Module 9 Flashcards
(58 cards)
1
Q
Cell division + goal
A
All eukaryotic cells undergo cell division, which includes 4 distinct and highly regulated phases
Goal of Cell Division: To take a single cell and produce 2 identical daughter cells
2
Q
4+1 cell division phases
A
→ [G0 - Quiescent State:]
Division does not occur and allows cell differentiation
→ G1 - First Gap Phase: [Interphase]
Cell is actively growing
Engages in gene expression and synthesis of new proteins to prepare the cell with resources for division
Duplication of centrosomes
→ S - DNA Synthesis Phase: [Interphase]
The cell now forms to have sister chromatids
Duplication of centrosomes
→ G2 - Second Gap Phase: [Interphase]
Readies the cell for mitosis
→ M - Mitotic Phase: [Mitosis]
Sister chromatids are separated into 2 daughter cells
3
Q
Implications of Unregulated Cell Growth
A
- Cell Death
- Over-proliferation of cells
- Tissues and cells cannot be repaired
- Cancer
4
Q
What can a stem cell do (2 options)
A
- Replicate to produce more stem cells
- Cease cell division and enter G0 (differentiate OR halt cell division until later)
5
Q
Prophase
A
- Chromosomes condense (early prophase) - 4n
- Assembly of mitotic spindle
- Dissolution of nuclear envelope
- Breakdown of endomembrane of cell into small vesicles
6
Q
Prometaphase
A
- Chromosomes are maximally condensed
- The centromeres of the chromosomes are in the process of attaching to the now fully formed bipolar microtubule spindle
- Kinetochore proteins help with this assembly to mediate the association of chromosomes to the plus-ends of spindle microtubules
7
Q
Metaphase
A
- Every chromosome is now attached to both poles of the mitotic spindle (bipolar attachment)
- The tension of the forces pulling towards the poles causes chromosomes to aggregate/align in the middle of the mitotic spindle - equator of the spindle
8
Q
Anaphase
A
- A signal releases the association between the replicates sister chromatids and each of the sister chromatids are pulled towards opposite poles in the spindle
9
Q
Telophase
A
- Cell begins to reverse the cellular changes that occurred in prophase
- Chromosomes decondense, mitotic spindle disassembles, and nuclear envelope and endomembrane systems reassemble
10
Q
Cytokinesis
A
- Pinching between cell membranes leads to full separation, as the cell elongates
- Driven by a contractile ring (composed of actin filaments and myosin II molecules at the equator of the cell
11
Q
Sequence regulators that allow mitosis to occur in the correct order
A
- CDK - Cyclin-Dependent Kinases
- E3 Ubiquitin Ligase Complexes
12
Q
CDK - Cyclin-Dependent Kinases - mitosis regulation
A
phosphorylation
- Heterodimeric protein complexes that facilitate regulated phosphorylation
- Kinase activity is regulated with the cyclin protein
- Activated kinases initiate cellular processes by phosphorylating target proteins
13
Q
E3 Ubiquitin Ligase Complexes - mitosis regulation
A
- Target specific proteins for degradation in the proteasome
- Cyclins can be degraded to turn off kinases
- Cell cycle inhibitors can be degraded when checkpoints are passed in the cell cycle
14
Q
4 Major Classes of Cyclin-CDK complexes
A
- G1 Cyclin-CDK
Active in G1
- leads to activation of S phase Cyclin-CDK - G1/S Phase Cyclin - CDK
- S phase Cyclin-CDK
- Mitotic Cyclin CDK
- Phosphorylates a collection of proteins for all cellular changes that occur in prophase
15
Q
How does G1 Cyclin-CDK prepare the cell for DNA synthesis
A
- DNA pre-replication complexes assemble at origins
- G1 cyclin CDK inactivates Cdh1
- activates expression of S-phase cyclin CDK components
- phosphorylates S-phase inhibitor
- SCF/proteasome degrades phosphorylated S-phase cyclin-CDK inhibitor
16
Q
G1 Cyclin-CDK phosphorylation targets (3)
A
- APC-Cdh1
- at the end of mitosis signals that mitosis is complete - Transcription factors to prepare for S-phase
