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Master course
Molecular and Cellular Life Sciences
16/10/2012
Cytokinesis
Cytokinesis
1)  How does it happen?
(formation and constriction actin contractile
ring)
1  How does it happen?
2  Where does it happen?
2)  Where does it happen?
(spindle determines cleavage plane position)
3 How is it controlled during development?
3) How is it controlled during development?
(spindle positioning, asymmetric cell division)
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Cytokinesis
Cytokinesis
Actin (cortex)
Microtubules
Sea urchin
Sea urchin
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The cytoskeleton is made up of three classes of
protein filaments:
Todayʼs topic: developmental control of the
cleavage plane position#
!
!
Players: actin and tubulin#
motor proteins (myosin)#
regulators (Rho GTPase)#
!
!
#
#
#
Model: C. elegans!
Microtubules
(α/β-tubulin)
 
Microfilaments
(actin filaments)
 
Intermediate filaments
(various, e.g. Keratin)
α/β-tubulin
G actin
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[email protected]
 
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Master course
Molecular and Cellular Life Sciences
16/10/2012
Dimers of α/β-tubulin assemble into large
microtubules
Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008)
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Actin monomers assemble into filaments
(microfilaments)
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Formin promotes formation of
actin filaments
Figure 16-36 Molecular Biology of the Cell (© Garland Science 2008)
Figure 16-12 Molecular Biology of the Cell (© Garland Science 2008)
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The ARP complex nucleates the formation of
side chains, which results in an actin web
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Myosin motor protein:
binds and moves in association with actin
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Figure 16-34c Molecular Biology of the Cell (© Garland Science 2008)
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Myosin forms bundles (filaments) and
is regulated by light chain phosphorylation
Regulation by phosphorylation of light chains
Figure 16-54a Molecular Biology of the Cell (© Garland Science 2008)
[email protected]
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Figure 16-72a Molecular Biology of the Cell (© Garland Science 2008)
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Master course
Molecular and Cellular Life Sciences
16/10/2012
The cytoskeleton is rapidly and dramatically
reorganized during cell division
Dynein
a minus-end directed
microtubule motor
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Figure 16-64 Molecular Biology of the Cell (© Garland Science 2008)
The small GTPases Rho, Rac and Cdc42
control actin dynamics
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Figure 16-2 Molecular Biology of the Cell (© Garland Science 2008)
Small GTPases (Rho, Rac, CDC-42)
switch between active and inactive states
GEF
Stress fibers
(exchange factor)
GTP
GDP
RhoA
RhoA
GAP
(GTPase Activating Protein)
Lamellipodia
Filopodia
Figure 16-97 Molecular Biology of the Cell (© Garland Science 2008)
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Activated Rac promotes formation
branched actin (meshwork)
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Activated Rho promotes formation actin fibers
(stress fibers and contractile ring)
Actin fiber formation (formin)
Myosin activation (ROCK)
Cytokinesis!
contractile ring formation and
contraction
Figure 16-98a Molecular Biology of the Cell (© Garland Science 2008)
[email protected]
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Figure 16-98b Molecular Biology of the Cell (© Garland Science 2008)
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Master course
Molecular and Cellular Life Sciences
16/10/2012
Rapid and complete reorganization of the
cytoskeleton during cell division
Microtubules of the mitotic spindle control#
the plane of cell division!
Actin
formation contractile ring:
local Rho
accumulation/activation
Microtubules
Figure 16-2 Molecular Biology of the Cell (© Garland Science 2008)
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Conly Rieder
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What is the role of microtubules in cleavage plane
determination, and which microtubules are involved?
“Equatorial stimulation” or “polar relaxation” ?
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Both astral- and midzone microtubules (between
segregating chromosomes) position cleavage plane
Rappaport’s famous experiments
with sand dollar eggs
Cell cleavage takes place between spindle poles
-> argument for “equatorial stimulation” by overlapping
astral microtubules
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In development, mitotic spindle positioning is used to
change the orientation and plane of cell division
change !
of axis!
asymmetric!
division!
Mechanism only partly understood
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[email protected]
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Master course
Molecular and Cellular Life Sciences
16/10/2012
How is cell cleavage controlled
in early C. elegans embryos?
How can asymmetric division
generate different daughter cells ?
Note: asymmetric division !
1)  Polarity
2) Localization of
Determinants
Polarity
determinants
Stem cell lineage
Chromosomes
Germ line
components
3) Correct plane of
cell cleavage
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The cell cleavage plane is determined by the spindle:
what determines the position of the spindle ?
!
Asymmetric cell division
 
