module 10 Flashcards
question: how is phosphorylation mediated?
- MPF
- bc MPF = heterodimer of mitotic cyclin and CDK
- active MPF = promotes phosphorylation
- inactive MPF = reverses phosphorylation
question: what events in prophase are mediated by MPF phosphorylation (what gets phosphorylated for each one)? (5)
- active MPF
- formation of mitotic spindle
- phosphorylate microtubule assoc. prot.
⤷ bc they promote microtubule instability - condensation of chromo.
- phosphorylate condensins and histone prot. - preparation for sister chromatid separation
- phosphorylate cohesins - break down of nuclear envelope
- phosphorylate nuclear lamins - fragmentation of golgi and ER
- phosphorylate GM130
question: what events in telophase are mediated by MPF dephosphorylation? (4)
- inactive MPF
- nuclear envelope reassembly
- chromo. decondensation
- mitotic spindle disassembly
- golgi and ER mem. reassemble
explain: histone prot. role in chromo. condensation
- 5 types of histones
- H1 and H3 = phosphorylated by Aurora B kinase during chromo. condensation
- H3 forms prot. core for nucleosome
- H1 links nucleosomes
- get packed tighter during condensation
explain: cohesin prot. role in chromo. organization
- form cohesin complex
⤷ holds sister chromatids together until anaphase - releasing them = 2 steps
question: how does the release of cohesins happen?
- release from chromo. arms
- keep cohesins in middle (centromere)
⤷ protected from phosphorylation by phosphotases
- makes the x shape
- release by phosphorylation (cyclin B-CDK, Aurora B) - release from centromere
- cleaved by separase
- in anaphase
- allows chromatid separation
explain: condensin role in chromo. condensation
- condensins get phosphorylated by cyclin B-CDK
- allow assembly and chromo. condensation
- MPF phosphorylation sites are on XCAP-D2 domain of condensins
question: how do chromo. decondense?
- dephosphorylate condensins and histones
question: what happens of the nuclear envelope throughout mitosis (nuclear envelope disassembly at interphase, prometaphase, metaphase)
- interphase = intact mem.
- prometaphase = chromo. condense and envelope fragments
- metaphase = no envelope
⤷ frag. into small vesicles and dist. throughout sytosol
explain: struc. of nuclear envelope
- 2 lipid bilayers
- outer = cont.d w/ rough ER
- inner = assoc. w/ IF called nuclear lamina
- nuclear pore complexes everywhere to allow transport
- lamina = lamin A, B, C
explain: phosphorylation of lamin prot.
- phosphorylated at serine by cyclin B-CDK
- initiates envelope disassembly
- only lamin B stays assoc. w/ nuclear mem.
⤷ A and C are dispersed
question: what happens when lamin A doesn’t get phosphorylated (explain w/ hamster exp.)?
HAMSTER + HUMAN LAMIN A
- interphase = intact envelope
- prophase = lamina breaking down
⤷ can see lamin A diffusing into cytosol
- metaphase = lamin no longer organized + chromo. condensed
HAMSTER + VARIANT OF HUMAN LAMIN A
- lamin A couldn’t be phosphorylated
⤷ serine became alanine
- interphase = same (intact lamina)
- prophase = lamin A not diffusing into cytosol
- metaphase = chromo. condensed but still has ring of lamin A
**lamin B and C could still be phosphorylated
⤷ so lamina intact in metaphase means lamin A needs to be phosphorylated to break down lamina
question: how does the nuclear envelope reassemble?
- inactivation of cyclin B-CDK + phosphatase activity
- dephosphorylation of lamins A, B, C
- lamins reassemble and reform lamina
- B still assoc. w/ vesicles from before
⤷ so it brings vesicles to lamina to form inner nuclear envelope - nuclear pore complexes dephosphorylated
explain: golgi fragmentation
- before separation, golgi = fragmented to each pole of spindle
- GM130 (golgi prot.) = phosphorylated by cyclin B-CDK
question: what is the purpose of golgi and mitochondria fragmentation?
- to ensure organelles are distributed to both daughter cells
question: what are the cyclins that control each part of the cell cycle for vertebrates?
EARLY G1
- D type cyclins
- CDK4 or 6
S PHASE (trigger)
- cyclin E-CDK2
S PHASE (completion)
- cyclin A-CDK2
- cyclin A-CDK1
MITOSIS
- cyclin B CDK1
G0
- none
question: what is the restriction point?
- time late in G1 when passage through cell cycle = indep. of presence of the mitogen
- cell continues into S phase even w/out mitogen
question: what are mitogens?
- sig. molecules that induce cell div.
- cause expression of G1 cyclin-CDK
question: what is the diff. between early and delayed resp. genes in re-entry to the cell cycle?
- adding mitogen starts early gene expression
- peaks at 1 hour and declines
- delayed starts when early declines
question: what regulates early resp. gene expression?
- transcription factors activated by MAP kinase
⤷ SRF
⤷ TCF - already in cell so only need to be phosphorylated
⤷ also means its not affected by inhibitors
question: what do the early resp. genes code for?
- c-Fos
- c-Jun
(transcription factors)
^these activate delayed resp. genes
question: what do the delayed resp. genes code for?
- cyclin D
- cyclin E
- CDK2
- CDK4
- CDK6
question: how does c-Fos activate delayed gene resp.?
- c-Fos = early resp. gene
- induces exp. of CDKs needed for cell division
question: what happens if mitogens and inhibitors of prot. synthesis are added into cell?
- translation inhibitors have no effect on early resp. gene exp bc already in cell (SGF, TCF)
- but affects c-Fos and C-Jun -> affects delayed resp. gene
question: how do you turn off early resp. genes?
- expression and translation of transcriptional inhibitors encoded by early resp. genes
- they turn themselves off
question: what happens when early genes turn off?
