Chemotaxis of Eukaryotic Cells:2. Comparing Neutrophils to Dictyostelium

QuickTime™ and aNone decompressor

are needed to see this picture.

QuickTime™ and aNone decompressorare needed to see this picture.

Dictyostelium chasing cAMP Neutrophil chasing fMLP

Van Haastert and Devreotes 2004 Nat Rev Mol Cell Biol 5, 626

Receptor

Outside

Cytosol

GG ?

PIP2

PI3K

PIP2 PIP2PIP2 PIP3 PIP3

PH PH

Recruitment of cortical actin to drive cell polarization and motility

cAMP

G

G

?PIP3

PIP3

Adenylyl Cyclase

cAMP (secreted)

PTEN

PIP2 PIP2

??

BSPI3KBSAPI3K

R

L

S

L

PI3K

BSAPI3K

I IA

EEA

PI3K

BSPI3KBSAPI3K

PI3KIA

Cytosolic PI3K, I and IA are freely diffusable throughout the cytosol.Membrane imbedded components (R, S, E, BS) have more restricted movement

Membrane Membrane

PI3K half of the LEGI model

Local

Global

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

Is Ras the PI3K upstream activator in response to cAMP?Sasaki et al.,(Firtel) 2004 JCB 167, 505

Dictyostelium (like neutrophils) has three Ras genes (RasG, RasB and RasD) that are close homologs of mammalian K-Ras and H-Ras. Activated Ras (GTP loaded) binds to both mammalian and Dictyostelium PI3K.

PI3K 110catalyticPIKRas-bindingp85binding

PI3K 110catalyticPIKRas-binding

catalyticPIKRas-binding PI3K1N-terminal

C2

C2

C2

catalyticPIKRas-binding PI3K2C2

Human

Dicty

Note that he N-terminal domain of Dictyostelium PI3K1 (which is recruited to the membrane within 5 sec after cAMP addition) is not conserved in human PI3K.

Deletion of RasG has a chemotactic defect similar to loss of PI3K1 and PI3K2.AKT activation in response to cAMP is reduced in RasG deleted cells. Sasaki et al.,(Firtel) 2004 JCB 167, 505

RasG is uniformly distributed on the plasma membrane whether or not cells are stimulated.

Within 5 seconds of stimulation with cAMP, RasG is activated as judged by its ability to bind to the Ras-binding domain (RBD) of Raf.

Uniform localization of GFP-Rasduring chemotaxis

The initial activation of Ras by cAMP does not require PI3K activity, although sustained activation of Ras does appear to be attenuated in the presence of PI3K inhibitors.

GFP-RBDlocalization

Activation of Ras at the membrane occurs in parallel to PI3K recruitment and prior to PI3,4,5P3 production (judged by PH domain recruitment).

Time after cAMP (sec)

Five seconds after cAMP, Ras is activated at the plasma membrane

GFP-RBD, Wild TypeGFP-RBDPI3K1/2 -/-

GFP-RBD PTEN-

Ras is activated at the leading edge of the migrating cell whether or not PI3K is active, but activation of PI3K enhances Ras activation at this location

Inhibition of actin polymerization with latrunculin dramatically reduces the recruitment of the N-terminal domain of PI3K1 to the membrane in response to cAMP, but it does not have a major effect on Ras activation and only partially inhibits activation of AKT

AKT activation

Ras activation

Model: 1. Ras is activated downstream of the cAMP receptor rapidly (less than 5 seconds)

and independent of cortical actin polymerization and PI3K activity (e.g. Ras is the BSPI3K in the LEGI model).

2. Ras recruits and activates a small fraction of PI3K at the plasma membrane, resulting in AKT activation and other responses (including a secondary Rac activation-see below).

3. In parallel to Ras activation, actin polymerization also is initiated at the plasma membrane. This initial actin polymerization (at 5 seconds) is independent of PI3K and probably mediated by a GDP/GTP exchange factor for Rac that is directly regulated by a heterotrimeric G protein.

4. Cortical actin causes further recruitment of PI3K to the plasma membrane via the N-terminal domain of PI3K, enhancing the Ras-dependent activation of PI3K.

5. Products of PI3K result in further stimulation of Ras activation (by an unknown mechanism), which further activates PI3K (positive feedback loop).

6. A combination of the positive feedback loop and the LEGI model ultimately results in global inhibition and local amplification of PI3K.

