module 9 Flashcards
name: steps to mitosis (5)
- interphase
- prophase
- prometaphase
- metaphase
- anaphase
- telophase
question: what happens in interphase?
- chromo. replicated into sister chromatids in S phase
- centrosomes duplicated in G1 and S phase
question: what happens in prophase?
- chromo. begin condensation
- mitotic spindle begins assembling
- duplicated centrosomes start to separate to opp. sides
- nuclear envelope dissolves
- endomembranes breakdown into small vesicles
question: what happens in prometaphase?
- chromo. attach via centromeres to microtubule spindle
- kinetochore prot. assemble at centromere of chromo.
question: what happens in metaphase?
- all chromo. attached to spindle from both poles
⤷ bipolar attachment - chromo. align at middle of spindle
question: what happens in anaphase
- sig. breaks assoc. between sister chromatids
- chromatids pulled to opp. poles of spindle
question: what happens in telophase?
- cell reverses everything done in prophase
⤷ chromo. decondense
⤷ spindle disassembles
⤷ nuclear envelop reforms
question: what regulates the sequence of events of mitosis?
- 2 classes of prot.
- CDK (cyclin dep. kinases)
- E3 ubiquitin ligase complexes
explain: role of CDKs (+ struc.)
- hetero dimeric prot. complex
- facilitates phosphorylation
- CDK = regulated by cyclin
explain: role of E3 ligases
- target specific prot. for degradation in proteasome
- helps degrade cyclin to turn off kinases
- helps degrade cell cycle inhibitors
name: major classes of cyclin-CDK kinases (4)
- G1 cyclin-CDK
- G1/S-phase cyclin-CDK
- S-phase cyclin-CDK
- mitotic cyclin-CDK
question: what’s the difference between the types of cyclin-CDK?
- diff. target prot.
- diff. timing
⤷ ex. G1 cyclin-CDK = active in G1
name: main E3 ligase complexes (3)
- SCF
- APC-Cdc20
- APC-Cdh1
explain: roles of each E3 ligase complex
SCF
- allows transition to S-phase
APC-Cdc20
- regulates transition from meta- to ana-
APC-Cdh1
- mediates exit from mitosis
question: what is happening in G1 phase (explain w/ cyclin-CDK and E3 ligases)
- G1 cyclin-CDK and SCF are involved
- G1 cyclin-CDK has 3 major targets
⤷ phosphorylating prot. at end of mitosis
⤷ prepare for DNA rep.
⤷ phosphorylate S-phase inhibitors - prepares for the next cyclin-CDK
- phosphorylating S-phase inhibitors makes them a target for SCF
- activates S-phase cyclin-CDK
question: what is happening in between G1 and M phases (explain w/ cyclin-CDK and E3 ligases)
- G1/S-phase cyclin-CDK and S-phase cyclin-CDK involved
- G1/S targets transcription factors that regulate exp. of genes coding for mitosis
⤷ ex. M-phase cyclin CDK - phosphorylating M-phase CDK inhibits it’s activation until cell is ready to start mitosis
- S-phase cyclin-CDK needed to activate and assemble pre-replication complex
question: what is happening in M-phase (explain w/ cyclin-CDK and E3 ligases)
- M-phase cyclin-CDK involved
- many phosphorylation targets
⤷ chromosomal prot. (for condensation)
⤷ nuclear lamins (for envelope breakdown)
⤷ MAPs (for assembly of spindle)
⤷ kinetochore prot. in centromeres (for assoc. between chromo. and spindle)
⤷ APC complex (to prep. cell to go through mitosis phases) - regulated degradation
question: what is the regulated degradation that takes place during the M phase?
- ubiquination and degradation at 2 points
- anaphase inhibitors = degraded to allow metaphase to anaphase transition (MAT)
- mitotic cyclin = degraded to allow cell to exit mitosis via mitotic exit network (MEN)
question: what is MPF?
