T7

Question 0

Define interphase.

Answer 0

G1, S phase and G2 combined.

Question 1

The four main stages of mitosis and their functions are…

Answer 1

1. G1, gap phase
2. S phase, chromosome duplication
3. G2, gap phase
4. M phase, chromosome segragation, cytokinesis

Question 2

What is G0?

Answer 2

A resting state.

Question 3

What determines when a cell starts proceeding towards mitosis?

Answer 3

Passing of, in favourable conditions, the restriction point (Start) which commits the cell to DNA replication but not necessarily division.

Question 4

What is the function of a Cdc gene (cell-division-cycle gene) and what does its mutation entail?

Answer 4

Genes encoding vital components for progression of cell towards division. Mutations may cause ex. temperature dependance (conditionalism, permissive/restrictive, cannot divide at high temperature) and arresting of cell in late G2 phase with signature look of large undivided cells.

Question 5

What is the phenomenon of replicative cell senescence?

Answer 5

The arresting of division of a cell after a given number.

Question 6

What principles makes the timer/oscillatorlike cell-cycle control system reliable?

Answer 6

Its independence of the events it controls and the irreversible and on/off nature of the transitions it controls.

Question 7

What are the tree checkpoints in the cell cycle, what are their criterias and reults?

Answer 7

1. Start checkpoint, is environment favourable? Start of cell cycle.
2. G2/M checkpoint, is all DNA replicated/is environment favourable? Entering into mitosis.
3. Metaphase-to-Anaphase transition, are all chromosomes attached to the spindle? Trigger anaphase and proceeding to kinesis.

Question 8

What are the effectors of the cell-cycle control system?

Answer 8

Cdks (cyclin-dependent kibases).

Question 9

What’s the function of Cdks?

Answer 9

They lead to “cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle”.

Question 10

Cdks (cyclin-dependent protein kinases) are controlled by a multitude of other proteins and enzymes. Which one is the most essential?

Answer 10

Cyclin.

Question 11

Outline the four classes of cyclins in vertebrates.

Answer 11

1. G1/S-cyclins: activation of Cdks in late G1, help trigger progression through Start, result in commitment to entry into cell-cycle, levels fall in S phase
2. S-cyclins: binds Cdks after Start, stimulate chromosome duplication, levels elevated until first parts of mitosis
3. M-cyclins: “activate Cdks that stimulate entry into mitosis at G2/M checkpoint”, destroyed in mid-mitosis
4. G1 cyclins: govern activities of G1/S cyclins

Question 12

What are the different cyclin-Cdk complexes and when are they active?

Answer 12

G1-Cdk: “through Start in late G1”
G1/S-Cdk: mid G1 to late G1 phase
S-Cdk:late G1 to early M phase
M-Cdk: beginning of M phase to mid-M phase

Question 13

What is the function of the cyclin-Cdk complexes?

Answer 13

To phosphorylate specific sets of substrate proteins.

Question 14

What are the activation steps of Cdks?

Answer 14

1. Partial activation when cyclin is bound and moves T-loop

2. Full activation when CAK (Cdk-activating kinase) phosphorylates threonine on T-loop, near active site

Question 15

How is the activity of the cyclin-Cdk complex regulated?

Answer 15

1. By diphosphorylation/dephosphorylation by kinase Wee1 and phosphatase Cdc25
2. Binding of CDIs (CDK inhibitory proteins), ex. p27

Question 16

Does Cdk contain ATP?

Answer 16

Yes. One ATP is bound to the protein.

Question 17

How does progression through the Start and G2/M checkpoint differ from profression through the metaphase-anaphase-transition?

Answer 17

The former are triggered by protein activation through phosphorylation, whilst the latter is triggered through proteolysis.

Question 18

Describe the mechanism of progression through the metaphase-to-anaphase transition.

Answer 18

APC/C (anaphase-promoting complex, cyclosome) is activated through process activated by Cdc20/Cdh1. It catalyzes ubiquitylation of securin, causing release of sister chromatids from each other. It also causes ubiquitylation of cdk-bound cyclins, which together with dephosphorylation inactivates most Cdks.

Question 19

Why does APC/C action persist also in the former part of F1?

Answer 19

It provides a stable period of Cdk inactivity.

Question 20

What is the role of SCF?

Answer 20

It ubiquitylates certain CKIs in late G1, controlling activation of S-Cdks and DNA replication.

Question 21

How does APC/C and SCF activity periods differ, and how does protein phosphorylation play a part in this?

