1
Q
Why is cell cycle important
A
- highly organized and regulated
- cell cycle= repeated rounds of cell growth and division
- primary goal= to divide the genetic materials and organelles accurately and equally
- for reproduction in prokaryotes and eukaryotes
- for replacing old cells with new cells
- during would healing or cell damage
2
Q
Why do we need to understand cell cycle/cell division
A
- continuity and diversity
- mitosis ensures continuity
3
Q
What is the basic role of the cell cycle
A
- maintain chromosome number
- increase diversity among individuals and species
4
Q
What is the overall theme of the cell cycle
A
-double, align, separate
5
Q
M phase
A
- mitosis (nuclear division)
- cytokinesis (cytoplasmic division)
- approx 30 mins
6
Q
Interphase
A
- growth phase
- G1 (gap)
- S (synthesis - DNA replicated)
- G2 (gap)
7
Q
S phase
A
- DNA replication occurs
- after replication, each chromosome consists of 2 sister chromatids, held together at the centromere
- 2 sister chromatids will be separated during mitosis
8
Q
Appearance of cell after S phase
A
- cell material in synthesized
- cell mass is higher
- DNA has replicated
- chromatin are diffuse and decondensed
- DNA not organized
- nuclear envelope intact
- centrosomes have also duplicated
- and double the organelles
9
Q
Key experiment to deduce features of cell cycle
A
- flow cytometry
- add radioactive thymidine to an asynchronous cell culture (ie. cells at different stages of cycle)
- lead 3Hthymidine in culture media for 30 mins
- cells will incorporate it into DNA that is being replicated
- refresh media and wait
- use autoradiography to look for labeled DNA
- similar to pulse-chase but with DNA
- only cells that were going through DNA replication in that 30 min pulse will show up (only in S phase)
10
Q
Observations of flow cytometry experiment
A
- if cell was gong through mitosis, the DNA was not labeled
- therefore, cells in mitosis are not replicating DNA
- only a fraction of the cells were labeled
- therefore, S is only a single, short phase of the cycle
- there was a gap of at least 30 mind between the end of labelling and when the labelled DNA showed up in compact chromosomes
- therefore there must be a G2 phase o at least 30 mind between S and M phase
11
Q
Phases of mitosis
A
- Prophase
- Prometaphase
- Metaphase
- Anaphase
- Telophase
- MPF = maturation promoting factor
- initiates mitosis
12
Q
Prophase
A
- chromosomes start to condense
- Centrosomes move to opposite poles
- nuclear lamina breaks down (phosphorylation by kinases)
- each chromosome consists of:
- 2 identical strands (chromatids)
- Joined at centromere
- Terminal regions (telomeres)
- 2 proteins important in maintains compacted mitotic chromosomes
- condensin: organizes DNA to maintain a condensed state. Activated by phosphorylation by MPF
- cohesin: holds 2 sister chromatids together. Run entire length of chromosome but lost fro the arms in prophase (remains concentrated in the centromere)
- condensin and cohesin work together during chromosome condensation and segregation
13
Q
Centromeres
A
- also called primary constriction
- location of highly repeated DNA sequences
- this DNA is not translated
- repeated are an indication to cell that this is where kinetochore needs to be assembled
14
Q
Kinetochores
A
- structure on outer surface of centromere
- more than 100 proteins
- roles:
1. Attachment site between chromosome and microtubules
2. Location of some motor proteins involved in anaphase
3. Involved in mitotic checkpoint
-MT plus end attaches to kinetochore
- kinesin13 (depolymerase) found in kinetochore
- plus a regular kinesin and cytoplasmic dynein
15
Q
Centrosomes
A
- composed of 2 perpendicular centrioles
- each centriole made of 9 triplet MTs + pericentriolar material (PCM)
16
Q
S phase
A
- DNA duplicates
- centrosome duplicates
17
Q
Centrosome cycle
A
- normally in G1 there is only 1 centrosome per cell
- during S phase new baby centrioles will emerge at right angles from each parent centriole
18
Q
Prometaphase
A
- chromosomes have finished condensing
- nuclear envelope is gone
- centrosomes at opposite poles
- mitotic spindles start to form
- attachment of MTs to chromosomes and the movement of chromosomes to equator
19
Q
Mitotic spindle
A
- composed of:
- Astral MT: radiate outward (shorter)
- Kinetochore MT: attach to sister chromatids
- Polar MTs: overlap and do not attach to sister chromatids
-plus end of MT associates with kinetochore
20
Q
