Regulation of cytokinesis
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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.

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Regulation of Cytokinesis

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Regulation of cytokinesis

Regulation of Cytokinesis

Corinna Benz, PhD, Biology Centre


Overview

Overview

  • Cell cycle and cytokinesis in mammalian cells

  • Cell cycle and cytokinesis in trypanosomes

  • NDR kinase – MOB signalling in different organisms


General idea

General idea


The eukaryotic cell cycle

The eukaryotic cell cycle

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


Different microtubules in a mammalian spindle

Different microtubules in a mammalian spindle

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


A condensed chromosome with attached kinetochore microtubules

A condensed chromosome with attached kinetochore microtubules

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


Cell cycle control system

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

Cell cycle checkpoints

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


The major players cyclin dependent protein kinases cdks

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


Regulation of cytokinesis

Cyclin-dependent protein kinases (Cdks) and cyclins

M-Cdk promotes

mitotic events

G1-Cdk promotes passage

through a restriction point

G1/S-Cdk commits cell

to DNA replication

S-Cdk is required for

initiation of DNA replication

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


The major cyclins and cdks of vertebrates and budding yeast

The major cyclins and Cdks of vertebrates and budding yeast

Vertebrates

Budding Yeast

  • * 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

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


Regulation via phosphorylation

Regulation via phosphorylation

  • Cdk-cyclin complexes are regulated through phosphorylation

  • Particularly important at onset of mitosis

  • Further regulation through Cdk inhibitor proteins (CKIs)

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


Control through proteolysis

Control through proteolysis

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

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

APC/C complex

SCF complex


Regulation of cytokinesis

Control through proteolysis

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


The specifics

The specifics


Exit from g 0 and g 1 events

Exit from G0 and G1 events

Cdk2:cyclin E

G1/S-Cdk

Cdk7

P

Rb

E2f

+

G1

S

growth

factor

stimulus

cyclin D ↑

G0 to G1 transition

Cdk4:cyclin D/Cdk6:cyclin D

G1-Cdk

Rb-

negative feedback

loop

cyclin A/B

transcription of e.g.

cyclin A

cyclin B

cyclin E

E2f

cyclin E ↑

APC/CCdh1

cyclin A/B-ubiquitin

degradation


Exit from g 0 and g 1 events1

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)

  • PhosphorylatedRb 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

Genome duplication: Replication licensing

1)

Helicase loader

Mcmhelicase (inactive)

DePamphilis et al, Frontiers in PHYSIOLOGY, 2012


Genome duplication

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 Mcmhelicase (inactive): Mcm2-7 (double

    hexamer)


Replication initiation

Replication initiation

1)

2)

DePamphilis et al, Frontiers in PHYSIOLOGY, 2012


Replication initiation1

Replication initiation

3)

4)

DePamphilis et al, Frontiers in PHYSIOLOGY, 2012


Regulation of cytokinesis

Replication initiation

5)

DePamphilis et al, Frontiers in PHYSIOLOGY, 2012


Replication initiation2

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)

  • ssDNA binding protein RPA required to facilitate DNA unwinding

  • Formation of two active replisomes upon addition of Pol-α:primase and Pol-δ

  • Mcm10 stabilises final replisome structure


Regulation of cytokinesis

DNA replication checkpoints

2)

3)

DePamphilis et al, Frontiers in PHYSIOLOGY, 2012


Dna replication checkpoints

DNA replication checkpoints

  • Effectorkinase: 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

DePamphilis et al, Frontiers in PHYSIOLOGY, 2012


Prevention of dna re replication1

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-phosphorylatesOrc subunits and Cdc6 allowing preRC assembly

  • Geminin degraded  Cdt1 becomes available for preRC assembly


Regulation of cytokinesis

DNA damage response

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

Exit from S phase

P

P

P

CRL1:Skp2

cyclin E-P

cyclin E-P

-ubiquitin

DNA replication proteins ↓

preRC proteins ↓

Cdk2:cyclin A

S-Cdk

E2f-P

translocates to nucleus

phosphorylates substrates to initiate

mitosis

Cdk1:cyclin B

M-Cdk

Cdk1:cyclin B-P


Exit from s phase1

Exit from S phase

  • Cyclin E inactivated through phosphorylation by Cdk2:cyclin A (S-Cdk)  becomes a target for CRL1:Skp2 ubiquitinligase 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

Entry into mitosis – M-Cdk activation

P

P

Wee1

Myt1

Y15-P

Y15-P

Cdc25

Cdk1

Cdk1

Cdk1:cyclin B

M-Cdk

Positive feedback loop

T14-P

T14-P

G1, S, G2

M

G1


Entry into mitosis apc c activation

Entry into mitosis – APC/C activation

P

P

S/G2:

APC subunits: * Cdc20-P active

* Cdh1-P inactive

Emi1

Cdc20-P

Cdk2:cyclin A

S-Cdk

APC/C

Cdh1 binding

degradation

Emi1

Prometaphase:

