SP-HC4: Alberts h17 (Cell Cycle) Flashcards
The cell cycle checkpoints
-Metaphase to anaphase: are all chromosomes attached to spindle? proceed to cytokinesis
-G1/S checkpoint: is the environment favorable? proceed to S-phase
-G2/M checkpoint: is the DNA replicated and is the environment favorable? proceed to mitosis
The cell cycle is regulated by cyclins. Name the types of cyclins.
-G1/S cyclins: activate Cdks in late G1
-S cyclins: start/help DNA duplication
-M cyclins: stimulate entry into mitosis
To which enzymes do cyclins bind?
Cyclin-dependent kinases (Cdks) > checkpoint molecules
Which cyclins are active in which cell cycle phases?
G1/S cyclin: during G1 phase
S-cyclin: during late G1, S, G2 and M until metaphase-anaphase
M-cyclin: during late G2 phase until metaphase-anaphase
How are Cdks activated?
A cyclin binds to a Cdk > the T-loop will expose and a CAK (Cdk-activating kinase) will phosphorylate the loop for activation
Wee1-kinase
A kinase which can add an additional phosphate group to an active Cdk-cyclin complex, which inactivates the complex (inhibitory phosphate)
Cdc25 phosphatase
Cdc25 can remove the additional inhibitory phosphate on a Cdk-cyclin complex > reactivation
p27
p27 can bind an active Cdk complex which inactivates it > safety mechanism (like a clamp on the complex)
> p27 is mutated in cancer cells > proliferation
> p27 regulation through gene expression
Positive feedback mechanism of M-Cdk
PP2A-B55 phosphatase dephosphorylates Cdk-substrates for inactivation/
- Active M-Cdk activates Greatwall kinase
> Greatwall kinase activates Ensa
> Ensa inactivates PP2A-B55
- M-Cdk activated Cdc25 by phosphorylation and removes inhibitory phosphates from M-Cdk molecules
PP2A-B55 inhibitory mechanisms
-Dephosphorylate Cdk-targets
-Inactivate Greatwall kinase
-Dephosphorylation Wee1 > activation (inhibitory phosphate)
> Wee1 adds inhibitory phosphate to M-Cdk
M-Cdk main function
Phosphorylate substrates which lead to mitosis
Function APC/C
Anaphase-promoting complex or cyclosome > ubiquitin ligase > initiation proteolysis in proteasome
Targets APC/C
-Securin: securin inhibits separase, the sister chromatid seperator enzyme
-S- & M-cyclins: Cdk inactivation by breakdown of cyclins
APC/C pathway with Cdc20 / Cdh1
In M-phase
-M-Cdk phosphorylates APC/C and Cdh1
-APC/C binds Cdc20 > activation
-APC/C-Cdc20 initiates anaphase and inactivaties M-Cdk by ubiquitination of M-cyclin
-APC/C is dephosphorylated and inactive (unbinding of Cdc20 due to conformation)
-Cdh1(P) is inactive
Onset G1-phase
-M-Cdk inactivation by APC/C leads to dephosphorylation of Cdh1 > activation
-Dephosphorylated APC/C can bind active Cdh1 > activation
-APC/C-Cdh1 keeps destructing M-cyclin and thereby inhibiting M-Cdk
DNA damage can influence checkpoints. How?
Inhibit G1/S-Cdk, S-Cdk, M-Cdk
> S-Cdk and M-Cdk prevent DNA re-replication
How is one time replication regulated?
Assembly of PreRCs (block the replication origins) > inhibited by Cdks and activated by APC/C
>Low PreRCs during S-phase and G2-phase
S-Cdk DNA replication pathway
Prevention new duplication: phosphorylation of Orc (origin recognition complex) during S/G2/M
- S-Cdk activation stimulates assembly of Cdc45 and GINS at each Mcm helicase (inactive)
-DDK (a kinase) phosphorylates Mcm helicase forming an active CMG helicase (complex of Cdc45-Mcm-GINS) > unwinding DNA > trigger DNA-polymerase activity
Prophase
Condensation of sister chromatids which are still closely associated. Assembly mitotic spindle between 2 centrosomes.
Prometaphase
Breakdown nuclear envelope > microtubuli can reach the chromatids. And further condensation of sister chromatids
Metaphase
Chromosomes are localised in the equatorial axis and the kinetochore MTs bind the sister chromatids at opposite poles of the spindle
Anaphase
Sister chromatids seperate in sinc because of shrinkage of kinetochore MTs.
