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
Alberts et al., Molecular Biology of the Cell, 4th edition
A condensed chromosome with
attached kinetochore microtubules
Alberts et al., Molecular Biology of the Cell, 4th edition
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
Alberts et al., Molecular Biology of the Cell, 4th edition
The major players – Cyclindependent 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
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
Vertebrates
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
* 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
• 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
• 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
APC/C complex
SCF complex
Control through proteolysis
Alberts et al., Molecular Biology of the Cell, 4th edition
The specifics
Exit from G0 and G1 events
Cdk7
Rb
+
growth
factor
stimulus
cyclin D ↑
G0 to G1 transition
E2f
Cdk4:cyclin D/Cdk6:cyclin D
G1-Cdk
Rb- P
negative feedback
loop
E2f
transcription of e.g.
cyclin A
cyclin B
cyclin E
cyclin A/B
cyclin E ↑
APC/CCdh1
cyclin A/B-ubiquitin
Cdk2:cyclin E
G1/S-Cdk
G1
S
degradation
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
1)
Helicase loader
Mcm helicase (inactive)
DePamphilis et al, Frontiers in PHYSIOLOGY, 2012
Genome duplication
• Initiated at replication origin, where a preRC (prereplication 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)
DePamphilis et al, Frontiers in PHYSIOLOGY, 2012
Replication initiation
3)
4)
DePamphilis et al, Frontiers in PHYSIOLOGY, 2012
Replication initiation
5)
DePamphilis et al, Frontiers in PHYSIOLOGY, 2012
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/SCdk)
• 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 Polα:primase and Pol-δ
• Mcm10 stabilises final replisome structure
DNA replication checkpoints
2)
3)
DePamphilis et al, Frontiers in PHYSIOLOGY, 2012
DNA replication checkpoints
• 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
DePamphilis et al, Frontiers in PHYSIOLOGY, 2012
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
DNA damage response
Wang et al., Molecular Cancer, 2009
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
CRL1:Skp2
P
cyclin E-P
Cdk2:cyclin A
S-Cdk
E2f-P
P
P
Cdk1:cyclin B
M-Cdk
Cdk1:cyclin B-P
cyclin E-P
-ubiquitin
DNA replication proteins ↓
preRC proteins ↓
translocates to nucleus
phosphorylates substrates to initiate
mitosis
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
P
P
Wee1
Y15-P
Myt1
Cdc25
Cdk1
Cdk1
Cdk1:cyclin B
M-Cdk
Y15-P
T14-P
T14-P
G1, S, G2
M
G1
Positive feedback loop
Entry into mitosis – APC/C activation
S/G2:
Emi1
APC subunits: * Cdc20-P active
* Cdh1-P inactive
Cdc20-P
Cdk2:cyclin A
S-Cdk
P
APC/C
Cdh1 binding
degradation
Prometaphase:
Emi1
APC/CCdc20
Cdk1:cyclin B
M-Cdk
P
Cdc20-P
APC/CCdc20
Cdk2  Entry into mitosis
cyclin A
p21/p27
cyclin B
Cdk1  Anaphase progression
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/Cmediated 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
• 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
Alberts et al., Molecular Biology of the Cell, 4th edition
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
Alberts et al., Molecular Biology of the Cell, 4th edition
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) (+
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)
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)
formation
• Ect2 (GEF for RhoA) converts RhoAGDP 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
• 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
Green et al, Annu. Rev. Cell Dev. Biol., 2012
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
Agromayor and Martin-Serrano, TRENDS in Cell Biology, 2013
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
Agromayor and Martin-Serrano, TRENDS in Cell Biology, 2013
Abscission checkpoint
• CHMP4C (ESCRT-III subunit) regulates Aurora Bmediated 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
• 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 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
Ziyin Li, Eukaryotic Cell, 2012
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, PK50,
PK53 and Cdc14?
Ziyin Li, Eukaryotic Cell, 2012
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
• 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
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 =
Cdc fourteen early anaphase release)
• Cdc14 dephosphorylates/activates Cdc15 (STE20-like kinase)
• Cdc15 phosphorylates Dbf2-Mob1 and activates the complex
• Dbf2-Mob1 phosphorylates Cdc14 and activates it
Role of PLK, NDR kinases, MOB1
and Cdc14 in T. brucei
PLK (polo) RNAi
PK50 RNAi
 post-mitotic cells that are not dividing
MOB1 RNAi
PK53 RNAi
• PLK, the NDR kinases
PK50 and PK53, MOB1 and
Cdc14 are essential in
trypanosomes
• Depletion results in specific
cytokinesis defects
Cdc14 RNAi
 post-mitotic cells that are arrested during cytokinetic furrow ingression
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
• NDR kinases, MOB proteins and Cdc14 are not
substrates of each other
• Regulation and signalling pathways???
•  Conserved proteins ≠ conserved functions!
Questions?