Microfluidic devices for studying chemotaxis and electrotaxis

Directed cell migration plays important roles in physiological processes such as host defense, wound healing, cancer metastasis and embryogenesis. Many organisms are capable of directional migration, which can be guided by diverse cellular factors including chemical and electrical cues. Recently, microfluidic devices that consist of small channels with micrometer dimensions are being developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus can be used to study the complex guiding mechanisms for cell migration. In this paper we highlight recent applications of microfluidic devices for cell migration research, with a focus on electric field-directed cell migration, to provide important and timely updates of this rapidly developing research field.     

On the role of calcium in chemotaxis and oscillations of dictyostelium cells

Migration of differentiated cells to a capillary containing cyclic AMP was enhanced in the presence of 1 mM CaCl2 and was virtually absent in the presence of 1 mM EGTA. Furthermore, the cells contracted and extended pseudopods to a capillary filled with the calcium ionophore A 23187. At short distances, migration to the tip of the capillary was observed, THe ionophore also induced transient decreases of the optical density of suspended cells indicating changes of cell shape. These findings support the hypothesis that cyclic AMP-binding to cell surface receptors causes a local influx of calcium ions. These in turn lead to an increase of the cytosolic calcium concentration and subsequently to an activation of cell migration. Perturbing pulses of the ionophore induced permanent phase shifts of free-running light scattering oscillations. This result indicates that cytosolic calcium is an intrinsic component of the oscillatory system.     

On the role of calcium in chemotaxis and oscillations of dictyostelium cells

Migration of differentiated cells to a capillary containing cyclic AMP was enhanced in the presence of 1 mM CaCl₂ and was virtually absent in the presence of 1 mM EGTA. Furthermore, the cells contracted and extended pseudopods to a capillary filled with the calcium ionophore A 23187. At short distances, migration to the tip of the capillary was observed. The ionophore also induced transient decreases of the optical density of suspended cells indicating changes of cell shape. These findings support the hypothesis that cyclic AMP-binding to cell surface receptors causes a local influx of calcium ions. These in turn lead to an increase of the cytosolic calcium concentration and subsequently to an activation of cell migration. Perturbing pulses of the ionophore induced permanent phase shifts of free-running light scattering oscillations. This result indicates that cytosolic calcium is an intrinsic component of the oscillatory system.Based on material presented at the Symposium Intercellular Communication, Stuttgart, September 16–17, 1982     

Superoxide plays critical roles in electrotaxis of fibrosarcoma cells via activation of ERK and reorganization of the cytoskeleton

Direct-current electrical field (DCEF) induces directional migration in many cell types by activating intracellular signaling pathways. However, the mechanisms coupling the extracellular electric stimulation to the intracellular signals remain largely unknown. In this study, we show that DCEF directs migration of HT-1080 fibrosarcoma cells to the cathode, stimulates generation of hydrogen peroxide and superoxide through the activation of NADPH oxidase, induces anode-facing cytoskeleton polarization, and activates ERK signaling. Subsequent studies demonstrate that the electrotaxis of HT-1080 fibrosarcoma cells is abolished by NADPH oxidase inhibitor and overexpression of manganese superoxide dismutase (MnSOD), an enzyme that hydrolyzes superoxide. In contrast, overexpression of catalases, which hydrolyze hydrogen peroxide, does not affect electrotaxis. MnSOD overexpression also eliminates cytoskeleton polarization as well as the activation of AKT, ERKs, and p38. In contrast, under catalase overexpression, the cytoskeleton still polarizes and p38 activation is affected. Finally, we show that inhibition of ERK activation also abolishes DCEF-induced directional migration and cytoskeleton polarization. Collectively, our results indicate that superoxide plays critical roles in DCEF-induced directional migration of fibrosarcoma cells, possibly by regulating the activation of ERKs. This study provides novel insights into the current understanding of DCEF-mediated cancer cell directional migration and metastasis.     

Ggamma in dictyostelium: its role in localization of gbetagamma to the membrane is required for chemotaxis in shallow gradients

G-protein-mediated signal transduction pathways play an essential role in the developmental program of the simple eukaryotic organism Dictyostelium discoideum. Database searches have yielded 11 Galpha-subunits, a single Gbeta-subunit, but no Ggamma-subunits. We report here the purification, cDNA isolation, and functional analysis of a Ggamma-subunit. Like Gbeta, the Ggamma appears to be unique and hybridization studies show that Ggamma and Gbeta are expressed in parallel during development. Species-wide sequence comparisons of Ggamma-subunits and gamma-like domains of RGS proteins reveal short stretches of highly conserved residues as well as the common CXXL motif at the COOH-terminal of Ggammas that target Gbetagammas to plasma membrane. Overexpression of a CSVL-deleted Ggamma (GgammaDelta) in wild-type cells shifts Gbetagamma to the cytosol and selectively impairs certain G-protein-mediated signal transduction pathways. These cells are able to respond to increments in the stimulus, but are unable to sense chemoattractant gradients. They neither move directionally nor recruit PH-domains to their leading edge. Thus, a full complement of membrane-tethered Gbetagamma is required for sensing shallow gradients, but is not essential for responses to increments in extracellular stimuli.     

