Transcript How migration occurs

Cell migration:
Rho GTPases lead the way
Xia Fan
How migration occurs?
• The major driving force of
migration is the extension
of a leading edge
protrusion or
lamellipodium, the
establishment of new
adhesion sites at the
front, cell body
contraction, and
detachment of adhesions
at the cell rear.
The Rho GTPase switch
(Rho,Rac,Cdc42)
•
•
o
o
o
Cycle between a GDP-bound, inactive form
GTP-bound, active form.
The cycle is regulated by
o
GEF:promote the exchange of GDP for GTP
GAP:enchance the intrinsic GTPase activity
GDI:block the cycle by sequestering and solubilizing
he GDP-bound form
Predominantly through GEF.
•
How they regulate migration:
o
o
o
Rho:assembly of contractile actin:myosin filaments
Rac:polymerization of actin to form lamellipodial
Cdc42:polymerization of actin to form filopodial
protrusion
all promotes the assembly of integrin-based, matrix
adhesion complex
o
Regulation of Rho GTPases during
cell migration
• Observation: Rac can be visualized with highest
concentrations at the leading edge.
• Focus: Rac activation-two pathways
PI-3-kinase and PI(3,4,5)P3
• Extracellular signal activated receptor protein
activated PI-3-kinase PI(3,4,5)P3 docked Rac GEF
• How Rac GEF promotes GTP loading
o PI(3,4,5)P3 binding relieves interaction between PH and DH domain
to stimulate activity
o Loss of activity due to deletion of PH domain is restored by addition
of a CAAX motif to stable the binding.(The DH domain induces Rac to
displace GDP and bind to GTP. The DH domain is invariably proceeded by
a PH domain, which greatly increase catalytic efficiency.)
• PI(3,4,5)P3 regulates PIX, which activates Rac and
interacts with PAK
• Rac activation stimulates PI 3-kinase, resulting a positive
feedback loop
p130Cas/CrkII/DOCK80
•
•
o
Extracellular signal activated receptor protein
activated PI-3-kinase PI(3,4,5)P3 docked
DOCK180
How DOCK180 promotes GTP loading
o
Bind to the adaptor protein Crk, which in turn associates with
another adapor p130Cas. The Crk/p130Cas/DOCK180 can lead
to Rac activation.
Two plausible explanation of the biochemical mechanism:
1.
DOCK180 recruits a DH-containing GEF to the complex.
2.
The C-terminus of DOCK180 can stimulate GTP loading on Rac.
o
The basic region of DOCK180 seems to gunction in the same way
as the PH domain of DH-domain-containing GEF
Downstream effects of Rho
GTPases during cell migration
• Regulation of the actin cytoskeleton
o Rac: generates a protrusive force through the localized
polymerization of actin.
o Cdc42: generates filopodia through the localized polymerization of
actin.
o Rho: generates focal adhesion and cell contractility by across-linking
actin:myosin filaments.
• Regulation of the microtubule and polarity
o Efficient and persistent long-range migration requires stabilizeion of
cell polarity and is achieved through reorganization of the
microtubule cytoskeleton.
o Cdc42 plays a crucial role in polarized migration by reorientation of
microtubules and centrosome.
Regulation of the actin cytoskeleton
•
•
•
Either Rac or Cdc42 can activate p65PAK; Cdc42 activates WASp and NWASp;Rac activates the Scar/WAVE.
p65PAK
1.regulates focal adhesion turnover with help of PIX and GIT1(mechanism is
unkonwn)
2.phosphorlates and activates LIMK, which in turn phosphorylates and
inactivates cofilin.(confilin faciliates subunit dissociation from the pointed
end of actin filaments and induces filament severing and is essential fro
promoting filament treadmilling at the front)
WASP/SCAR/WAVE
1.stimulates the Arp2/3 complex, which can initiate actin polymerization.
2.WASP/WAVE can bind to profilin, which acts synergistically with Arp2/3 to
speed up actin polymerization.
Regulation of the actin cytoskeleton
•
Rho can activates p160ROCK and mDia
• p160ROCK
1.works like p65PAK,leading to stabilization of actin filaments within
actin:myosin filament bundles.
2.interacts with and phosphorylates the myosin binding subunit of
myosin light chain phosphatase and inactivates it,leading to
increased levels of myosin phosphorylation, which then can crosslink actin filaments.
• mDia is linked to actin filament assembly. (mechanism is unknown)
Regulation of the actin cytoskeleton
The microtubule cytoskeleton and
polarity
• Rho:activates mDia, which directly interacts with microtubules and
promotes their capping, leading the stabilization of microtubules.
• Rac:promotes microtubule elongation through
1. p65PAK-dependent phosphorylation by activating p65PAK
2.inactivating stathmin, the microtubule destabilizing protein.
• Cdc42 can activates the complex Par6/PKC.
PKC phosphorylates and inactivates GSK-3.
This induces the association of APC with the plus ends of
microtubules specifically at the leading edge.
Through a dynein-or dynactin-dependent mechanism, this results in
microtubule reorganization and centrosome reorientation.
(APC could also serve to localize Asef, a Rac-specific GEF, to sites
of Rac-dependent actin polymerization)
The microtubule cytoskeleton and
polarity