Cellular Signaling and the Cell Cycle

L18 - Intro to the Cell Cycle

• The cell cycle is a series of events that allows a cell to reproduce itself
• Key regulatory steps in the cell cycle include cyclin-dependent kinases (Cdks) as master regulators and cell cycle checkpoints
• Cell cycle checkpoints monitor if conditions are right before the next phase of the cycle
• Checkpoints such as G1-S, G2-M, and metaphase-anaphase transitions ensure DNA replication is complete and conditions are favorable
• If a checkpoint is not satisfied, the cell may enter senescence, a state of irreversible growth arrest, or apoptosis, programmed cell death
• Defects in checkpoints can lead to diseases like cancer

Chromosome, Centrosome and Organelle Duplication (Recap)

• Chromosomes condense and segregate in mitosis phase
• Decondensation occurs in G1
• DNA replication occurs in S phase
• Sister chromatid cohesion occurs in G2
• Centrosomes are involved in organizing microtubules and separating chromatids during cell division

What Drives the Cell Cycle

• Cyclin-dependent kinases (Cdks) are important proteins that control the cell cycle by transferring a phosphate to their substrates
• Cdks act as master regulators, controlling numerous processes in the cell cycle
• Cdks have little activity on their own; they are activated by cyclin proteins and also influence the substrate specificity of Cdks
• Cyclin levels oscillate to order cell cycle phases

Additional Cdk Regulators

• Cdks are inactivated by phosphorylation by upstream kinases and phosphatases
• Cdk regulatory proteins (CKIs) can bind onto the cyclin-cdk complex to push it to an inactive complex

Discovery of Cdks and Cyclins

• Work in model organisms is crucial to understand the cell cycle
• Yoshio Masui identified a cytoplasmic factor (MPF) that induces cell division in frog oocytes
• Leland Hartwell conducted screens in budding yeast to identify cell division cycle (cdc) mutants such as cdk1 (Cdc28)
• Sir Paul Nurse identified and characterized cdk1 (Cdc2) in fission yeast, and cloned human Cdk1; complementation

Cell Division Cycle (Cdc) Mutants

• Yeast cells that are defective in the cell cycle are important for studying the cell cycle.
• Phenotype of such mutants is typically an inability to grow and divide
• These cells can be studied using temperature-sensitive mutations, which only disable the gene at high temperatures, or by observing the yeast's size and budding patterns to track cell cycle progression

Discovery of Cyclins

• Disocvered by Sir Tim Hunt at Woods Hole Marine Biological Laboratory in 1982,
• Radiolabelled extracts of sea urchin eggs showed a protein that appeared after fertilization and disappeared each time the cells divided

What Makes Cyclin Levels Oscillate

• Cyclin synthesis is crucial to drive the cell cycle
• Mechanisms controlling synthesis include changes in transcription and translation rate, varying by cell type
• Important is the control of cyclin destruction, like that of M-cyclin triggered by the APC/C

The APC/C

• APC/C is a ubiquitin ligase that triggers the degradation of M-cyclin to end mitosis and initiate the next cell cycle.
• Ubiquitination (addition of ubiquitin proteins) is a tag for protein degradation by the proteasome.
• The APC/C choses when to degrade cyclin

How Cell Cycle Fidelity is Maintained

• Cyclin oscillations provide the timing for successive phases.
• The cells have monitoring systems (checkpoints) which check whether conditions are correct before allowing the next phase to occur
• The G1 checkpoint, for example, checks if the environment is favorable, if DNA damage has been repaired, and if the cell is receiving proliferation signals
• The G2 checkpoint checks if DNA replication is complete and if any DNA damage has been repaired
• The mitotic checkpoint, or spindle assembly checkpoint, ensures that all chromosomes are effectively attached to spindles before they segregate during mitosis
• Important is a mechanism which ensures conditions are correct before each step

Discovery of the DNA Damage Checkpoint

• Budding yeast cells typically arrest in G2 when their DNA is damaged (e.g., by x-rays)
• Rad9 mutant cells do not delay in G2 after DNA damage and continue to proliferate, even with damaged DNA.

Metaphase-Anaphase Transition

• Mitotic (or spindle assembly checkpoint, SAC). It is important to ensure correct bi-orientation of chromosomes before anaphase commences
• Checks if all chromosomes are properly attached to spindle fibers.

If a Checkpoint Cannot Be Satisfied

• If errors or damage can be fixed, the cell can resume its cell cycle.
• However, if damage is too extensive or cannot be fixed in a timely way, the cell may enter senescence, a terminal exit from the cell cycle.