Answer 21

Whilst APC/C is periodically active, SCF is constantly active but its interaction depends on the F-box recognizing target proteins based on their phosphorylation.

Question 22

How big an impact on cell-cycle control does transcriptional regulation impart?

Answer 22

The impact is not great but still substantial, ex. for cyclin level regulation. The method is used in the more complex cell-cycles of most cell types.

Question 23

Outline the initiation of replication and its regulation.

Answer 23

1. Assembling of pre-RC, pre-replicate complex, (ORC, origin recognition complex + 6 Mcm proteins loaded by Cdc6 and Cdt1, load the helicases) origin of replication is inhibited by Cdk activity, stimulated by APC/C.
2. Forming of preinitiation complex through activation of S-Cdk. Also attracts other proteins, forming giant preinitiation complex). Partial dismantling of pre-RC due to high S- and M-Cdk until late mitosis. Cdks phosphorylate ORC and Cdc6 resulting in inhibition.
3. Unwinding of DNA and loading of polymerases etc.
   …
4. APC/C triggers destruction of geminin that inhibits Cdt1, thus allowing replication to start over.

Question 24

How are Cdks involved in control of chromosome replication?

Answer 24

S-Cdk stimulates synthesis of histone momomers.

Question 25

What is the function of the SMC family protrins cohesins in chromosome replication?

Answer 25

Non-covalent shepherding of sister chromatids, keeping them close inside the ring structure.

Question 26

What is DNA catenation?

Answer 26

Intertwining of sister DNA molecules at replication forks. Sorter by topoisomerase IIs.

Question 27

Summerize the two main steps of mitosis.

Answer 27

1. Abrupt increase in M-Cdk at G2/M checkpoint and subsequent early mitosis 2. At metaphase-to-anaphase transition APC/C triggers ercurin destruction, cohesin cleaving proteases liberated and subsequent separation of sister chromatids. APC/C triggered destruction leads to Cdk inactivation and dephosphorylation of target proteins, leading to late M ohase events.

Question 28

In addition to M-Cdk two kinases are responsible for phosphorylation of key proteins needed for early mitosis. Ehich are these?

Answer 28

Polo-like kinases and Aurora kinases.

Question 29

Outline the steps to activation of M-cyclin.

Answer 29

1. Increased M-cyclin produced during G2/M phase leads to increase in M-Cdk 2. CAK activates the M-Cdk complex 3. Wee1 inactivates it through diphosphorylation 4. Protein phosphatase Cdc25 is activated, ending M-Cdk inhibition 5. Positive feedback from M-Cdk to Cdc25 + inhibition of Wee1

Question 30

How does condensin action relate to M-Cdk?

Answer 30

Increase in concentration of M-Cdk increases chromayin coiling.

Question 31

What three types of microtubules are present in the mitotic spindle?

Answer 31

Astral and interpolar microtubules along with kinetochore microtubules.

Question 32

How does M-Cdk initiate spindle assembly and stimulate centrosome separation and maturation?

Answer 32

The sharp increase in M-Cdk in early M phase initiates spindle assembly and centrosome maturayion. M-Cdk and aurora-A phosphorylate kinesin-5 motors and that induce separation.

Question 33

A why is nuclear breakdown critical for mitotic spindle assembly?

Answer 33

Because the nucleus contains protein vital for the process?

Question 34

Describe the process of nuclear envelope breakdown.

Answer 34

1. Phosphorylation by M-Cdk of several subunits of nuclear pore complex and components of nuclear lamina. 2. Nuclear pore complex disassembly initiates, as well as diassembly of nuclear lamina into vesicles.

Question 35

What part does M-Cdk play in the dynamic instability of tubule formation in mitosis?

Answer 35

M-Cdk initiates reworking of microtubule structure through catastrophea and rescues, to suit chromatid separation. It phosphorylates two protein classes that control microtubule dynamics, one being MAPs (microtubule associated proteins).

Question 36

What is a catastrophe factor?

Answer 36

A microtubule/mitotic spindle destabilizing protein.

Question 37

Describe the regulation of chromosome separation by ACP/C.

Answer 37

1. M-Cdk activates APC/C by binding Cdc20 2. Activated APC/C ubiquitylates securin of securin-separase complex, thus activating separase 3. Separase cleaves and dissociates cohesins

Question 38

Differentiate between the two stages of anaphase.

Answer 38

Anaphase A: separation of chromatids through shortening of kinetochore microtubules. Force generated mainly at kinetochore. Anaphase B: Separation of spindle poles themselves. Sliding forces generated between interpolar microtubules and pulling forces at polar tubules.

