Dynamic Distribution of Chemoattractant Receptors in Living Cells During Chemotaxis and Persistent Stimulation

Abstract

While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattractant receptors. Some newly extended pseudopods at migration fronts showed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to “cap” rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.

Novel Mechanism of PTEN Regulation by Its Phosphatidylinositol 4,5-Bisphosphate Binding Motif Is Critical for Chemotaxis

“…Therefore, the PI(4,5)P 2 binding motif is essential for PI(3,4,5)P 3 and PI(3,4)P 2 -directed phosphatase activity of both Dictyostelium and human PTEN. PTEN 16 -515 Preferentially Dephosphorylates Ins (1,3,4,5)P 4 -The observations that PTEN 16 -515 is unable to dephosphorylate PI(3,4,5)P 3 and PI(3,4)P 2 suggest that the PI(4,5)P 2 binding motif regulates the specificity of the phosphatase activity of PTEN. We reasoned that the PI(4,5)P 2 binding motif is important for interaction with phosphatidylinositol lipids such as PI(3,4,5)P 3 and PI(3,4)P 2 .…”

“…Inositol phosphates were identified by their co-elution with radiolabeled standards of inositol phosphate. (3,4,5)P 3 and PI(3,4)P 2 in VitroTo determine the lipid phosphatase activity of Dictyostelium PTEN, we expressed PTEN fused to GFP in pten Ϫ cells and purified the protein by immunoprecipitation with protein A beads coupled to anti-GFP antibody. Immunopurified PTEN and a GFP control were incubated with PI(3,4,5)P 3 or PI(3,4)P 2 for 30 min.…”

“…Thus, intracellular signaling events involved chemotaxis are activated locally at the leading edge. However, neither the receptors nor the receptor-coupled G-proteins are concentrated in this region but are found uniformly distributed along the plasma membrane (3)(4)(5).…”

A PtdInsP3- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity

“…1i; 5 out of 52 cells uniformly translocated PH-Akt-GFP in 30-60 sec, and 0 out of 56 cells exhibited polarized PH-Akt-GFP recruitment at 2-3 min. A GFP-tagged chemoattractant receptor 14 remained uniformly distributed throughout the plasma membrane in polarized cells, as previously described for neutrophils 14 and Dictyostelium 15 . This suggests that the PH-Akt-GFP asymmetry we observed after PtdInsP 3 -histone stimulation is not a reflection of cell morphology (Fig.…”

Oscillatory signaling and network responses during the development of Dictyostelium discoideum

“…CAR1 is distributed uniformly around the cell periphery (Figure 3), allowing all regions of the cell to be equally responsive to a progressing cAMP wave (Xiao et al, 1997). Cells lacking CAR1 fail to initiate cAMP signaling, respond chemotactically, or form multicellular aggregates (Klein et al, 1988).…”