Spindle poles move apart from each other due to elongation of the spindle
Telophase
Sets of daughter chromosomes arrive at the poles and decondensate, a new nuclear envelope is made around the set of chromosomes and the contractile ring starts forming.
What characteristic means the end of mitosis
Forming of 2 new nuclei
Cytokinesis
Division of the cytoplasm by the contractile ring consisting of actine and myosine
Centrosome division
Early in cell cycle
-G1: procentriole site selection
-S: centriole duplication
-Entry into M-phase:
> Removal tether between centrosomes
> centriole and centrosome maturation
> centrosome seperation
-Licensing (start G1)
> contriole disengagement and centriole-to-centrosome conversion
Where are the kinetochores located most of the times?
At the centromeres
How many winds of DNA around a nucleosome core particle?
2.5x
Cohesins
Cohesins hold the sister chromatids toghether tightly
> Smc molecules with ATPase domain > dimer binds ATP
>Scc molecules as a clamp around Smc-heterodimer
>Smc dimer forms a loop around the sister chromatids
Condensin
Can get the DNA into a more spiral conformation> more condense
Where are cohesins located?
At the places where the sister chromatids make contact
Three classes of microtubules form the mitotic spindle. Name them
-Kinetochore MTs: connect to the chromosomes
-Interpolar MTs: making bipolar connection with each other in the spindle (plus and minus ends connection)
-Astral MTs: polar MTs radiating outside
Kracht maken van microtubules
Multimere motoreiwitten induceren minus-end foci > kracht via sliding antiparallel microtubules
Krachten via kinesines
Lopen naar plus-end
> Kinesin-5 loopt met 2 uiteinden naar plus end > antiparallele crosslinking (door op 2 MTs naar plus te lopen zorgen ze voor wegduwen van minus ends van interpolar MTs)
>Kinesin-14 heeft 1 stabiele binding site en 1 motor domein van 2 heads, beweegt naar plus-end (zorgt voor push door binden een MT van ene pole en dan op MT van andere pole naar plus lopen waardoor plus van de motor domain push)
> Kinesin-4/10 > outward push van zusterchromatiden (bind zusterchromatide die al wordt getrokken en loopt naar plus end voor duwen microtubule naar het midden toe voor push)
Krachten van dyneines
Lopen naar minus end > tegenkracht (houden de cell cortex vast en door lopen naar minus-end een kracht naar de plus end en ruimte maken)
Ran-GTP is vooral actief rond de chromosomen, noem de functie en reden tot lokalisatie
Functie: stimulatie microtubule nucleatie en stabilisatie
- Ran-GEF is geassocieerd met de chromosomen
-Gradiënt richting periferie door delay tot hydrolyse tot Ran-GDP
Ran-GTP en spindle formation
Met Ran-GTP wordt er bias aangelegd voor MT nucleatie rond de chromosomen, voor de rest is vorming spindle random proces. > spindle formatie door zelforganisatie met motoreiwitten.
Search-and-capture mechanism
De kans van kinetochore-MTs om tegen een kinetochore te botsen is een kansproces via dynamische instabiliteit > kinetochore de stabiliserende structuur.
Kinetochore opbouw
Inner kinetochore: centromeric nucleosome
Outer kinetochroe: Ndc80 complex die MTs stabiliseren (filamenten die MT attachment sites vormen)
Oriëntatie kinetochoren bij metafase
Bi-oriëntatie van de zusterchromatiden, kinetochoren naar aparte centrosoom polen
Hoe ontstaat de bi-oriëntatie van zusterchromatiden (tijdens mid prometafase)
Trial-and-error: tension sensing door Aurora-B kinase > missen tension geeft inhibitoir signaal: loslaten fosforylatie Ndc80)
> stabiel wanneer er tension van de kinetochoren vanuit beide kanten is.
