Comprehensive Cell Cycle Notes

Cell Cycle

  • Cell Cycle Definition: An orderly sequence of events in which a cell duplicates its contents and divides.
  • Purpose:
    • Unicellular organisms: Reproduction to create a new organism.
    • Multicellular organisms: Production of new organisms through complex divisions, survival by replacing dying cells.

Cell Cycle Tasks

  • Fundamental Tasks:
    • Faithful DNA replication.
    • Equal distribution of replicated chromosomes.
    • Division of the whole cell.
  • Additional Duplication: Most cells also duplicate organelles and macromolecules.
  • Coordination: Cells must coordinate growth and division to maintain size.

Historical Context: Cell Theory

  • Developed in the mid-19th century.
    • Every living organism consists of one or more cells.
    • Cells are the basic unit of life.
    • Cells arise from pre-existing cells.

Eukaryotic Cell Cycle Phases

  • Interphase:
    • G1 (Rapid Growth and Metabolic Activity): 8-10 hours.
    • S (DNA Replication): 10-12 hours.
    • G2 (Growth and Preparation for Division): 1-4 hours.
  • M Phase (Cell Division): 1 hour.
  • Interphase Duration: Typical human cells spend 23 hours in interphase and 1 hour in mitosis, totaling a 24-hour cycle.
  • Cell Growth: Occurs throughout the cell cycle, except in M phase.
  • Gap Phases (G1 and G2): Allow time for cell growth and monitoring of internal and external stimuli.

G0 Phase

  • Entry: Cells enter G0 if external stimuli are inappropriate, delaying progress through G1.
  • Specialized Resting State: Cells can remain in G0 for extended periods (days, weeks, or years) before re-entering the cell cycle.
  • Cell Death: Some cells remain in G0 until they undergo cell death.

M Phase (Mitosis) Stages

  • Prophase: DNA condenses into sister chromatids.
  • Prometaphase: Sister chromatids attach to the mitotic spindle.
  • Metaphase: Sister chromatids align at the spindle equator.
  • Anaphase: Sister chromatids separate.
  • Telophase: Spindles disintegrate, and chromosomes pack into separate nuclei.
  • Cytokinesis: Cell divides into two daughter cells.

Cell Cycle Regulation

  • Basic Organization: Similar in all eukaryotes.
  • Control Machinery: Eukaryotic cells use similar machinery to control the cell cycle.
  • Conserved Proteins: Proteins involved in cell cycle regulation are well-conserved from yeast to humans.

Model Organisms for Cell Cycle Research

  • Yeast:
    • Fission yeast (Schizosaccharomyces pombe).
    • Budding yeast (Saccharomyces cerevisiae).
    • Reproduce rapidly, have small genomes (1% of human genome), are easily manipulated genetically, and can proliferate in a haploid state.
    • Cell division cycle genes (Cdc genes) identified in yeast are regulators of the cell cycle.
  • Xenopus:
    • Large eggs (1 mm diameter) with 100,000 times more cytoplasm than average human cells, making them easy to inject.
    • Rapid cleavage divisions upon fertilization (12 divisions within 7 hours) are easy to observe.
    • Easy to prepare pure cytoplasm and work under cell-free conditions.
  • Mammalian Cells:
    • Cells isolated from mammals and cultured in vitro.
    • Replicative cell senescence: Cells stop dividing after 25-40 divisions.
    • Immortalized cell lines: Acquire indefinite division capacity but have mutations, making them unlike normal/healthy cells.

Cell Cycle Regulation Signals

  • External Signals: Growth factors.
  • Internal Signals: DNA damage.
  • Proliferation: CDK on.
  • Quiescence: CDK off.

External Signals

  • Physical and chemical signals from outside the cell.
  • Contact Inhibition: Mammalian cells stop dividing when they form a single layer and touch each other.
  • Growth Factors: Chemical signals acting on growth factor receptors to induce cell division.

Contact Inhibition

  • Noncancerous cells stop proliferation when they contact each other.
  • Associated with a halt in cell division and initiation of differentiation.
  • Reversed in physiological conditions requiring rapid cell growth like embryonic development, wound healing, and tissue regeneration.