7. The local PI3,4,5P3 production activates a distinct GDP/GTP exchange factor for Rac that produces the second, polarized cortical actin polymerization. This results in a second positive feedback loop by actin-dependent recruitment of PI3K.

Effect of various Ras mutants on chemotaxis

Sasaki et al.,(Firtel) 2004 JCB 167, 505

Sasaki et al.,(Firtel) 2004 JCB 167, 505

QuickTime™ and aCinepak decompressor

are needed to see this picture.

Neutrophils migrating into a site of injury in a live ZebrafishMione and Redd

PH

PDK1AKTPH

PI-3,4,5-P3

Phosphoinositide 3-kinase Signaling in chemokine or fMLP stimulated neutrophils

p110 PI3Kp101

Chemokine

GTPRas

GEF?

GTPRac

Cell Migration

GPCR

G

P

? ?

PTEN

PI-3,4,5-P3

p110 PI3Kp101

Chemokine

GTPRas

PH

PDK1AKTPH

GEF?

GTPRac

GPCRPI-4,5-P2PTE

N

Phosphoinositide 3-kinase Signaling in chemokine or fMLP stimulated neutrophils

/Cdc42

PI3K

GEFs

Actin polymerizes

Arp2/3

R* PIP3

Finding the Compass: Where in thepathway does asymmetry first appear?

WASP/WAVE

Rac

Sensing Amplification/Polarization Migration

PHAKT-GFP translocates to leadingedge in responseto a point source ofchemoattractant

QuickTime™ and aNone decompressor

are needed to see this picture.

The following results with neutrophils are primarily from Weiner et al., 2002 Nat. Cell Biol. 4, 509 and Wang et al., 2002 Nat. Cell Biol. 4, 513

PHAKT-GFPasymmetry exceedsthat of the externalgradient duringchemotaxis

External

Internal

Amplifying the Gradient

Asymmetric AKT recruitment doesnot require new actin polymerization

/Cdc42

PI3K

GEFsActin polymerizes

Arp2/3

UniformPolarized

R* PIP3WASP/WAVE

Rac

What is uniform/polarized?

Cdc42GTP

Cdc42GTP Cdc42

GTP

Cdc42GDP

Cdc42GDP

Cdc42GDP

WASPGBD/GFP

WASPGBD/GFPWASP

GBD/GFP

Activated Cdc42 accumulatesat the leading edge of chemoattractant-stimulatedneutrophils

?

How do cells establish gradients of PI3K lipid products during chemotaxis?

GFP-AKT-PH Domain

PIP3 distribution for uniform stimulation by FMLP

Unstim 30 sec 2 min

GFP-AKT-PH probe

Exogenous membrane-permeable PIP3 is sufficient to induce neutrophil polarity

PIP3

F-Actin staining F-Actin staining

PIP3 distribution for uniform stimulation by membrane-permeable PIP3

Unstim 30 sec 2 min

GFP-AKT-PH probe

160

PIP2/histunstim PIP3 alone

A B C

D E F

G H I

30s 60s 90s

120s 150s 180s

Time course for cell polarization in response to PIP3 addition

GFP-AKTPH

PI3K and Rho GTPase activity are required for exogenousPIP3-induced PHAKT-GFP translocation

control

PI3K and Rho GTPase activity are required for exogenousPIP3-induced GFP-AKT-PH probe PH-AKT translocation

control

200 M LY or200 nM wortmannin

PIP3

Ly294002/wortmannin

PIP3

PIP3

PI3K

PIP3 positive feedback loop

PI3K and Rho GTPase activity are required for exogenousPIP3-induced PHAKT-GFP translocation

control

C. difficile Tox. B

200 M LY or200 nM wortmannin

PIP3

PIP3

PI3K

Rac/Cdc42

PIP3 positive feedback loop

Requirements for eukaryotic chemotaxis

1. Sensing 2. Amplification 3. Migration

R*

Cdc42Rac

PI3K PIP3

GEFs

Actin polymerizes

WASP/WAVE

Arp2/3/

PI3K

feedback

PIP3 positive feedback loopin chemotactic signaling

Self-organizing pattern formation systemfor polarity

Local positive feedback(PIP3-RhoGTPases)

Global inhibitionPTEN? SHIP?RhoGEF inhibition?