>Bi-oriëntatie
Te weinig trekking gedetecteert door Aurora-B pathway
Te weinig tension
> Aurora-B kinase fosforyleert Ndc80 complexes
> Inhibitie microtubule attachment
> Tension laat Aurora-B uitgaan
Dynamiek van mitotic spindle
-Continue groei bij plus-end
-Continue krimp bij minus-end
-Sliding movements bij overlappende MTs
-Treadmilling
Chromosoombeweging tijdens anafase (2 mechanismen)
-Kinetochore MT krimp (pull)
-Astral ejection force: interpolar of astral MTs met plus-end directed kinesin-4/10 (sliding force/afstoting richting minus-end door groei aan plus-end) zorgt voor duwbeweging centrosomen van elkaar af (push) (> eerst trekken centrosomen naar de periferie door vasthouden aan cell cortex, dan duwbeweging)
Anafase 1 en Anafase 2
Anafase 1: kinetochore pulling
Anafase 2: interpolar/astral sliding pushing force
APC/C-Cdc20 stimulatie van anafase
Checkpoint voor de anafase: wanneer de chromosomen verbonden zijn aan de kinetochoren dan zorgt Cdk-M via APC/C voor relief van inhibitory signal
> APC/C zorgt voor ubiquitilation en degradatie van securin die als inhibitor van seperase werkt
> Separase maakt cohesines kapot > scheiding zusterchromatiden
Snelheid chromosoom segragatie
Snelheid segregatie is gelijk aan flux snelheid van de microtubuli = constant (niet versnellend)
Stadia van cytokinese
Initiatie
> Contractie actine en myosine filament in contracile ring
> Membraan insertie
> Completie
Waaruit bestaat de contractile ring en waardoor wordt de vorming gestimuleerd?
Actine en myosine-II
> Stimulatie door RhoA
Completie van de cytokinese
Vorming mid-body, gemaakt uit de overlap van MTs en actine.
Actieve RhoA functies
-Activatie formine: actine filament formatie
-Activatie Rho-associated kinases incl ROCK> myosin-II activatie
-Totale assembly en contractie van actine-myosine contractile ring
Welke kinase uit de anafase kan RhoA-GEF activeren?
Aurora-B kinase
Modellen voor positioneren contractile ring
-Astral stimulation model
-Central spindle stimulation model
-Astral relaxation model
Hoe wordt het vlak van cytokinese bepaald bij dierlijke cellen?
Door de microtubules van de mitotic spindle tussen de polen in
Verschil metafase plaat bij mitose en meiose-I
Mitose: homologe chromosomen liggen los van elkaar
Meiose-I: homologe chromosomen zijn gepaard > segregatie maternale en paternale chromosmen at random
Welk genetisch diversiteits mechanisme kan opspelen bij meiose-I?
Crossing-over
Vind er na meiose-I en voor de meiose-II nog nieuwe DNA replicatie plaats?
Nee
Meiose-II scheiding
Segragatie zusterchromatiden per 1 van de 2 homologe chromosomen
Hybride chromosomen kunnen bij meiose ontstaan indien….
crossing-over
Hoe worden homologe chromosomen gepaard?
Via transverse filaments van het synaptonemale complex
Synapsis
Alignment van homologe chromosomen > synaptenemal complex
5 phases of synapsis
-Leptotene; seperated homologous chromosomes
-Zygotene: assembly synaptonemal complex
-Pachytene: assembled synaptonemal complex
-Diplotene: disassembling synaptonemal complex
-Diakinesis: seperated homologous chromosomes
Mitogens can stimulate the activity of these Cdks
G1-Cdk and G1/S-Cdk
Name Cdk activating mitogens
PDGF, EG
Mitogen pathway
Mitogen (PDGF/EG) binds receptor
> Ras / MAPK route
> Expression of Myc
> Myc upregulates G1-cyclins (D-cyclins)
> Activation G1-Cdk
> G1-Cdk inactivates Rb protein (an inhibitor of E2F)
> Active E2F stimulates expression of G1/S cyclin (E-cyclin) and S-cyclin (A-cyclin)
> Activation S-Cdk > DNA synthesis
Loss of both Rb alleles leads to
Retinoblastoma (inherited eye cancer)
p21 tumor suppressor pathway
DNA damage
> Phosphorylation p53 due to certain kinase activations
> Mdm2 (inhibitor of p53) released from p53
> p53 binds regulatory region of the p21 gene
> expression p21
> p21 inhibits G1/S-Cdk and S-Cdk by binding their Cdk-cyclin complexes as a clamp
Excessive Myc production and p21-route
Excessive Myc
> Synthesis Arf
> Arf binds Mdm2 > inactivation Mdm2
> p53 stable and active
> Expression p21
> cell cycle arrest or apoptosis
Function of active Mdm2
Degradation of p53
mTORC1 complex
Is regulated by growth factor receptor (enzyme coupled) cascade or by incoming diffusing amino acids (factors for cell growth and proliferation)
> mTORC1 complex regulates protein synthesis, lipid synthesis, protein turnover
Cel proliferatie gaat gepaard met …
Celgroei (celgroei gemedieerd door mTORC1 complex)
