Neutrophil Polarity

Question: Upon exposure to chemoattractants, neutrophils take on a polarized morphology, with F-actin-rich protrusions (pseudopods) at the front and distinctive actin-myosin contractile complexes at the sides and back. A concentration gradient of the attractant is not necessary: the polarization response occurs even when the attractant concentration is uniform and apparently stimulating all portions of the plasma membrane at the same intensity; in the absence of a gradient, the direction of polarity is random, but all cells can be induced to polarize. Over the past five years this laboratory has begun to dissect intracellular signals responsible for neutrophil polarity.

HL-60 model and roles of PIP3, Rho GTPases, and F-actin:
 Five years ago Guy Servant and Orion Weiner adapted the HL-60 human leukemia cell line as a model neutrophil in which we could express dominant-interfering proteins and fluorescent (GFP-tagged) protein probes. After DMSO-induced differentiation, HL-60 cells look like neutrophils, polarize in response to attractant, and migrate in gradients (or in uniform attractant) at rates comparable to those of neutrophils from peripheral blood.

Seeking to determine where in the signaling pathway asymmetry first appears, Guy and Orion found that GFP-tagged C5a receptors are distributed uniformly throughout the plasma membrane in both resting and polarized cells, but that the PH domain of AKT, tagged with GFP (PH-Akt-GFP) Ñ a probe for PIP3, a membrane lipid Ñ translocates from the cytoplasm to the plasma membrane at the leading edge of polarized HL-60 cells. (Other laboratories had used different fluorescent probes to show that PIP3 accumulates at the leading edge of a soil amoeba, Dictyostelium discoideum, when it polarizes and crawls up an attractant gradient.) Further experiments revealed that polarity of HL-60 cells require Rho GTPases, which regulate actin polymerization and accumulation of PIP3 at the leading edge and myosin distribution at the trailing edge. Orion and Fei Wang obtained evidence indicating that Rho GTPases and PIP3 participate in an F-actin-dependent positive feedback loop that is necessary for creating asymmetry. Although translocation of PH-Akt-GFP to the up-gradient edge does not absolutely require polymerization of new actin polymers, Wang found that inhibiting actin polymerization completely blocked PH-Akt-GFP asymmetry in a uniform concentration of attractant and markedly reduced this asymmetry in gradients.

Supriya Srinivasan explored the roles of individual Rho GTPases in controlling polarity and migration. By expressing dominant negative mutants of Rho GTPases in HL-60 cells, she found that Rac is necessary for PIP3 accumulation at the leading edge and that a second GTPase, Cdc42, is required to consolidate, stabilize, and focus the leading edge.

Role of Rho at sides and trailing edge: Together, Jingsong Xu and Fei Wang used pharmacological inhibitors, toxins, and mutant proteins to show that attractant-induced polarity depends on divergent, opposing sets of 'frontness' and 'backness' signals generated by different GPCR-activated trimeric G proteins. The frontness pathway Ñ which is responsible for the positive feedback loop involving PIP3, Rac, and F-actin, as described above Ñ is mediated by the trimeric G protein, Gi, and is blocked by treating cells with pertussis toxin. The same GPCR population triggers backness via two pertussis toxin-resistant trimeric G proteins, G12 and G13, which activate a signaling cascade that includes a second GTPase, Rho, a Rho-dependent kinase (ROCK), and myosin II. Functional incompatibility of actin assemblies triggered by the two pathways causes them to aggregate into separate domains.

In keeping with these findings, unpublished studies by Kit Wong have shown that the accumulation of Rho-GTP (detected with a FRET probe) triggered by attractants is localized at the back of polarized HL-60 cells. She is presently exploring the relation of Rho-GTP accumulation to morphologic 'backness.'

What are the consequences of using two separate pathways to promote polarity? The positive feedback loop greatly increases responsiveness of pseudopods Ñ relative to back and sides Ñ to actin-polymerizing effects of attractant. This phenomenon explains both the neutrophil's ability to polarize in uniform concentrations of chemoattractant and its response to reversal of an attractant gradient by performing a U-turn. Stimulation of physically and mechanistically distinct frontness and backness pathways creates a 'four-wheel drive' machine that allows neutrophils to crawl fast and to maneuver rapidly in response to changing gradients of attractant (see Neutrophil chemotaxis)

 

 


F-actin (red) in the pseudopod, RhoA (green) at the rear. Cells treated with a uniform concentration of attractant for 2 min, fixed and stained.

Polarization: Exposure of cell expressing a fluorescent PIP3 probe (PH-Akt-GFP) to a uniform concentration of attractant. In the basal (unstimulated) state the PIP3 probe is cytoplasmic. After addition of attractant the PIP3 probe initially translocates to the entire plasma membrane, but then localizes in discrete membrane regions. Finally, the PIP3 probe is polarized (that is, primarily in the pseudopod at one end of the cell) and the cell crawls.