Regulation of Cytokinesis Corinna Benz, PhD, Biology Centre.

Regulation of Cytokinesis

Corinna Benz, PhD, Biology Centre

Overview

• Cell cycle and cytokinesis in mammalian cells

• Cell cycle and cytokinesis in trypanosomes

• NDR kinase – MOB signalling in different organisms

General idea

The eukaryotic cell cycle

Alberts et al., Molecular Biology of the Cell, 4th edition

Different microtubules in a mammalian spindle


A condensed chromosome with attached kinetochore microtubules


Cell cycle control system

• Events must occur in a timely fashion• Correct order• Only once per cell cycle• Backup system if things go wrong

(checkpoints)• Adaptability

Cell cycle checkpoints


The major players – Cyclin-dependent protein kinases (Cdks)

• Oscillation between active and inactive states cyclical phosphorylation of cellular proteins that initiate or regulate cell cycle events

• Activity regulated by binding to a cyclin partner (with exceptions)

• Cyclins are degraded cyclically while Cdks are constitutively present

G1/S-Cdk commits cell to DNA replication

S-Cdk is required for initiation of DNA replication

M-Cdk promotes mitotic events

G1-Cdk promotes passagethrough a restriction point


Cyclin-dependent protein kinases (Cdks) and cyclins

Adapted from Alberts et al., Molecular Biology of the Cell, 4th edition

* There are three D cyclins in mammals (cyclins D1, D2, and D3).** The original name of Cdk1 was Cdc2 in both vertebrates and fission yeast, and Cdc28 in budding yeast

The major cyclins and Cdks of vertebrates and budding yeast

Cyclin-Cdk complex

Cyclin Cdk partner Cyclin Cdk partner

G1-Cdk cyclin D* Cdk4, Cdk6 Cln3 Cdk1**

G1/S-Cdk cyclin E Cdk2 Cln1,2 Cdk1

S-Cdk cyclin A Cdk2 Cln5,6 Cdk1

M-Cdk cyclin B Cdk1** Clb1,2,3,4 Cdk1

Vertebrates Budding Yeast

Regulation via phosphorylation

• Cdk-cyclin complexes are regulated through phosphorylation

• Particularly important at onset of mitosis

• Further regulation through Cdk inhibitor proteins (CKIs)


Control through proteolysis

• SCF (Skp1-Cullin-F-box protein): Ubiquitin ligase, active in G1/S, constitutively active, substrates become available through phosphorylation

• APC/C (anaphase-promoting complex): Ubiquitin ligase, active in G1 (subunit Cdh1) and M (subunit Cdc20), activated by addition of subunits

SCF complexAPC/C complex


Control through proteolysis

The specifics

Exit from G0 and G1 events

growthfactorstimulus

cyclin D ↑G0 to G1 transition

Cdk4:cyclin D/Cdk6:cyclin DG1-Cdk

Cdk7

Rb

Rb-

E2f

E2f

transcription of e.g. cyclin Acyclin Bcyclin E

cyclin E ↑

cyclin A/B

APC/CCdh1

cyclin A/B-ubiquitin

degradation

negative feedback loop

G1 S

Cdk2:cyclin EG1/S-Cdk

+

P

Exit from G0 and G1 events

• Most cells in adults are in quiescent G0 stage

• Growth factor stimulus cyclin D levels increase and Cdk4:cyclin D/Cdk6:cyclin D (G1-Cdk) drives cells into G1 by phosphorylating retinoblastoma tumor suppressor protein (Rb)

• Phosphorylated Rb can no longer inhibit transcription factor E2f expression of genes important for DNA replication is induced

• These genes include cyclin A, B and E: Only cyclin E accumulates in G1 since cyclin A and B are marked for degradation by APC/CCdh1

• Cdk2:cyclin E (G1/S-Cdk) promotes G1-S transition

Genome duplication: Replication licensing

Helicase loader Mcm helicase (inactive)

1)

DePamphilis et al, Frontiers in PHYSIOLOGY, 2012

Genome duplication

• Initiated at replication origin, where a preRC (pre-replication complex) is assembled which is then converted to a preIC (pre-initiation complex)

• Replication licensing = Assembly of preRCs on chromatin• Replisome: DNA replication proteins• preRC assembly occurs only when Cdk activity is low

(anaphase to G1/S transition)

• 1) Assembly of helicase loader: Orc1-6, Cdc6 and Cdt1• 2) Assembly of Mcm helicase (inactive): Mcm2-7 (double

hexamer)

