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. 2007 Nov 5;179(3):403-10.
doi: 10.1083/jcb.200704169.

Positive Feedback Between Cdc42 Activity and H+ Efflux by the Na-H Exchanger NHE1 for Polarity of Migrating Cells

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Free PMC article

Positive Feedback Between Cdc42 Activity and H+ Efflux by the Na-H Exchanger NHE1 for Polarity of Migrating Cells

Christian Frantz et al. J Cell Biol. .
Free PMC article

Abstract

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H(+) efflux by Na-H(+) exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H(+) efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H(+) efflux by NHE1 is not necessary for release of Cdc42-guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5-bisphosphate is pH dependent, suggesting a mechanism for how H(+) efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells.

Figures

Figure 1.
Figure 1.
H+ efflux by NHE1 is necessary for activation of Cdc42 by different extracellular cues. (A) Time course of Cdc42 activity in WT and E266I cells after multiple wounding of a confluent monolayer. At the indicated times, Cdc42-GTP was determined by affinity precipitation with GST-PAK-CRIB and immunoblotting for Cdc42. Total Cdc42 was determined in cell lysates. Representative immunoblots are shown, and data, expressed relative to the abundance of Cdc42-GTP in WT monolayers (t = 0), represent means ± SEM of six cell preparations. (B) Total Cdc42 and Cdc42-GTP in cells in suspension (susp) or at the indicated times after plating on 10 μg/ml fibronectin were determined as in A. Data are expressed as means ± SEM of three separate cell preparations. (C and D) Abundance of Cdc42-GTP and total Cdc42 in subconfluent quiescent cells (t = 0) and at the indicated times after treating with PDGF were determined as described in A for WT and E266I cells (C) and for CCL39 and PS120 cells (D). Data for Cdc42-GTP are expressed relative to the abundance of precipitated Cdc42 in quiescent WT cells (t = 0) and represent means ± SEM of four separate cell preparations. White lines indicate that intervening lanes have been spliced out.
Figure 2.
Figure 2.
H+ efflux by NHE1 is necessary for active Cdc42-GTP at the front of migrating cells. (A) Probe used as a biosensor for endogenous Cdc42-GTP. MeroCBD is composed of the CRIB domain of neural Wiskott-Aldrich syndrome protein (blue), covalently labeled with the merocyanine dye Mero (red), and fused to EGFP (green). (B) Representative ratio images of EGFP fluorescence and MeroCBD probe fluorescence in cells at the edge of a wounded monolayer as determined by microinjecting the MeroCBD probe 15 h after wounding. Arrows indicate direction of migration. (C) Percentage of WT (n = 22) and E266I (n = 52) cells with active Cdc42-GTP in wound-edge protrusions. (D) Relative abundance of Cdc42-GTP in wound-edge cells determined by the Mero/EGFP fluorescence ratio. Data are expressed as means ± SEM of fluorescence ratios in WT (n = 12) and E266I (n = 18) cells. Bar, 15 μm.
Figure 3.
Figure 3.
H+ efflux by NHE1 is necessary for GEF activity but not for Cdc42 dissociation from RhoGDI or translocation to particulate fraction. (A) Lysates prepared from quiescent cells (t = 0) and cells treated with PDGF for the indicated times were incubated with antibodies directed against RhoGDIα, and coprecipitating proteins were immunoblotted for Cdc42 and RhoGDIα. Cdc42 abundance normalized to the abundance of RhoGDIα in immune complexes is expressed relative to values in WT cells at time 0. Data represent means ± SEM of four separate cell preparations. (B) Proteins in S100 and P100 fractions obtained from cell lysates were separated by SDS-PAGE and immunoblotted for Cdc42. The abundance of Cdc42 in P100 fractions was normalized to total Cdc42 in cell lysates and expressed relative to the abundance of Cdc42 in the particulate fraction at time 0. Data are the mean of two separate cell preparations. (C) Lysates from quiescent cells (control) and cells treated for 2 min with PDGF were incubated with recombinant [32P]GTP–preloaded Cdc42-GST in the presence of excess unlabeled GTP. At the indicated incubation times, radioactivity in aliquots of the incubation mix was determined by scintillation counting.
Figure 4.
Figure 4.
PI(4,5)P2 binding, but not guanine nucleotide exchange activity, for Cdc42 by the DH-PH domain of Dbs is pH sensitive. (A) Recombinant Cdc42-GST was loaded with mant-GDP and mixed with GTP alone (dashed lines) or with the recombinant DH-PH domain of Dbs and GTP (solid lines) in exchange buffer at the indicated pH values. Exchange of mant-GDP with GTP was measured every 8 s for 5 min, and data are representative of four separate preparations. (B and C) Recombinant DH-PH domains of Dbs (B) and intersectin (C) were incubated with lipid micelles without PI(4,5)P2 or containing 40 μM PI(4,5)P2 at the indicated pH values. The abundance of the DH-PH domain in pellets was normalized to nonspecific binding with lipid micelles in the absence of PI(4,5)P2 and calculated relative to the sum of DH-PH domain in supernatant and pellet. Data are expressed as a percentage of PI(4,5)P2 binding at pH 6.5 and represent means ± SEM of four preparations.

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