Eukaryotic Cell Cycle Notes 1-9, 20, 22, 29

Chapter 19 – The Eukaryotic Cell Cycle

19.1 Overview of the Cell Cycle and Its Control

• Cell cycle consists of 4 phases: G1, S, G2, and M.
• Cells commit to division at the G1 START/Restriction point.
• Cyclin-CDK complexes drive cell cycle progression.
• Positive and negative feedback loops drive CDK activity oscillations.
• Checkpoint pathway surveillance mechanisms ensure each cell cycle step is completed correctly before initiating the next one.

Cell Cycle Timing

• Human cells: ~24 hours (G1 ~ 9 hrs, S ~ 10 hrs, G2 ~ 4.5 hrs, M ~ 30 min).
• Yeast cells: ~90 min.
• Embryonic/developmental cells: can be as fast as ~8 min.

Stages of Mitosis

Prophase

• Chromosomes condense.
• Nuclear envelope breaks down.
• Spindle poles duplicate.
• Microtubules form the mitotic spindle apparatus.

Metaphase

• Spindle microtubules from each pole attach to chromosome kinetochores.
• Sister chromatid pairs center in the spindle.

Anaphase

• Spindle microtubule shortening and motor proteins pull each sister chromatid toward an opposite spindle pole.

Telophase

• Chromosomes decondense.
• Each presumptive daughter cell reassembles a nuclear membrane around its chromosomes.

Cytokinesis

• Cell divides into two daughter cells.

19.3 Regulation of CDK Activity

• Different cyclins, present only in the cell cycle stage they promote, activate CDKs at different cell cycle stages.
• The ubiquitin-proteasome system limits the presence of a cyclin to the appropriate cell cycle stage.
• Activating and inhibitory phosphorylation of the CDK subunit regulates CDK activity.
• CDK inhibitors (CKIs) inhibit CDK activity by binding directly to the cyclin-CDK complex.
• CDKs initiate every aspect of each cell cycle stage by phosphorylating many different target proteins.

Cyclin-Dependent Kinases (CDKs)

• Small serine/threonine kinases.
• Require a regulatory cyclin subunit for activity.
• The activity and substrate specificity of any given CDK are defined by the particular cyclin to which it is bound.
• Each CDK activity is cell-cycle-stage-specific.

Regulators of Cyclin-CDK Activity

• Kinases and Phosphatases:
  • CAK kinase: Activates CDKs.
  • Wee1 kinase: Inhibits CDKs.
  • Cdc25 phosphatase: Activates CDKs.
  • Cdc14 phosphatase: Activates Cdh1 to degrade mitotic cyclins.
  • Cdc25A phosphatase: Activates vertebrate S phase CDKs.
  • Cdc25C phosphatase: Activates vertebrate mitotic CDKs.
• Inhibitory Proteins:
  • Sic1: Binds and inhibits S phase CDKs.
  • CKIs (p27KIP1, p57KIP2, p21CIP): Bind and inhibit CDKs.
  • INK4: Binds and inhibits G1 CDKs.
  • Rb: Binds E2Fs, preventing transcription of multiple cell cycle genes.
• Ubiquitin-Protein Ligases:
  • SCF: Degradation of phosphorylated Sic1 or p27 KIP1 to activate S phase CDKs.
  • APC/CCdc20: Degradation of securin, initiating anaphase. Induces degradation of B-type cyclins.
  • APC/CCdh1: Degradation of B-type cyclins in G1 and geminin in metazoans to allow loading of replicative helicases on DNA replication origins.

Regulation of Cell Cycle Transitions

• G1 CDKs and G1/S phase CDKs prepare cells for S phase.
• S phase CDKs activate DNA replication.
• Mitotic CDKs induce mitosis.
• APC/C ubiquitin-protein ligase induces anaphase.
• APC/C and phosphatases induce late steps in mitosis.
• SCF ubiquitin-protein ligase induces S phase.

19.4 Commitment to the Cell Cycle and DNA Replication

• Extracellular signals, such as nutritional state (in yeast) and presence of mitogens and anti-mitogens (in vertebrates), regulate cell cycle entry.
• Molecular events promoting entry into the cell cycle are conserved across species.
• G1/S CDKs trigger chromosome duplication at DNA origin of replication sites.
• Cohesins link replicated DNA molecules to ensure accurate segregation during mitosis.

Control of G1-S Phase Transition

• In S. cerevisiae, nutrients influence Whi5.
• In metazoans, growth factors influence Rb.
• G1/S phase CDKs assembled in late G1 phosphorylate Sic1 at multiple sites, marking it for SCF ubiquitin-protein ligase ubiquitinylation and subsequent proteasomal degradation.
• Active S phase CDKs trigger initiation of DNA synthesis by phosphorylating and recruiting MCM helicase activators to DNA replication origins.