- activates them - S-phase inhibitors (SCF)
- become targets for E3 ubi = S phase continues
17
Q
How does G1/S Cyclin-CDK Complex prepare the cell for DNA synthesis
A
Prepares cell for upcoming M-phase
18
Q
G1/S Cyclin-CDK Complex targets
A
- Transcription factors that regulate the expression of genes coding for mitosis (ie. M-phase cyclins)
- Proteins that mediate the process of centrosome replication
19
Q
How does S-phase Cyclin-CDK Complex prepare the cell for DNA synthesis
A
Necessary for activation and assembly of the pre-replication complex at sites of origins of replication
20
Q
S-phase Cyclin-CDK Complex targets
A
Pre-replication complex
- activates it
- Ensure phosphorylation of proteins associated with origins of replication are “fired” only once per cell cycle
- There must be only one replication complex/origin in each cell cycle
- Phosphorylates the M-phase CDK to inhibit the cell until it is prepared for mitosis (Until DNA is properly replicated)
21
Q
M-phase Cyclin-CDK Complex targets
A
Phosphorylates:
- chromosomal proteins allows chromosome condensation
- nuclear lamins initiates nuclear envelope breakdown
- microtubule associated proteins (MAPs) allows assembly of mitotic spindle
- kinetochore proteins at chromosome centromeres allows chromosome-spindle association
- APC complex prepares the cell for serial procession through phases of mitosis
22
Q
During mitosis, ubiquitination and degradation of proteins happens at these 2 points…
A
Metaphase → Anaphase required anaphase inhibitors
- MAT - Metaphase to Anaphase Transition
Mitotic cyclins are degraded to allow the cell to exit mitosis
- Mitotic Exit Network - MEN
23
Q
Masui and Market discovery
A
Identified a factor called the Maturation/Mitosis Promoting Factor (MPF)
- induced cells to complete meiosis and initiate a series of 11 mitotic divisions to form a blastocyst
- the blastocyst undergoes cell division and differentiation to become a tadpole
- this factor is shown to be the M-phase Cyclin CDK
24
Q
Tim Hunt and Joan Ruderman discovery
A
Discovered cyclic nature
- using sea urchin embryos
- Identified a collection of proteins that undergo cyclical synthesis and degradation during embryonic cell division cycles
- Isolated radiolabeled proteins at different time points after fertilization and then separated these on a polyacrylamide gel
Every increase in cyclin conc. is followed by an increase in cells that are in mitosis
25
Anaphase impact on Cyclin B concentration
There is a rapid drop in Cyclin B associated with anaphase → mitosis exit
26
How can cyclin regulate the cell cycle? - Andrew Murray experiment
- Performed in vitro experiment to study cyclin proteins’ role
- Isolated extracts from fertilized eggs that contained mRNAs and proteins needed for cell division
Consisted of 3 assays
1. Assay for MPF activity
- Identified by phosphorylation of histone H1 protein
2. Measured Cyclin B protein conc. in the gel extracts
- Antibody to cyclin B
3. Looked for behaviors typical of a cell undergoing mitosis
- Looked at behavior of sperm nuclei added to in-vitro cell extracts
27
Murray experiment 1
- Nuclei underwent cyclical behaviours typical of embryonic cell division
- Sperm nucleus underwent events typical of early mitosis and late mitosis
- As cyclin B concentration goes down, MPF also goes down
Allows for chromosome decondensation (decrease)
and v.v.
Synchronized cycling can continue in the absence of a cell for as many as 10 cycles
28
Cyclin B and RNase experiments
Cell Treated with RNase
- Removes mRNAs but leaves tRNAs and rRNAs needed for protein synthesis
- Leads to no increase in cyclin B concentration and no increase in MPF activity
- There are no mitotic events
(flat graph)
Cyclin B mRNA and RNase added
- Cyclic activity and mitosis was resumed!