Movement of chromosomes to equator (congression)
A
- chromosomes usually originally attach to the MT along its side
- but then a kinesin will move it to the plus end
- of that kinesin is missing, the chromosome will not get to the end of MT (ie. wont get to equator)
- once both kinetochores are attached to MT from opposite poles, the chromosomes will be moved to the centre of the cell (congression)
- MTs will either grow or shrink
- whichever is required to get the chromosome to the middle
- depolymerize on one side, while adding subunits on the other side
21
Q
Metaphase
A
- chromosomes are aligned at the metaphase plate
- longest part of mitosis
- once at equator, cell confirms that each chromosome is under tension, and bi-oriented (spindle assembly checkpoint)
22
Q
Anaphase
A
- cohesin is cleaved by separase
- sister chromatids pulled to opposite poles of the cell
- normally separase (protease) is inhibited by securin (until needed)
- mechanism to control when cohesin gets cleaved
- MAD and BUB must confirm that every chromosome is bi-oriented and under tension
- once all chromosomes are attached, mad and bub activate anaphase promoting complex (APC)
- APC degrades securin once the chromosomes are attached
- separase now active, and cuts cohesin
23
Q
Anaphase A
A
- movement of chromosomes to poles
- kinetochore MTs get shorter
- depends on motor proteins
- kinesin13 (depolymerase) to disassemble MT
- cytoplasmic dynein just ahead of where MT is falling apart to walk to minus end
24
Q
Anaphase B
A
- spindle poles move further apart
- polar MTs get longer
- depends on motor proteins:
- 2 kinesin5 attached to each other (one on back of the other)
- they bind between antiparallel polar MTs and slide apart to elongate the spindle
25
Telophase
- chromosomes decondense
- nuclear envelope reforms (dephosphorylation by phosphatases)
- spindle disassembles
26
Cytokinesis
- splitting of cytoplasm
- contractile ring theory:
- myosin II moves along a ring of actin in the cortex (just below cell membrane)
- actin concentrated in a cleavage furrow
- midbody: transient dense structure connecting 2 daughter cells at end of cytokinesis
- abscission: final step of cytokinesis - splitting into 2
- midbody is the last to be cut
27
Cancer
-happens when the cell cycle becomes unregulated
28
Growing cancerous cells in culture
- dont require growth factors
- will divide indefinitely
- will pile all over eachother
29
HeLa cells
- first cancer cells to be grown in culture
| - from Henrietta Lacks
30
Cell cycle length
- varies in different cells
- usually depends on G1
- constantly replicating cells have short G1
- mature nerve cells almost always in G1
- hard to repair
- liver cells live in G0 but then divide if activated by trauma
31
Cell cycle check points
- metaphase-anaphase transition
- restriction point
- G2-M transition
32
Cell fusion experiment
- helped us to figure out how the cycle was controlled
| - heterokaryon=a single cell with 2 genetically different nuclei
33
Fusion experiment 1
- fuse G1-phase cells and S-phase cells
- DNA in G1 nucleus immediately started to replicate
- ie. there is a molecule in the S phase that triggers replication
34
Fusion experiment 2
- fuse G1 cells and M phase cells
- DNA in G1 nucleus immediately started to compact and nuclear envelope started to disintegrate
- ie. there must be a molecule in M phase cells that triggers prophase
35
Progression through cell cycle
- relies on cdks and cyclins
- CDk is a kinases and phosphorylates other proteins to trigger different phases of the cell cycle to start
- only when activated by a cyclin
- CDK= cyclin dependent kinase
- concentration of cyclin varies throughout the cell cycle
- CDk usually always same concentration
36
Work on control of cell cycle
- Hartwell
- Nurse
- Hunt
- Nobel prize 2001
37
G2-M transition
- G2 checkpoint
- mitotic cyclin + mitotic CDK = MPF (maturation promoting factor)
- once activated, CDK will phosphorylate other proteins to cause cell to progress into mitosis
- M phase cyclin peaks just before M phase
-MPF complex must be phosphorylated before it is active
- ATR recognizes and binds to broken DNA
- ATR phosphorylates Chk1
- Chk1 phosphorylates cdc25
- a phosphorylated cdc25 is retained in the nucleus
- therefore cdc25 cannot removed the inhibitory phosphate
- mitosis will not start, cell must repair DNA
38
Fission yeast experiment
- fission yeast reproduce by growing and then splitting into 2 equal sized cells
