Bourne lab Home

Click for a QuickTime Movie:
This differentiated HL60 cell expresses a PIP3-binding PH domain fused to GFP (PH-Akt-GFP). At zero time the cell was exposed to fMLP, a chemoattractant, in a micropipette located at the lower edge of the picture. The movie shows PH-Akt-GFP moving from the cytoplasm to the edge of the cell nearest the micropipette.

Neutrophil chemotaxis:

Question: Extensive studies by many investigators have not achieved a rigorous understanding of the mechanism(s) used by any cell Ñ yeast, Dictyostelium, or vertebrate cell, including the neutrophil Ñ to choose its direction of polarity in response to environmental clues, such as gradients of attractant. We know many fascinating details of the neutrophil cytoskeleton, its crawling mechanism, and the myriad ligands and receptors that initiate and maintain its polarity, but little more than we did 25 years ago about how neutrophils interpret gradients of chemoattractant. Recent technological advances are beginning to open productive avenues.

PIP3 and Cdc42: A key role for PIP3 in neutrophil direction-finding was underscored by demonstrations, in several laboratories, that transgenic knockout of a phosphatidyl inositol 3'-kinase (PI3K) isoform, PI3Kgamma, makes neutrophils virtually unable to interpret pipette-generated gradients of attractant, while the cells' ability to crawl is much less severely impaired. We strongly suspect that Cdc42, a Rho GTPase, also plays a major role in neutrophil direction-finding, based on its important role in mediating polarity of budding yeast and on our observations that dominant-negative Cdc42 destabilizes polarity and prevents direction-finding in HL-60 cells (see Neutrophil polarity). Alexandra Van Keymeulen's investigations of these two molecules shows that polarity can be experimentally dissected from directionality.

F-actin plays a key role in a PIP3- and Rac-GTP-dependent positive feedback loop that is responsible for forming pseudopods in response to fMLP (see Neutrophil polarity). In contrast, Alexandra found that F-actin is not required for fMLP-stimulated accumulation of Cdc42-GTP. In addition, in collaboration with Zach Knight in Kevan Shokat's laboratory, Alexandra has applied a number of recently reported PI3K inhibitors to HL-60 cells. Of these, two abrogate direction-finding without inhibiting polarity or migration responses to fMLP. Moreover, in keeping with the mouse knockout studies described above, the same compounds selectively inhibit the activity of PI3Kgamma Ñ again suggesting that this PI3K isoform plays an important role in neutrophil directionality. Alexandra finds that the same PI3K inhibitors reduce fMLP-stimulated accumulation of Cdc42-GTP, at concentrations that do not impair migration.

In summary, we are beginning to suspect that fMLP-stimulated PIP3 accumulation triggers two separate but superimposed responses. One of these involves PIP3/Rac/actin-dependent positive feedback, and results in protrusion of F-actin-containing pseudopods. The other response may involve a separate signaling pathway in which PIP3 activates Cdc42 and Cdc42-GTP in turn feeds back to increase PIP3 accumulation. In this scenario the two 'pools' of PIP3 reinforce one another, because they do not accumulate in separate membrane regions. Presumably the Cdc42-associated PIP3 pool acts to bias the direction of Rac- and F-actin-dependent pseudopods.

Microtubules: Neutrophil directionality and polarity can be separated in a second way: nocodazole and other microtubule-inhibitors markedly impair directionality in micropipette assays, but do not block polarity or slow migration. Indeed, as described by others, nocodazole can actually induce polarity in cells that have not been exposed to fMLP or other attractants. Experiments by Jingsong Xu and Fei Wang suggest that nocodazole impairs direction-finding in two ways: (a) by stimulating actin-myosin contraction, triggered by Rho-GTP at the cells' back and sides of cells; (b) by reducing accumulation of PIP3 and Rac-GTP, as well as F-actin, in pseudopods. With Alexandra Van Keymeulen they are exploring possible roles for PI3K isoforms and Cdc42 in these effects.

Directionality in shallow gradients: Zigmond reported more than 20 years ago that neutrophils can orient and crawl correctly in very shallow gradients (in which the attractant concentration differs by less than 2% at front vs. back). In the interim, progress in understanding this exquisite sensitivity to shallow gradients has been extremely slow. Now, however, custom-designed microfluidic devices make it possible to create stable, reliable gradients and to impose or remove them almost instantaneously. Paul Herzmark is using state-of-the-art devices fabricated in the laboratory of Alex Groisman at UCSD to determine precisely the effects of different experimental conditions (attractant concentrations, gradient slopes, and effects of inhibitors PI3K, microtubules, actin polymerization, mediators of 'backness,' etc.) on directionality of HL-60 cells.