VL 33 (Ralph Gräf) Flashcards
S-Phase
Inition:
- ORCs bind ORIs during entire cell cycle (“landing platform”)
- ORCs initially dephosphorylated
- Cdt1, Cdc6 recruit Mcm protein to ORC → prereplication complex
- kinases (Cdk2-cyclinE) phosphorylate ORC + Cdc6 (→Cdc6 dissociation, degradation)
- geminin binds Cdt1
→ prereplication complex in G1 only (APC/CCdh1 activity from anaphase till late G1→ geminin degradation) - Cdk2-CyclinA phosphorylates Cdt1
→ Cdt1 degraded by SCFSkp1 - Assembly of DNA Pol + replication proteins (= preinitiation complex)
- Mcm proteins act as helicases, DNA replication initiated, proceed
Regulation of G2/M transition:
- G2 phase: timer after S-phase termination, CDK1-CyclinB1 activation
- inhibitory CDK1 phosphorylation by Wee1 (within nucleus), Myt1 (within cytosol)
- activating phosphorylation by CAK (= cyclin activating kinase CDK7-cyclinH); initially compensated by Wee1/Myt1
- Cdk1-CyclinB activation through Cdc25A phosphorylation at Wee1/Myt1 sites
- Cdc25A activation by polo kinase Plk1
- Positive feed back by activating phosphorylations of Cdc25A, inhibiting phosphorylations of Wee1 by CDK1-CyclinB + Plk1 → burst of active CDK1-cyclinB
Prophase:
- Chromosome condensation (condensing translocates into nucleus) Histone
- H3 phosphorylation
- Nucleolus decomposition
- Transcription halt
- Centrosome separation with 2 separate MT asters
- IF disassembly (cells round up)
- Golgi, ER fragmentation
- Reduced exocytosis
→ internalization of membrane anchors, receptors
Prometaphase
Early prometaphase:
* 1. NEBD
* 2. MT grow, shrink in aster
* 3. Kinetochore captures MT
Late prometaphase:
* 4. Chromosome slides rapidly poleward along MT
* 5. MT from opposite pole captured by sister kinetochore
* 6. Chromosome attached to both poles congresses to spindle middle
Spindle formation in prometaphase
- MT exhibit increased dynamics, start to capture chromosomes
- Dynein transports chromosome → pole
- Connection of sister kinetochore with opposite pole balances this movement
- → chromosome congression at equator → metaphase
Thee Types of microtubules during spindle formation
- Non-kinetochore MTs; including pole-to-pole (=interpolar) MTs.
–> they partly interdigitate (contribute to spindle formation and elongation; interact with chromosomes via chromokinesin) - Kinetochore-Mts
–> bind chromosomes at their kinetochores - Astral Mts:
–> mediate contact with cortical dynein (centrosome separation, spindle orientation/elongation)
MT nucleation during spindle formation:
1.Centrosomes /Spindle poles
2.Chromosomes
* TPX2
–> MT-associated protein; one of several spindle assembly factors (SAFs) required for acentrosomal MT nucleation
–> activated by Ran-GTP triggered dissociation of its complex with importin α/β
–> Aurora A (TPX2-activated) phosphorylates→activates SAFs (e.g. NEDD1, XMAP215, TACC, Eg5) → promote MT assembly
* Ran-GTP releases Y-complex NPC-components (including ELYS) from importin α
* ELYS anchors complex at kinetochores→NUP-complex binds γ-TuRCs
3.Other microtubules
* Augmin = (HAUS) complex: aids MTs nucleation at pre-existing MTs; 2 components HICE1 binds MT, FAM29A binds NEDD1, γ-TuRC
* Regulation by Plk1, Aurora A
* Augmin-dependent MTs contribute to K-fiber formation
* MT linkage to augmin = weak
* After their release MT (-)-ends with bound γ-TuRCs are directed toward the poles by dynein
Motor proteins in spindel formation
1a: chromosome capture at its end or
1b: its side
2: polewards migration through dynein activity
3: capture of sister kinetochore, MT end stabilization, force balance
- bidirectional chromosome oscillations due to MT growth/shrinkage; MT-ends stay in contact with kinetochores
- shrinking mediated by kinesin-13 (MCAK)
- dynein promotes poleward movement
- ends of growing MT held in place by kinesin-7 (CENP-E)
- chromokinesin (kinesin-4) causes telomer orientation towards equatorial plane
Picture:
Eg5
* bipolar kinesin
* connection at stalk domains o 4 motor domains
* linkage of pole-to-pole MT o migration (+) direction
* pushing apart the MT compensation by Ncd
Ncd
* Motor domain at the C-terminus
* migration (-)-direction
* do not walk along, only motor domains
* Anaphase Eg5 dominates poles apart
* Dynein
* spindle MT bundled by dynein
* Transport of chromosomes poleward * Astral MTs associated
* Dynactin bound to cell cortex
* Actin network – dynactin
* Tension on MT
➔ Pole
➔ Plasma membrane
➔ pathway movement in anaphase supporting
Metaphase
- checking of intact, bipolar connections between chromosomes, poles
- constant MT flux despite a relatively static appearance of this phase
How does the spindle checkpoint work?