Hippo Pathway

  • Regulates the expression of genes controlling growth.
  • Serine kinase cascade with regulatory/scaffolding proteins acting on a transcriptional complex.
  • Ser/Thr kinases receive signals from outside the cells and regulate cytoplasmic kinase activity.
  • Cytoplasmic kinase regulates degradation or nuclear translocation of transcription activators.
  • Contact inhibition is one of the regulators of the Hippo Pathway.
  • Adherens junctions and cadherin-catenin complex activate the Hippo signaling pathway, inhibiting cell growth.
  • Tight-junction-associated proteins, especially angiomotin, activate Hippo pathway signaling.

Growth Factors

  • Proteins secreted by cells to communicate with each other.
  • Regulate cell proliferation, differentiation, migration/pathfinding, and survival/death both positively and negatively.
  • Bind to specific transmembrane receptors, starting intracellular signaling cascades that cause transcription-dependent and transcription-independent changes.
  • Target cells:
    • Autocrine signaling: same cell that releases the growth factor.
    • Paracrine signaling: a neighboring cell.
    • Endocrine signaling: a distant cell, requiring transport through circulation.

Types of Growth Factors

  • Extracellular regulatory proteins including cytokines, chemokines, and some hormones.
  • Play an important role in promoting cell division and differentiation in insects, amphibians, humans, and plants.
  • Examples:
    • Epidermal growth factor: epithelial cells.
    • Platelet-derived growth factor: muscle and connective tissue cells.
    • Nerve growth factor: neuronal cells.

Cytokines

  • Subset of growth factors.
  • Initially described signaling proteins associated with hematopoietic or immune cells.
  • Terms cytokines and growth factors have become interchangeable.
  • Common families: interleukin (IL), interferon (IFN), and tumor necrosis factor (TNF) families.

Hormones

  • Signaling molecules exerting effects in an endocrine manner.
  • Classifications:
    • Amine hormones: modified amino acids (tyrosine).
    • Steroid hormones: derived from cholesterol.
    • Protein hormones: e.g., IGF-1, also classified as growth factors.

Mitogens vs. Growth Factors

  • Mitogen: Small protein that induces the cell to begin cell division, triggering mitosis.
  • Growth Factor: Naturally occurring substance (secreted protein or a steroid hormone) that stimulates cell proliferation, wound healing, and cellular differentiation.

Growth Factor Receptors

  • Each GF binds to a specific cell surface receptor with tyrosine kinase activity, named Receptor Tyrosine Kinases (RTKs).
  • RTKs are classified into 20 families.
  • Most studied RTKs in cell cycle:
    • Epidermal growth factor receptor (EGFR) family.
    • Insulin receptor family.
    • Platelet-derived growth factor receptor (PDGFR) family.
    • Nerve growth factor receptor (NGFR).
  • GFs drive cell cycle by activating RTKs and downstream signaling pathways, regulating cyclin-Cdk complexes.

Activation of Receptor Tyrosine Kinases (RTKs)

  • GF binding to the extracellular ligand-binding domain allows the intracellular tyrosine kinase domain to phosphorylate tyrosine side chains on receptors and downstream signaling proteins.
  • Ligand binding leads to dimerization of receptor chains, bringing kinase domains into proximity for cross-phosphorylation, also known as transautophosphorylation.

Intracellular Signaling

  • Phosphorylation enhances kinase activity and creates high-affinity docking sites for intracellular signaling proteins.
  • Intracellular signaling proteins bind to phosphorylated sites via their phosphotyrosine binding domain.
  • Bound intracellular signaling proteins are also phosphorylated on tyrosines and become active.

SH2/3 and PTB Domains

  • Intracellular signaling proteins involved in various structures and functions can bind to RTKs and become activated, sharing conserved binding domains such as SH2/3 (Src Homology Region) or PTB (Phosphotyrosine Binding) Domain.
  • Some proteins consist entirely of SH2 and SH3 domains, serving as adaptors for other intracellular proteins lacking these domains.
  • Adaptor proteins help the activity of Ras proteins, and PI3K can bind to the intracellular tail of RTKs.

