Topic 7 - Cell Cycle Flashcards
1
Q
What does inhibiting TSN nuclease activity has with inhibiting cell proliferation?
A
- functional miRNAs are degraded by TSN will promote G1/S phase transition in a cell
- by using CRISPR-Cas9 knockout of TSN inhibited cell cycle progression by upregulating miRNAs which down regulate mRNAs which normally encode proteins used in G1-S phase transition
2
Q
The cell cycle consists of 3 main events:
A
- Cell Growth and Chromosome Replication
- Chromosome Segregation
- Cell division
3
Q
How is the cell cycle regulated? and what is its goal?
A
- though internal and external signals (environmental conditions)
- controlled duplication producing identical DNA, organelles, membranes
4
Q
What is Mitosis? How many N before and after mitsosis
A
- 4N to 2N (duplicated chromosomes segregated into daughter nuclei) - not separate cells yet
5
Q
What is cytokinesis? What makes it different from mitosis?
A
- the cell with two sets of daughter nuclei already separated, will now divide itself into TWO CYTOPLASMIC DIVISIONS creating two cells
6
Q
How long does the S phase take? what does it stand for?
A
- DNA Synthesis
- 10-12 hours in humans (half the time of cell cycle)
7
Q
How long does M phase take? What does it stand for?
A
- Mitosis
- consists of TWO events: mitosis and cytokinesis
- 1 hour in mammals
8
Q
What are the 4 phases in the Cell Cycle in Order?
A
- G1 (Interphase), S (Synthesis), G2 (Interphase 2,) M (mitosis and cytokinesis)
9
Q
What is the speed of DNA replication during DNA (S)ynthesis?
A
- 83,333 bp/second
10
Q
What is the role of Gap 1 and Gap 2?
A
- the interphase stages are there for environmental monitoring, to decide whether or not to go and replicate
11
Q
What occurs after you START at the end of G1, start S phase?
A
- the cell is committed to replication; even if conditions become unfavourable or stimulating factors for the progression of cell cycle is removed
12
Q
What are 3 reasons why Yeast are the perfect model system to study?
A
- divide rapidly with a small genome
- able to proliferate in the haploid state (each cell has one copy of the each gene) - advantage for mutation analysis
- Cdc genes have been identified (Cell-division-cycle genes)
13
Q
What do conditional mutants refer to?
A
- permissive environmental conditions are met OR restrictive conditions allows the cell to replicate, or prevents replication
14
Q
What are other eukaryotic model systems?
A
- animal embryos, cultured mammalian cells
15
Q
Why is it tricky to isolate mutants affecting cell cycle?
A
- mutant propagation@ permissive temperature
- switch off function of mutant genes (restrictive temperature)
16
Q
What are the advantages of working with early embryos? example?
A
- there is no G1 G2 phases as the cells divide nearly every 30 minutes
- Frog embryos produce large eggs with lots of proteins where the 1st division takes 90minutes but the next 11 divisions occur every 30 minutes but there is NO cell growth as the egg stays the same size
17
Q
Why are mammalian model systems cultured in tubes? Problems and Solutions?
A
- difficult to study individual cells in an intact organism
Problem: replicative senescence
Solution: immortalized cell lines & tissue cultures
18
Q
Can you study the cell cycle in a free system?
A
Yes; large eggs used
- allows for the manipulation of cell cycle by controlling their growth conditions; ex drugs block & unblock pathways OR remove check points OR replace normal host proteins with other proteins
19
Q
How do you monitor model systems of mammalian cells during the cell cycle? What phase the cells are in
A
- BrdU is an artificial thymidine dye for DNA (antibodies or stains) - used to label 40% of cells in the S phase
- flow cytometer with DNA binding dyes to observe the stages of the cell cycle these cells are
20
Q
What does immortalized cell lines refer to?
A
- induce a mutation so the cell evades normal cellular senescence to continue undergoing cell division
21
Q
What occurs when a cell reaches one of the three main check points? - some questions to ask.
A
- progression is blocked if a problem is detected
1. Start Transition - the end of G1 phase before S phase (DNA replication) - Is the environment favourable
2. G2/M transition - Enter Mitosis - is the DNA replicated, is the environment favourable?
3. Meta-to- Ana phase Transition - Triggers anaphase and proceed to cytokinesis - are all the chromosomes attached to the spindle
22
Q
What is the role of Checkpoints in a cell cycle?