Replication initiation

1)

2)



3)

4)



5)


Replication initiation• DDK (Dbf4-dependent Cdc7 kinase) phosphorylates

Mcm4 and 6• Sld3 and Cdc45 assemble into preRC

• Sld2 and 3 are phosphorylated by Cdk2:cyclin E (G1/S-Cdk)

• Sld2-P and Sld3-P bind to Dpb11 recruitment of GINS complex

• Recruitment of Pol-ε (leading strand synthesis)• Formation of two active replisomes upon addition of Pol-

α:primase and Pol-δ• Mcm10 stabilises final replisome structure

DNA replication checkpoint

2)

3)


DNA replication checkpoint

• Effector kinase: Chk1• Inhibits activation of preRCs by:• Phosphorylating and inhibiting phosphatase Cdc25,

which then prevents activation of Cdk2• Phosphorylating Sld3 (at a site different from the one

phosphorylated by Cdk2:cyclin E (G1/S-Cdk)), which prevents interaction with Dpb11 (prevents formation of Cdc45:Mcm:GINS complex)

Prevention of DNA re-replication


Prevention of DNA re-replication

• Cdk2:cyclin A (S-Cdk) phosphorylates Orc1, Cdc6 and Cdt1 export to cytoplasm and/or degradation

• Absence of Orc1 causes other Orc subunits to leave the complex

• Non-phosphorylated Cdt1 bound to PCNA (polymerase processivity factor) is ubiquitinated by CRL4:Cdt2

• Re-expression of preRC proteins in G2 and M phase, but in modified forms which are kept inactive

• Metaphase exit: Cdc14 de-phosphorylates Orc subunits and Cdc6 allowing preRC assembly

• Geminin degraded Cdt1 becomes available for preRC assembly

Wang et al., Molecular Cancer, 2009

DNA damage response

DNA damage response• Checkpoint kinases Chk1 and Chk2, activated by ATR

and ATM kinases• ATM activated by ionising radiation• Chk2 in ATM-Chk2-Cdc25 pathway that senses double

strand breaks, not essential in mammals• ATR activated by UV radiation• Chk1 in Atr-Chk1-Cdc25 pathway that senses single

strand breaks, bulky lesions and stalled replication forks, essential in mammals

• Both pathways result in Cdc25 phosphorylation sequestered in cytoplasm by 14-3-3 M-Cdk inactive

• p53 phosphorylation causes activation of p21 (M-Cdk inhibitor) and upregulation of 14-3-3

Exit from S phase

Cdk2:cyclin AS-Cdk

Cdk1:cyclin BM-Cdk

E2f-P

cyclin E-PCRL1:Skp2

cyclin E-P-ubiquitin

DNA replication proteins ↓preRC proteins ↓

Cdk1:cyclin B-Ptranslocates to nucleusphosphorylates substrates to initiatemitosis

P

P

P

Exit from S phase

• Cyclin E inactivated through phosphorylation by Cdk2:cyclin A (S-Cdk) becomes a target for CRL1:Skp2 ubiquitin ligase and is degraded

• Cdk2:cyclin A (S-Cdk) also phosphorylates and inactivates E2f (downregulation of expression of genes required for DNA replication, also prevents re-licensing of origins)

• Cyclin B levels increase Cdk1:cyclin B (M-Cdk) complex formed, translocates to nucleus to phosphorylate substrates important for entry into mitosis

Entry into mitosis – M-Cdk activation

Cdk1

Y15-P

T14-P

Cdc25Cdk1:cyclin B

M-Cdk

Wee1Myt1

Cdk1

Y15-P

T14-P

Positive feedback loop

G1, S, G2 M G1

PP

Entry into mitosis – APC/C activation

Prometaphase:

Cdk1:cyclin BM-Cdk

APC subunits: * Cdc20-P active * Cdh1-P inactive

S/G2:

Cdc20-P

Cdk2:cyclin AS-Cdk

APC/C Cdh1 binding degradation

Emi1

Emi1

Cdc20-P APC/CCdc20

cyclin A

cyclin B

P

P

APC/CCdc20

Entry into mitosis• Positive feedback loop increases activity of Cdk1:cyclinB (M-Cdk)• APC/C regulation:• Cdc20 subunit is active when phosphorylated• Cdh1 subunit is inactive when phosphorylated

• During S and G2: APC/C inactive, Emi1 prevents phosphorylation of Cdc20 and Cdk2:cyclin A (S-Cdk) phosphorylates the APC/C, which inhibits binding of Cdh1 and results in its degradation

