Cell Bio Exam 3

What is the cell doctrine? What is required for it to exist?

• All living things are composed of one or more cells; the cell is the basic unit of life, and all cells arise from pre-existing cells (a cell divides into two identical cells)

• Cell division is high fidelity; controlled by the cell cycle

What are the stages of the cell cycle?

• Interphase:

  • G1: cell grows and becomes bigger, monitoring to find and repair DNA damage

  • S: DNA replication

  • G2:

• Mitosis

• G0: cells can decide if they should divide; this phase is outside of the cell cycle, where a cell is not dividing

Note: this applies to stem cells, once a cell is differentiated, it remains in G0

What does it mean to say that the cell cycle is evolutionarily conserved? How was this proven?

• All cells on Earth divide the same way; there is no variation

• In yeast, scientists deleted the gene that encoded the Cdk protein of the M-Cdk complex

  • Resulted in cell death (cell cycle does not occur)

• Then put the human Cdk gene into the yeast

  • Cells stayed alive, showing Cdk gene is same in humans and yeast

Why is yeast a good organism to use to study the cell cycle?

• Yeast is haploid; if you mutate a single gene, you immediately see a phenotype change

• Morphology reveals the cell cycle stage

  • Allowed for scientists to identify the genes involved in the cell cycle

What are temperature-sensitive mutants?

• Conditional mutation where the protein product functions normally at a low ("permissive") temperature but becomes unstable, inactive, or denatured at a high ("restrictive") temperature

What is replicative cell senescence? What are immortalized cell lines?

• Human fibroblasts stop dividing after 25-40 divisions

• Immortalized cell lines proliferate indefinitely (eg tumor cells)

What is a fluorescence-activated cell (FAC) sorter?

• Sorts heterogeneous cell mixtures into distinct, pure populations based on their fluorescent labels and light-scattering characteristics

• Can be used to determine DNA content of cells

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What are the elements of the cell cycle control system?

• Clock: determined when the cell transitions to the next stage

• Switches: trigger the cell cycle transitions (e.g., for G2 → mitosis transition, M-Cdk is activated)

• Mechanisms: monitor problems (e.g., DNA damage), checkpoints

When a mitotic cell and a G2 cells are fused, what happens?

• The G2 cell also enters mitosis

• Upon the fusion of the cells together, the G2 now has Cdk activity, inducing mitosis

  • M-Cdk is dominant

What happens if you fuse a G1 and S cell?

• G1 stars replicating its genome, entering S phase

• Upon the fusion of the cells together, the G1 now has Cdk activity, inducing mitosis

  • S-Cdk is dominant

What makes up the cell cycle clock?

Cdks

What is S-Cdk?

• What the Cdks phosphorylate

• That triggers the initiation of DNA replication during the S-phase of the cell cycle

  • Replication factors → DNA replication

What is cyclin? Does its abundance remain constant? What about Cdk?

Cyclin:

• Activates Cdk

• Gives Cdk substrate specificity

• Protein abundance oscillates through the cell cycle (by txn and proteolysis)

• E.g.:

  • M-Cdk = M cyclin + Cdk

  • S-Cdk = S cyclin + Cdk

Note: protein abundance of Cdk = kinase is constant through the cell cycle

What stages of the cell cycle do M cyclin and S cyclin correspond with?

• The cyclins are made by transcription and translation, and degraded through proteolysis

What is the structural basis of Cdk activation by cyclins?

• When inactive: active site covered up by T-loop

• Binding of cyclin induces a conformational change that moves T-loop, allowing it to be partially active

• Cdk-activating kinase (CAK) phosphorylates the T-loop, inducing another conformational change that moves the T-loop further away from the active site, allowing for full activity

  • “activating phosphorylation”

What happens if there is no cyclin?

There is no Cdk activity, even if Cdk-activating kinase (CAK) is present

What is ubiquitin-dependent proteolysis/degradation?

• Ubiquitin (Ub) is a small protein that can covalently attach to other proteins in chains (Ub-Ub-Ub-Ub) by a ubiquitin ligase

  • This flags the protein for degradation by the proteasome

• This is how cyclins get degraded

• Done by ubiquitin ligase APC/C

What is ubiquitin ligase APC/C (anaphase promoting complex/cyclosome)

• APC/C is present throughout the whole cell cycle

• It gets activated at specific cell cycle transitions by activating subunits

  • cdh1 → APC/C—cdh1 → the active APC/C ubiquitinates M Cyclin

  • cdc20 → APC/C—cdc20 → the active APC/C ubiquitinates securin

• Activating subunits (cdh1 and cdc20) active APC/C and provide substrate specificity

What does this figure show?

• M-cyclin levels decrease at end of mitosis because Cdh1-APC/C activity levels increases to degrade M-cyclin

• Cdc20-APC/C activity increases, which triggers the transition from metaphase to anaphase

What are the different Cdks and APC/C complexes and what transitions do they correspond to?

• S-Cdk activation: triggers the initiation of DNA replication during the S-phase of the cell cycle

• M-Cdk activation: triggers transition into mitosis

• Cdc20-APC/C: triggers transition from methaphase to anaphase (degradation of securin)

• Cdh1-APC/C: triggers transition from metaphase to G1 (degradation of M-cyclin)