MCDB XL 100 Chapter 17

Which of the following is more directly driven by active M-Cdk? A. Centrosome maturation B. Centrosome duplication C. Nuclear envelope reassembly D. Inactivation of APC/C E. Cell cleavage

A The M-Cdk is responsible for most events in early mitosis, including spindle assembly and centrosome maturation. It is inactivated later in mitosis, resulting in the events of telophase and cytokinesis.

Treatment of dividing cells with a low dose of the antifungal drug benomyl, which destabilizes microtubules, slows down correct spindle assembly. But at such doses, the spindle is eventually formed and the cells survive. However, mutations in some genes confer benomyl sensitivity: the mutant cells die because they fail to arrest the cell cycle in the presence of unattached kinetochores and progress through anaphase, with disastrous consequences. Which of the following would you expect to be a benomyl-sensitive mutant?

A. A loss-of-function mutant in the gene encoding Mad2. B. A mutation causing the overexpression of Cdc20. C. A loss-of-function mutation in the gene encoding a kinase that inhibits Cdc20-APC/C. D. A loss-of-function mutation in the gene encoding a tubulin subunit.

How is centrosome duplication similar to DNA replication?

A. They both use a semiconservative mechanism. B. They are initiated at around the same time in the cell cycle, near the G1/S transition. C. They are both controlled in such a way that they replicate once and only once per cell cycle. D. They are both separated from their sister copies in mitosis.

Indicate whether each of the following phosphorylation events typically activates (A) or inactivates (I) the protein that is being phosphorylated. ( ) Phosphorylation of Cdc25 by M-Cdk ( ) Phosphorylation of condensin subunits by M-Cdk ( ) Phosphorylation of kinesin-5 by Aurora-A ( ) Phosphorylation of Ndc80 subunits by Aurora-B

AAAI M-Cdk activates the Cdc25 phosphatase in a positive feedback loop. It also phosphorylates condensin subunits, stimulating DNA binding and supercoiling activity. Along with Aurora-A, it also phosphorylates kinesins such as kinesin-5, stimulating microtubule binding and motor activity. Without tension in the kinetochore, Aurora-B phosphorylates and inactivates components of Ndc80, lowering its affinity for microtubule plus ends.

Indicate whether each of the following, when active, tends to activate (A) or inactivate (I) M-Cdks. ( ) M-Cdk ( ) Cdc20-APC/C ( ) Cdh1-APC/C ( ) Sic1

AIII

Loss of the gene encoding shugoshin in many multicellular organisms leads to sterility, suggesting defects in meiosis. What would you expect to occur in meiotic cells lacking shugoshin?

All chromatids separate prematurely in anaphase I. In the absence of shugoshin, cohesin complexes at the centromeric regions are no longer protected from phosphorylation. The phosphorylated cohesin subunits are then cleaved by separase in anaphase I. Consequently, the sister chromatids segregate randomly in anaphase II.

In the fission yeast Schizosaccharomyces pombe, the proteins Cut1 and Cut2 form a complex that is catalytically inactive. At the onset of anaphase, Cut2 is polyubiquitylated by a large E3 complex containing Cut4, Cut9, Cut23, and other proteins, and is subsequently destroyed. Cut1 then cleaves Rad21, a non-SMC component of a complex that also contains two SMC proteins, thus allowing sister-chromatid separation. Mutations in the cut genes lead to the cut phenotype, in which the cell attempts cytokinesis without chromosome segregation. Indicate which of the following proteins or protein complexes corresponds to or contains the product of the genes cut1 (A), cut2 (B), cut4 (C), and rad21 (D). ( ) Securin ( ) Cohesin ( ) Anaphase-promoting complex ( ) Separase

BDCA Rad21 is a Scc1 homolog, which is cleaved by separase in the metaphase-anaphase transition.

Consider two mammalian cells, one in G1 and the other in G0 (stationary) phase. If they are stimulated to pass the restriction point by the addition of an extracellular proliferation signal, but the signal is then immediately removed, what would you expect to happen?

Both cells will replicate their DNA. Once a cell passes the restriction point, it is committed to DNA replication (and cell division) even if the stimulating signals are removed.

