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Address correspondence to Min Zhao, Dept. of Biomedical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK. Tel.: 44-1224-273001. Fax: 44-1224-273019. E-mail: m.zhao{at}abdn.ac.uk
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Abstract |
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Key Words: Dictyostelium; cell migration; electrotaxis; electric fields; G proteincoupled receptor signaling
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Introduction |
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Motile cells also detect physiological gradients in electrical potential and show directional migration (electrotaxis or galvanotaxis) or growth (galvanotropism) (Robinson, 1985). This includes bacteria (Rajnicek et al., 1994), fungi (Gow, 1994), amoeba (Korohoda et al., 2000), amphibian neuronal growth cones (Hinkle et al., 1981), fish and human keratinocytes (Cooper and Schliwa, 1986; Nishimura et al., 1996), and bovine and human corneal epithelial cells (Zhao et al., 1996). There is some subtlety in their responsiveness (McCaig et al., 2002). Most cells migrate cathodally, but corneal fibroblasts and lens epithelial cells migrate anodally (Soong et al., 1990; Wang et al., 2000). Neuronal growth cones are attracted cathodally and repelled anodally, but this is reversed when grown on positively charged polylysine (Rajnicek et al., 1998). In hippocampal neurones, dendrites are attracted cathodally, but axons are not (Davenport and McCaig, 1993). Corneal epithelial cells migrate cathodally, and this is enhanced on fibronectin or laminin (Zhao et al., 1999), but lens epithelial cells are attracted or repelled cathodally at electric field (EF) strengths that vary only twofold (E. Wang, personal communication; unpublished data). Polarized signaling of the EGFRMAP kinase pathway underpins directional migration of corneal epithelial cells in a physiological electric fields (Zhao et al., 2002).
Direct current (DC) EFs similar to those inducing these effects have been measured in areas where cells migrate developmentally and during wound healing (Jaffe and Stern, 1979; Chiang et al., 1992; Nuccitelli, 1992; Shi and Borgens, 1995). These arise because of spatial and temporal variations in epithelial transport or spatial variations in the tightness (electrical resistance) of epithelial sheets (Robinson and Messerli, 1996). Experimental disruption of such endogenous EFs indicates their physiological importance, since this both impairs wound healing and results in gross developmental abnormalities of embryonic nervous and skeletal systems. In each case, these defects could be the consequence of impaired directional cell migration (Hotary and Robinson, 1992; Sta Iglesia and Vanable, 1998). One interesting question is whether cells use similar directional sensing mechanisms to detect an EF and a chemical gradient.
Dictyostelium discoideum is a well-used model in which the pathway driving chemotaxis has been characterized. Dictyostelium also show robust electrotaxis. Although these amoebae would not naturally encounter DC EFs, they offer a powerful system in which to make molecular and genetic analyses of the mechanisms underpinning electrotaxis. In chemotaxis, G proteincoupled receptors sense chemoattractants and regulate pseudopod extension at the leading edge (Parent and Devreotes, 1999; Chung et al., 2001). We have investigated whether electrotaxis and chemotaxis share signaling mechanisms through G proteincoupled receptors. We show that the early stages of signal reception and transduction are not shared but that the respective signaling strategies converge somewhere upstream of directed actin polymerization.
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Results and discussion |
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No clear drop in response was found during several hours of exposure to an EF. Successive directedness values for the same cells were 0.96 ± 0.02 and 0.88 ± 0.04 for 1 and 2 h, respectively (20 V/cm). Trajectory and displacement speeds also were identical (7.6 ± 0.2, 7.6 ± 0.2 µm/min for trajectory speed and 2.5 ± 0.3, 2.5 ± 0.3 µm/min for displacement speed at both 1 and 2 h; n = 1124).
