Cell Cycle Notes

OVERVIEW OF THE CELL CYCLE

• The eukaryotic cell cycle typically comprises four phases.
• The cell-cycle control system is responsible for initiating the primary processes within the cell cycle.
• Cell-cycle control mechanisms are conserved across all eukaryotes.

CELL CYCLE

• The cell cycle involves:
  • Cell growth.
  • Chromosome duplication.
  • Chromosome segregation.
  • Cell division.

TABLE 18–1 SOME EUKARYOTIC CELL-CYCLE DURATIONS

• Early fly embryo cells: 8 minutes
• Early frog embryo cells: 30 minutes
• Mammalian intestinal epithelial cells: ~12 hours
• Mammalian fibroblasts in culture: ~20 hours

CELL CYCLE PHASES

• M Phase:
  • Mitosis (nuclear division).
  • Cytokinesis (cytoplasmic division).
• Interphase:
  • G1 Phase.
  • S Phase (DNA replication).
  • G2 Phase.

CELL CYCLE CHECKPOINTS

• Start (G1 Checkpoint):
  • Determines if the environment is favorable to proceed to S phase.
• G2 Checkpoint:
  • Checks if all DNA has been replicated and if any DNA damage has been repaired before entering mitosis.
• Checks if all chromosomes are properly attached to the mitotic spindle before pulling duplicated chromosomes apart.

CYCLIN-DEPENDENT PROTEIN KINASES (CDKS)

• Progression through the cell cycle depends on cyclin-dependent protein kinases (Cdks).
• Cdks must associate with cyclins to become active.
• Cyclin + Cdk \rightarrow Active Cyclin-Cdk

M-CDK ACTIVITY

• M-Cdk activity oscillates during the cell cycle.
• M-cyclin concentration also oscillates, correlating with M-Cdk activity.
• The complex formed triggers mitosis.

Cyclins and Cdks

• Cdks associate with different cyclins to trigger the different events of the cell cycle:
  • G1-Cdk
  • G1/S-Cdk
  • S-Cdk
  • M-Cdk

Major Cyclins and Cdks of Vertebrates

• G1-Cdk: Cyclin D, Cdk4/6
• G1/S-Cdk: Cyclin E, Cdk2
• S-Cdk: Cyclin A, Cdk2
• M-Cdk: Cyclin B, Cdk1

Regulation of Cyclin Concentrations

• Cyclin concentrations are regulated by transcription and proteolysis.
• The activity of some Cdks is regulated by cyclin degradation.

M-Cdk Activation

• Inhibitory phosphates must be removed for M-Cdk to be active.
• Inhibitory kinase (Wee1) adds inhibitory phosphates, inactivating M-Cdk.
• Activating phosphatase (Cdc25) removes the inhibitory phosphates, activating M-Cdk.

Regulation by Cdk Inhibitors

• Cdk and cyclin form an active complex.
• Cdk inhibitor proteins (e.g., p27) can bind to the complex, inactivating it.

M-Cdk Positive Feedback Loop

• Active M-Cdk shuts down a protein phosphatase (PP2A-B55) that opposes its activity.
• This positive feedback loop helps to fully activate M-Cdk, driving the cell into mitosis.

Cell-Cycle Arrest

• Mechanisms exist to halt progression through the cell cycle under certain conditions.
• DNA Replication Not Complete/DNA Damage:
  • Inhibition of activating phosphatase (Cdc25) blocks entry to mitosis.
• Chromosomes Not Properly Attached to Spindle:
  • Inhibition of APC/C activation delays completion of mitosis.
• Unfavorable Environment:
  • Cdk inhibitors block entry into the cell cycle.

G1 PHASE

• Cdks are stably inactivated in early G1.
• Mitogens promote the production of cyclins, stimulating cell division.
• DNA damage can temporarily halt progression through G1.
• Cells can delay division for prolonged periods by entering specialized non-dividing states (G0).

G1 Checkpoint Decisions

• Proceed to S phase?
• Pause?
• Withdraw to G0?
• Withdraw permanently and terminally differentiate?

Mitogens and Rb Protein

• Mitogens activate intracellular signaling pathways.
• Active Rb protein binds to a transcription regulator, inhibiting transcription of genes required for entry into S phase.
• Mitogen-activated G1-Cdk and G1/S-Cdk phosphorylate Rb, inactivating it.
• Inactivated Rb releases the transcription regulator, allowing transcription of genes for entry into S phase.

DNA Damage and p53

• DNA damage activates protein kinases that phosphorylate p53, stabilizing and activating it.
• In the absence of DNA damage, p53 is degraded in proteasomes.
• Active p53 binds to the regulatory region of the p21 gene.
• p21 is a Cdk inhibitor protein that inactivates G1/S-Cdk and S-Cdk, halting the cell cycle.

S PHASE

• Cdc6 binds to the ORC (origin recognition complex), and together these proteins load a pair of DNA helicases onto the DNA.
• At the start of S phase, S-Cdk triggers the firing of this loaded replication origin by guiding the assembly of the DNA polymerase and other proteins that initiate DNA synthesis at the replication fork.
• S-Cdk also blocks re-replication by phosphorylating Cdc6 and the ORC. This phosphorylation keeps these proteins inactive and prevents the helicases from reloading onto the origin until the Cdks are turned off in the next G1.