• Prometaphase: Selective Emi1 degradation• Negative feedback loops regulate Cdk1/2 activity through APC/C-

mediated ubiqitination of cyclin A and B

M-Cdk – functions

• Induces assembly of mitotic spindle• Ensures that replicated chromosomes attach to spindle• In many organisms also triggers chromosome

condensation, actin cytoskeleton rearrangement, reorganisation of Golgi and ER and nuclear envelope breakdown; e.g. through phosphorylation of lamins dismantling of nuclear envelope


Metaphase-anaphase transition

Metaphase-anaphase transition

• M-Cdk phosphorylates condensin complex resulting in chromosome condensation at prometaphase

• M-Cdk activates APC/CCdc20 triggers anaphase by promoting degradation of securin

• Securin no longer inhibits the protease separase• Separase becomes active and cleaves cohesin subunits

resulting in sister chromatid separation


Spindle attachment checkpoint

• Mitotic spindle in green• Mad2 in red

• Sister chromatids only separated when all chromosomes properly attached to spindle

• State of kinetochore monitored

• Several proteins e.g. Mad2 recruited to unattached kinetochores inhibition of APC/CCdc20

Dai and Grant, Clinical Cancer Research, 2010

Cytokinesis

Agromayor and Martin-Serrano, TRENDS in Cell Biology, 2013

Cytokinesis initiation• Signalling between anaphase spindle and cortex• Spindle recruits narrow zone of active RhoA (GTPase)• Active RhoA recruits effector contractile ring proteins

(cytokinesis formin, Rho kinase, Citron kinase)• RhoA flux model: Global GAP-mediated RhoA inhibition

versus localised GEF-mediated RhoA activation (e.g. Ect2 at cell equator)

GAP=GTPase activating protein, GEF=Guanine nucleotide exchange factor Green et al, Annu. Rev. Cell Dev. Biol., 2012

Central spindle (spindle midzone) formation

• Spindle midzone: Overlapping, antiparallel microtubules (MTs)

• Formation requires PRC1 and kinesins Kif4 and MKLP1

• PRC1: antiparallel MT cross linker• Chromosomal Passenger Complex

(CPC): Aurora B (kinase) and three additional subunits phosphorylates and recruits

• Centralspindlin: Heterotetramer, consists of two molecules MKLP1 and two molecules CYK-4 (GAP)

Green et al, Annu. Rev. Cell Dev. Biol., 2012


• PRC1 recruits Kif4 to overlap zones where it slows down MT dynamics

• Polo-like kinase 1 (Plk1) required for spindle elongation

• Plk1 recruits itself by phosphorylating substrates (e.g. PRC1, MKLP2) and thus creating binding sites

• Plk1 phosphorylates CYK-4 subunit of centralspindlin, which then scaffolds recruitment of Ect2



• Ect2 (GEF for RhoA) converts RhoA-GDP into RhoA-GTP, which triggers contractile ring assembly

• Contractile ring consists of actin, myosin II and septin filaments (recruited by anillin which as a crosslinker binds to all three)

• Ring is disassembled as it constricts


Spindle midzone midbody• Upon contractile ring constriction, midzone-localised proteins

are redistributed:• PRC1 and Kif4 stay at MT overlap zone• Centralspindlin and Ect2 concentrate in midbody ring, where

they colocalise with anillin, RhoA, ARF6 and Cep55• CENP-E, MKLP2 and Aurora B colocalise with tightly packed,

parallel midbody MTs in regions flanking the midbody core• Plk1 essential for these relocalisations


Contractile ring midbody ring• Anillin required for assembly of midbody ring and

anchoring to plasma membrane• Citron kinase essential for abscission, required for

localisation of anillin and RhoA• RhoA required for anillin localisation to midbody ring


Abscission


Abscission• CEP55, TSG101 and ALIX are translocated to midbody and

mediate recruitment and polymerisation of ESCRT-III (complex for scission)

• MIT-domain containing protein 1 (MITD1) coordinates activity of ESCRT-III

• Rab35 recruits OCRL and Rab11/FIP3-positive endosomes deliver p50RhoGAP to midbody resulting in changes of membrane lipid composition and clearing of actin

• FYVE Cent interacts with PtdInsP and recruits TTC19 and CHMP4B

• ESCRT-III filaments constrict midbody• Spastin (MT severing enyzme) cleaves microtubules• AAA ATPase VPS4 disassembles ESCRT-III

