Cell Exam 2 Study Guide

47. list the Cdk-Cyclin complexes present at each phase of the cell cycle? On the same diagram show the cell cycle checkpoints.

- 4 2 2 1 - D E A B - G1 S G2&M - Check points = G1/S, G2/M, M (kinetochore attachment)

13. How is procaspase 9 induced?

- APAF-1 molecules that oligomerize, forming the apoptosome, expose CARD domains, which match up with the CARD domains on procaspase 9. This causes the procaspase 9 to dimerize and become activated by induced proximity

26. To prevent cells with damaged DNA from passing through the G1 checkpoint, what are ATR or ATM doing on Chk1 or Chk2?, which then modulate protein phosphatase Cdc25

- ATR and ATM phosphorylate Chk1 and Chk2. Once phosphorylated, the Chk1 and Chk2 become activated and phosphorylate Cdc25, marking it for proteolysis. Cdc25 is necessary for activation of the CDK 2-cylin E complex. Since Cdc25 is destroyed by the ATR and ATM pathways, CDK 2-cyclin E remains inactive and the cell cannot proceed into S phase.

25. List the different Ubiquitination events that occur over the cell cycle. Give their names and roles and regulatory mechanism(s).

- Anaphase promoting complex/cyclosome (APC/C): the APC + cdc20 ubiquitinates securin causing its desruction, which liberates separase so that the cohesins holding sister chromatids together can be broken down. - SCF complex: SCF ubiquitinates CKI (cyclin-dependent kinase inhibitors), marking them for degradation so that CDK/cyclin complexes can be activated. - Cyclin can also be ubiquinated for degradation in order to progress the cell cycle or inactivate CDKs

14. How are Apoptotic cells completely removed from the tissue they reside in?

- Apoptotic cells produce a "find me" and a "eat me" signal - Find me = lysophosphatidyl-choline - Eat me = externalization of phosphatidylserine - They are then located and phagocytized by other, healthy cells.

36. How is the position of the spindle used to determine the formation of the contractile ring?

- As the spindle pull sister chromatids apart, new MTs called stem bodies form in between the separating sister chromatids (at the middle of the cell where they were before separation). These stem bodies produce a signal that stimulates the formation of the contractile ring.

31. What are astral microtubule? What is their origin?

- Astral MTs radiate outward in all directions from the centrosome, but they are often thought of as only the MTs that point toward the cortex (away from the chromosomes). - They originate at the centrosome (aka MTOC) and are often attached to the cell's cortex by dynein proteins

7. What is the complex found in the mitochondria that drives apoptosis?

- Bax/Bak complex

58. What is one function of the Cdk1-cyclin B complex in mammalian cells?

- CDK 1/B phosphorylates APC allowing for the progression into anaphase - CDK 1/B also phosphorylates lamin filaments leading to nuclear envelope break down

35. How the ORI (origin of replication) being licensed to fire?

- CDK 2/A activates the binding of Cdc45 to the MCM, which activates its helicase activity - Once replication has begun, CDK 2/A phosphorylates Cdc6 on the ORC, marking it for degradation. - Without the Cdc6, another MCM cannot bind and begin replication a second time.

56. There are several mechanisms for regulating Cdk activity. List three distinct mechanisms of Cdk activation or inhibition.

- CDKs can be phosphorylated in an inactivation site (ex. Wee1 inactivates CDK1) - CDKs can be inactivated by the removal/degradation of their cyclin counterparts (ex. Ubiquitination of cyclin molecules) - CDKs can be activated by phosphorylation by CAK

20. Explain how the protein Bid links the death receptor pathway and the MOMP pathway to apoptosis.

- Caspase 8, which is activated in the death receptor pathway, activates Bid. Then Bid activates Bax/Bak to form the pores in the MOMP, which release cytochrome c and activate the apoptosome and caspase 9.

9. What is the functional role of CARD domains?

- Caspase recruitment domains are a part of the death fold of caspases. They interact with the CARD domains found on other proteins to define an apoptotic pathway.

55. The isolation of conditional cell cycle mutations in yeast helped identify many of the key proteins involved in cell cycle control. Many of these mutations were given the name cdc (cell division cycle) and are temperature sensitive. Hypothesize about the outcomes of a cdc temperature sensitive mutant.

- Cdc molecules are involved in activating many different proteins, such as the APC, so if a cdc were mutated, it would not be able to activate different proteins, and the cell cycle would have many errors.

