Cells expressing the F-actin binding protein LimE-GFP and myosin-RFP were stimulated with a gradient of cAMP or 8CPT-cAMP from the right. Cells move towards cAMP and are repulsed from 8CPT-cAMP.

Image courtesy of Dr. Peter J.M. van Haastert, University of Groningen, Haren, Netherlands.

Directed cell migration can be guided by either chemoattractants or repellents and is an essential biological process involved in both normal development and pathological conditions. Chemorepulsion is important in early embryogenesis and while there has been a considerable advance in the understanding of the mechanisms of chemoattraction, little is known about how repellents work. In the Journal of Cell Biology, Peter Van Haastert and colleagues now report that repellent-induced cell migration reversal is dependent on phospholipase C (PLC) in Dictyostelium discoideum.

The lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) accumulates at the leading edge of chemotacting cells. It is involved in the establishment of cell polarity, and induces actin polymerization and pseudopod extension. PIP3 is produced at the cell front by the phosphorylation of phosphatidylinositol-4,5-biphosphate (PIP2) by phosphatidylinositol 3-kinases (PI3Ks), and is degraded at the rear and sides by the phosphatase and tensin homologue PTEN. Because PI3K accumulates at sites of F-actin polymerization and PTEN binds to PIP2, the gradients of these proteins and their products stabilise once established. As well as activating PI3K, chemoattractants also activate PLC, which hydrolyses and reduces PIP2 levels. At the front of cells, PLC would inhibit PTEN recruitment and lead to further PIP3 accumulation at the leading edge.

Peter Van Haastert and colleagues studied the mechanisms by which Dyctiostelium cells reverse direction when they face a repellent. In the presence of both the chemoattractant cAMP and the repellent 8-para-chlorphenylthio-cAMP (8CPT-cAMP), cells moved in random directions. However, when 8CPT-cAMP was removed, cells began to migrate towards the cAMP source, indicating that the repellent can reversibly inhibit the response to cAMP. Cell treatment with 8CPT-cAMP resulted in the formation of F-actin at the front and actomyosin filaments at the rear. As the front is furthest from the chemorepellent source, this indicates that chemorepellents induce reversal of the intracellular motility machinery.

The authors also investigated PIP3 distribution using the PIP3 detector PHcracGFP. 8CPT-cAMP induced strong localization of PHcracGFP at the plasma membrane similar to cAMP but with opposite polarity; PIP3 levels increased at the opposite side to the chemorepellent. In 8CPT-cAMP gradients, PI3K-GFP and PTEN-GFP localizations were also reversed compared to cAMP gradients. Impaired PIP3 production, such as in pi3k1/2^-/- mutant cells, resulted in an abolished chemotactic response to 8CPT-cAMP. However, these cells were still able to move towards cAMP even in the presence of 8CPT-cAMP, suggesting that PI3K is required for the repellent activity of 8CPT-cAMP. Similarly, plc-null cells moved randomly in the presence of 8CPT-cAMP but were still able to migrate towards cAMP, showing that PLC is also essential for the inhibitory effect of repellents. These observations also imply that PI3K and PLC are not essential for chemotaxis towards cAMP, which is in accordance with recent reports of parallel pathways mediating positive chemotaxis.

The authors propose that the mechanism for negative chemotaxis control relies on PLC inhibition. Inactivation of PLC leads to the local accumulation of PIP2, recruitment of PTEN and degradation of PIP3. PIP3 consequently accumulates at the opposite side of cells, inducing a reversal of cell migration. In this model, the polarity switch depends upon PLC. As PLC activity is regulated by activating and inhibitory G proteins, a single compound could act as either an attractant or a repellent depending on whether it is bound to an activating or inhibitory G protein-coupled receptor. Recent discoveries have shown that PIP3 plays an important role in chemotacting mammalian cells, but whether PLC may be involved in polarity reversal in other organisms has yet to be elucidated.

Author: Kim Baumann