Electrotaxis has been reported in cells from humans to amoeba, and many experience endogenous EFs in vivo (see Introduction). The importance of studying the effects of DC EFs on cell migration includes (a) using EFs as a tool to understand direction sensing, (b) determining the involvement of endogenous EFs in directing cells, and (c) shaping tissues in vivo and using EFs to spatially control the engineering of cells and tissues. Because chemotaxis in Dictyostelium is well understood, this is an excellent model to study the molecular and genetic basis of electrotaxis and to compare and contrast the signaling strategies underpinning electrotaxis and chemotaxis.
cAR1-/cAR3-, G2-, Gß- cells maintained directional migration toward the cathode
Dictyostelium cells that enter the development stage use G proteincoupled receptor signaling to direct chemotactic migration to a source of cAMP. The most important receptor for this is cAR1. To test whether cAMP receptors were involved in electrotaxis, cAR1-/cAR3- cells (RI9) were used. These cells, which show no response to cAMP, maintained significant directional migration toward the cathode when stimulated with DC EFs (Fig. 2, A and G). The displacement speed and the directedness were comparable to wild-type cells (2.7 ± 0.2 µm/min, 0.8 ± 0.1, respectively; 20 V/cm, n = 36).
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The Gß subunit is essential for chemotaxis to all chemoattractants (Wu et al., 1995; Jin et al., 1998). However, although markedly suppressed, significant directional migration remained in Gß- cells in an EF (Figs. 2, C, D, F, and G). These data are robust. Gß- cells were processed exactly as wild-type cells (starved for 1 h, cAMP pulsed for an additional 12 h). The same cells were subjected to an EF of 20 V/cm. EF polarity was reversed twice (Fig. 2 C, C', and C''). Significant directional migration is seen in the composite trajectories of the cells. Cells moved to the left (cathode) at the beginning (Fig. 2 C), then to the right on reversing the polarity (Fig. 2 C'), and left again with a further polarity reversal (Fig. 2 C''). In each case, there was significant directedness toward the cathode (Fig. 2 D), and trajectory and displacement speeds remained virtually the same (Fig. 2 E). At 7 V/cm, Gß- cells maintained a directional migration of 0.25 ± 0.07 (n = 69; four experiments, significantly different from no field control), which was only half that of wild-type cells. Compared with wild-type, cAR1-/cAR3- and G2-, Gß- cells had significantly lower trajectory and displacement speed (P < 0.01) (Fig. 2 E compared with Fig. 1, F and G).
Heterotrimeric G proteins have crucial functions in Dictyostelium development. There are twelve G, one Gß, and one G
subunits (Devreotes, 1994; Brzostowski and Kimmel, 2001; Zhang et al., 2001). G
subunits are expressed at different stages, whereas Gß and G
are expressed throughout development. The Gß-null mutants are unable to form heterotrimeric G proteins, and their development is blocked at an early stage because of defects in gene induction (Wu et al., 1995; Jin et al., 1998). The G
2 deletion mutants are impaired in cAMP-mediated responses in the aggregation stage. Gß- cells display a slower speed in random movements compared with wild-type, cAR1-/cAR3- or G
2- cells, which may account for their lower trajectory and displacement speeds in an EF.
Electrotaxis does not engage G proteincoupled receptor signaling
Membrane recruitment of the PH domaincontaining protein CRAC (cytosolic regulator of adenylate cyclase, PHcrac) is an indicator of G protein signaling in cells. Using PHcrac fused with green fluorescent protein (GFP) (PHcrac-GFP) has provided significant insight in understanding the molecular mechanisms of chemotaxis. Asymmetry in signaling drives polarized actin changes needed for lamellipodium or pseudopodium extension and motility (Zigmond, 1996; Parent and Devreotes, 1999; Chung et al., 2001). Using GFP fusion constructs in Dictyostelium (PHcrac-GFP) and neutrophils (PHAkt-GFP) has shown that phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol-3,4-trisphosphate generated upon activation of the G proteincoupled receptors are polarized toward the chemoattractant source (Parent et al., 1998; Meili et al., 1999; Jin et al., 2000; Servant et al., 2000). Spatial regulation of PI 3-kinase and phosphatase activities therefore are crucial for directional sensing during G proteinmediated chemotaxis (Rickert et al., 2000; Chung et al., 2001).
In wild-type cells expressing PHcrac-GFP, there was no redistribution of PHcrac-GFP during electrotaxis or after polarity reversal (Fig. 3; Table S1 available at http://www.jcb.org/cgi/content/full/jcb.200112070/DC1 for quantitative results), indicating that the EF did not act upon the G protein subunits or their immediate effectors to direct movement.