33. How does cdc25 affect commitment to Mitosis?

- Cdc25 is a phosphatase that removes the inactivating phosphate from CDK 1-cyclin B complex to activate it. - The CDK1/B complex then initiates the break down of the nuclear envelope.

42. What are centromeres? What are their structure and roles?

- Centromeres are the site where the two kinetochores form on each chromosome - They are made up of highly condensed heterochromatin composed of highly repetitive DNA sequences that bind CENP and MCAK proteins. - It functions to attach the two sister chromatids together, and direct the formation of the kinetochore for spindle attachment

27. What are the events of prophase?

- Chromosomes begin to condense inside the nucleus - The centrosomes appear and the spindle begins to form - The nuclear envelop begins to break down (prometaphase)

45. What is congression (that really important part of metaphase) during mitosis?

- Congression is the balancing of forces which align the chromosomes in the center of the cell at the metaphase plate prior to anaphase

39. What are the critical events for M phase to occur in animal cells?

- Cyclin B begins to accumulate and complex with CDK 1, but Wii1 adds an inactivating phosphate to CDK 1. - Cdc25 will remove the inhibitory phosphate from CDK 1 to activate it. - Then the CDK 1/B complex will activate more Cdc25 (to induce more CDK 1/B activation), and inhibit Wee1 (to prevent CDK 1 deactivation).

16. What happens when cytochrome C binds APAF-1?

- Cytochrome C binding to APAF-1 allows the APAF-1 molecules to oligomerize and form the apoptosome

19. Briefly explain how cytochrome c activates caspase-9.

- During MOMP, cytochrome c is released form the intermembrane space of the mitochondria. It this binds to APAF-1, causing it to oligomerize and form the apoptosome. The CARD domains on the apoptosome then dimerize with caspase 9 to activate it.

17. List the features of executioner caspases that distinguish them from other caspase types.

- Executioner caspases exist as dimers in the inactive form. The are different from other caspases in that cleavage between their two subunits is both necessary and sufficient for activation - Note: the cleavage is usually mediated by initiator caspases

6. Which events does take place during the death receptor pathway of the tumor necrosis factor receptor (TNFR1) signaling?

- FADD adaptor proteins bind with the DD-DD interaction to death receptors. Then procaspase 8 binds with DED-DED interactions to the FADD. Procaspase 8 become activated to the initiator caspase 8. Initiator caspase 8 then cleaves and activates executioner caspases 3,6,7 which cleave substrates and induce apoptosis.

49. Stable, bipolar attachment of sister chromatids is assessed by kinetochore passenger proteins and the MCAK motor (that depolymerizes microtubules) so that the SAC is not passed too soon. True or false?

- False. The SAC senses when there is improper kinetochore attachment and becomes activated to inhibit the APC from targeting securin for degradation, therefore preventing separase activity and progression into anaphase.

9. The nucleus is completely filled with chromatin. True or false?

- False. The nucleus is comprtmentalized. There are chromosome domains, interchromosomal domains, and the nucleolus.

38. The signal for the spindle assembly checkpoint comes from the spindle poles. True or false?

- False. This is regulated by sensing tension in the kinetochores Note: the spindle assembly checkpoint = kinetochore attachment checkpoint

44. What type of molecular sensors does a cell possess to respond to the late outcome of the absence of functional Retinoblastoma protein?

- If proliferative cues are provided to cells in the absence of sufficient nutrients to ensure a successful round of cell division, apoptosis is triggered.

18. Define the term "induced proximity", and give one example.

- Induced proximity is a mechanism of caspase activation whereby two monomers are brought close to one another, forming active dimers - Ex. Procaspase 9 is activated by induced proximity and dimerization with the apoptosome's CARD domains.

8. The protein IκB functions in two independent and complimentary roles to control the import and export of the transcription factor NFκB. Explain these two roles.

- IκB binds to NFκB and disables its NLS so that it cannot be transported into the nucleus. When IκB is degraded by IKKs, the NLS on NFκB activates and can be imported into the cell. - The NFκB induces gene expression once inside the nucleus. Then the IκB (which also has a NLS that is inactivated when bound to NFκB) binds to the NFκB inside the nucleus and transports it out of the nucleus because IκB has a NES and NFκB does not.

22. What are the forces in Anaphase B that move sister chromatids and poles further apart?

- Kinesins on the MTs push the overlapping MTs apart, poleward - Dynein anchored to the cell cortex pulls on the astral MTs

7. Leptomycin is a compound that inhibits the growth of Human Immunodeficieny Virus in human cells, yet it cannot be used to treat HIV patients because it is toxic to all of a patient's cells. Explain how leptomycin functions in human cells.