Ras Superfamily

  • Consists of various families of small GTPases, but only Rho and Ras rely on cell surface receptors for activation.

Ras Activation

  • Contains lipid groups that anchor the protein to the cytoplasmic surface of the plasma membrane.
  • Functions as a molecular switch, cycling between active/GTP-bound and inactive/GDP-bound forms.
  • Ras Guanidine Nucleotide Exchange Factors (Ras-GEFs) stimulate GDP dissociation and GTP association.
  • Ras GPTase Activating Proteins (Ras-GAPs) increase the rate of bound GTP hydrolysis.

MAPK Module

  • Ras activation is short-lived, requiring a longer-lasting signaling event to regulate gene expression and cell proliferation.
  • Mitogen-activated protein kinase (MAPK) module:
    • MAP Kinase Kinase Kinase/MAPKKK (Raf).
    • MAP Kinase Kinase/MAPKK (Mek).
    • MAP Kinase (MAPK).
  • Activated MAPK transcribes immediate early genes to induce cell proliferation and G1 Cyclins.

PI3K-Akt Pathway

  • PI3K binds to intracellular tails of RTKs and phosphorylates inositol phospholipids (PIP2).
  • Once activated via RTKs, PI3K produces PI(3,4,5)P3/PIP3, allowing various intracellular signaling proteins to bind through specific interaction domains like the plekstrin homology (PH) domain.
  • PIP3 recruits Akt (Protein Kinase B/PKB) and phosphoinositide-dependent protein kinase 1 (PDK1), leading to Akt activation.

Akt and mTOR

  • Activated Akt stimulates cell growth through the mammalian target of rapamycin (mTOR) kinase, a serine/threonine kinase.
  • Active Akt phosphorylates and inhibits Tsc2, freeing Rheb and activating mTOR on the mTORC1 complex.
  • mTOR then stimulates cell growth.

Cytokine Receptors

  • Serve as receptors to local mediators (cytokines) and some growth hormones.
  • Associated with cytoplasmic tyrosine kinases called Janus Kinases (JAKs).
  • JAKs phosphorylate and activate Signal Transducers and Activators of Transcription (STATs).
  • Activated STATs translocate into the nucleus and activate transcription.

Transforming Growth Factor β (TGF-β)

  • A potent antimitogen in a wide variety of cells, inhibiting cell cycle progression.
  • Acts through enzyme-coupled receptors, single-pass transmembrane proteins with a serine/threonine kinase domain.
  • Each TGF-β family member binds to a characteristic combination of Type-I and Type-II receptor dimers, bringing kinase domains together.
  • Type-II receptors activate Type-I receptors, forming an active tetrameric receptor complex.

TGF-β Signaling

  • Activated Type I receptors bind to and activate Smad family proteins (Smad 2/3).
  • Phosphorylated Smad2/3 dissociates from the receptor and oligomerizes with Smad4.
  • Oligomer translocates to the nucleus, recruits other gene regulator proteins, and activates gene expression.
  • Enhances p21 & p16 expression, blocking cyclin-dependent kinase activation and cell cycle progression.

RTK & Cytokine Signaling

  • RTK Signaling:
    • Ligand (e.g., EGFR) binds to RTK.
    • Activation of RAS via SOS and GRB2.
    • PIP2 converted to PIP3 by PI3K.
    • PTEN dephosphorylates PIP3 back to PIP2.
    • Akt (PKB) and PDK1 activation.
    • mTORC1 activation.
    • Activation of RAF, MEK, and ERK1/2.
  • Cytokine Signaling:
    • Cytokine (e.g., IL-6) binds to cytokine receptor.
    • JAKs are activated and phosphorylate the receptor.
    • STATs bind to the phosphorylated receptor, are phosphorylated by JAKs, dimerize, and translocate to the nucleus to activate transcription.