Cdk1:cyclin B

M-Cdk

Cdc20-P

APC/CCdc20

cyclin A

Cdk2

 Entry into mitosis

APC/CCdc20

p21/p27

cyclin B

Cdk1

 Anaphase progression


Entry into mitosis

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

  • Cdk1 and Cdk2 also regulated by interaction with Cdk inhibitors (CKIs) p21 and p17 whose expression follows that of the APC/C


M cdk functions

M-Cdk – functions

  • Induces assembly of mitotic spindle

  • Ensures that replicated chromosomes attach to spindle

  • In many organisms also triggers chromosome condensation, nuclear envelope breakdown, actin cytoskeleton rearrangement, reorganisation of Golgi and ER; e.g. through phosphorylation of lamins dismantling of nuclear envelope


Metaphase anaphase transition

Metaphase-anaphase transition

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


Metaphase anaphase transition1

Metaphase-anaphase transition

  • M-Cdkphosphorylatescondensin 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

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

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


Regulation of cytokinesis

Dai and Grant, Clinical Cancer Research, 2010


Cytokinesis

Cytokinesis

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


Cytokinesis initiation

Cytokinesis initiation

  • Signalling between anaphase spindle and cortex

  • Spindle recruits narrow zone of active RhoA (GTPase)

  • Active RhoA recruits effector contractile ring proteins (cytokinesisformin, Rhokinase, Citron kinase)

  • RhoA flux model: Global GAP-mediated RhoA inhibition versus localised GEF-mediated RhoA activation (e.g. Ect2 at cell equator)

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

GAP=GTPase activating protein, GEF=Guanine nucleotide exchange factor


Central spindle spindle midzone formation

Central spindle (spindle midzone) formation

  • Spindle midzone: Overlapping, antiparallel microtubules (MTs) (+ ends facing each other)

  • 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


Central spindle spindle midzone formation1

Central spindle (spindle midzone) formation

  • 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

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


Central spindle spindle midzone formation2

Central spindle (spindle midzone) formation

  • 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

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


Spindle midzone midbody

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 midbodyMTs in regions flanking the midbody core

  • Plk1 essential for these relocalisations

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


Contractile ring midbody ring

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

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


Abscission

Abscission

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


Abscission1

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

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


Abscission checkpoint1

Abscission checkpoint

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

  • Trapped chromatin at midbody CHMP4C binds to Borealin 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

The cell cycle and cytokinesis in Trypanosomabrucei

  • 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 cytokinesis

Wheeler et al, Molecular Microbiology, 2013


Cdk cyclin system in t brucei

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

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

T. bruceimitosis

  • 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

Ziyin Li, Eukaryotic Cell, 2012


Differential localisation of trypanosome cpc during mitosis

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

Cytokinesis signalling in trypanosomes vsmetazoa

  • 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, PK50, PK53 and Cdc14?

Ziyin Li, Eukaryotic Cell, 2012


A closer look at mob ndr signalling

A closer look at Mob-NDR signalling

S. cerevisiae

D. melanogaster

H. sapiens

yellow: STE20-like kinase

green: NDR kinase

red: MOB protein

Hergovich, Cellular Signalling, 2011


Common elements of regulation

Common Elements of Regulation

  • STE20-like kinasesphosphorylate MOB proteins

  • Phosphorylated MOB proteins can interact with and activate NDR kinases

  • NDR kinasesphosphorylate 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


Fear and men in budding yeast

FEAR and MEN in budding yeast

Bub2-Bub1: GAP

Tem1: GTPase

Lte1: GEF

D'Amours and Amon,

Genes and Development, 2004

  • Cdc14 is needed for mitotic exit in budding yeast

  • Cdc14 is kept inactive throughout the cell cycle by binding to Net1

  • Polo kinase (Plk) activates Cdc14 in early anaphase (FEAR =

  • Cdcfourteen early anaphase release)

  • Cdc14dephosphorylates/activates Cdc15 (STE20-like kinase)

  • Cdc15 phosphorylatesDbf2-Mob1 and activates the complex

  • Dbf2-Mob1phosphorylatesCdc14 and activates it


Role of plk ndr kinases mob1 and cdc14 in t brucei

Role of PLK, NDR kinases, MOB1 and Cdc14 in T. brucei

  • PLK, the NDR kinases

  • PK50 and PK53, MOB1 and

  • Cdc14 are essential in

  • trypanosomes

  • Depletion results in specific

  • cytokinesis defects

PLK (polo) RNAi

PK50 RNAi

 post-mitotic cells that are not dividing

MOB1 RNAi

PK53 RNAi

Cdc14 RNAi

 post-mitotic cells that are arrested during cytokinetic furrow ingression

Hammarton et al, MolecularMicrobiology, 2005; Hammarton et al, MolecularMicrobiology, 2007; Benz, Ma et al, JBC, 2010


Differential regulation of trypanosome ndr kinases

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 kinases1

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

  • NDR kinases, MOB proteins and Cdc14 are not substrates of each other

  • Regulation and signalling pathways???

  •  Conserved proteins ≠ conserved functions!


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