- Leptomycin binds to the Rev molecule and prevents it from binding NESs. This completely stops all NES-dependent nuclear export.

15. Explain how death receptors activate initiator caspases.

- Ligation of the death receptors causes the recruitment of the adaptor protein, FADD, via death domain (DD)-DD interactions. Then the FADD dimerizes with initiator caspases via DED-DED (death effector domain) interactions, causing the initiator caspases to be activated through induced proximity.

1. What is apoptosis?

- Morphological features of programmed cell death

2. Which features appear in cells undergoing apoptosis but not in cells undergoing necrosis?

- Necrosis is the direct result of acute cell injury, and often invokes an inflammatory response because the cells burst. - Apoptosis is "silent" (no inflammation) because the cells do not burst, but are broken into fragments and engulfed by surrounding cells and recycled.

1. Is all protein trafficking through nuclear pores unidirectional?

- No. They nuclear pores are channels that allow movement both into and out of the nucleus. Some proteins have to complete full circles (in and out) of the nucleus to performs their functions where they are needed.

11. What are nuclear lamin proteins making up?

- Nuclear lamin is assembled together to form the nuclear lamina, which plays a role is nuclear envelope assembly and support. - *lamin may even play a role in organizing the chromatin for DNA replication

6. Explain the property of the nuclear pore and how it was discovered.

- Nuclear pores are symmetric channels that are constructed of nucleoporins - They were discovered by tracing mRNA exports

54. What prevents an origin of replication from being used more than once per cell cycle?

- Once the Pre-RC is formed, cyclin A concentration rises in the cell. - Once replication is begun, the CDK 2/A complex removes Cdc6 from the RC. - Without Cdc6, MCM cannot bind to the ORC and replication cannot begin again. (this is true as long as cyclin A concentration is high in the cell)

21. How mRNA processing is linked to mRNA export from the nucleus?

- Proteins bind to mRNA as it is synthesized in the nucleus. This complex is known as heterogeneous nuclear ribonucleoprotein particles (hnRNPs) - Those proteins help to package the mRNA for nuclear export, and also structure/mark it for processing.

19. What are the features of the transport of proteins across the nuclear envelope?

- Proteins can be transported in and out of the nucleus, but they must be carried by Ran/karyopherin (importin) complexes, or some can move by themselves if they have NLS and NES sequences. - **Importins bind NLS-cargo in the cytoplasm and can enter the nucleus without RAN, then Ran-GTP binds to the importins and causes disassociation of the cargo. The importin/Ran-GTP complex can then exit the nucleus.

50. A cell in quiescence is?

- Quinescence (aka G0) is a temporary and reversible withdrawal from the cell cycle. It is a non-dividing state which is caused by signals that the environment is not good for dividing.

16. Know the cargo with the protein(s) necessary for nuclear export or import.

- Ran-GDP can enter the nucleus - Ran-GEF exchanges GDP for GTP - Ran-GTP can exit the nucleus - Ran-GAP hydrolyzes GTP on Ran-GTP to GDP so that Ran-GDP can enter the nucleus again - **RAN molecules bind karyopherins and affect their ability to bind their cargoes

18. What is the definition of heterochromatin?

- Regions of the genome that are highly condensed, are not transcribed, and are late-replicating. Heterochromatin is divided into two types, which are called constitutive and facultative

41. Hypothesize the early and late outcome of a cell without a functional Retinoblastoma protein. How oncologist classify this protein?

- Retinoblastoma protein (Rb) is classified as a tumor suppressor protein - Rb is the "off" switch for E2F, a transcription factor, which turns on cyclin E - Since cyclin E would not be regulated the cell would proceed through M1/S checkpoint and would inevitably proceed through the rest of the cell cycle, causing endless cell proliferation and eventually lead to cancer and tumors would form.

52. What is the phenotype of a cell that loses the function of Wee kinase, the inhibitory kinase of Cdk1?

- Since CDK1 will not be inhibited, the cell will progress into mitosis every time that cyclin B is present. This will cause continuous proliferation of the cell.

8. Explain how activation of tumor necrosis factor receptor (TNFR1) can induce antiapoptotic signals?

- TNFR1 can trigger the activation of NF-kB, which is a transcription factors that will promote the expression of several proteins that prevent the formation of the death-inducing signalling pathway and the activation of caspase-8

29. What is the function of the anaphase-promoting complex (APC)?

- The APC acts with the cdc20 activator in order to ubiquitinate securin, marking it for degradation. This liberates separase, which cleave cohesin and allos sister chromatids to separate in anaphase.