Different roles of CheY1 and CheY2 in the chemotaxis of Rhizobium meliloti

Cells of Rhizobium meliloti swim by the unidirectional, clockwise rotation of their right-handed helical flagella and respond to tactic stimuli by modulating the flagellar rotary speed. We have shown that wild-type cells respond to the addition of proline, a strong chemoattractant, by a sustained increase in free-swimming speed (chemokinesis). We have examined the role of two response regulators, CheY1 and CheY2, and of CheA autokinase in the chemotaxis and chemokinesis of R. meliloti by comparing wild-type and mutant strains that carry deletions in the corresponding genes. Swarm tests, capillary assays, and computerized motion analysis revealed that (i) CheY2 alone mediates 60 to 70% of wild-type taxis, whereas CheY1 alone mediates no taxis, but is needed for the full tactic response; (ii) CheY2 is the main response regulator directing chemokinesis and smooth swimming in response to attractant, whereas CheY1 contributes little to chemokinesis, but interferes with smooth swimming; (iii) in a CheY2-overproducing strain, flagellar rotary speed increases upon addition and decreases upon removal of attractant; (iv) both CheY2 and CheY1 require phosphorylation by CheA for activity. We conclude that addition of attractant causes inhibition of CheA kinase and removal causes activation, and that consequent production of CheY1-P and CheY2-P acts to slow the flagellar motor. The action of the chief regulator, CheY2-P, on flagellar rotation is modulated by CheY1, probably by competition for phosphate from CheA.     

A regulator of G protein signaling-containing kinase is important for chemotaxis and multicellular development in dictyostelium

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax approximately 50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by approximately 40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at approximately 10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Galpha2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.     

Gravity and intracellular differences in membrane potentials of plant cells

The electrical potentials across the cell membranes of the lower parts of Zea mays coleoptile cells are about 2 millivolts more negative than across the upper parts. This electrical polarization with respect to gravity occurs when coleoptiles are oriented with their apical ends either up or down and seems independent of the magnitude of the potential when the potential is modified by other treatments.     

Key roles of enzyme positions and membrane surface potentials in the properties of biomimetic membranes

The coupling of diffusion, reaction, and electrostatic interactions existing in the microenvironment of permeable and charged bi-enzymatic membranes has led to the concept of biomimetic membranes. These membranes permit the active and specific transport of small hydrophilic molecules against their concentration gradients, at constant temperature and pressure. This study shows that such membranes behave in totally different ways, depending both on the relative position of the two enzymes, either within or outside the ionic double layer, and on the membrane surface potentials.     

Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate that in response to chemoattractants three PH domain-containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.     

Glycoproteins of the plasma membrane of dictyostelium discoideum during development

Differential binding of ¹²⁵I-Con A to whole cells throughout the life cycle of Dictyostelium discoideum indicates that the appearance of Con A binding proteins on the plasma membrane is under developmental regulation. Con A-Sepharose affinity chromatography of radio-iodinated plasma membrane preparations, followed by analysis with SDS-PAGE, revealed that there are at least 15 Con A binding proteins associated with the surface of 0 hr cells. Between 6 and 18 hr of development, the relative intensity of one of the bands, which corresponds to a protein of molecular weight of 150,000 daltons, increases dramatically.     

Cyanine dye as monitor of membrane potentials in Escherichia coli cells and membrane vesicles.

The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential.     

Lymphocyte electrotaxis in vitro and in vivo

Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.     

Assessment of membrane potentials of mitochondrial populations in living cells.