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cAR1 receptor did not redistribute during steady state electrotaxis and reversal of EF
Membrane receptor redistribution may be involved in cell responses to EFs and has been demonstrated in several cell types (Poo and Robinson, 1977; Brown and Loew, 1994; McCaig and Zhao, 1997; Fang et al., 1999; Zhao et al., 1999, 2002). Cathodally directed migration of corneal epithelial cells involved induced asymmetry of membrane lipids and associated EGF receptors and asymmetric activation of MAP kinase signaling shown by leading edge asymmetry of dual phosphorylated extracellular signalregulated kinase (Zhao et al., 2002). Using cAR1-GFPexpressing cells, we monitored the dynamic distribution of receptors during electrotaxis in Dictyostelium. Neither obvious redistribution nor accumulation at the leading edge was observed during electrotaxis or during field polarity reversal (Fig. 4; Table S2 available at http://www.jcb.org/cgi/content/full/jcb.200112070/DC1). Therefore, EF-induced receptor asymmetry is a selective event, perhaps depending in part on the balance between the charge carried by the receptor and that on the membrane surface.
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Materials and methods |
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Cell migration was analyzed with a frame interval of 30 s using MetaMorph (Universal Imaging Corp.). Trajectories of cells were pooled to make composite graphs. The directedness of migration was assessed as cosine (Zhao et al., 1996), where
is the angle between the EF vector and a straight line connecting start and end positions of a cell. A cell moving directly to the left (cathodally) would have a directedness of 1; a cell moving directly to the right (anodally) would have a directedness of 1. A value close to 0 represents random cell movement. Therefore, the average directedness of a population of cells gives an objective quantification of how directionally cells have moved. The trajectory speed is the total length traveled by the cells divided by time, and the displacement speed is the straight line distance between the start and end positions of a cell, divided by time.
Fluorescence microscopy on live cells was performed as described previously (Parent et al., 1998). Statistical analysis was performed using Student's t tests. Data were mean ± SEM.
Online supplemental material
Details of cells used are available online at http://www.jcb.org/cgi/content/full/jcb.200112070/DC1. Video 1 shows wild-type cell electrotaxis. Videos 2, 3, and 4 show electrotactic responses of cAR1-/cAR3-, G2-, and Gß- cells, respectively. Videos 5 and 6 show PHcrac-GFP and cAR1-GFP distribution in wild-type Dictyostelium undergoing electrotaxis. Videos 7 and 8 show coronin-GFP distribution in wild-type and Gß- cells responding to DC EF.
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Footnotes |
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T. Jin's present address is Laboratory of Immunogenetics, NIH-NIAID, Twinbrook II Facility, 12441 Parklawn Dr., Rockville, MD 20852.
* Abbreviations used in this paper: DC, direct current; EF, electric field; GFP, green fluorescent protein; PH, pleckstrin homology.
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Acknowledgments |
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A Wellcome Trust Short Term Travel Award (062375/Z/00/Z) supported this work. M. Zhao is a Wellcome Trust University Award Lecturer (058551).
Submitted: 17 December 2001
Revised: 16 April 2002
Accepted: 16 April 2002
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References |
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Brown, M.J., and L.M. Loew. 1994. Electric field-directed fibroblast locomotion involves cell surface molecular reorganization and is calcium independent. J. Cell Biol. 127:117128.[Abstract]
Chiang, M., K.R. Robinson, and J.W. Vanable, Jr. 1992. Electrical fields in the vicinity of epithelial wounds in the isolated bovine eye. Exp. Eye Res. 54:9991003.[Medline]
Cooper, M.S., and M. Schliwa. 1986. Motility of cultured fish epidermal cells in the presence and absence of direct current electric fields. J. Cell Biol. 102:13841399.[Abstract]
Devreotes, P.N. 1994. G protein-linked signaling pathways control the developmental program of Dictyostelium. Neuron. 12:235241.[Medline]
Fang, K.S., E. Ionides, G. Oster, R. Nuccitelli, and R.R. Isseroff. 1999. Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields. J. Cell Sci. 112:19671978.