- Synchronized behaviors were restored
- Therefore, Cyclin B oscillations are sufficient to restore cycling CDK activity
(normal graph)
mRNA that codes for Cyclin B was added with RNase
- This means the Cyclin B does not oscillate and its concentration is high
- Cells enter mitotic arrest - stay in early mitosis as chromosomes are not able to decondense
(graph increase but no decrease)
| Needs cyclin b for mitosis
29
Sperm Nuclei Experiments - degradation
RNase treated extracts with added Cyclin B mRNA
- Mitotic spindle can be seen assembling and disassembling
RNAse-treated extracts with added mRNA for non-degradable Cyclin B
- Chromosomes undergo anaphase but fail to decondense and the mitotic spindle does not disassemble
- Therefore, we can deduce that cyclin B must be degraded to allow a cell to exit mitosis
30
3 E3 ligase complexes
1. SCF complex
- Releases cell from G1 and allows the transition to S-phase
Anaphase Promoting Complex: (and accessory proteins)
2. APC-Cdc20
- regulates the transition from metaphase into anaphase
3. APC-Cdh1
- mediates exit from mitosis
31
How is Cyclin B degraded?
APC-Cdc20 and APC-CDh1 complexes are the E3 ligases which target Cyclin B for ubiquitination and then degradation.
- Destruction of Cyclin B inactivates CDK activity and allows the cell to exit mitosis and prepare for the next stage of the cell cycle
- At anaphase, CB degradation is mediated through APC-Cdc20 but allows exit from cell cycle via APC-Cdh1
32
How does APC recognize Cyclin B?
Study by Marc Kirschner:
- There is a short peptide sequence found NEAR the N-terminus of Cyclin B
- Identified as the recognition sequence for ADC-Cdc20
- Called the destruction/D-box
- Contains the following conserved AAs: RxxLxxxxN/Q
33
Is D-box necessary and/or sufficient for degradation
yes to both
34
Other targets of APC
APC has a second target in addition to Cyclin B - an anaphase inhibitor
- When APC binds to an anaphase inhibitor, it would allow anaphase to happen
- When the D-box peptide was added, it would not allow the APC to bind to the anaphase inhibitor, which is why the cells are stuck in metaphase!
35
What is the anaphase inhibitor?
SECURIN protein
- Indirectly ensures two replicated sister chromatids are secured together prior to anaphase
36
Cohesin Complex
- Complex of a collection of cohesin proteins between sister chromatids
- Includes Smc1, Smc3 and Scc1 which attach the sister chromatids together at replication
- Association persists as the cell enters mitosis and the chromosomes condense
During metaphase:
Cohesion complex holds together the 2 replicated sister DNA molecules
37
Separase
- Protein able to initiate separation of sister chromatids by cleaving Scc1
- Type of protease
- Kept inactive through its association with the securin protein
38
Securin and cohesin complex
- Activation of APC-Cdc20 enables E3 ligase to target securin for ubiquitination and degradation
- When securin is removed, separase is activated and cleaves Scc1 at a single peptide recognition sequence to break apart the cohesin complex
39
APC-Cdc20 Ubiquitinylates ...
APC-Cdh1 Ubiquitinylates ...
APC-Cdc20 Ubiquitinylates Securin
APC-Cdh1 Ubiquitinylates Cyclin B
40
SCF Ligase Complex
- Required for progression through the cell cycle
- Ubiquitinylates the S-phase inhibitor (Sic1)
- Phosphorylation of Sic1 allows it to be recognized by the SCF E3 ligase and leads to Sic1 ubiquitination and degradation
- Removal of Sic1 activates the S-phase-CDK and the cell proceeds through into S-phase
SCF - Skp, Cullin, F-box Containing Complex
41
What is a genetic screen?