A
- ensure that the preceding phase is complete before the next phase of the cell cycle begins
- check points may delay this advance to ensure repairs are done or the environmental conditions improve
23
Q
We know what the Control System (Checkpoints does) - but was is key to its success?
A
- Switches are binary
- robust & reliable
- adaptable (to different signals - inter/intra cellular)
24
Q
What are cyclins? where does the name come from?
A
- a regulator of CDKinases
- they will undergo a cycle of synthesis and degradation with each turn of the cell cycle
- each new round of cell cycle synthesis calls for new cyclins, so after each round of the cell cycle old cyclins are degraded
25
What are the two main roles for cyclins?
1. to activate its specific Cdk partners
| 2. to direct this active Cdk partner to the specific target protein
26
At what four stages are cyclins or another regulatory protein required?
1. START for G1/S-Cdk
2. S-Cdk
3. M-Cdk
4. Between Metaphase-Anaphase via APC/C to initiate this transition
27
What is the role of Cdks? - and their concentration
- are protein kinases which are controlled by cyclin activation
- the concentration of Cdks DO NOT change
- they are present in a higher concentration than cyclins
28
How does cyclin activate a Cdk?
- Cdk contains a bound ATP with an unexposed T loop
- a cyclin will come in and expose the T-loop partially activating it
- now cyclin phosphorylates the T loop on the Cdk which causes a conformational change in the active site allowing tighter binding - Cdk is now Fully Active
29
How do different cyclin-Cdk complexes trigger different cell-cycle events? one catch though is...
- cyclin activates Cdk AND directs it to a specific target protein
- so each Cdk-cyclin complex activates a different set of substrate proteins BUT the same cyclin-Cdk complexes can have different properties at different times during the cell cycle
30
Since Cdk has an important function of directing cell-cycle progression - how do you regulate/inhibit this process?
- through regulating the activation of Cdk by preventing Cyclin from binding and exposing the T loop for activation
- ex Wee1 adds a Phosphate group on Cdk preventing cyclin from binding while Cdc25 will remove this phosphate and allow Cyclin to bind the Cdk
- ex. Cdk inhibitor proteins (CKIs) alter the strucutre of the entire Cdk-cyclin complex
31
What do Wee1 and Cdc25 regulate?
- since Wee1 kinase inhibits Cdk activity by adding P to a sequence of AA at the Cdk's active site -- thus blocking cyclin
- since Cdc25 works to dephosphorylate these sites -- it activates Cdk activity
- therefore Wee1 and Cdc25 regulate the onset of MITOSIS
32
What affect does CKI have on the cyclin activation of Cdks?
| - what are two examples?
- Cdk Inhibitory Protein alter the structure of Cdk-cyclin complex
- P21 and P27
33
What regulates the 3 stages of Cdk's in a cell?
- G1/S-Cdk - CKIs
- S-Cdk - CKIs
- M-Cdk - Wee1 (inactive) vs Cdc25 (inactive)
34
What are three questions a Cdk asks before it progresses to is action? if all these are yes?
- has this phosphate been removed (the inhibitory one by Wee1)
- has this phosphate been added (the activating one on the T-loop by cyclin)
- is cyclin present
- if these answer as YES; Cdk activity turns on
35
What is the one stage in the cell cycle where a cyclin or Cdk functions
- stage between the transition from Metaphase-to-Anaphase (and cytokinesis)
- this stage is controlled by the APC/C complex
36
What does the APC/C complex refer to?
- Anaphase-promoting-complex/cyclosome
37
What function does APC/C have? of what two proteins degraded?
- this stage is regulated through proteolysis
| - APC/C will catalyze the ubiquityltation and degradation of 1) Securin 2) S- & M-cyclins
38
Where and how does Securin function? and how does APC/C degrade this protein? and what Cdc is important in regulating what cell stage?
- Cdc20 activates APC/C during the metaphase to anaphase stage
- the now Cdc20 bound to APC/C will target Securin
- Securin functions by binding and inactivating the protein Separase
- once the active APC/C & Cdc20 ubiquitylation and degradation of securin can Separase Activate
39
What is Separase?
