Chemotaxis of Eukaryotic Cells: 2. Comparing Neutrophils to Dictyostelium Dictyostelium chasing cAMP Neutrophil chasing fMLP

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

Outside ? ? cAMP Receptor PIP3 PIP3 PI3K ? ? Gbg Gbg PH PH PTEN Ga Ga Adenylyl Cyclase cAMP (secreted) Recruitment of cortical actin to drive cell polarization and motility Cytosol

PI3K half of the LEGI model L R S Membrane Membrane EA E Local BSAPI3K BSPI3K BSAPI3K BSPI3K BSAPI3K PI3K I IA PI3K PI3K IA Global Cytosolic PI3K, I and IA are freely diffusable throughout the cytosol. Membrane imbedded components (R, S, E, BS) have more restricted movement

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. Ras-binding C2 PI3K 110a p85binding PIK catalytic Human Ras-binding C2 PI3K 110g PIK catalytic Ras-binding C2 PI3K1 N-terminal PIK catalytic Dicty Ras-binding C2 PI3K2 PIK catalytic 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-Ras during 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.

Activation of Ras at the membrane occurs in parallel to PI3K recruitment and prior to PI3,4,5P3 production (judged by PH domain recruitment). GFP-RBD localization Five seconds after cAMP, Ras is activated at the plasma membrane Time after cAMP (sec)

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 GFP-RBD PTEN- GFP-RBD PI3K1/2 -/- GFP-RBD, Wild Type

AKT activation Ras activation 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: 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). 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). 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. 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. Products of PI3K result in further stimulation of Ras activation (by an unknown mechanism), which further activates PI3K (positive feedback loop). A combination of the positive feedback loop and the LEGI model ultimately results in global inhibition and local amplification of PI3K. 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

Neutrophils migrating into a site of injury in a live Zebrafish Mione and Redd

Phosphoinositide 3-kinase Signaling in chemokine or fMLP stimulated neutrophils Chemokine GPCR Ga Ras PI-3,4,5-P3 b/g PTEN p110g PI3K p101 GTP PH PDK1 AKT GEF? GTP Rac P ? Cell Migration

Phosphoinositide 3-kinase Signaling in chemokine or fMLP stimulated neutrophils Chemokine GPCR PI-4,5-P2 Ras PI-3,4,5-P3 b/g PTEN p110g PI3K p101 GTP PH PDK1 AKT GEF? GTP Rac

Finding the Compass: Where in the pathway does asymmetry first appear? Sensing Amplification/Polarization Migration WASP/WAVE PI3Kg R* PIP3 Cdc42 a/bg Rac Arp2/3 GEFs Actin polymerizes

translocates to leading edge in response to a point source of 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-GFP translocates to leading edge in response to a point source of chemoattractant

Amplifying the Gradient External PHAKT-GFP asymmetry exceeds that of the external gradient during chemotaxis Internal

Asymmetric AKT recruitment does not require new actin polymerization

What is uniform/polarized? WASP/WAVE PI3Kg R* PIP3 Cdc42 a/bg Rac Arp2/3 GEFs Actin polymerizes Uniform Polarized

Activated Cdc42 accumulates at the leading edge of chemoattractant-stimulated neutrophils Cdc42 GTP Cdc42 GTP Cdc42 GTP WASP GBD/GFP WASP GBD/GFP WASP GBD/GFP Cdc42 GDP Cdc42 GDP Cdc42 GDP

? 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

Time course for cell polarization in response to PIP3 addition B C GFP-AKTPH 30s 60s 90s D E F 120s 150s 180s G H I 160 unstim PIP3 alone PIP2/hist

PI3K and Rho GTPase activity are required for exogenous PIP3-induced PHAKT-GFP translocation control

PI3K and Rho GTPase activity are required for exogenous PIP3-induced GFP-AKT-PH probe PH-AKT translocation control 200 mM LY or 200 nM wortmannin

Ly294002/wortmannin PIP3

PIP3 positive feedback loop PI3K PIP3

PI3K and Rho GTPase activity are required for exogenous PIP3-induced PHAKT-GFP translocation control 200 mM LY or 200 nM wortmannin C. difficile Tox. B

PIP3 positive feedback loop Rac/ Cdc42 PI3K PIP3

Requirements for eukaryotic chemotaxis 1. Sensing 2. Amplification 3. Migration

PIP3 positive feedback loop in chemotactic signaling PI3Kg PIP3 Actin polymerizes WASP/WAVE Arp2/3 R* PI3K feedback Cdc42 Rac a/bg GEFs

Self-organizing pattern formation system for polarity Local positive feedback (PIP3-RhoGTPases) Global inhibition PTEN? SHIP? RhoGEF inhibition?