Abscission checkpoint


Abscission checkpoint

• CHMP4C (ESCRT-III subunit) regulates Aurora B-mediated abscission checkpoint

• Trapped chromatin at midbody CHMP4C binds to Borealin (CPC subunit) and is phosphorylated by Aurora B

• CHMP4C-P relocalises to midbody preventing abscission until chromatin is removed

The cell cycle and cytokinesis in Trypanosoma brucei

• Several single copy organelles/structures (e.g. flagellum) that need duplicated and segregated

• Cell division achieved by lateral ingression of a cleavage furrow rather than medial constriction of actin filaments

• Actin is dispensable for cytokinesisWheeler et al, Molecular Microbiology, 2013

Cdk-cyclin system in T. brucei

• Cdk-cyclin system is conserved

• 10 cyclins and 11 CRKs (Cdc2-related kinases)

• 26 potential interactions between these have been identified

• Not all of them exclusively involved in cell cycle regulation (CRK12, CYC2 and CYC7)

Ziyin Li, Eukaryotic Cell, 2012

DNA replication and licensing• Orc components:

Orc1/Cdc6 related protein, Orc1b, Orc4, Tb3120 and Tb7980

• Well-conserved CMG (Cdc45-Mcm-GINS) complex, but homologs of Cdt1, Sld2, Sld3 and Cdc7-Dbf4 missing

• Additional licensing system for mitochondrial DNA (kDNA)

Yeast T. brucei Ziyin Li, Eukaryotic Cell, 2012

T. brucei mitosis• Closed mitosis (no

nuclear envelope break

down)• MTOCs (flagellar basal

bodies) not involved in

spindle formation• Mitotic kinesin Kif13-1• APC/C components

expressed, only APC1

and Cdc27 essential

for mitosis, substrates?• Aurora B (AUK1) forms unique CPC with two novel proteins• Conserved proteins like INCENP, Survivin and Borealin absent


Differential localisation of trypanosome CPC during mitosis

• Trypanosome CPC shows dynamic localisation:

• Chromosomes to central spindle

• Central spindle to anterior tip of the new flagellar attachment zone (FAZ)

• Travels along cleavage furrow to the posterior end of the cell

Li et al, PLoS ONE, 2008

Cytokinesis signalling in trypanosomes vs metazoa

• AUK1 and PLK implicated in cytokinesis

• Both proteins found at anterior tip of new FAZ during late stages of cell cycle

• Common target?• Rho-like small GTPase, RHP

present and involved in cytokinesis, but doesn’t localise to cleavage furrow

• Role of MOB1, and NDR kinases PK50 and PK53?


A closer look at MOB-NDR signalling

S. cerevisiae

D. melanogaster

H. sapiens

yellow: STE20-like kinasegreen: NDR kinasered: MOB protein

Hergovich, Cellular Signalling, 2011

Common Elements of Regulation

• STE20-like kinases phosphorylate MOB proteins• Phosphorylated MOB proteins can interact with and

activate NDR kinases• NDR kinases phosphorylate downstream targets and

function in mitotic exit and morphogenesis in yeast, morphogenesis and cell proliferation in Drosophila and centrosome duplication and cell proliferation in mammalian cells

PK53 RNAi

PK50 RNAi

MOB1 RNAi

PLK (polo) RNAi

Role of PLK, NDR kinases, and MOB1 in T. brucei

post-mitotic cells that are not dividing

post-mitotic cells that are arrested during cytokinetic furrow ingression

• PLK, the NDR kinases PK50 and PK53, and MOB1 are essential in trypanosomes• Depletion results in specific cytokinesis defects

Hammarton et al, Molecular Microbiology, 2005; Hammarton et al, Molecular Microbiology, 2007; Benz, Ma et al, JBC, 2010

Differential regulation of trypanosome NDR kinases

Recombinant trypanosome NDR kinases are active

Trypanosome NDR kinases do not interact with MOB1 proteins

Benz, Ma et al, JBC, 2010

Differential regulation of trypanosome NDR kinases

• Recombinant trypanosome NDR kinases are active and not further activated in the presence of MOB proteins

• Endogenous trypanosome kinases don’t interact with MOB proteins

• PLK is excluded from the nucleus during the entire cell cycle and does not function in mitosis

• Regulation and signalling pathways???• Conserved proteins ≠ conserved functions!

Questions?

Regulation of Cytokinesis Corinna Benz, PhD, Biology Centre.

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