Establishment of cell polarity: reorganization
cytoskeleton#
#
 
Localization of cell determinants along the polarity
axis#
#
 
GFP::Tubulin
A. Desai
#
 
Asymmetric division in the early C. elegans
embryo provides an attractive model
Positioning of the mitotic spindle, which instructs #
the plane of cell cleavage#
-> Cell division generates unequal daughters #
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Mutations in par (partitioning abnormal) genes
disrupt early embryonic asymmetries!
Asymmetric cell division
 
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Establishment of cell polarity: reorganization
cytoskeleton: #
-> MECHANISMS RELATED TO CYTOKINESIS#
#
 
Localization of cell determinants along the polarity
axis#
WT
par-2
par-3
par-5
#
 
Positioning of the mitotic spindle#
#
 
Division generates unequal daughters #
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[email protected]
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SvdH 1
Master course
Molecular and Cellular Life Sciences
16/10/2012
Several PAR proteins localize
to the cell cortex
anterior-posterior!
polarity
In the fertilized egg, PAR-2
localizes to the posterior
cortex, together with PAR-1#
!
PAR-2::GFP
Levitan et al. PNAS 2004
Movies:
Cuenta et al. Dev 2003
 
Localization critical and dynamically controlled !
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PAR-3/6 and PAR-2 localize to opposite domains
and counteract each other
Anterior PAR complex:
PAR-3, PAR-6, PKC-3 and CDC-42
CDC-42
PAR-6
Conserved
roles
in polarized
cells!!
PKC-3
PAR-6
PAR-2
PAR-3
Counteract PAR-2, PAR-1 localization in C. elegans
one-cell embryo
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PAR-3/6 and PAR-2 localize to opposite domains
and counteract each other
PAR-2::GFP
  Initially:
#
PAR-3/PAR-6 are at the cortex, #
PAR-2 cytoplasmic#
  When
PAR-3/PAR-6 retract,#
PAR-2 appears at the cortex #
#
  In the absence of PAR-6, #
PAR-2 is all over the cortex
#
WT par-6 mutant
[email protected]
#
Cuenta et al. Dev 2003
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The one-cell C. elegans embryos is polarized
by a conserved set of PAR proteins
 