Imagine a prometaphase chromosome pair that is attached to one spindle pole. Which of the following would happen if both arms of the chromosome are severed with a strong laser beam? A. All chromosome fragments (the centromere-containing fragment and the arm fragments) would be pushed away from the pole. B. Only the two arm fragments would be pulled toward the pole. The kinetochore-containing fragment would remain stationary. C. The kinetochore-containing fragment would be pulled toward the pole. The two arm fragments would move away from the pole. D. The kinetochore-containing fragment would move away from the pole, but the two arm fragments would be pulled toward the pole. E. All chromosome fragments would be pulled toward the pole.

C

In most mammalian cells, low M-cyclin protein levels are maintained during G1 phase. What is mainly responsible for maintaining these low levels?

Cdh1-APC/C Cdh1-APC/C activity increases in late mitosis and keeps the M-Cdk activity low via M-cyclin degradation during G1.

Which of the following events occurs in mitotic metaphase? [13] #5 A. Nuclear envelope breakdown B. Nuclear envelope reassembly C. Chromosome attachment to spindle microtubules for the first time D. Chromosome alignment at the spindle equator E. Mitotic spindle assembly

D

A cell has been subjected to ultraviolet irradiation, causing a significant number of mutations in the genome. Which of the following would you NOT expect to occur as a result? [54] A. Activation of the protein kinase ATR B. Activation of the protein kinase Chk1 C. Inactivation of the protein phosphatase Cdc25 D. Binding of p53 to Mdm2 E. Stabilization of p53

D Activation of the DNA damage response involves the two upstream kinases ATM and ATR, which phosphorylate and activate the Chk1 and Chk2 kinases. Cdc25 phosphatases can be inhibited by phosphorylation in this pathway, whereas p53 phosphorylation stabilizes the protein by reducing its interaction with its inhibitor Mdm2.

Indicate true (T) and false (F) statements below regarding cellular control of growth and division. ( ) Replicative cell senescence usually arises due to the accumulation of mutations in genes encoding S- and M-cyclins. ( ) Replicative cell senescence is caused by the induction of apoptosis by p53. ( ) Excessive mitogenic stimulation can result in Mdm2 activation and the induction of apoptosis or cell-cycle arrest. ( ) The Mdm2 inhibitor Arf induces cell-cycle progression.

FFFF

Indicate true (T) and false (F) statements below regarding cytokinesis in animal cells. ( ) The force for cytokinesis is generated by kinesin motors on microtubule bundles that form the contractile ring. ( ) As the contractile ring constricts, its thickness increases to keep a constant volume. ( ) The midbody forms from bundles of actin and myosin II. ( ) Local activation of Ran GTPase triggers the assembly and contraction of the contractile ring.

FFFF

Indicate true (T) and false (F) statements below regarding meiosis in eukaryotic cells. ( ) Bivalents form before prophase I. ( ) Crossing-over begins before the synaptonemal complex assembly. ( ) Chiasmata can first be seen under the microscope after the disassembly of the synaptonemal complexes. ( ) All recombination events lead to crossovers.

FTTF

Indicate true (T) and false (F) statements below regarding mitosis and the changes that it brings about compared to interphase. Your answer would be a four-letter string composed of letters T and F only, e.g. TFTF. [17] ( ) Microtubules become greatly stabilized in mitosis compared to interphase. ( ) The number of γ-tubulin ring complexes in the centrosomes increases greatly during mitosis compared to interphase. ( ) Chromosomes stabilize mitotic microtubules through activation of Ran monomeric G protein. ( ) A bipolar spindle can be formed even in the absence of centrosomes.

FTTT

Mammalian Cdk inhibitor proteins (CKIs) can be grouped into two families based on their structural and functional differences. The Cip/Kip family proteins (e.g. p21) have a broad binding specificity. These proteins bind preferentially to already formed cyclin-Cdk complexes and thus enhance complex formation. However, they inhibit the kinase activity of most complexes (e.g. S-Cdks), except in the case of G1-Cdk complexes where no inhibition occurs. Consequently, Cip/Kip family proteins have an overall positive effect on Cdk4/6 activity due to their help in bringing the subunits together. In contrast, the inhibitors of the INK4 family (e.g. p16) bind only to the Cdk subunit of G1-Cdks and prevent binding of both the G1 cyclins and the Cip/Kip family CKIs. Based solely on these findings, would you expect p16 to activate (A) or inactivate (I) the S-Cdks in the presence of limited amounts of p21?