Cell Cycle Regulation by Size

  • Amoeba Experiment:
    • Two Amoeba proteus cells grown under identical conditions.
    • One amoeba had cytoplasm amputated daily; the other was untouched.
    • The amputated amoeba didn't divide for 20 days, while the control divided 11 times.
    • When amputations stopped, the operated amoeba divided within 38 hours.
  • Interpretation:
    • Repeated amputations prevented the experimental amoeba from reaching a sufficient size to undergo division.
    • Some human cells have similar size control mechanisms, while others do not.

Internal Signals

  • Cyclins and Cyclin Dependent Kinases (Cdks).

Cyclin-Dependent Kinases (Cdks)

  • Are components of cell cycle control systems.
  • Their activities rise and fall as the cell progresses through the cell cycle.
  • They have no catalytic activity unless bound tightly to a cyclin.

Cyclins

  • Cyclical change in Cdk activity is regulated by proteins named cyclins.
  • Undergo a cycle of synthesis and degradation as the cell progresses through the cell cycle.
  • Cyclical changes in cyclin protein levels result in cyclic assembly and activation of cyclin-Cdk complexes and this activation in turn triggers the cell cycle events.

Cyclin Classification

  • Four classes of cyclins are defined by the stage of the cell cycle at which they bind to Cdks and function:
    • G1/S Cyclins: activate Cdks in late G1 and thereby trigger progression through cell cycle entry; their levels decrease in S phase.
    • S Cyclins: bind Cdk soon after progression through start and stimulate chromosome replication; their levels remain high until mitosis and also contribute to some early mitotic events.
    • M Cyclins: activate Cdks that stimulate entry to mitosis at G2/M phase; they are destroyed in mid-mitosis.
    • G1 Cyclins: help govern the activities of G1/S cyclins.

Cyclin-Cdk Complexes

  • All eukaryotic cells require three or four cyclin classes.
  • In yeast cells, a single Cdk protein binds to all cyclins and triggers different cell cycle events by changing the cyclin partner at different stages of the cell cycle.
  • In vertebrates, four types of Cdks exist; two interact with G1 cyclins, one interacts with G1/S and S cyclins, and one interacts with M cyclins.

Cyclin-Cdk Complex Vertebrates and Budding Yeast

  • Vertebrates
    • G1-Cdk: Cyclin D, Cdk4, Cdk6
    • G1/S-Cdk: Cyclin E, Cdk2
    • S-Cdk: Cyclin A, Cdk2 and Cdk1
    • M-Cdk: Cyclin B, Cdk1
  • Budding Yeast
    • G1-Cdk: Cln3, Cdk1
    • G1/S-Cdk: Cln1,2, Cdk1
    • S-Cdk: Clb5,6, Cdk1
    • M-Cdk: Clb1,2, 3, 4, Cdk1

Cyclin-Cdk Complex Activation

  • Each cyclin-Cdk complex phosphorylates a different set of substrate proteins.
  • The same cyclin-Cdk complex can induce different effects at different times in the cycle due to changing accessibility of some Cdk substrates through the cell cycles.
  • In the absence of cyclins, slab proteins obscure the active sites of Cdks to prevent their activity.
  • Cyclin binding causes slab protein to dissociate from the active sites which leads a partial activation.
  • Cdk-Activating Kinase (CAK) phosphorylates an amino acid located nearby the entry of active site, achieving full activation of the cyclin-Cdk complex.

Cyclin-Cdk Complex Inhibition

  • Phosphorylation of a pair of amino acids in the roof of the Cdk active sites can inhibit the catalytic activity of the cyclin-Cdk complexes.
  • Wee1 phosphorylates these sites and inhibits the Cdk complex activity, while dephosphorylation of these sites by Cdc25 (dephosphatase) enhances the Cdk complex activity.

Cdk Inhibitor Proteins (CKIs)

  • CKI binding stimulates a rearrangement of the active site of Cdk structure, rendering it inactive.
  • Mainly involved in the regulation of G1/S- and S-Cdks.