- early gene mRNA lvls stay high
- no translation for early gene
- delayed genes = never transcribed bc dep. on early genes
question: what happens when mitogen is added and removed before and after RP?
- adding growth factor/mitogen induces exit from G0 and entry into G1
- removing mitogen before RP passes -> decrease in cyclin D/CDK4-6 -> failure to proceed to S phase
⤷ retreat back to G0 - removing mitogen after RP passes -> decrease in cyclin D/CDK4-6 has no effect -> progresses to S phase
⤷ no effect bc cyclin E/CDJ2 is high enough
question: why can cell progress to S phase if mitogen removed after RP?
- E2F = regulates exp. of genes needed in S-phase
- initially, E2f = inactive bc inhibitory prot. Rb present
- cyclin D-CDK can phosphorylate Rb
- if mitogen removed before RP, cyclin D lvls aren’t high enough
- but E2f also induces cyclin E/CDK which can also target Rb
- if enough E2F is activated, induces exp. of E2f
⤷ +ive feedback loop - even if cyclin D decreases, cyclin E is self-sustaining and cell proceeds to S-phase
explain: functions of checkpoints
- recog. error and delay progression until its fixed
- look for:
⤷ damaged DNA
⤷ unreplicated DNA
⤷ assembly of spindle
⤷ chromo. attachment during metaphase
⤷ completion of anaphase - if can’t fix, cell does apoptosis
question: what are the types of damage found at damage checkpoints? (2)
- ionizing radiation
- creates double-stranded DNA breaks - ultraviolet radiation
- creates thymine nucleotide dimers
question: what prot. recog. which types of damage? (2)
- ATR recog. UV damage (thymine dimers)
- ATM recog. double strand breaks
question: how does ATR recog. + fix damage?
- targets Chk1
- Chk1 targets cdc25
**cdc25 regulates MPF between G2 to M-phase (reverses inhibitory phosphorylation) - Chk1 phosphorylates cdc25
- inactive cdc25 -> no MPF
- arrests cell at G2
⤷ gives time for cell to fix problem - when repaired, ATR dissociates
⤷ reverses everything
^^works for thymine dimers but also DNA replication checkpoints
question: how does ATM recog. + fix damage?
- targets Chk2
- Chk2 targets p53 prot.
- p53 usually unstable but if phosphorylated, becomes stable
- p53 activates p21 (CIP)
⤷ cyclin inhibitor - p21 binds to G1 cyclin-CDK to inhibit activity
- cell stalls in G1
- when repaired, ATM dissociates
⤷ reverses everything
question: what happens if p53 can’t fix the damage?
- constantly activated
- induces pro-apoptotic genes to kill cell
question: what does spindle assembly checkpoint do?
- prevents entry into anaphase if chromo. aren’t assoc. properly to spindle
- delays cell in metaphase
question: how does cell fix error at spindle assembly checkpoint?
- wildtype cell reassembles the spindle when cell is arrested
question: what happens if SAC not functioning
- cell proceeds to anaphase even if chromo. aren’t attached to spindle
- chromatids don’t get segregated to separate poles
- nondisjunction and aneuploidy
- one daughter cell has an extra chromo.
- other cell missing a homologue
question: what regulates SAC?
- Mad2
- inactivates APC-Cdc20
⤷ securins can stay
⤷ prevents separase from working - chromatids can’t separate
- loss of func. mut. in Mad2 -> cells proceed to anaphase early
question: what is Mad2 (what is it regulated by?)?
- can be open or close conformation
- open Mad2 assoc. w/ kinetochores of chromo. that aren’t on microtubules
- Mad1 binding to Mad2 makes it close
- closed Mad2 leaves chromo. and interacts w/ cdc20
⤷ blocks cdc20 from associating with APC - keeps cell in metaphase
question: what happens when SAC error is fixed?
- Mad1 and Mad2 release
- Mad2 back to open
⤷ releases the cdc20 - APC binds to cdc20
- APC-cdc20 reactivates
⤷ targets securin
⤷ separase can work - allows anaphase
question: what does mitotic exit network do?
- monitor completion of anaphase
- activates cdc14
⤷ to dephosphorylate cdh1 so it can bind to APC - activate APC-cdh1
⤷ to inactivate MPF
question: where is cdc14?
- in nucleolus
- can’t reach it unless sister chromatids separate
⤷ means anaphase needs to happen
question: what happens in metaphase vs anaphase of budding yeast (tem1, cdc14)?
METAPHASE
- Tem1 = GTPase
- Tem1-GAP = GAP
⤷ keeps Tem1 in GDP bound form (inactive)
- Tem1-GEF = on mem. (far)
- when Tem1-GDP is inactive, cdc14 is hidden in nucleolus
ANAPHASE
- spindle gets longer
- Tem1 gets closer to Tem1-GEF
⤷ exchanges GDP for GTP in Tem1
- allows passage through MEN checkpoint
- allows release and activation od cdc14
question: what happens when cdc14 is released at anaphase?
- cdc14 dephosphorylates cdh1
- cdh1 activates APC
- APC-cdh1 inhibits MPF
- exit from mitosis can occur
question: what happens if chromo. segregation and anaphase don’t happen?
- can’t pass MEN
- Tem1 stays inactive
- cdc14 never gets released
- MPF stays active
- cell never exits mitosis
question: what does cdc14 do to inhibit S-phase?
- targets cdh1 (dephosphorylates)
- inactivates MPF (degrades cyclin B)
- keeps Sic1 unphosphorylated
- Sic1 binds to S-phase cyclin-CDK
- inhibits progression into S-phase
**opposite can allow progression to S-phase if cdc14 is phosphorylated by G1 cyclin-CDK