I In the absence of p16, p21 is sequestered by the G1-Cdk complex. Active p16, however, prevents p21 from binding, making it available to inhibit the S-Cdk complexes.

3

In the early embryos of Caenorhabditis elegans, defects in the formation of astral microtubules increase myosin activity throughout the cell cortex. If the spindle is forced to one side of the cell, the cortical myosin activity is observed mostly at the opposite side of the cell. Which model (1, 2, or 3) better predicts these observations?

NNEN

In the following schematic diagram of a meiotic bivalent in diplotene, indicate whether each of the following chromatid pairs have undergone DNA exchange (E) or not (N). The different color of the final form of chromatid 3 is for clarity only. Your answer would be a four-letter string composed of letters E and N only, e.g. NENE. [44] ( ) Chromatids 1 and 3 ( ) Chromatids 1 and 4 ( ) Chromatids 2 and 3 ( ) Chromatids 2 and 4

BC

In the following schematic diagram of a typical eukaryotic cell cycle, choose two major time points (among A to E) at which the cell-cycle control system normally arrests the cycle if DNA damage is detected

B Cdh1-APC/C activity increases in late mitosis and keeps the M-Cdk activity low via M-cyclin degradation during G1.

In the following schematic diagram of an early Drosophila embryo, which step (A to C) corresponds to cellularization?

A

In the following schematic drawing of a vertebrate prometaphase chromosome as seen under a microscope, indicate whether the chromosome is more likely to be closer to pole A or pole B.

Which of the following motor proteins are more directly involved in anaphase B?

Kinesin-5 on interpolar microtubules and dynein on astral microtubules

Consider two kinesin motor proteins at the mitotic spindle midzone: kinesin-5 is a tetrameric motor that walks toward the plus end of both microtubules to which it is attached via its motor domains; kinesin-14, on the other hand, walks toward the minus end of one microtubule while it is attached to another microtubule via its tail domain. How do these motors affect the length of the spindle?

Kinesin-5 works to lengthen the spindle whereas kinesin-14 works to shorten it.

Fill in the blank: "The is a large structure formed at the centromeric region of each eukaryotic chromosome. It captures spindle microtubules in mitosis, and therefore serves to attach the chromosomes to the spindle poles."

Kinetochore

Indicate whether each of the following descriptions better applies to mitogens (M), growth factors (G), or survival factors (S). ( ) They unblock cell-cycle progression ( ) They suppress apoptosis ( ) They trigger a wave of G1/S-Cdk activity ( ) They inhibit the degradation of cellular macromolecules

MSMG

Which division in meiosis is more similar to mitosis? In which division do sister chromatids separate from each other?

Meiosis II; meiosis II

Which of the following events contributes to driving the mammalian cell past the restriction point of the cell cycle?

Phosphorylation of Rb by G1-Cdk, G1/S-Cdk, and S-Cdk

Which stage is usually the longest in meiosis?

Prophase I

B S-phase arrest will increase the proportion of cells in this phase; that is, cells with a relative cellular DNA content of between 1 and 2.

Resveratrol is a natural compound found in red grapes (and red wine) and is thought to have beneficial effects in mammals, such as preventing tumor growth and delaying age-related diseases. In vitro, resveratrol and its derivatives have been shown to cause cell-cycle arrest in S phase and induce apoptosis. You have analyzed the DNA content of cultured cells in the presence and absence of these drugs using fluorescence-activated cell sorting. Compared with control cells (dashed line), which of the following curves do you think better represents the DNA content of cells treated with these compounds?

Indicate whether each of the following occurs mainly in G1 phase (G), S phase (S), or G2 phase (H) of the cell cycle. ( ) DNA helicase activation ( ) DNA helicase deposition on DNA at the replication origins ( ) ORC phosphorylation ( ) Licensing of replication origins

SGSG

CFEDAB

Sort the following schematic diagrams (A to F) to reflect the order of events in a typical eukaryotic M phase. An interphase cell in G2 phase is drawn on the left for comparison.