- first seen in frog eggs
- mitosis promoting factor
- a cyclin and CDK heterodimer
- MPF = M-phase cyclin CDK complex
question: what is cyclin B?
- cycling prot.
- regulates M-phase cyclin-CDK activity
- seen from sea urchins
⤷ saw that a prot. had varying lvls of conc.
⤷ kept going up and down -> cycling - saw that increase in cyclin was coordinated w/ increase in number of cells engaging in mitosis
⤷ less cyclin = less cells in mitosis
explain: in vitro experiment for cyclin
- used assay for MPF activity
⤷ looked for phosphorylation of target - measured cyclin B conc. on a gel
- looked for beha. typical for a cell that does mitosis
question: what happened in the in vitro experiment when treated w/ and w/out low RNase? what about when adding back in mRNA for cyclin B? what about adding non-degradable cyclin B?
WITHOUT RNASE
- cells did mitosis
- cyclin B and MPF matched mitotic events
⤷ increase in cyclin B -> increase in MPF activity
- less cyclin B = less MPF activity = cells did mitosis exiting beha.
WITH RNASE
- removed mRNA but leaves tRNA and rRNA
- no increase in cyclin B
- no increase in MPF activity
ADDING BACK CYLIN B
- mitosis restored the same
- MPF activity returned
NON-DEGRADABLE CYCLIN B
- cyclin B lvls stay high
- cells stay in condensed state and can’t complete mitosis
conclusion = cyclin B is necessary for MPF activity
⤷ and need to be able to remove/degrade cyclin B
question: how is cyclin B degraded?
- APC-Cdc20 and APC-Cdh1 target cyclin B
- ubiquitination and degradation via proteasome
- degradation begins at anaphase
- APC-Cdc20 mediates w/ ubiquitinylation
- degradation actually happens via APC-Cdh1 ubiquitinylation at exit from cell cycle
question: how is cyclin B recognized by APC-Cdc20?
- recognizes short pep. seq. at N-term of cyclin B
- pep. seq. = destruction box
⤷ arginine in pos. 1
⤷ leucine in pos. 4
⤷ asparagine or glutamine in pos. 9
** RxxLxxxxN/Q - any mutations to seq. -> undegradable form of cyclin B
- adding D-box to other prot. will make them degrade the same was as cyclin B
question: does APC-Cdc20 have other targets other than cyclin B? if so, what?
- yes
- targets an anaphase inhibitor = securin
⤷ ensures the replicated chromatids are secured before anaphase
question: how is securin removed?
- activation of APC-Cdc20 -> targets securin
- when securin = removed, separase activates
- metaphase has a prot. = separase
- cleaves one of the cohesin prot. (Scc1)
- breaks apart cohesin complex + chromatids = pulled apart
explain: role of SCF
- SCF = Skp, Cullin, F-box containing complex
- SCF can recog. Sic1 (S-phase inhibitor)
⤷ only recog. when it gets phosphorylated by G1 cyclin-CDK - SCF degrades Sic1 -> activates S-phase CDK -> proceed to S-phase
recap: steps of cell cycle
- early G1
⤷ DNA pre-replication complex assembles - mid/late G1
G1 cyclin-CDK
⤷ inactivates APC-Cdh1
⤷ activates S-phase cyclin-CDK
⤷ phosphorylates S-phase inhibitors - S
SCF
⤷ degrades phosphorylated S-phase cyclin-CDK (Sic1)
S-phase cyclin-CDK
⤷ activates pre-replicaiton complex
- G2
M cyclin-CDK
⤷ activates early mitotic events - M
METAPHASE
APC-Cdc20 (in between)
⤷ degrades securin
ANAPHASE
APC-Cdh1 (in between)
⤷ degrades mitotic cyclin
TELOPHASE AND CYTOKINESIS
define: genetic screen
- unbiased search for genes that are involved in a certain mechanism
- make mutations in every gene + look at phenotypic effect
define: temperature sensitive mutation
- TS mut.
- mutated gene codes for a TS prot.