Mitochondrial membrane potentials (MMP) reflect the functional status of mitochondria within cells. Fluorescent probes to estimate these potentials within cells have been available for some time, but measurements of populations of mitochondria are not possible by existing methods. Therefore, comparisons between different cell types (e.g., fibroblasts and neuroblastoma), fibroblast cell lines from different patients, or even the same cell following various experimental paradigms are not feasible. The current approach estimates populations of MMP within living cells at 37 degrees C using the combination of conventional fluorescence microscopy and three-dimensional deconvolution by exhaustive photon reassignment. With this method, raw images are acquired rapidly with low-intensity (nonlaser) light with minimal concentrations of fluorescent dye. The method uses the fluorescent dye tetramethylrhodamine methyl ester, which equilibrates in cells according to the Nernst equation and provides a numerical, replicable estimate of MMP for populations of cellular mitochondria. This method can detect either increases or decreases in MMP as small as 5%. Furthermore, MMP in different cell types appear distinct. Values in fibroblasts (-105 +/- 0.9 mV) and N2a cells (-81 +/- 0.7 mV) were very different by this method. This approach bridges investigations of individual mitochondria to those that assess MMP by examining global fluorescence from cells.     

Mitogenic stimulation in isoproterenol treated cells of dictyostelium discoideum.

Amoebae of the cellular slime mould Dictyostelium discoideum showed stimulated mitogenic activity when exposed to 200 microM isoproterenol, an activator of adenyl cyclase, for 30 min. Approximately 40% increase in cell proliferation was found at 48 h after isoproterenol treatment. A faster and larger plaque formation as well as higher uptake of FITC-labelled E. coli indicates greater phagocytotic activity in the treated cells. A concurrent increase in DNA and protein syntheses was also recorded in the treated cells. Administration of 400 microM caffeine or 200 microM (+) propranolol brought down the isoproterenol-induced elevation in the cell division rate to control levels. These results are discussed in relation to a precocious activation of adenyl cyclase in the treated cells leading to a transient but significant increase in cell division in this organism.     

Compatible and incompatible rhizobia alter membrane potentials of soybean root cells

Inoculation with Rhizobium japonicum or R. meliloti reduced the electrical transmembrane potential (E_m) of soybean (Glycine max [L.] Merr.) root cells within 1 day. The response could be attributed to altered diffusion potential (E_D). E_m values return to control levels by the second day after inoculation, but again were reduced in R. meliloti-inoculated tissue on the seventh day. Increased concentrations of sodium phosphate in the perfusion solution magnified the effects of inoculation on E_m. Neither heat-killed rhizobia nor living cells of Pseudomonas fluorescens elicited the response. The E_m and E_D of nodule cells were nearly 20% lower than corresponding values from adjacent cortical cells of the root.     

Requirement of a vasodilator-stimulated phosphoprotein family member for cell adhesion,the formation of filopodia,and chemotaxis in dictyostelium

We have examined the function of a member of the vasodilator-stimulated phosphoprotein family of proteins (DdVASP) in Dictyostelium. Ddvasp null cells lack filopodia, whereas targeting DdVASP to the plasma membrane with a myristoyl tag results in a significant increase in filopodia. The proline-rich domain-Ena/VASP homology 2 structure is required for both actin polymerization activity and filopodia formation. Ddvasp null cells exhibit a chemotaxis defect, which appears to be due to a defect in the ability of the cells to properly adhere to the substratum and to suppress lateral pseudopod extension. We demonstrate that during chemotaxis, the anterior approximately 50% of the cell lifts from the substratum and remains elevated for up to 1 min. These defects lead to a significant decrease in chemotaxis efficiency. DdVASP localizes to the leading edge in migrating cells and to the tips of filopodia. In addition, Ddvasp null cells have a defect in particle adhesion but internalize particles normally. Our results provide new insights into the function of DdVASP in controlling the actin cytoskeleton during chemotaxis and filopodia formation.     

Elucidation of the multiple roles of CheD in Bacillus subtilis chemotaxis

Chemotaxis by Bacillus subtilis requires the CheD protein for proper function. In a cheD mutant when McpB was the sole chemoreceptor in B. subtilis, chemotaxis to asparagine was quite good. When McpC was the sole chemoreceptor in a cheD mutant, chemotaxis to proline was very poor. The reason for the difference between the chemoreceptors is because CheD deamidates Q609 in McpC and does not deamidate McpB. When mcpC‐Q609E is expressed as the sole chemoreceptor in a cheD background, chemotaxis is almost fully restored. Concomitantly, in vitro McpC activates the CheA kinase poorly, whereas McpC‐Q609E activates it much more. Moreover, CheD, which activates chemoreceptors, binds better to McpC‐Q609E compared with unmodified McpC. Using hydroxyl radical susceptibility in the presence or absence of CheD, the most likely sites of CheD binding were the modification sites where CheD, CheB and CheR carry out their catalytic activities. Thus, CheD appears to have two separate roles in B. subtilis chemotaxis – to bind to chemoreceptors to activate them as part of the CheC/CheD/CheYp adaptation system and to deamidate selected residues to activate the chemoreceptors and enable them to mediate amino acid chemotaxis.