Gow, N.A. 1994. Growth and guidance of the fungal hypha. Microbiology. 140:31933205.[Medline]
Hinkle, L., C.D. McCaig, and K.R. Robinson. 1981. The direction of growth of differentiating neurones and myoblasts from frog embryos in an applied electric field. J. Physiol. 314:121135.[Abstract]
Hotary, K.B., and K.R. Robinson. 1992. Evidence of a role for endogenous electrical fields in chick embryo development. Development. 114:985996.[Abstract]
Jin, T., D.M. Soede, J. Liu, A.R. Kimmel, P.N. Devreotes, and P. Schaap. 1998. Temperature-sensitive Gß mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors. EMBO J. 17:50765084.
Jin, T., N. Zhang, Y. Long, C.A. Parent, and P.N. Devreotes. 2000. Localization of the G protein betagamma complex in living cells during chemotaxis. Science. 287:10341036.
McCaig, C.D., and M. Zhao. 1997. Physiological electrical fields modify cell behaviour. Bioessays. 19:819826.[Medline]
Meili, R., C. Ellsworth, S. Lee, T.B. Reddy, H. Ma, and R.A. Firtel. 1999. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J. 18:20922105.
Nishimura, K.Y., R.R. Isseroff, and R. Nuccitelli. 1996. Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds. J. Cell Sci. 109:199207.
Nuccitelli, R. 1992. Endogenous ionic currents and DC electric fields in multicellular animal tissues. Bioelectromagnetics. (Suppl 1):147157.
Parent, C.A., and P.N. Devreotes. 1999. A cell's sense of direction. Science. 284:765770.
Poo, M., and K.R. Robinson. 1977. Electrophoresis of concanavalin A receptors along embryonic muscle cell membrane. Nature. 265:602605.[Medline]
Rajnicek, A.M., C.D. McCaig, and N.A.R. Gow. 1994. Electric fields induce curved growth of Enterobacter cloacae and Escherichia coli cells: implications for mechanisms of galvanotropism and bacterial growth. J. Bacteriol. 176:702713.[Abstract]
Rickert, P., O.D. Weiner, F. Wang, H.R. Bourne, and G. Servant. 2000. Leukocytes navigate by compass: roles of PI3Kgamma and its lipid products. Trends Cell Biol. 10:466473.[CrossRef][Medline]
Robinson, K.R., and M.A. Messerli. 1996. Electric embryos: the embryonic epithelium as a generator of developmental information. Nerve Growth and Nerve Guidance. C.D. McCaig, editor. Portland Press, London. 131150.
Servant, G., O.D. Weiner, P. Herzmark, T. Balla, J.W. Sedat, and H.R. Bourne. 2000. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science. 287:10371040.
Soong, H.K., W.C. Parkinson, S. Bafna, G.L. Sulik, and S.C. Huang. 1990. Movements of cultured corneal epithelial cells and stromal fibroblasts in electric fields. Invest. Ophthalmol. Vis. Sci. 31:22782282.[Abstract]
Wang, E., M. Zhao, J.V. Forrester, and C.D. McCaig. 2000. Re-orientation and faster, directed migration of lens epithelial cells in a physiological electric field. Exp. Eye Res. 71:9198.[CrossRef][Medline]
Wu, L., R. Valkema, P.J. Van Haastert, and P.N. Devreotes. 1995. The G protein ß subunit is essential for multiple responses to chemoattractants in Dictyostelium. J. Cell Biol. 129:16671675.[Abstract]
Zhang, N., Y. Long, and P.N. Devreotes. 2001. G in Dictyostelium: its role in localization of gbetagamma to the membrane is required for chemotaxis in shallow gradients. Mol. Biol. Cell. 12:32043213.
Zhao, M., A. Agius-Fernandez, J.V. Forrester, and C.D. McCaig. 1996. Orientation and directed migration of cultured corneal epithelial cells in small electric fields are serum dependent. J. Cell. Sci. 109:14051414.
Zhao, M., A. Dick, J.V. Forrester, and C.D. McCaig. 1999. Electric fields directed cell motility involves upregulation and asymmetric redistribution of EGF receptor and is enhanced by fibronectin or laminin. Mol. Biol. Cell. 10:12591276.
Zhao, M., J. Pu, J.V. Forrester, and C.D. McCaig. 2002. Membrane lipids, EGF receptors and intracellular signals co-localize and are polarized in epithelial cells moving directionally in a physiological electric field. FASEB. J. 10.1096/fj.01-0811fje.