Question 39

What concludes mitosis?

Answer 39

Cdk inactivation / phosphatase action

Question 40

Ehat function does dephosphorylation of Cdks at the end of mitosis have, other than actually ending mitosis?

Answer 40

Starting of cytokinesis.

Question 41

What is the cleavage furrow?

Answer 41

The radial surface indentation during cytokinesis, caused by the cobtractile ring which is made up of "actin filaments, myosin II filaments and many structural and regulatory proteins"

Question 42

What is the role of RhoA in cytokinesis?

Answer 42

It promotes myosin II activity.

Question 43

Describe the reaction steps of RhoA mediated cytokinesis.

Answer 43

1. Activation-deactivation (binding of GTP/GDP) by RhoGEF/RhoGAP 2. Active RhoA activates formins and Rho-activated kinases including Rock 3. Formins induce actin filament formation. Rock et.al. inhibits myosin phosphatase and induces regulatory mysosin light-chain phosphorylation 4. Myosin II is activated 5. Actin-myosin ring assembled and contracted

Question 44

What are the three hypothesized models for mitotic spindle plane-deciding of cytokinesis?

Answer 44

Astral and central spindle stimulation model, and astral relaxation model.

Question 45

Outline the control mechanisms of end of M phase that allows a cell to enter G1.

Answer 45

1. Dropping of M-cyclin levels due to Cdc20-APC/C activity 2. APC/C levels drop due to lack of stimulus from M-Cdk 3. Cdh1-APC/C levels rise, keeping Cdk from reactivating. Cdh1-APC/C is usually inactivated by Cdc20-APC/C 4. CKI protein Sic1/p27 also increases as M-Cdk activity drops, inhibiting Cdk activity further in G1 phase

Question 46

What three categories can extracellular signals that regulate size and numbers of cells be divided into?

Answer 46

1. Mitogens, trigger wave of G1/S-Cdk activity 2. Growth factors, stimulate increase in cell math through macromolecular/synthesis 3. Survival factors, inhibit entering into apoptosis

Question 47

Give two examples of mitogens. How do they differ in types of cells they affect?

Answer 47

PDGF (platelet derived growthfactor) is very general whilst erytropoietin is very specific, only affecting erythrocytes.

Question 48

Is G0 permanent for all cells entering it?

Answer 48

No.

Question 49

Describe the mechanisms that control cell-cycle entry and S phase initiation.

Answer 49

1. Mitogen receptor binds ligand and activates Ras 2. Ras triggers MAP kinase reaction which induces immediate early gene expression, producing Myc gene regulatory protein 3. Delayed response gene expression produce G1-cyclins 4. Active G1-Cdk inactivates Rb protein, releasing and activating E2F protein (gene regulatory factor) 5. E2F activates S phase gene transcription producing G1/S-cyclins and S-cyclins 6. S-Cdk activated initiates DNA synthesis 7. Positive feedback loops from E2F to itself, and from G1/S-cyclin and active S-cyclin to E2F activation.

Question 50

At what points can a cell arrest the cell cyfle upon detecting DNA damage?

Answer 50

At Start in late G1 and at G2/M.

Question 51

Describe arresting of the cell-cycle upon detecting DNA damage.

Answer 51

1. Kinase ATM/ATR at site of damage are activated 2. They activate Chk1/Chk2 (kinases) transcription 3. p53 tumour controlling factor is phosphorylated and thus activated 4. Binding of p53 to regulatory region of p21 5. p21 is transcribed and translated 6. Resulting p21 protein (CKI) inactivates G1/S-Cdk and S-Cdk

Question 52

Relate p53 action to aging.

Answer 52

Deficiency of telomerase in human fibroblasts and other somatic cells makes for gradually shorter telomeres. This DNA damage is eventuslly detected by p53 and the cell-cycle is thus arrested, leading to decreased renewal of cells.

Question 53

How si overexpression of Myc regulated?

Answer 53

Abnormally high levels of Myc causes Arf to activated. Arf binds Mdm2 (p53 binding/inactivating protein) and thus activates p53.

Question 54

Explain density-dependent inhibition of cell division.

Answer 54

Cells will not grow past a certain population density without adequate supply of nutrients. Petri dish exsmple.

Question 55

How do polyploidi and cell size correlate?

Answer 55

The greater the number of copies of genomes in a cell, the greater the cell volume. Organism size remains constant though since the number of cells are correspondingly lower.