Molecular players
* Cohesin: all along chromatids
* Separase: protease cleaving cohesin
* Securin: separase inhibitor protein
* MCC: mitotic checkpoint complex including Mad2
* APC/C: Ub ligase for cyclin B, securin
* Cdc20: S-binding factor for APC/C; regulated by MCC
Free kinetochores
* (w/o MT) → checkpoint/Mad2 active → MCC inhibits APC/C-regulator Cdc20
MT-bound kinetochores
→ SAC inactivation → APC/C active → separase activation → chromatid separation enabled
Summary: regulation of kinetochore/microtubule connections
Effect of tension on MT/kinetochore-
connections
Current hypothesis:
- Activation of the SAC protein Mad2
transmits a waiting signal to the cell cycle machinery until all kinetochores are bound to microtubules - Tension at opposing sister kinetochores results in increased distance between these kinetochores
- Aurora B, which is fixed at the inner centromere, does not reach its substrates anymore for sterical reasons. Then dynein transports MCC components away from the kinetochore region
Anaphase
- SAC inactivation
- Key event: APC/CCdc20 activation
→securin, CyclinB degradation
→Cdk1 inactivation
→Cdh1 activation (initially inhibited by Cdk1 phosphorylation)
→APC/CCdh1 formation
→factor degradation blocking mitotic exit - Ana A and B
Chromosome movements in anaphase A and B
Anaphase A:
1. migration of kinetochore-bound dynein to MT minus ends and simultaneous depolymerisation of MT dimers at plus ends by kinesin-13 (MCAK)
2. depolymerization of tubulin dimers at minus ends at the spindle pole by kinesin-13
Anaphase B:
1. antiparallel sliding of pole-to-pole MTs by Eg5 activity, inactivation of antagonizing kinesin-14 (Ncd)
2. pulling forces of cortical dynein exerted on astral MTs
Cytokinesis
- Formation: contractile actin/myosin-ring in equatorial region
- Midbody formation
- Membrane vesicle transport to cleavage furrow
- Translocation of regulatory chromosomal passenger proteins to cleavage furrow
- Actomyosin ring constriction in Ana B
- Abscission at constriction site by mass vesicle fusion employing a v/t-SNARE mechanism, ESCRT complex
Role of MTs in cleavage furrow formation:
- Centralspindlin consisting of Rac-GAP (CYK-4 = MgcRacGAP), kinesin (Pavarotti = MKLP1, kinesin 6) migrates through its kinesin activity towards MT (+)-ends → form: ring-like arrangement;
activated by Aurora B within CPC - Centralspindlin Rac-GAP recruits Rho-GEF (Ect2) → local RhoA activation → actomyosin ring assembly
Actin and myosin in cleavage furrow formation:
- RhoA activated formin → actin filaments → activates myosin II
- Membrane-associated septin filaments are connected with actin, myosin filaments through anillin
Role of the midbody in abscission
- Midbody: ring-shaped protein ensemble at the central spindle in the interdigitation zone of pole-to-pole MTs (> 160 proteins including many vesicle
trafficking proteins)
- Centrosomal protein Cep55 recruits ESCRT-I, ALIX, ESCRT-III
- ESCRT-III forms helical filaments (regulated by AAA-ATPase VPS4)