Unbiased search for genes that are involved in a particular mechanism
42
Temperature-Sensitive (TS) Mutants
Mutated gene codes for a temperature-sensitive protein:
- Protein folds at permissive temperature (ie. 24)
- Protein misfolds at the restrictive temperature (ie. 37)
→ This allows the researcher to turn the protein function on and off
However, wild-type yeast cells (no mutation) can grow and divide at both temperatures
43
Model organism:
Schizosaccharomyces pombe
TS mutations in Cdc2 phenotypes
Phenotype #1: Elongated Cell
(Recessive Mutation)
- Cells are deleted in G2 and keep growing instead of entering mitosis
- Cells grow longer than they should
Cdc2-
Phenotype #2: Wee
(Dominant mutation)
- Cells transition into mitosis prematurely and result in smaller cells
CdcD
44
Cdc2 importance for cell division
- In the absence of Cdc2, cells fail to divide
- When Cdc2 protein function is increased, cell divides too early and too frequently
- Suggests Cdc2 is a key regulator for entry into mitosis and acts to promote cell division
45
Cdc2 general characteristics
- 34 kDa protein with kinase activity
- Forms heterodimer with Cdc 13
- Might be the CDK of the MPF
46
Cdc13
- cell cycle regulator
- Loss of function/recessive mutations in cdc13 cause the elongated phenotype
- Dominant mutation = wee phenotype
Cdc 13 is found to increase and decrease in concentration during the cell cycle
= homologous to the Xenopus cyclin B protein
47
Cdc2-Cdc13 heterodimer
- 2 proteins in the S.pombe MPF
- Cdc2 is a CDK in yeast
- Cdc13 is a cyclin in yeast
48
Other TS Mutations - Cdc25
Cdc25 mutation also causes elongated and wee phenotype:
- Cdc25 is normally an activator of MPF and promotes entry into M-phase
- Supported by observation that the gain of function mutation causes premature entry into M-phase
49
Wee1 gene
Loss of function/recessive = wee phenotype
Dominant = elongated phenotype
(Therefore switched)
Lack of Wee1 = premature entry into M-phase
→ is it therefore likely that Wee1 is normally an inhibitor of MPF and delays entry into M-phase
50
Wee1 vs. Cdc25
Wee 1:
A tyrosine kinase that phosphorylates Tyr 15 on Cdc2 and inactivates the MPF complex
Cdc25:
A phosphatase that reverses this phosphorylation by Wee1 on Tyr 15 to activate MPF
51
3 regulators of MPF activity
1. Wee1 Kinase
2. CAK kinase
3. Cdc25 phosphatase
52
Wee1 Kinase
Mitotic cyclin and CDK associate
- The Cdk contains Y15 (Tyrosine Residue) and T161 (Threonine)
Wee1 Kinase phosphorylates Tyr 15
- Inhibitory phosphorylation that keeps MPF inactive
53
CAK kinase
CAK kinase phosphorylates T161
- This is an activating phosphorylation, but MPF is inactive because of Tyr 15 phosphorylation
- Phosphorylation of Y15 seems to increase affinity of CAK
54
Cdc 25
Cdc 25 dephosphorylates Tyr 15
- Activates Cdc2 kinase
55
Model System: Saccharomyces cerevisiae
Type of budding yeast:
- Formation of daughter cell buds off of the mother cell in G1 before the cell enters S-phase
- Cdc28 is the budding yeast homologue of the fission yeast Cdc2
56
Functional Complementation - explanation
Technique used to identify genes coding for cell cycle regulators:
- Screen through genes to identify one that can “rescue” the phenotype caused by mutation and restore wild-type behaviour
Usually, the rescuing gene is added to the wild-type version to cause phenotype
57
Functional Complementation - steps for experiments
Step 1: TS Mutation that causes cell cycle defect
Step 2: Since the mutation which caused the phenotype is unknown, genes are added back at random to restore the ability of cells to divide
- use library of cDNAs
Step 3: Identifying Gene Z
- Isolate plasmid from the bacterial cell to sequence the cDNA
58
Cdc28
- Codes for the single CDK in budding yeast S.cerevisiae.
- Interacts with a cyclin to form an active heterodimer which targets for phosphorylation proteins necessary for DNA synthesis
- MPF → S phase promoting factor (SPF)
There are multiple cyclins:
- Cln1,2 and 3 (G1/S-phase cyclins which associate with Cdc28 to form SPF)
- Clb1 and 29M phase cyclins similar to Cyclin B to form MPF)