Cyclin-Cdk Regulation via Cyclical Proteolysis

  • Progression through start and G2/M phases is driven by the cyclin-Cdk complex.
  • Progression through metaphase to anaphase, on the other hand, is not only driven by protein phosphorylation but also by protein destruction.
  • The key element is the anaphase-promoting complex or cyclosome (APC/C), a member of the ubiquitin ligase family of enzymes.
  • APC/C catalyzes the ubiquitinylation and degradation of two major proteins: securin and S-&M Cyclins.
  • APC/C activity changes during the cell cycle via changes in its association with an activating subunit Cdc20 (anaphase) or Cdh1 (late mitosis to G1). These subunits help APC/C to recognize its targets.

Securin Proteolysis

  • Securin protects protein linkages that hold sister chromatid pairs together in early mitosis.
  • Destruction of securin via APC/C leads to separation of sister chromatids and unleashes anaphase.

Proteolysis of S- and M- Cyclins

  • Destruction of these cyclins inactivates most Cdks.
  • As a result, many proteins phosphorylated by the Cdks from S phase to early mitosis are dephosphorylated with various phosphatases present at anaphase.
  • This phosphorylation is required for mitotic progression.
  • Following its activation at mid-mitosis, APC/C remains active in G1 until activated G1/S Cdks turn off the APC/C.

SCF Proteolysis

  • SCF ubiquitylates certain CKI proteins in late G1, helping activate S-Cdks and DNA replication.

Table 17-2 Summary of the Major Cell-Cycle Regulatory Proteins

  • Cdk-activating kinase (CAK) phosphorylates an activating site in Cdks
  • Wee1 kinase phosphorylates inhibitory sites in Cdks; primarily involved in suppressing mitosis
  • Cdc25 phosphatase removes inhibitory phosphates from Cdks; three family members (Cdc25A, B, C) in mammals;
    primarily involved in controlling Cdk1 activation at the onset of mitosis
  • Sic1 (budding yeast) suppresses Cdk1 activity in G.; phosphorylation by Cdk1 at the end of G, triggers its destruction
  • p27 (mammals) suppresses G,/S-Cdk and S-Cdk activities in G,; helps cells withdraw from cell cycle when they
    terminally differentiate; phosphorylation by Cdk2 triggers its ubiquitylation by SCF
  • p21 (mammals) suppresses G,/S-Cdk and S-Cdk activities following DNA damage
  • p16 (mammals) suppresses G,-Cdk activity in G,; frequently inactivated in cancer
  • APC/C catalyses ubiquitylation of regulatory proteins involved primarily in exit from mitosis, includingSecurin and S- and M-cyclins; regulated by association with activating subunits
  • Cdc20 APC/C-activating subunit in all cells; triggers initial activation of APC/C at metaphase-to-anaphase transition; stimulated by M-Cdk activity
  • Cdh1 APC/C-activating subunit that maintains APC/C activity after anaphase and throughout G,; inhibited by Cdk activity
  • SCF catalyses ubiquitylation of regulatory proteins involved in G, control, including someCKIs (Sic1 inbudding yeast, p27 in mammals); phosphorylation of target protein usually required for thisactivity

Cyclins-Cdk Complex Regulation via Transcriptional Regulation

  • Cell cycle regulations largely depend on post-transcriptional mechanisms, but more complex organisms use transcriptional regulation to add additional modulation.
  • Changing transcription levels of cyclins is one example.

Cell Cycle Control Systems as Biochemical Switches

  • When conditions for cell proliferation are met, various external and internal signals stimulate the activation of G1-Cdk, which stimulates the expression of genes encoding G1/S- and S-cyclins.
  • Resulting activation of G1/S Cdks drives progression through the start check point, unleashes chromosome duplication in S phase, and contributes to early events in mitosis.
  • M-Cdk activation triggers progression through the G2/M check point and early events in mitosis, leading to the alignment of sister chromatids at the equator of the mitotic spindle.
  • Finally, APC/C together with its activator Cdc20 triggers the destruction of securin and cyclins, unleashing sister chromatid segregation.

Cell Cycle Checkpoints

  • Cell progress through the cell cycle in a regulated way, checking at several points whether the progression is appropriate.
  • They use information about their own internal state and cues from the environment to decide whether to proceed with cell division.
  • This regulation ensures cells don't divide under unfavorable conditions.
  • A checkpoint is a stage in the eukaryotic cell cycle at which the cell examines internal and external cues and