Disease Due to Defective Checkpoints

• Aberrant mitogen signaling inappropriately drives cells through the G1 checkpoint, a common occurrence in cancer
• Defects in the G2 checkpoint can allow cells with damaged DNA to proliferate
• Defects in mitotic checkpoints can lead to cells with the wrong number of chromosomes, a problem common in cancer, or other health problems

L19 - Cell Cycle Entry and DNA Replication

• The cell cycle is a series of events that cells undergo to reproduce.
• Mitogens promote the G1 to S transition.

G1-S Transition

• Nutrients
• Growth signals
• DNA damage
• Previous mitosis

Mechanism of DNA replication

• Semiconservative
• Origin of replication
• Replication fork

Initiation of Replication in Bacteria

• Circular chromosomes with a single origin of replication
• ORC (Origin Recognition Complex) initiates DNA synthesis. Replication is in about 30 minutes

Initiation of Replication in Eukaryotes

• Linear chromosomes with multiple origins of replication
• Licensing ensures DNA is only copied once.
• ORCs only activate DNA replication initiation in S phase and then are deactivated

The DNA Replication Machine

• Helicase separates the DNA double helix
• Single-strand binding protein maintains the separation of single strands
• DNA primase initiates DNA polymerization
• Two DNA polymerases synthesize the two new strands of DNA, adding new nucleotides onto primer strands.
• Sliding clamp (PCNA) keeps polymerase on DNA
• Topoisomerases remove supercoils and tangles ahead of the replication fork for removal.

DNA Polymerases are accurate

• Proofreading activity
• Mismatch repair to correct mis-incorporated nucleotides
• Exonuclease editing

DNA Replication Needs RNA Primers

• DNA polymerases cannot start new DNA synthesis, instead using RNA primers from which to extend the new DNA strand. DNA primase creates these primers.

Telomeres

• Ends of linear chromosomes
• Repeating GGGTTA units
• Telomerase creates these primers for the lagging strand to allow a 1-to-1 strand replication

DNA Damage Repair

• Detection of nucleotide mis-incorporation during replication
• Detection of damaged nucleotides or bases (e.g., from chemicals, UV exposure).
• Detection of breaks in DNA
• Mismatch repair
• Base/nucleotide excision repair
• Double-strand break repair

G2 Checkpoint

• Checks if DNA replication is complete
• Checks for DNA damage

L20 - Mitosis and Meiosis

• Mitosis and meiosis are cell division processes producing two and four daughter cells respectively,
• Mitosis produces genetically identical diploid cells,
• Meiosis produces genetically unique haploid gametes.

Chromosome Basics

• Human somatic cells have 46 chromosomes (2 copies each of chromosomes 1 to 22 (autosomes) + X and Y sex chromosomes).
• Human gametes (sperm and egg) contain 23 chromosomes.

Replicated Chromosome Basics

• Two sister chromatids are held together by the cohesion complex.

During Mitosis

• Chromosomes condense
• Chromosomes attach to spindle microtubules
• Sister chromatids separate.
• Chromosomes decondense and cell divides into two daughter cells.

The Stages of Mitosis

• Prophase
• Prometaphase
• Metaphase
• Anaphase
• Telophase
• Cytokinesis

Entry into Mitosis (Prophase)

• Underlying regulatory processes control mitotic entry.
• It's driven by the master regulator of mitosis, M-Cdk.

Kinetochore Assembly (Prophase)

• The kinetochore is the microtubule binding site on a chromosome
• Composed of a complex that assembles at the centromere. M-Cdk (Cyclin B-Cdk1), and Aurora B kinases are required to produce it.

Cohesion and Cohesion Release - The Prophase Pathway

Regulation of cohesion - cohesion is a protein complex that physically links two bits of DNA, keeping sister chromatids together. Chromosome condense (prophase) - condensins I and II condense chromosomes, condensin 1 and 2 have similar structural properties, important for condensing DNA

Prophase - Summary

• Interphase chromosome structure is lost.

Prometaphase

• Nuclear envelope breaks down
• Microtubules attach to chromosomes

Mitotic Spindle

• A microtubule-based machine required to align and segregate chromosomes.

Microtubules

• Nucleated at the minus end
• Dynamic instability (can grow and shrink at the plus end).

Mitotic Spindle - MAPs and Motors

• Microtubule-associated proteins (MAPs) modulate the stability of microtubules.
• Motors such as Kinesin-5 and Dynein move components along microtubules

Spindle Checkpoint - Metaphase to Anaphase Transition

• Chromosome attachment to the spindle is checked by a surveillance mechanism to prevent the initiation of anaphase until all chromosomes are attached properly.