Molecular Mechanisms Governing the Initiation of DNA Replication

• ORC (Origin Recognition Complex) binds to DNA.
• Cdc6 and Cdt1 load MCM helicase onto the DNA.
• S phase CDK and DDK (Dbf4-dependent kinase) activate MCM helicase.
• Polymerase loading and replication occur.

Establishment of Cohesin Linkage of Sister Chromatids

• Chromosomes duplicated during S phase form sister chromatids held together by cohesin complex rings.

19.5 Entry into Mitosis

• Mitotic CDKs induce entry into mitosis in all eukaryotes by inducing chromosome condensation, nuclear envelope breakdown, and spindle formation.
• Mitotic CDKs:
  • are inactivated by inhibitory phosphorylation of the CDK subunit until completion of DNA replication.
  • promote their own activation through positive feedback loops that inactivate Wee1 kinase and activate Cdc25 phosphatase.
• The kinetochore on each compacted sister chromatid attaches to microtubules emanating from opposite spindle poles.
• Cells ensure bi-orientation of sister chromatids in the spindle by a tension-based mechanism.

Regulation of Mitotic CDK Activity

• Inhibitory phosphorylation of the CDK subunit restrains mitotic CDK activity during S phase and G2.
• Wee1 kinase phosphorylates CDK at Y15 and T14, inactivating it.
• Cdc25 phosphatase removes these phosphates, activating mitotic CDKs.

Chromosome Attachment to the Mitotic Spindle

• Sister chromatids must be stably bi-oriented on the mitotic spindle to be accurately segregated during mitosis.
• Dynamically assembling-disassembling microtubules anchored by their minus (-) ends to each spindle pole search-and-capture chromosomes: chromosome motor proteins propel chromosomes to the plus (+) end of microtubules, where each of the two sister chromatid kinetochore attaches to several microtubules from opposite poles.
• Sensing mechanisms correct inappropriate attachments.

Stable and Unstable Chromosome Attachments

• Amphitelic attachment: Stable attachment with microtubules from opposite poles.
• Merotelic attachment: Unstable attachment with microtubules from both poles to one kinetochore.
• Syntelic attachment: Unstable attachment with both sister chromatids attached to the same pole.
• Monotelic attachment: Unstable attachment with only one sister chromatid attached to a pole.

19.6 Completion of Mitosis: Chromosome Segregation and Exit from Mitosis

• Cohesin cleavage by separase initiates chromosome segregation during anaphase.
• Exit from mitosis is triggered by mitotic cyclin degradation and requires protein phosphatase reversal of mitotic CDK phosphorylation of many different proteins, permitting mitotic spindle disassembly, decondensation of chromosomes, and reassembly of the nuclear envelope.
• Cytokinesis position is coordinated with spindle position.

Regulation of Cohesin Cleavage

• From S phase to M metaphase, sister chromatids are held together by cohesins.
• Separase protease activity is inhibited by CDK phosphorylation and securin binding.
• APC/CCdc20 degrades securin, activating separase and initiating anaphase.

Exit from Mitosis and Re-entry into the Cell Cycle

• APC/CCdc20 activates mitotic CDKs, initiating early mitotic events.
• Phosphatases activate Cdh1 and APC/CCdh1, leading to the degradation of mitotic cyclins.
• G1/S phase CDKs inactivate Cdh1 and activate the expression of S phase cyclin CDKs components.
• G1/S phase CDKs phosphorylate S phase inhibitors, and SCF-proteasome degrades phosphorylated S phase CDK inhibitor, promoting DNA replication.

19.8 Meiosis: A Special Type of Cell Division

• Meiosis involves one cycle of chromosome replication followed by two cycles of cell division to produce haploid germ cells.
• Meiosis-specific gene products and activities modulate the mitotic cell division program to perform meiosis.

Mitosis vs. Meiosis

Steps of Meiosis

1. Pre-meiotic cells:
   • Contain two copies of each chromosome (2n), one from each parent.
2. Chromosome replication:
   • Yields 4n chromosomal complement. Cohesin complexes link sister chromatids together.
3. Meiosis I:
   • Prophase I:
     • Chromosomes condense, replicated homologs pair and undergo homologous recombination, leading to at least one crossover event.
   • Metaphase I:
     • Both chromatids of one chromosome associate with microtubules emanating from one spindle pole, while each member of a homologous chromosome pair associates with microtubules emanating from opposite poles.
   • Anaphase I:
     • Homologous pairs of chromatids are pulled to opposite spindle poles.
   • Cytokinesis I:
     • Yields two daughter 2n cells, which enter meiosis II without DNA replication.
4. Meiosis II:
   • Metaphase II:
     • Sister chromatids associate with spindle microtubules from opposite spindle poles, similar to mitosis.
   • Anaphase II:
     • Segregates sister chromatids to opposite spindle poles.
   • Cytokinesis II:
     • Generates haploid (1n) gametes containing one copy of each chromosome.