15. The nuclear localization signal (NLS) is recognized by and binds to which protein in the process of nuclear protein transport?

- The NLS (usually a short stretch of basic Aas {arg/lys}) is recognized and bound by karyopherins (importins) in most cases. The NLS can also bind directly to the NPC (nuclear pore complex) in some cases for direct import.

4. The transport of proteins across the nuclear envelope has some distinct mechanisms compared to the transport of proteins across the mitochondrial membrane, although both are mediated by the presence of a signal sequence. What are the other differences?

- The NLS is not cleaved, but remains an integral part of the protein in nuclear transport.

40. What is the pre-Replication Complex (RC)?

- The Pre-RC is the protein complex that forms on the Origin of replication (ORI) prior to DNA replication - It consists of the Origin recognition complex (ORC), Cdc6 and Cdt1 (licensing proteins), and the MCM helicase

53. What is the role of the spindle assembly checkpoint (SAC)?

- The SAC (aka kinetochore attachment checkpoint) ensures that there is proper kinetochore attachment prior to anaphase. When the kinetochores are not attached properly, SAC deactivates APC so that it cannot target securin for degradation and liberate Separase.

23. How does centrosome reproduce?

- The centrosome is made up of 2 centrioles (at right angles) - The centrioles reproduce during S phase, at the same time that DNA replication is taking place. - It seems that the CDK2 and cyclins E and A play a role in stimulating centriole duplication

12. What is a complex of multimers of APAF-1 doing?

- The complex is called the apoptosome, and it exposes CARD domains which bind to, and dimerize initiator caspases, activating them to cleave and activate executioner caspases, leading to apoptosis.

21. Explain how a flippase enzyme assists in the clearance of apoptotic cells in mammals.

- The flippase switches the positions of two phospholipids in the plasma membrane, moving outside lipid to the inside leaflet. This moves the phosphatidylserine to the outer leaflet, but it is still inside the membrane. The a translocase will "flop" it to expose it outside of the cell.

20. What mechanism ensures that cargo-protein complex does not exit the nuclear pore complex (NPC) once it has passed through the nuclear pore and into the nucleus?

- The importin-cargo complex cannot exit the nucleus without associating with a Ran-GTP complex. One this association occurs, the cargo disassociates from the importin protein and cannot be carried back to the cytoplasm with the importin.

32. What is the mitotic spindle?

- The mitotic spindle consists of the centrosome and the MTs that extend outward from it to attach to chromosomes. Its function is to move the chromosomes and direct where in the cell they go.

14. What is the nucleolus?

- The nucleolus is located at the very center of the nucleus and is the site of rRNA synthesis and ribosomal subunit assembly

17. What is one advantage to a cell of having a nucleus?

- The nucleus acts a protective barrier which can select which molecules/proteins can interact with the DNA

13. What advantage does possession of a nucleus offer a cell?

- The nucleus allows for more controlled protection of the DNA.

11. During mitochondrial outer membrane permeabilization (MOMP) what is allowed to escape from the mitochondria?

- The proteins that are found in the intermembrane space. Things in the mitochondrial matrix are contained by the inner membrane and therefore do not escape.

37. Initiation of DNA synthesis and replication of centrioles are tightly coordinated events. Briefly explain how this coordination is achieved.

- The same regulator, CDK 2/E&A, initiates the replication of both DNA and centrioles.

30. How many kinetochores are there in a human cell at mitosis?

- There are 2 kinetochores per centromere, and one centromere per chromosome in humans cells. - 46 humans chromosomes = 92 kinetochores present in mitosis

5. How are executioner caspases activated?

- They are cleaved at a specific area (aspartate residues) between the large and small subunits to expose the catalytic cysteine-histidine dimer.

10. MOMP, mitochondrial outer membrane permeabilization is a key event in apoptosis. What role do the apoptotic Bcl-2 proteins Bax and Bak play in MOMP?

- They associate together in the outer membrane of the mitochondria to form a pore through which the proteins in the intermembrane space can diffuse out of the mitochondria. In other words, they cause the membrane permeabilization.

3. How are initiator caspases activated?

- They dimerize with the same "death folds" on adaptor proteins

28. what are Chk1 or Chk2 doing on Cdc25?