- allows researchers to change temp. to turn prot. on and off
- ex. folding at permissive temp. vs misfolding at restrictive temps.
question: what are the mutations for s.pombe (fission yeast)?
- mut. to cdc2 gene
- mut. to cdc13 gene
- (TS) mut. to cdc25
- wee1 gene
question: what are the mutations from cdc2 in s.pombe?
- mut. in cell cycle regulators - cell division cycle mutants (cdc mutants)
- 2 effects on cells
⤷ elongated cell
⤷ wee phenotype - elongated = delayed in G2 -> kept growing instead of going to mitosis
- wee = went to mitosis too early -> smaller than normal
question: which s.pomble mutation is recessive vs dominant?
- wildtype = cdc2+
- elongated = recessive = cdc2-
- wee = dominant = cdcD
question: what is the role of the cdc2 prot. in the cell cycle?
- absence of cdc2 = cell fails to divide
- cdc2 increased = cell divides too early and too often
- cdc2 = key regulator for entry into mitosis
- cdc2 = CDK for MPF
question: what is the role of cdc13 prot. in cell cycle?
- also a cell cycle regulator
- forms heterodimer w/ cdc2
- increases and decreases like cyclin B
explain: MPF of s.pombe
- heterodimer of cdc2 and cdc13
- CDK = cdc2
- cyclin = cdc13
- works as M, S, and G phase cyclin-CDK
question: what are the mutations from cdc25 in s.pombe?
- loss of func. cdc25 mut. -> elongated phenotype
- (gain of func.) cdc25 dominant mut. -> wee
question: what is the role of cdc25 in cell cycle?
- lack of cdc25 inhibits entry into M-phase
⤷ meaning its an MPF activator - ex. gain of func. mut. -> early entry to mitosis
question: what are the mutations from wee1 in s.pombe?
- loss of func. mut. = wee- -> wee phenotype
- gain of func. mut. = weeD -> elongated
question: what is the role of wee1 in the cell cycle?
- lack of wee1 causes premature entry to M-phase
⤷ meaning wee1 = inhibitor to MPF
question: how do cdc25 and wee1 regulate MPF?
- cdc25 = MPF activator
⤷ encodes a phosphatase that dephosphorylates tyrosine on cdc2 to activate MPF - wee1 = MPF inhibitor
⤷ encodes a tyrosine kinase that phosphorylates tyrosine on cdc2 to inactivate MPF
**both affect same tyrosine on cdc2 (tyrosine 15)
question: what is the diff. between s.pombe and s.cerevisiae? similarities?
- s.pombe = fission yeast
- sp. cerevisiae = budding yeast
- budding yeast has formation of daughter cell that buds off mother in G1
- still have same cell cycle regulators
- s.pombe’s cdc2 = s.cerivisiae’s cdc28
explain: impact of TS loss of func. mut. in G1 on s.cerevisaie
- mut. disrupts cell cycle causing cell to arrest in G1
- forms daughter bud but cannot enter S-phase
- caused by mut. in cdc28
define: functional complementation (+ func.)
- technique to ID genes coding for cell cycle regulators
- screens through genes to ID one that can fix the mut. and restore wild-type
explain: step 1 in functional complementation
- start w/ a TS mut.
- permissive T = cells can grow
- restrictive T = cells are arrested at G1
explain: step 2 in functional complementation
- add genes back in randomly to see which one restores the ability to divide
- use genes from cDNA library
- see which one makes a change in phenotype
question: what is a cDNA library and how is it made and used?
- complementary DNA
- cDNA genes = copies of mRNAs of an org.
- library made by isolating mRNAs using reverse transcriptase
- means library isn’t the whole genome
⤷ bits of mRNA expressed at a certain time
explain: step 3 in functional complementation
- ID the gene that makes the phenotypic change
- go back to cDNA library
⤷ isolate the plasmid and sequence the cDNA - for s.cerevisaie = cdc28
- for s.pombe = cdc2
