C3 transferase is a bacterial toxin that inactivates Rho. What aspect of cell division would be blocked if you treated cells with C3?
The spindle checkpoint monitors kinetochore attachment to microtubules, as well as tension placed on the kinetochore by bound kinetochore microtubules. During normal mitosis in animal cells, when would you expect the spindle checkpoint to be activated?
What would you predict would happen if you were to express a mutant of cyclin B that is missing the D box? The cell would arrest in:
Anaphase (with high CDK activity)
If the proteasome was inhibited in a dividing cell just before the onset of mitosis: the cell would arrest in:
Duplication of centrosome occurs during:
A cell with a non-functional mutant of Cdc25: would arrest in:
Chromatin organization is not typically friendly to RNA polymerase and transcription. How is chromatin remodeled to promote transcription?
Histones are modified by histone deacetylase, which weakens the interaction between the nucleosome and the DNA backbone.
What are the factors that remodel chromatin, and how they recruited to the promoter?
SWI/SNF factors use ATP to physically slide nucleosomes “out of the way”, which allows for the binding of general transcription factors and recruitment of RNA polymerase.
If you had a yeast strain that had a temperature-sensitive mutation in Cdc25, and you shifted the cells to the non-permissive temperature, what phase of the cell cycle would the cells arrest, and what would be the phosphorylation state of Cdk1?
The cell cycle would arrest in G2 with replicated chromosomes. However, because Cdk1 (cdc2 in fission yeast) would remain phosphorylated on Tyrosine 15 (by Wee1), the cell wouldn’t be able to enter M phase.
What are the potential consequences to cell cycle regulation if a cell loses both functional copies of a small CDK inhibitor like p21?
Small CDK inhibitors such as p21CIP1/WAF1 are critical mediators of p53-mediated cell cycle arrest. If both copies of p21 are lost or inactive, then p53 would not be able to arrest the cell cycle in response to DNA damage. This would result in cells entering S phase with DNA damage, increasing the likelihood of mutations or translocations.
You are studying a cell type whose entry into the cell cycle requires Epidermal Growth factor (EGF) signaling. Be sure you can draw out all the factors involved starting with receptor ligand binding and ending with passage through the Restriction point on to S phase. If there is a phosphorylation event, make sure you indicate who gets phosphorylated, and what the effect is (activating or inhibitory).
EGF-EGFR -> receptor autophosphorylation -> Grb2/Sos recruitment -> Ras activation -> Raf -> Mek -> Mapk -> ELK1/transcription factors -> Fos/Jun (early response genes) transcription/translation -> CDK4/6 and Cyclin D (delayed response genes) -> Rb phosphorylation -> E2F transcription of Cdk2/Cyclin E and other S phase genes.
The DNA damage response is capable of stopping the cell cycle throughout interphase. Describe how this checkpoint stops the cell cycle during A) G1
DNA damage induces the activation of ATM/ATR, which phosphorylate Chk1/2, which in turn phosphorylate p53. Phosphorylated p53 is now stabilized (ie not degraded), and can accumulate in the nucleus, where it mediates the transcription of p21, a small CDK inhibitor. P21 then binds and inactivates the G1/S Cdk’s (Cdk4/6, Cdk2)
The DNA damage response is capable of stopping the cell cycle throughout interphase. Describe how this checkpoint stops the cell cycle during B) G2
DNA damage induces the activation of ATM/ATR, which phosphorylate Chk1/2, which in turn phosphorylates Cdc25. Phosphorylated Cdc25 is bound by 14-3-3, a phospho-specific binding protein which prevents Cdc25 from activating Cdk1, thus keeping the cell arrested in G2.
30 years ago, biologists took out a small amount of cytoplasm from a meiotic oocyte, and injected it back into an oocyte that was still in G2, and the G2 oocyte would then proceed directly into meiosis I. How might a small amount of active CDK1 activity push a cell into M phase? In other words, describe or draw the Cdk1 positive feedback loop.
A cell in late G2 has cyclin B/Cdk1 complexes in an inactive state due to Wee1 phosphorylation of Cdk1 on Y15. If you were to introduce a small amount of active complex, then the active complex would promote full activation in the cell by phosphorylating (and inactivating) Wee1, and phosphorylating (and activating) Cdc25. Thus by inactivating the “inactivator” and activating the “activator”, a small amt of active Cdk1 could create a positive feedback loop that results in full activation and entry into mitosis.
You are studying a cell line that experiences a rise in intracellular calcium in response to EGF stimulation. Where is the source of calcium (internal, external, etc), and how is it mobilized in the cell?
One of the SH2-containing proteins recruited to tyrosinse kinase receptors is the Phospholipase C (gamma). PLC cleaves phosphatidylinostitide 4, 5 bisphosphate (PIP2) into diacylglycerol (DAG) and Inositol triphosphate (IP3). Soluble IP3 binds a receptor on the Endoplasmic reticulum, which opens and releases calcium ions stored in the ER into the cytoplasm.
Describe three mechanisms by which the Receptor tyrosine kinase /Ras/MAPK pathway maybe inactivated or down regulated.
The receptor may be internalized or degraded; Tyrosine phosphatases may degrade the receptor - thus removing binding sites for SH2-domain containing proteins; RAS-GAP (which contains an SH2 domain itself) may facilitate the inactivation of Ras; MAPK phosphatase, which is downstream of MAPK signalling, may dephosphorylate and inactivate MAPK
Knowing what you know about the ATP-dependent maintenance of ion gradients across cells (such as calcium) and the myosin ATPase cycle, explain the molecular basis for rigor mortis (the stiffening of the corpse following death).
When cells run out of ATP, one of the consequences is that they can no longer maintain ion gradients (such as calcium), and calcium levels will increase in the muscle, allowing troponin to move tropomyosin from the myosin binding site on the actin filament. In addition, in the absence of nucleotide, myosin binds actin in what is called the rigor conformation. Thus, while the muscle is not contractile, the muscle is locked with myosin bound to actin. Thus rigor mortis.
Cholera patients suffer from dehydration. Based on your encyclopedic knowledge of signal transduction and ion transport, explain how cholera toxin can lead to water loss.
Cholera toxin modifies Gαs such that it cannot hydrolyze GTP, and thus is permanently active. This leads to overproduction of cAMP, which activates protein kinase A. Protein kinase A phosphorylates and activates CFTR, the chloride transporter involved in cystic fibrosis, The chronically activated CFTR leads to chloride transport, and in turn, water transport out of the intestinal epithelium. This leads to water loss, and in the case of children, sometimes death.
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