- They phosphorylate the Cdc25, marking it for proteolysis by a proteosome

2. Importin bound to NLS-containing cargo is transported into the nucleus where the complex binds Ran-GTP and gets dissociated. True or false?

- True. Importin bind to NLS-cargo in the cytoplasm (no Ran needed), gets transported into the nucleus, where [Ran-GTP] is high and binds and causes disassociation from the cargo. The importin, no bound to Ran-GTP goes back to the cytoplasm.

46. Chromosomes are pushed away from the poles (polar winds) by chromokinesins and spindle microtubule interactions. True or false?

- True. Polar winds are in the interactions between chromokinesins (on the arms of chromosomes) and spindle MTs, which push the chromosomes back toward to center of the cell and away from the cortex.

51. CENP-E protein in an unattached kinetochore can slide along an adjacent, attached kinetochore fiber toward a centered chromosome to help the unattached chromosome get closer to the center. This may help shorten the distance for the microtubules searching for an unattached kinetochore. True or false?

- True. The CENP-E on the unattached kinetochore will find an adjacent kinetochore that is attached and grab onto its kinetochore fiber and pull the chromosome back toward the middle of the cell.

43. After the nuclear envelope breaks down, microtubules gain access to the chromosomes and, every so often, a randomly probing microtubule connects with a kinetochore and captures the chromosome. True or false?

- True. The MTs capture the Kinetochores by randomly searching. Once this happens, the attachment of a second MT to the other kinetochore is expedited.

10. Active genes are found adjacent to interchromosomal domains. True or false?

- True. They are more easily accessible for transcription at this region. Active genes are also found near the poles.

24. Cohesins help the cell remember the gene expression pattern that existed before the cell divided by marking transcription factor binding, helping the transcription factors find their correct places. True or false?

- True. This is a role of cohesins, but we were told that we didn't need to know it in class. - We were told to know: Cohesins are involved in a protein complex that hold the sister chromatids together until separase is liberated from securin and proteolyzes them so the sister chromatids can separate in anaphase.

48. Each kinetochore fiber exerts a poleward-pulling force that is proportional to its length (until the pull forces from each pole are equal). True or false?

- True. This is the hypothesis for how congression occurs. The longer kinetochore fibers have a stronger pulling force than the shorter ones. So they pull back an forth until both kinetochore fibers are the same length because then they have the same pulling force and the net force is zero.

5. Both U snRNAs and microRNAs are transcribed by polymerase II and then further modified including cleavage of the RNAs. What are RNAs?

- U snRNA: involved in RNA processing; they are developed to maturity into their protein complexes outside of the nucleus and then imported back in - miRNA: involved in gene expression; either upregulate or stop mRNA from being translated - RNA: messenger molecules that carry information from DNA to be translated into proteins

4. XIAP is an inhibitor of caspases. How does it work?

- XIAP functions as a ubiquitin ligase, binding to and ubiquitinating inhibitor caspases for degradation.

34. Does cdc25 expression oscillate during cell cycle?

- Yes, because Cdc25 is degraded before the S phase when there is DNA damage found during the G1 checkpoint.

3. How would you design an experiment to demonstrate that the NLS (nuclear localization signal) of the yeast protein Swi5 is sufficient for the transport of this protein into the nucleus?

- You would need to remove or inhibit the carrier proteins (such as Ran-GDP) to show that it does not require a protein to move into the nucleus. Then you would need to destroy the NLS in the Swi5 protein to show that it cannot enter the nucleus without it. - Note: Swi5 can enter the nucleus when it is dephosphorylated and it cannot enter when it is phosphorylated

57. Explain why cells that lack functional p53 are more prone to accumulate mutations in their DNA?

- p53 is a tumor suppressor gene that is involved in checking that DNA is replicated and repaired correctly. When it is not functional, the cells escape cell cycle regulatory control and can proliferate in the presence of DNA damage

12. Your textbook describes a simple experiment illustrating that export of transfer RNA (tRNA) from the nucleus to the cytoplasm is under the control of the small GTP binding protein Ran. Summarize the results of this experiment and explain how Ran is functioning in this process.

- tRNA export became saturated when the cell was continuously injected with tRNA, which suggested that its transport requires a protein - By injecting the nucleus with Ran-GAP to hydrolyze the Ran-GTP, it was shown that tRNA transport was stopped, which proved that tRNA transport is Ran-dependent - Ran-GTP carries tRNA/exportin complexes out of the nucleus