- we can predict exactly when they should divide (we know their normal length)
- yeast cdks and cyclins and human cdks and cyclins are almost identical
- yeast have cdk called cdc2 (cell division cycle)
- CAK = kinase
- Wee1 = kinase
- cdc25 = phosphatase
39
Regulation of MPF activity by phosphorylation
1. MPF is phosphorylated by CAK and Wee1
- CAK (CDK activating kinase) puts 1 activating phosphate on cdc2
- Wee1 puts 2 inhibitory phosphates on cdc2 (inactivates it)
2. Cdc25 will remove the 2 inhibitory phosphates
3. MPF now active
4. Once cell has gone through mitosis the activating phosphate is removed and mitotic cyclin is degraded
40
Mutating Wee1 and cdc25
- if Wee1 mutated, there is no brake applied and mitosis begins prematurely
- daughter cells smaller than normal
- if cdc 25 mutated, the brake is not removed and mitosis is delayed
- cells longer than normal
41
Ras-MAP pathway and cdk/cyclins
- Ras-MAP pathway results in transcription of cdks and cyclins
- the G1 cdk-cyclin complex phosphorylates Rb so E2F is able to transcribe other genes required for S phase
42
P53 and restriction point
- ATM = protein that detects and binds to damaged DNA (similar to ATR)
- this leads to phosphorylation of p53
- if p53 phosphorylated it will:
1. Activate transcription of p21 which will inactivate G1 cdk
2. Trigger apoptosis if damage is irreparable
- p53 gene mutated in 50% of all tumors
- if p53 doesnt halt cell cycle or induce apoptosis the cell becomes permissive and allows sick cells to divide
43
Apoptosis
- Greek=falling off
- programmed cell death
- apoptosis clears out cells between mammalian digits, in plans
44
C. Elegant
- used to study apoptosis
- transparent round worm about 1mm long
- lifespan 3 days
- only 1090 cells produced during development so each can be followed with precision
- 131 cells programmed to die at specific time
- key proteins in apoptotic pathway named ced (ie cell death)
- homologous proteins found in mammals (caspases)
45
Features of apoptosis
- cytoplasm shrinks, cell shrivels and becomes lobed
- loss of adhesion
- nucleus and organelles fragment
- DNA digested by a DNAse in regular intervals (laddering) 200bp
- flippant inserts phosphotidylserine into the outer leaflet of PM
- usually in cytoplasmic leaflet
- signal for macrophage to cut it
- blebbing
- cell dismantled into small apoptotic bodied
- phagocytic cells ingest the apoptotic bodies
- neighbouring cells protected from damage by potentially harmful digestive enzymes
46
Necrosis
- cell swells
- organelles swill
- cell contents leak into surrounding environment
- leads to inflammation
- bad for tissues
47
Caspases
- cysteine proteases (cysteine in active site)
- cleaves at an aspartic acid residue
- produced as inactive PROcaspases
- activated by cleavage
- leads to proteolytic cascade
48
Caspase targets
- lamins
- cytoskeletal proteins
- endonucleases (cut DNA)
49
Extrinsic pathways
- direct, death signal
- death signal (eg. TNF) binds to a dearth receptor in PM
- 2 adaptor proteins (FADD and TRADD) are recruited
- 2 procaspases-8 also recruited
- procaspases-8 cleave eachother to make the mature enzyme (caspase-8)
- caspase-8 is an initiator caspase
- caspase-8 initiates apoptosis by cleaving and activating downstream executioner caspases like caspase-3
50
Intrinsic pathway
- indirect
- mitochondria mediated
1. survival factor (trophic factor) binds a RTK
2. causes protein called BAD to be phosphorylated (keeps cell alive)
3. Mitochondrial outer membrane contains channel proteins called BAX
- normally anti-apoptotic Bcl protein will keep this channel closed
- if BAD loses is phosphate it will inhibit Bcl proteins and BAX channel will open
4. Cytochrome c will be released from mitochondria
- triggers apoptosis
5. In the cytosol, cytochrome c recruits an adaptor protein Apaf-1 and another initiator caspase (procaspase-9) to form an apoptosome
6. The apoptosome will activate (cleave) caspase-3 (executioner caspase)
51
P53 in apoptosis
1. P53 will bind to puma
2. puma will bind to Bcl-2
3. This allows cytochrome c to be released into cytoplasm... forms apoptosome with Apaf-1 and procaspase-9
4. Apoptosome will activate caspase-3
52
Anastasis
- “rising to life”
- cells avoiding suicide may play role in spread of cancer
- even while undergoing apoptosis, cell holds onto a life line that could bring it back if situation improves/cell becomes healthier
BIOL 2020
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