Indicate true (T) and false (F) statements below regarding cytokinesis in eukaryotic cells. ( ) The preprophase band, composed of microtubules and actin filaments, marks the site of cytokinesis in plant cells. ( ) In some cell types, the site of contractile-ring formation is determined before mitosis. ( ) The early cell plate in dividing plant cells appears after phragmoplast formation. ( ) The membrane required for the newly formed cell plate in plant cells is provided by endocytosis from the equatorial plasma membrane.

TTTF

If cells that have started mitosis are treated with nocodazole, a drug that depolymerizes microtubules, what would you expect to happen? Where would you expect Mad2 protein to be localized?

The cells would arrest in prometaphase; Mad2 would be localized to almost all kinetochores

CBDA

The following schematic diagram shows the activation of M-Cdk in mitosis. Indicate which proteins below correspond to those indicated as A to D in the diagram. ( ) Wee1 ( ) CAK ( ) Cdc25 ( ) M-cyclin

How is Cdc20-APC/C similar to Cdh1-APC/C?

They both inhibit M-Cdk activity. Although regulated differently, both APC/C complexes target M-cyclins for degradation.

Indicate whether each of the following descriptions better applies to a Wee1 protein (W) or a Cdc25 protein (C). ( ) It is a protein kinase. ( ) It activates M-Cdk complexes. ( ) It is activated by M-Cdk complexes. ( ) Its loss in fission yeast results in small cell size.

WCCW

A The G1/S-Cdk activity (solid black curve) peaks in late G1 and falls in S phase. The S-Cdk levels (solid gray curve) remain elevated through S and G2 until early M phase. M-Cdk (dashed gray curve) accumulates before entry into mitosis, but their level falls in mid-mitosis.

Which of the following simplified diagrams better shows the changes in the concentrations of three major cyclin-Cdk complexes (G1/S-Cdk, S-Cdk, and M-Cdk) in the cell in different stages of the cell cycle?

B

Which of the time points (A to E) in the following schematic drawing of the mammalian cell cycle represents the restriction point?

C

Which one of the following chromosomes has formed stable attachment(s) to the spindle microtubules?

213

You have been studying the effect of loss-of-function mutations in the Cdk inhibitor protein (CKI) p21. You add the drug fucoxanthin to cell cultures harboring either wild-type or mutant versions of the p21 gene. Fucoxanthin is known to induce cell-cycle arrest in G1. After a day, you add the thymidine analog BrdU to the culture media, collect the cells after an hour, treat them with anti-BrdU antibody and the fluorescent DNA stain DAPI, and finally subject them to fluorescence-activated cell sorting (FACS). The FACS data can be viewed as a two-dimensional dot plot composed of thousands of dots, in which each cell is represented by one dot at coordinates that correspond to the intensities of the DAPI fluorescence signal (X axis) and BrdU fluorescence signal (Y axis) for that cell. Answer the following question(s) according to the simplified dot plot below, generated from your experiment. Indicate which boxed region (1, 2, or 3) in the FACS plots corresponds better to each of the following phases of the cell cycle. ( ) G1 phase ( ) S phase ( ) G2 and M phases

Mutant Saccharomyces cerevisiae cells that lack the gene encoding securin can divide more or less normally by mitosis, without significant chromosome segregation defects. Cells harboring a nondegradable version of securin, on the other hand, arrest in metaphase as expected, since they cannot activate separase to enter anaphase. Similarly, cells lacking Cdc20 arrest in metaphase, since they cannot activate APC/C. Finally, cells lacking both securin and Cdc20 arrest in anaphase: they manage to separate sister chromatids, but do not progress much further. These results suggest that in wild-type cells:

degradation of securin is necessary to trigger sister-chromatid separation Both Cdc20 activation and securin destruction are necessary for sister-chromatid separation because cells in which either of these fail to occur are observed to arrest in metaphase. Securin degradation does not appear to be sufficient for sister-chromatid separation, as the cells lacking securin are more or less healthy, meaning other mechanisms also control the separation. Cdc20 appears to be necessary for anaphase beyond sister-chromatid separation, since the double knockouts still arrest in anaphase despite lacking securin.

The cell cycle can be arrested in mitosis when a single sister-chromatid pair is mono-oriented on the mitotic spindle. If at this point a glass microneedle is used to pull the mono-oriented chromosome toward the pole to which it is not attached, the cell proceeds to anaphase. This observation confirms that:

lack of mechanical tension at the kinetochore in at least one chromosome prevents entry into anaphase.