- a protease that is inactivated by securin
| - once Separase is active it will separate the chromatids at metaphase-anaphase
40
What is the intended function of the S-cyclin and M-cyclin? which APC/C protein performs this function?
- these regulate the cell cycle and activate Cdk's S-Cdk and M-Cdk respectively
- APC/C targets these cyclins to inactivate Cdk
- Cdh1 (controls after anaphase to G1) when the APC/C complex activates it will form a polyubiquitin chain on the cyclin which is targeted by the proteosome
41
How do you regulate the CKI inhibitory proteins to allow the cell cycle to proceed?
- this regulation occurs in late G1 to allow S-Cdks and DNA replication
- a combination of SCF complex & Skp1 & F-box protein & cullin will ubiquitylase an active CKI
42
How does ubiquitylation of the CKI regulate activity of Cdks?
- by ubiquitylation of CKI proteins @ late G1 it helps with the activation of S-Cdks and DNA replication
43
Summarize the transition from G1 to G1/S-Cdk to S-Cdk to M-Cdk to the APC/C complex and any reasons for roadblocks?
1. G1: if conditions are favourable to proliferation, internal or external signals stimulate the activation of G1-Cdk. This stimulates gene transcription for G1/S cyclins and S cyclins
2. G1/S-Cdk: must first asses DNA damage before increasing S-Cdk activity to promote DNA replication in S phase
3. M-Cdk: must first assess DNA damage, unreplicated DNA and initiate DNA repair from DNA synthesis. Then M-Cdk leads the cell to aligning the sister chromatids at the equator of the mitotic spindle
4. APC/C will initiate the ubiquitylation of securin protein and the S-phase and M-phase cyclins to direct the Metaphase to Anaphase transition
44
What occurs during S phase? & two important levels of accuracy are required?
- DNA synthesis for the inheritance of two daughter cells
1. DNA forms a long correct molecule
2. Each nucleotide is copied ONCE
45
How are the two levels of accuracy maintained in S phase?
To maintain accurate chromosome duplication (DNA replication) is cone by EXTREME ACCURACY to minimize mutations
- protein packing around each region of DNA
46
How is S phase initiated?
- at the origin of replication a pre-replicative complex forms as initiator proteins
- stimulated by the APC/C complex and activates the S-Cdk
47
What two steps are involved in the initiation of S phase?
1. Pre-replicative Complexes form at the origins of replication (during G1)
2. at the onset of S phase - this pre-replicative complex nucleates the DNA forming a larger complex: PRE-INITIATION COMPLEX (DNA unwound, polymerase is loaded on)
- now the pre-replicative complex is dismantled till the next G1
48
Explain the formation of the Pre-replicative complex for the start of DNA synthesis
- This complex is assembled during the G1 phase where the Origin of Replication marks this site
- at the Origin of Replication complex these proteins bind at the site: Cdc6 & Cdt1 loads 6 Mcm proteins to the site = pre-replicative complex
- this Mcm6 helicase unwinds the DNA @ the origin and DNA synthesis will begin
49
What is the purpose of the Pre-replicative complex? who activates this process to inhibit who?
- to load the DNA helicase for DNA synthesis
- this complex consists of: Mcm via Cdt1 and Cdc6
- an active S-Cdk-cyclin will inhbit the ORC, Cdc6, Cdt1
50
How is the DNA synthesis officially activated
- protein kinase DDK phosphorylate the helicase and it now begins the synthesis
51
What resets the system in S phase?
| and what prevents the Pre-replicative complex from forming?
- at the end of Mitosis APC/C will inactivate Cdks
- this allows the pre-replicative complex to assemble; Cdt activated, Cdc6
- when APC/C is switched off, the precursor proteins for the complex are inhibited from forming
52
Since replication occurs during S phase: accurately and only once - what is required for Pre-Replicative Complex assembly?
- the Pre-Replicative assembly can only occur during APC/C activation
- this requires Cdk inactivation; dephosphorylation of ORC and Cdc6; the activation of Cdt1
- when the pre-replicative complex disassembles the Mcm helicase remains on the DNA
53
If during S-phase duplication of DNA and chromatin proteins occurs:
1 - what is the product at the end of S phase
2 - how are sister chromatids held together
3. - what is cohesion?