Positive Feedback in M-Cdk Activation

• Inactive Cdc25 phosphatase is activated to active Cdc25.
• Active Cdc25 removes inhibitory phosphates from inactive M-Cdk to activate it.

CHROMOSOME CONFIGURATION

• Cohesins and condensins help configure duplicated chromosomes for segregation.
  • Cohesin rings hold sister chromatids together.
  • Condensin rings help condense chromosomes.

M PHASE OVERVIEW

• M phase involves mitosis (nuclear division) and cytokinesis (cytoplasmic division).
• Mitosis is divided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase.

The Cell Before M Phase

• The cell increases in size.
• The DNA of the chromosomes is replicated.
• The centrosome is duplicated.

Mitosis Stages

• Prophase:
  • Duplicated chromosomes condense.
  • The mitotic spindle assembles between the two centrosomes.
• Prometaphase:
  • The nuclear envelope breaks down.
  • Chromosomes attach to spindle microtubules via their kinetochores and undergo active movement.
• Metaphase:
  • The chromosomes are aligned at the equator of the spindle.
  • Kinetochore microtubules on each sister chromatid attach to opposite poles of the spindle.
• Anaphase:
  • Sister chromatids synchronously separate and are pulled slowly toward the spindle pole to which they are attached.
  • Kinetochore microtubules get shorter, and the spindle poles also move apart.
• Telophase:
  • The two sets of chromosomes arrive at the poles of the spindle.
  • A new nuclear envelope reassembles around each set, completing the formation of two nuclei.
  • The division of the cytoplasm begins with the assembly of the contractile ring.

Cytokinesis

• The cytoplasm is divided in two by a contractile ring of actin and myosin filaments, which pinches the cell into two daughters, each with one nucleus.

Centrosomes and Spindles

• Asters are formed.
• Centrioles are within the centrosome.
• The duplicated centrosome forms the mitotic spindle.

Spindle Microtubules

• Astral microtubules.
• Kinetochore microtubules.
• Non-kinetochore microtubules.

Kinetochores

• Kinetochores attach chromosomes to the mitotic spindle.

APC/C

• APC/C (anaphase-promoting complex/cyclosome) triggers the separation of sister chromatids by promoting the destruction of cohesins.

Chromosome Segregation at Anaphase

• Anaphase A:
  • Chromosomes are pulled poleward.
  • Kinetochore microtubules shorten, dragging chromosomes toward their spindle pole.
• Anaphase B:
  • Poles are pushed and pulled apart.
    • A sliding force between non-kinetochore microtubules from opposite poles pushes the poles apart.
    • A pulling force at the cell cortex drags the two poles apart.
    • Microtubule growth at plus ends of non-kinetochore microtubules helps push the poles apart.

Nuclear Envelope Dynamics

• The nuclear envelope breaks down and re-forms during mitosis.
  • Phosphorylation of nuclear pore proteins and lamins leads to nuclear envelope breakdown.
  • Dephosphorylation of nuclear pore proteins and lamins leads to reassembly of the nuclear envelope.

Cytokinesis in Detail

• The contractile ring of actin and myosin filaments forms the cleavage furrow.
• The remaining non-kinetochore microtubules form the central spindle.

CONTROL OF CELL GROWTH, CELL DIVISION, AND CELL SURVIVAL

• Animal cells require extracellular signals to survive, grow, and divide.
• Mitogens stimulate cell division by promoting entry into the cell cycle.
• Growth factors promote an increase in cell size.
• Apoptosis helps regulate animal cell numbers.
• Apoptosis is mediated by an intracellular proteolytic cascade.
• The intrinsic apoptotic death program is regulated by the Bcl2 family of intracellular proteins.
• Signals from other cells activate the extrinsic apoptotic death program.
• Survival factors promote cell survival by suppressing apoptosis.
• Some extracellular signal proteins inhibit cell survival, division, or growth.

Growth Factors and Cell Growth

• Growth factors activate receptor tyrosine kinases (RTKs).
• Activated RTKs activate PI 3-kinase.
• Activated PI 3-kinase activates Akt.
• Activated Akt activates Tor.
• Tor stimulates protein synthesis and inhibits protein degradation, leading to cell growth.

Apoptosis

• Apoptosis in the embryonic mouse paw sculpts the developing digits.

Apoptosis Mechanism

• An apoptotic stimulus activates an initiator caspase.
• The initiator caspase cleaves and activates an executioner caspase.
• The executioner caspase cleaves multiple substrates, leading to apoptosis.

Intrinsic Apoptotic Pathway

• Bax and Bak molecules are activated and aggregate in the mitochondrial outer membrane.
• Cytochrome c is released from the intermembrane space of the mitochondria.
• Cytochrome c activates an adaptor protein, which forms an apoptosome with caspase-9.
• Caspase-9 is activated within the apoptosome, initiating a caspase cascade leading to apoptosis.

Survival Factors and Apoptosis

• Survival factors often suppress apoptosis by regulating Bcl2 family members.
• Survival factors activate a receptor.
• The activated receptor activates a transcription regulator.
• The transcription regulator increases transcription of the Bcl2 gene.
• Bcl2 protein blocks apoptosis.