuclear envelope.
• During mitosis, phosphorylation of lamins breaks down the nuclear lamina and dissolves the nuclear envelope.

MPF-induced Condensation

• MPF-induced chromosome condensation involves individualization of DNA strands (separated chromosomes), followed by folding of chromatin fibres into rod-like structures for organization and resolution or clarity of sister chromatids.
• Chromosome condensation depends on Condensin, a protein complex regulated by MPF.

Centrosomes/Centrioles/Mitotic Spindle

• Centrosomes (containing centrioles) serve as organizing centers for spindle microtubule formation during cell division.
• Centrosomes duplicate during the S phase, migrating to opposite poles of the nucleus to guide chromosome movement.
• The mitotic spindle comprises microtubules, associated proteins, and centrosomes, essential for sister chromatid segregation.

Metaphase Alignment

• The centromere region of each chromosome is vital for faithful segregation of chromosomes in mitosis.
• Within this region, kinetochores form, microtubules attach, and chromosome movement is facilitated.

Anaphase-Promoting Complex (APC)

• The APC acts as an ubiquitin ligase complex, targeting proteins for degradation by the proteasome. (this complex targets various proteins).
• It deactivates MPF by targeting cyclins for destruction, effectively initiating anaphase.
• The APC ensures successful completion of mitosis and is kept inactive till all chromosomes are properly attached to the mitotic spindle via kinetochores.
• The spindle assembly checkpoint controls APC activity, inhibiting it if chromosomes are not attached to the spindle.

Cohesion, Securin, and Separase

• Cohesin complexes hold sister chromatids together during S and G2 phases.
• Securin inhibits separase, an enzyme that cleaves cohesin, until anaphase.
• Separase activation leads to sister chromatid separation in anaphase.

Anaphase - Separating Sister Chromatids

• Sister chromatid separation involves the destruction of cohesin complexes along with motor proteins and microtubules of the mitotic spindle.
• Separation is crucial for ensuring chromosome segregation during cell division.

Anaphase Checkpoint

• This checkpoint monitors the proper attachment of every chromosome to the mitotic spindle fibers before allowing cell division.
• This crucial checkpoint avoids the production of daughter cells with an unequal number of chromosomes.

Karyotype

• A karyotype displays the chromosomes of an organism, used in diagnosing conditions with chromosomal abnormalities.

M Phase Cytokinesis (Cell Division)

• Cytokinesis is the final step of the M phase, distributing cellular components to daughter cells..
• It involves a contractile ring, formed by actin and myosin fibers, which pinches the cell membrane to create two daughter cells.

Cell Cycle Checkpoints

• Crucial regulatory points (G1, G2, and M checkpoints) check for conditions ensuring accurate and complete DNA replication and accurate chromosome segregation.
• Cells stop at these points and await appropriate conditions.
• Cells cycle through phases—G1, S, G2, and M.

G2/M Checkpoint

• The G2/M checkpoint monitors for complete DNA replication and cellular integrity (cell size) before allowing the cell to enter mitosis.
• A cell will not proceed to mitosis if any abnormalities are detected.

Mitosis-Promoting Factor (MPF)

• MPF is responsible for driving the cell cycle into mitosis and inhibiting anaphase.
• MPF activation depends on dephosphorylation of CDK1 by phosphatases Cdc25A/C in response to appropriate cell condition.
• MPF drives cell condensation, nuclear envelope breakdown, and assembly of the mitotic spindle. (essential steps in mitosis).

CDK1 (Cdc2)

• CDK1 is a cyclin-dependent kinase crucial for the M-phase (mitosis) of the cell cycle.
• It is only active during the M-phase, regulated by phosphorylation and dephosphorylation of tyrosine 15.
• CDK1 activation requires phosphorylation by CAK complex, binding to cyclins B1/B2, and dephosphorylation by Cdc25A/C phosphatases.
• Once active, CDK1 promotes entry into mitosis and drives the cell cycle toward metaphase. Active CDK1 is MPF.

Feedback loops (Cyclin B/CDK1)

• The cyclin B/CDK1 complex's activity is governed by feedback loops, creating a bistable switch.
• These loops ensure the complex's activation only under precise conditions and promote reliable progress through the cell cycle.

Cyclins D/E and Cyclins A/B Degradation

• Cyclins D/E and A/B are targeted for degradation via ubiquitin ligase complexes for precise regulation during cell cycle progression.
• This ensures the degradation of relevant proteins at the appropriate checkpoints.
• Each cyclin has its own specific ubiquitin ligase.

CDK Inhibitors (CDKI)

• The INK4 and CIP/KIP proteins act as CDK inhibitors, preventing CDK activation prior to the correct cell cycle progression conditions.
• These inhibitors are necessary for proper cell cycle regulation.

G1 Checkpoint

• G1 checkpoint checks for suitable conditions, cell size, nutrient availability, growth factors, and DNA damage, ensuring correct decision-making at this juncture.

G1 Checkpoint: Activation of p53 and p16 induction

• The presence of DNA damage or other stressors activates p53, which further leads to the induction of p21 and p16 genes—inhibitors for cell cycle progression
• p16 directly inhibits CDK4 activities to halt the cell cycle.
• p21 inhibits several CDKs (including CDK2).
• This mechanism helps prevent cells with damaged DNA to enter the cell cycle.

Positive & Negative Regulators of G1/S Transition

• Growth factors positively regulate the G1/S transition.
• In contrast, negative regulators help prevent uncontrolled or premature cell cycle progression.

Geminin

• Geminin is a protein that acts as an inhibitor of DNA replication for a certain time period, only allowing re-licensing of origins of replication after the cell has successfully completed mitosis.
• The degradation of Geminin is a critical step in re-activating the cell's capability for DNA replication in the G1 phase.

Overview of Cell Cycle Regulation

• Cell cycle progression relies on precise regulation through various checkpoints and the coordinated activities of cyclins, CDKs, CDK inhibitors, and associated pathways. Precise regulation is crucial for generating functional cells.

Test your knowledge on the essential components and processes of the cell cycle, including the roles of cyclins, CDKs, and key checkpoints. The quiz covers mechanisms like cytokinesis, the function of the Anaphase-Promoting Complex, and the impact of growth factors on cell cycle progression.