- during S-phase when DNA is replicated so are chromatin proteins to hold DNA together (performed during S-phase regulation)
1. each replicated chromosome consists of a pair of sister chromatids held together
2. via cohesion; a ring by 2 Smc proteins
3. the Smc1 and Smc3 proteins are held together by a Scc3&1 connecting the ATPase heads
54
What is DNA catenation? (follows after sister chromatids are formed and held together by cohesion proteins) - who is needed now?
- this occurs when the replicated DNA is interlocked in double helices = requiring Topiosomerase II
- recognize the entanglement; forms reversible covalent attachment; forms a double stranded break and a protein gate to prevent shearing apart - addition of 2 ATP; a second gate open to move the second DNA helix through (untangling them) - step using energy releasing phosphate; gate shuts releasing one DNA strand (other still held together by the protein gate); reverse the initial covalent bond from the topiosomerase which reattaches the other double helix
55
Summarize the Main Points of Mitosis
M-phase (mitosis and cytokinesis)
- 2 Major stages will occur: M-Cdk activity increases (forming mitotic spindle and attaching the sister chromatids) & Metaphase-Anaphase Transition (APC/C complex triggers securin-separase causing chromatid separation -- finally APC/C triggers cyclin destruction via ubiquitylation)
56
What are the traditional 6 Stages of Mitosis
Prophase (centrosome migration - mitotic spindle formation - kinetochore form on the condensing sister chromatids)
Prometaphase (nuclear envelope degrades - mitotic spindles attach to the kinetochore on the chromatids)
Metaphase (Centromsome at the poles - kinetochore attached to the microtubules & aligned in the middle of the cell)
Anaphase (shorteing of the kinetochore microtuble - (spindle moves outwards)
Telophase - (nuclear envelope reassembly with the daughter chromosomes - contractile ring formation)
Cytokinesis - (contractile ring forms cleavage furrow - interphase at the nucleus)
57
What is active M-Cdk repsonsible for?
- all cell rearrangements during early mitosis
- mitotic spindle formation
- chromatids pairs are attached to the opposite pole on the spindle
- breakdown nuclear envelope, cytoskeleton, golgi
- ensure chromosome condensation
58
What inhibits or activates the M-Cdk and M-cyclin?
- Cdk activating kinase (CAK) while phosphorylate M-Cdk BUT Wee1 (the inhibitory Cdk inhibitory kinase) will also phosphorylating M-Cdk, ensuring M-cyclin cannot bind) -- however when Cdc25 is active it will be able to remove the inhibitory phosphate from Wee1 and M-cyclin binds and activates M-Cdk (FEEDBACK LOOPS)
59
What process is involved in chromosome condensation? and what components? and how are they activated
- Condensin, opposite to cohesin (works as a intra-cross-linker)
- two Smc proteins (structural maintenance of chromosomes) linked around the chromosome
- these must be phosphorylated by M-Cdk to stimulate coiling of the chromosome
60
What is the state of microtubules prior to Mitosis?
- larger number of shorter microtubules during interphase and entry of mitosis
61
What does M-Cdk need to do with microtubules and the motor proteins?
- M-Cdk will phosphorylate both MT-dependent motor proteins and MT-associated motor proteins
- M-Cdk also triggers the assembly of the spindle bipolaral
62
What is the structure of the mitotic spindle? and the centrosome
- Spindle is bipolar: mins microtubules at the poles (centrosome) and positive microtubules radiating out
- the centrosome at the pole holds the gamma-tubulin ring complex
63
What are the three types of microtubules and what are their function?
1. Astral Microtubules: radiate from the poles contacting the cells cortex to stabilize the centromeres position at the poles
2. Kinetochore Microtubules: attach to the chromatids at the kinetochores
3. Interpolar Microtubules: is the interaction of the plus end microtubules from each sides centromesome; form an antiparallel array
64
What motor proteins are involved to have the mitotic chromosomes promote bipolar spindle assembly?
- the kinesins: which will always walk towards the plus end of microtubules (interacting with the INTERPOLAR microtubules)
- the dyneins: which will always walk towards the negative end of microtubules (interacting with the ASTRAL microtutubles)
65
Explain in point form the process of attaching chromosomes to the mitotic spindle.
a) late prophase: spindle poles moved to the opposite side of the nuclear envelope
b) nuclear envelope breaks down - where the chromatids are exposed to the plus ends of microtutubles - kinetochore first attaches at on the sides of microtubules (with chromosome arms pushed outward)
c) laterally attached chromatids are rearranged into a ring
d) the dynamic plus end of the microtutuble will encounter the kinetochore in and end on orientation where it is stabilized
e) stable end on attachments result in orientation, and the microtubules are now attached to the kinetochore
66
Are chromosomes always attached to MT via the kinetochore?
| Are the MT always attached to chromosomes with their ends?