PAR-1 (ser/thr protein kinase)#
 
PAR-2 (a ring finger protein)#
 
PAR-3 (a PDZ domain protein)#
 
PAR-4 (ser/thr protein kinase related to LKB1)#
 
PAR-6 (a PDZ domain protein)#
 
PKC-3 (atypical protein kinase C)#
Localizations regulated, dynamic and
asymmetric!!
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SvdH 1
Master course
Molecular and Cellular Life Sciences
16/10/2012
!
Summary: model polarity
establishment
!
What triggers polarization of the fertilized egg?
Drugs that prevent actin polymerization
cause defects similar to those in
par mutants
Anterior PAR complex (PAR-3, PAR-6, PKC-3)#
Posterior PARʼs PAR-1/PAR-2#
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actin-myosin contracts towards anterior
following fertilization with PAR-3/PAR-6
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Fertilization disrupts actin-myosin network,
which triggers movement to opposite pole
Munro et al. SvdH2004
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Dev Cell,
PAR-3/PAR-6 move with actin-myosin
towards anterior pole
Munro et al. SvdH2004
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Dev Cell,
!
Summary: model polarity
establishment
!
  Sperm
entry somehow destabilizes acto-myosin network
collapses towards anterior
  Acto-myosin transports PAR-3/PAR-6/PKC-3 to anterior
  This allows PAR-2 to occupy posterior
  PAR-2 prevents the return of PAR-3/PAR-6
  Acto-myosin
Munro et al. SvdH2004
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Dev Cell,
[email protected]
  ->
A/P axis created and fixed
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SvdH 1
Master course
Molecular and Cellular Life Sciences
16/10/2012
!
What is contribution sperm
entry in polarity
!
establishment
Small GTPases (Rho, Rac, CDC-42)
switch between active and inactive states
GEF
(exchange factor)
GTP
GDP
Rho
1) 
2) 
3) 
Rho
GAP
Microtubule organizing centers#
Need to mature (Cyclin E/CDK-2 dependent)#
Regulation Rho GTPase -> Rho kinase -> #
Myosin light chain phosphorylation
(GTPase Activating Protein)
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The RHO-1 GTPase, ECT-2 GEF and
CYK-4 GAP drive actin-myosin reorganization
Small GTPases (Rho, Rac, CDC-42)
switch between active and inactive states
ECT-2
(exchange factor)
GTP
GDP
RHO-1
RHO-1
CYK-4
(GTPase Activating Protein)
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CYK-4 GAP is provided paternally and may trigger
actin-myosin reorganization
Jenkins et al. SvdH 2006
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Science,
Cortical exclusion of ECT-2 GEF by the centrosome
may trigger actin-myosin movements
CYK-4
RNAi
Jenkins et al. SvdH 2006
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Science,
[email protected]
Motegi and
Sugimoto
Nat. Cell Biol.,
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Master course
Molecular and Cellular Life Sciences
16/10/2012
RHO-1 drives actin-myosin reorganization, sperm
CYK-4 GAP provides cue
!
Summary: model polarity
establishment
!
ECT-2
(exchange factor)
GTP
GDP
RHO-1
RHO-1
1. define posterior
Rho
kinase
4. Position pronuclei
Light Chain
Phosphatase
CONTRACTION
Many aspects of the first cell division are asymmetric
 
 
Cortex: #
contractions become restricted to anterior domain #
Spindle: #
- moves to posterior, “rocks” and flattens#
- rotates in P1#
Cell division: unequal sizes #
- planes opposite during second division#
- timing different#
Fate determinants become asymmetrically localized #
5. Position spindle and cleavage plane
Anterior PAR complex: PAR-3, PAR-6, PKC-3#
Posterior PARs: PAR-1/PAR-2#
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 
3. Posterior PARs localize,
Stabilize asymmetry!
Myosin Light Chain
Phosphorylation
CYK-4
(GTPase Activating Protein)
 
2. actomyosin contraction,
anterior PARs move (asymmetry!)
Spindle positioning requires the function of the
LIN-5 and GPR-1/2 proteins
Meiosis
Spindle movement Asymmetric division
AB
Spindle rotation
P1
lin-5
RNAi
gpr-1/2
RNAi
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The LIN-5 and GPR proteins localize and function
together, downstream of PAR proteins
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GPR-1,2 proteins contain a specific domain for
interaction with Gαi/o subunits#
TPR
GPR/GoLoco
GPR-1
Function
spindle
movement
GPR-2
CTL
LIN-5
GPR -1/2
AGS3
(Hs)
PINS
(Dm)
Merge
lin-5
(RNAi)
LIN-5
GPR -1/2
The GoLoco peptide binds Gα⋅GDP!
and competes with Gβγ!
Merge
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[email protected]
GDI for Gαi
spindle
orientation, binds
Gαi
Siderovski!
Nature 2002!
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Master course
Molecular and Cellular Life Sciences
16/10/2012
Heterotrimeric G proteins are key components
of trans-membrane signaling
#
An unusual Gα pathway
controls the cell cleavage plane
Similar loss of function
phenotypes:#
#
LIN-5 (Mud, NuMA)#
#
GPR-1,2 (Pins, AGS3, LGN)#
#
GOA-1/GPA-16 Gαi/o
#
RIC-8 GEF#
!
G-protein coupled receptor
Gα • ! GDP!
Gβγ!
ligand
(GEF)!
Gα !• GTP! Gβγ!
effectors! effectors!
Wild type
ric-8(RNAi)
Gα(RNAi)
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…..but here: independent of ligand
and transmembrane receptor
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A conserved Gα/GPR/LIN-5/dynein pathway
controls spindle and cleavage plane positioning
Gα subunit
Dy
nei
n
mic
rot
ubu
le
LGN, AGS3
Dm Pins
NuMA/Mud
Dynein
Generates cortical pulling forces on astral microtubules
during chromosome segregation and spindle positioning
Van den Heuvel, Gonczy,
SvdH Gotta
[email protected]
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