Involvement of the cytoskeleton in controlling leading-edge function during chemotaxis

Abstract

In response to directional stimulation by a chemoattractant, cells rapidly activate a series of signaling pathways at the site closest to the chemoattractant source that leads to F-actin polymerization, pseudopod formation, and directional movement up the gradient. Ras proteins are major regulators of chemotaxis in Dictyostelium; they are activated at the leading edge, are required for chemoattractant-mediated activation of PI3K and TORC2, and are one of the most rapid responders, with activity peaking at approximately 3 s after stimulation. We demonstrate that in myosin II (MyoII) null cells, Ras activation is highly extended and is not restricted to the site closest to the chemoattractant source. This causes elevated, extended, and spatially misregulated activation of PI3K and TORC2 and their effectors Akt/PKB and PKBR1, as well as elevated F-actin polymerization. We further demonstrate that disruption of specific IQGAP/cortexillin complexes, which also regulate cortical mechanics, causes extended activation of PI3K and Akt/PKB but not Ras activation. Our findings suggest that MyoII and IQGAP/cortexillin play key roles in spatially and temporally regulating leading-edge activity and, through this, the ability of cells to restrict the site of pseudopod formation.

Figure 1.

Regulation of Ras by MyoII. (A) Ras activation in wild-type, mhcA⁻, and mhcA⁻/MyoII^S456L cells. (B) Comparative quantitation of Ras activation (see Materials and Methods). Maximum value of wild-type cells is taken as 1.0. (C) Translocation kinetics of RBD-GFP in KAx-3 and mhcA⁻ cells before and after stimulation. (D) Translocation kinetics of PhdA-GFP in KAx-3 and mhcA⁻ cells before and after cAMP stimulation. (E) Kinetics of F-actin polymerization of KAx3 and mhcA⁻ cells, and MyoII assembly of KAx-3 in the Triton-insoluble, cytoskeleton fraction.

Figure 2.

MyoII regulation of PKB. (A) Chemoattractant-mediated Akt/PKB activation of different cell lines using H2B as a substrate. Top lanes show the kinase activity, and bottom lanes show a Western blot of Akt/PKB protein. LA, cells treated with 5 μM LatB for 20 min before cAMP stimulation. (B) Comparative quantitation of PKB activation (see Materials and Methods). Maximum value of wild-type cells is taken as 1.0. Values normalized with loading levels of the kinase. (C) Chemoattractant-mediated PKBR1 activation. We used H2B as a substrate. Top lanes show the kinase activity, and bottom lanes show a Western blot of PKBR1 protein. (D) Comparative quantitation of PKBR1 activation (see Materials and Methods). For all experiments, the maximum value of wild-type cells is taken as 1.0. Values were normalized with loading levels of the kinase.

Figure 3.

Spatial misregulation of Ras and PI3K activity in MyoII null cells. (A) A wild-type cell and an mhcA⁻ cell expressing GFP-RBD were exposed to a chemoattractant gradient of cAMP emitted by a micropipette, and the localization of GFP-RBD was recorded. (B) A wild-type cell and mhcA⁻ cells expressing GFP-PH were exposed to a chemoattractant gradient of cAMP emitted from a micropipette, and we recorded the localization of GFP-PH.

Figure 4.

Isolation of IQGAP-containing complexes. (A) Silver staining of immunoprecipitated products with anti-Myc in vegetative KAx-3, Myc-IQGAP1/iqgA⁻, and Myc-IQGAP2/iqgB⁻ cells. Right panel depicts a Myc Western blot of the same samples. (B) Silver staining of pulsed KAx-3, Myc-IQGAP1/iqgA-, and IQGAP2/iqgB⁻ cells unstimulated (U) and stimulated (S) cells.

Figure 5.

Images of chemotaxing cells obtained using DIAS computer software. The overlapping images were taken at 1-min intervals.

Figure 6.

Kinetics of F-actin polymerization and MyoII assembly in IQGAP and ctx mutant strains. Kinetics of F-actin polymerization (A) and MyoII assembly (B) in the Triton-insoluble, cytoskeleton fraction. (C) Phalloidin staining of pulsed cells.

Figure 7.

Regulation of Ras and PI3K activities in IQGAP and ctx mutant strains. (A) Ras activation in response to cAMP stimulation in KAx-3, iqgA⁻, iqgB⁻, iqgA⁻/B⁻, and ctxA^−/B− cells. (B) Comparative quantitation of values in A. Maximum value of wild-type cells is taken as 1.0. (C) Akt/PKB activation in response to cAMP stimulation. We used H2B as a substrate. LA, cells treated with 5 μM LatB added 20 min before cAMP stimulation. Top lanes show the kinase activation, and bottom lanes show a Western blot of Akt/PKB protein. (D) Comparative quantitation of values in C. Maximum value of wild-type cells is taken as 1.0. (E) We obtained the translocation kinetics of PhdA-GFP from time-lapse recordings. We quantified the fluorescence intensity of membrane-localization GFP fusion protein as E(t) using the linescan module of MetaMorph software. E/Eo(t) is plotted as a measure of the amount of membrane-associated protein relative to the starting condition.

Figure 8.

Kinetics of PI3K translocation to the cell cortex. (A) We obtained the translocation kinetics of PhdA-GFP from time-lapse recordings. We quantified the fluorescence intensity of membrane-localization GFP fusion protein as E(t) using the linescan module of MetaMorph software. E/Eo(t) is plotted as a measure of the amount of membrane-associated protein relative to the starting condition. (B and C) Chemoattractant-stimulated translocation of PI3K-GFP to the plasma membrane in wild-type (B) and iqgA⁻/B⁻ (C) cells. The middle panel shows the image from a time-lapse recording. The bottom panel shows the biochemical assay of PI3K-GFP. Cells were pushed through a Millipore membrane, and the membrane fractions were blotted with GFP antibody. (D) The basal level of PI3K-GFP in wild-type and iqgA⁻/B⁻ cells. The vegetative and pulsed cells were pushed through a Millipore membrane, and the membrane fractions were blotted with GFP and quantified.