NO - first occurrence after the nuclear envelope is dissolved
NO - initial side on for orientation, then walked into position
67
How do opposing forces drive the chromosomes to the metaphase plate? (2 types)
- requires the cooperation between INTERPOLAR microtubule and KINETOCHORE microtubule
a) polar ejection force: the plus end directed kinesins on the chromosome arms interact with the interpolar microtubules to generate this force via pushing the chromosomes toward the spindle equator this allows sister-chromatid alignment at the metaphase plate
b) Microtubule Flux: results in depolymerization at each of the minus end followed by depolymerization at the kinetochore to pull chromosomes to the pole
68
What are the two forces that drive chromosomes to the metaphase plate? and are energized by?
- Polar Ejection Force
- Microtubule Flux
- through GTP hydrolysis
69
How do Microtubules attach themselves to a Kinetochore?
- DNA will wrap itself around histone proteins forming nucleosomes where they will form into chromosomes = chromatids
- these proteins will interact with the kinetochore on the chromosomes where the microtubules can interact
70
How does the microtubule bind to the kinetochore while the microtubule still polymerizes and depolymerizes?
The kinetochore will attach to the Ndc80 complex which binds to the plus end of the microtubule by forming a protein ring collar around the microtubule
- this provides room for the addition/removal of tubulin subunits
71
Since the success of Mitosis is measured by how well sister chromatids separate to opposite cells, how does this process occur?
- kinetochores are arranged back to back on the chromosome which allows for BI-ORIENTATION of the spindle attachment
72
How does the microtubule ensure successful Orientation at the kinetochore? - what proteins is sensing for this?
- attachment will be trail and error and selective through STABILITY via TENSION of the microtubule
- Kinase Aurora-B: which will phosphorylate components of the microtubule if tension is too low - this loosens the affinity for microtubules at the plus end
- so if tension is reached it inactivates Kinase-Aurora-B which stops phosphorylation of microtubules and increases affinity of the microtubules to the kinetochore
73
What does Bi-orientation do when high tension is reached?
- the inhibitory signal will be shut off
| - strengthened kinetochore attachment leading to more kinetochore attachment and thicker microtubule
74
What affect does Low Tension have on microtubule attachment and Aurora-B Kinase?
- the sister chromatid may only attach at one kinetochore, or remain unattached - so low tension between the inner and outer kinetochores
- this leads to protein kinase Aurora-B (tethered to the inner kinetochore) to be able to reach and phosphorylate the Microtubule attachment sites (the Ndc80 Complex) reducing the affinity for microtubule attachement - destabilizing rapidly
75
What affect does HIGH Tension have on microtubule attachment and Aurora-B Kinase?
- this occurs when bi-orientation is reached, allowing the tensions to pull the outer kinetochore away from the inner kinetochore
- this distance prevents Aurora-B from phosphorylating the microtutuble
- increasing affinity of microtubule to the kinetochore via the Ndc80 and allows stable attachment
76
What checkpoint occurs after M-Cdk?
the APC/C complex will ensure all chromosomes are stable
- if there is no tension negative signalling will block the interaction between Cdc20 and the APC/C complex (ie. kinetochore not properly attached or bi-orientation of all chromosomes)
77
When does APC/C promote the cell to move into Anaphase?
- the APC/C complex is a ubiquitin ligase that leads to the degradation of M- & S-cyclins
- however when M-Cdk phosphorylates APC/C, it allows Cdc20 to bind and activate itself
- Cdc20 will ubiquitilase the protein SECURIN releasing Separese
- APC/C will also phosphorylate the other Cdks
78
What does Separase do once activated?
- Separase will cleave the cohesion interaction between sister chromatids
79
What occurs during Anaphase A?
- shortening of the kinetochore & forces are also generated here
80
What occurs during Anaphase B?
