Transcript CantleyLecture4

Chemotaxis of Eukaryotic Cells:
1. Dictyostelium discoideum - Part 2
http://dictybase.org/Multimedia/index.html
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http://dictybase.org/Multimedia/development/development.html
1. At high density and low nutrient availability, a
random cell begins to release cAMP.
4. The cycle continues as the next set of cells
chemotax toward the center of mass of cells and in
turn release cAMP after a lag, recruiting yet more
cells
2. Adjacent cells chemotaxis up the cAMP gradient and
the phosphodiesterase they release degrades the cAMP.
3. After a lag, the chemotaxing cells release their own
cAMP. This creates a a second, greater cAMP gradient.
cAMP release from a pipett stimulates chemotaxis of a population
of Dictyostelium (from Peter Devreotes)
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Richard Firtel
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Outside
cAMP
PIP2 Receptor
PIP2 PIP2
Gbg
Gbg
Ga
Ga
PIP2
?? PI3K
PIP33
PIP
PH
PIP2 PIP2
PTEN
??
Recruitment of
cortical actin to
drive cell
polarization and
motility
Adenylyl
Cyclase
cAMP
(secreted)
PIP3
PIP
3
PH
Cytosol
A green fluorescent protein chimera of a pleckstrin homology
(PH) domain that binds to the lipid phosphatidylinositol-3,4,5trisphosphate (PIP3) reveals that this lipid is generated at the
leading edge of the cell in a dynamic fashion that rapidly
responds to changes in the extracellular cAMP gradient. (from
Richard Firtel)
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Iijima and Devreotes 2002 Cell 109, 599
B. GFP-PTEN localizes to
the rear of a
chemotaxing cell. The
diamond indicates the
location of the cAMPcontaining pipette.
C. A mutant of PTEN
lacking the N-terminal
16 amino acids fails to
localize to the rear right panel.
Van Haastert and Devreotes 2004 Nat Rev Mol Cell Biol 5, 626
PI3K is not critical for acute stimulation of cortical actin accumulation but
participates in remodeling of actin to from a polarized leading edge
Van Haastert and Devreotes 2004 Nat Rev Mol Cell Biol 5, 626
Van Haastert and Devreotes 2004 Nat Rev Mol Cell Biol 5, 626
?
?
Local Excitation, Global Inhibition (LEGI) Model
Ma et al., 2004 Biophysical J. 87, 3764
Assumptions: PI3K LEGI
1. Receptor occupation rapidly stimulates a
local, membrane imbedded (slowly
diffusable) component that activates a
membrane imbedded PI3K binding protein.
2. This recruits PI3K from the cytosol to the
membrane and results in local production of
PIP3.
3. Receptor occupation also stimulates a
cytosolic component that diffuses
throughout the cell and globally inactivates
the membrane imbedded PI3K binding
protein.
4. The activation of the global inhibitor is
slower than activation of the local activator.
Thus, PI3K is concentrated near
activated receptors
Local Excitation, Global Inhibition (LEGI) Model
Ma et al., 2004 Biophysical J. 87, 3764
Assumptions: PTEN Regulation
1. Receptor occupation rapidly stimulates a
local, membrane imbedded (slowly
diffusable) component that inactivates a
membrane imbedded PTEN binding protein.
2. This locally releases PTEN from the
membrane and allows local production of
PIP3.
3. Receptor occupation also stimulates a
cytosolic component that diffuses
throughout the cell and globally activates
the membrane imbedded PTEN binding
protein.
4. The activation of the global PTEN regulator
is slower than activation of the local
regulator.
Thus, PTEN levels are reduced near activated
receptors and elevated elsewhere.
Note: PTEN regulation and PI3K regulation are assumed to be uncoupled
PI3K half of the LEGI model
L
L
R
S
EA
E
Membrane
Membrane
Local
BSPI3K
BSAPI3K
BSAPI3K
PI3K
PI3K
BSPI3K
I
BSAPI3K
IA
Global
IA
PI3K
Cytosolic PI3K, I and IA are freely diffusable throughout the cytosol.
Membrane imbedded components (R, S, E, BS) have more restricted movement
PTEN half of the LEGI model
L
L
R
S
EA
E
Membrane
Membrane
Local
BSPTEN
BSAPTEN
BSAPTEN
PTEN
PTEN
BSPTEN
IPTEN
PTEN
IAPTEN
Global
BSAPTE
IAPTEN
Cytosolic PTEN, I and IA are freely diffusable throughout the cytosol.
Membrane imbedded components (R, S, E, BS) have more restricted movement
Virtual Cell Model Ma et al. (Devreotes & Iglesias)
Using Lowe and Schaff, 2001, Trends Biotechnol. 19, 401; Virtual Cell
http://www.nrcam.uchc.edu/applications/applications.html
Virtual Cell Model Ma et al. (Devreotes & Iglesias)
IAPTEN creates PTEN binding sites
IAPI3K eliminates PI3K binding sites
Virtual Cell Model Ma et al. (Devreotes & Iglesias)
wt
(2 fold)
(10% wt for each)
Virtual Cell Model Ma et al. (Devreotes & Iglesias)
PTEN
cAMP
PI3K
PIP3
Virtual Cell Model Ma et al. (Devreotes & Iglesias)
The PIP3 gradient depends on the
cAMP gradient but is relatively
insensitive to the absolute amount of
cAMP, allowing adaptation to higher
basal cAMP
Virtual Cell Model Ma et al. (Devreotes & Iglesias)
Van Haastert and Devreotes 2004 Nat Rev Mol Cell Biol 5, 626
Myosin II accumulates in the lateral and trailing
edge of the migrating cell. There it suppresses
formation of additional pseudopods and retracts
the trailing end of the cell. What is the
mechanism for polarized location of myosin II
and for activation of myosin II-dependent
contraction?
Van Haastert and Devreotes 2004 Nat Rev Mol Cell Biol 5, 626
Elevation in cGMP stimulates the
global formation of myosin II
filaments and also activates
myosin light chain kinase, which
enhances traction on actin
filaments. This drives retraction
of pseudopods and retraction of
the uropod tail.
However, it does not explain why
myosin II is excluded from the
anterior region of the cell.
Receptor
Guanylate
cyclase
cGMP-binding
protein
Myosin light
chain kinase
Van Haastert and Devreotes 2004 Nat Rev Mol Cell Biol 5, 626
cAMP cAR1
Model for exclusion of Myosin
II from the leading edge.
Local phosphorylation of Myosin II at
the leading edge drives
depolymerization of the filament. This
phosphorylation may be caused by
more than one protein kinase. Myosin
heavy chain kinase A is implicated.
Also, a kinase regulated by the low
molecular weight GTP binding protein,
rac and by AKT (called PAKa) is
implicated. AKT is locally activated at
the anterior of the cell where AKT and
Rac accumulate. However, PAKa
accumulates in the rear of the cell.
PI3K
PIP3
?
Rac AKT
PAKa
Chung et al., 2001
Molecular Cell 7, 937
From Richard Firtel