If a Checkpoint Cannot Be Satisfied

• If errors or damage can be fixed, the cell can resume its cell cycle.
• However, if damage is too extensive or cannot be fixed in a timely way, the cell may enter senescence, a terminal exit from the cell cycle

Securin and Cohesion Release

• M-cyclin(cyclin B) is not the only target of the APC/C.
• Securin blocks separase, and when APC/C is active, securin gets tagged for degradation
• Separase then releases cohesion, which allows chromatids to separate.

Metaphase

• Chromosomes are all bi-oriented, and they align accordingly on the metaphase plate

Telophase

• Chromosomes decondense
• Spindle components dismantle or detach.
• Nuclear envelope reforms and nuclear pores are inserted

Cytokinesis

• The cell divides into two daughter cells by forming a contractile ring.

L21 - Asymmetric Cell Division

• Importance of asymmetric cell division in development - important for tissue homeostasis (renewal of cells) and immortality of germ cells.
• Asymmetric cell divisions are crucial during development to create different cell types from a single initial cell
• Methods of polarisation are conserved in development
• Cell-cycle regulates and controls the differentiation of different cell types, by controlling different parts of the cell, like microtubules/cytoskeleton, which define apical/basal sides.

Polarisation

• Cell polarization depends on the asymmetric localization of polarity regulators.
• Every cell in the body is polarized – ability of the cell to organize components.

C. elegans

• Model system for studying cell polarity and asymmetric division.
• The first division of the embryo is asymmetric.

Gain Insight into their Function

• How genetic screens and molecular tools can help to find out how the mechanisms that control the polarity process work.

How Mitogens Drive Progression into S Phase

• Mitogens bind to cell surface receptors, triggering signalling pathways (Ras-Raf-MAPK) leading to the expression of genes regulating the cell cycle, such as Cyclin D.
• Cyclin D forms G1-Cdk complexes, driving the cell into S phase.

G1 Checkpoints

• Checkpoints are critical for maintaining cell cycle control and ensure cell cycle doesn't occur in unfavorable conditions.
• If conditions are not good, cells stay in G0 and don't enter the cell cycle
• Questions checked during checkpoints: Are conditions favorable? Is DNA damage good? Has previous mitosis been too long?

How Does G1-Cdk Drive Progression into S Phase

• A key target of G1-Cdk is Retinoblastoma (Rb) protein
• Rb binds to and inactivates the transcription factor E2F
• Phosphorylation of Rb by G1-Cdk inactivates Rb, releasing E2F to activate gene expression (including cyclins E and A), driving the cell into S phase

What Can Prevent G1-Cdk Drive Progression into S?

• P53 is a key regulator in responding to stress and preventing the cycle when conditions are not favorable.

Cell Fate Determination

• How cell polarization is translated to asymmetric segregation of cell fate determinants. This is mediated through several kinase reactions and reaction-diffusion in asymmetric cell localisation of cell factors
• How cell polarity is transferred for asymmetric segregation of cell fate determinants - Kinase reactions regulating molecular dynamics of the cytoplasm

How Cell Polarity Is Transferred for Asymmetric Segregation of Cell Fate Determinants

• Cell polarity is transferred to the mitotic spindle for asymmetric segregation

L22 - Cell Cycle and Diseases

• Cell proliferation during development
• Surveillance mechanisms ensure cell cycle exit
• Cancer: Cell keeps dividing with defective cell cycle checkpoints
• Chromosome instabilities

ABL Proto-oncogene Activation

• what happens when a cell is forced into the cell cycle.
• ABL is a tyrosine kinase that becomes aberrantly active through reciprocal translocation.
• BCR-ABL causes Chronic Myeloid Leukemia.

Chromosome Translocations

• Arise from aberrant double strand breaks in the DNA, and mis-guided repair
• Agents that promote double strand breaks and might increase risks for translocation
• Sequence homology at the chromosome breakpoint
• The 3D chromosomal organization in interphase

Rb Tumor Suppressor Inhibition

• Rb is a critical protein in controlling cell cycle progression. It is involved in the G1-S transition.
• Loss of Rb function can lead to excessive cell proliferation and genomic instability leading to cancer.
• Rb loss leads to inappropriate activation of E2F related genes, leading to unregulated cell cycle activity

Explore the critical concepts of cellular signaling and the cell cycle in this quiz. Delve into the roles of mitogens, signaling pathways, and checkpoints in cell division. Test your understanding of the functions and consequences of aberrant chromosome behavior.