2 Forces are generated
1. Microtubule depolymerization at the kinetochore: shortens the microtubules as they are lost at the plus end
2. Microtubule Flux: depolymerization of the microtubules at the negative centriole end causing the microtubule as a whole to pull away
81
What are the two major steps that occur during telophase?
1. Disassembly of the mitotic spindle
2. Reformation of the nuclear envelope
- nuclear fragments associate with the surface of chromosomes
- membrane fragments fuse to partly closed clusters of chromosomes
- NPC form
- nuclear lamina forms
- continuos ER forms
- chromosomes reorganize
82
During Telophase are any specific proteins phosphorylated or dephosphorylated?
- dephosphorylation of the spindle breakdown and nuclear envelope reassembly
83
Cytokinesis consists of 4 main stages.
1. Initiation
2. Contraction
3. Membrane Insertion
4. Completion
84
What 5 components sum up Cytokinesis?
- the 4 main stages
- division of the cytoplasm
- contractile ring (via actin myosin)
- membrane is added (via vesicles)
- Ends just after telophase
85
Who regulates the contractile ring?
the monomeric GTPase Rho-A is responsible for the formation of the contractile ring
- Rho-GEF activates Rho-A at the cell cortex, while Rho-GAP inactivates
86
What two pathways does Rho-A activate?
1. Activate Formins to assist in actin filament formation
| 2. Rho-activated kinases will activate the regulatory mysosin light-chain phosphorylation to activate Myosin II
87
How do higher plant cells produce daughter cells?
- compared to animal cells who can furrow and form a contractile ring
- plant cells are unable to do this due to rigid cell walls: do not change shape for mitosis - a cell plate grows centrifugally
88
What are 3 further facts about cytokinesis
- organelles MUST be distributed equally
- some cells divide asymmetrically
- mitosis can occur without cytokinesis (cellularization)
89
How do you reset the cell cycle after replication?
- there are multiple regulatory mechanisms in place
- during EARLY G1, all Cdk activity is suppressed
1. Cdh1-APC/C activity increases in late mitosis
2. increased expression of CKIs (Cdk inhibitory proteins)
3. decreased cyclin gene expression
90
How do cells escape a stable G1 State to initiate a new cell cycle?
- requires an increasing in G1/S-Cdk activity in late G1 & a the release of suppression from Cdk = allows S-phase entry
91
What is the total cell mass referring to?
- the total number of cells and their size
1. cell growth
2. cell division
3. cell death
92
What 3 extracellular signals regulate Cell Size and Number?
1. Mitogens: stimulate cell division by activating G1/S-Cdk
2. Release of Growth Factors: NOT MITOGENS
3. Survival Factors: promote cell survival by suppressing apoptosis
93
What are mitogens capable of doing to the cell cycle?
- able to stimulate cell division, cell growth, survival, differentiation, and migration
1 - they release the break of Cdk activity and its regulatory components
2 - activation of Ras monomeric GTPase and activation of MAP kinase kinase kianse to increase myc expression
3 - Myc levels can increase G1-cyclins which will activate more Cdk (Cdk levels never change)
94
There are two checkpoints for DNA damage; Start & G2/M phase - what is the normal response pathway?
- DNA damage will occur which activates ATM/ATR kinase which active the Chk1/Chk2's which will be able to phosphorylate P53, releasing it from Mdm2
- active P53 can bind to the regulatory region of P21 and transcribe and translate the P21 protein
- the active P21 protein can bind and conformationally change the interaction between the G1/S-Cdk & S-Cdk interaction with cyclin
95
What happens if the DNA damage cannor be fixed in a normal healthy cell: Unicellular or multicellular organisms?
1. Unicellular: initial cell cycle arrest, but cell will resume the cell cycle - no life vs life
2. Multicellular: cell cycle arrest & cell apoptosis if the damage cannot be fixed
96
Why is P53 such an important regulatory protein?
- mutations in p53 = 50% of cancers
- stimulates transcription of p21 to inhibit G1/S-Cdk and S-Cdk
- normally highly unstable due to Mdm2 ubiquitylation
97
What type of abnormal proliferation signal would cause cell cycle arrest of apoptosis?
- If Ras functioned signalled excessive Myc which could bind to the cell cycle inhibitor Arf which will inactivate Mdm2
- free p53 now able to initiate cell-cycle arrest or apoptosis
