Ion currents associated with root tips, emerging laterals and induced wound sites in Nicotians tabacum: spatial relationship proposed between resulting electrical fields and phytophthoran zoospore infection

Abstract Growing roots of Nicotiana tabacum var. Havana generate transcellular ion currents which traverse developing and wounded tissues. Positive current of around 10 mA m^?2 enters meristematic and elongating cells at the tip of primary roots. The growing tips of first order laterals are also traversed by a similar positive current with a density of around 2.0 mA m^?2, as are immature laterals emerging at the primary root surface. These self-generated ion currents flow basipetally through developing tissues and leave from mature non-elongating tissue. A large positive current of around 70 mA m^?2 also enters induced wound sites on the primary root surface. Motile zoospores of the fungal pathogen Phytophthora parasitica var. nicotianae have been reported to associate preferentially with these regions of the root. This might suggest that electrotaxis may be part of the mechanism by which zoospores locate root regions susceptable to fungal infection.     

Keratinocyte galvanotaxis in combined DC and AC electric fields supports an electromechanical transduction sensing mechanism

Sedentary keratinocytes at the edge of a skin wound migrate into the wound, guided by the generation of an endogenous electric field (EF) generated by the collapse of the transepithelial potential. The center of the wound quickly becomes more negative than the surrounding tissue and remains the cathode of the endogenous EF until the wound is completely re‐epithelialized. This endogenous guidance cue can be studied in vitro. When placed in a direct current (DC) EF of physiological strength, 100 V/m, keratinocytes migrate directionally toward the cathode in a process known as galvanotaxis. Although a number of membrane‐bound (e.g., epidermal growth factor receptor (EGFR), integrins) and cytosolic proteins (cAMP, ERK, PI3K) are known to play a role in the downstream signaling mechanisms underpinning galvanotaxis, the initial sensing mechanism for this response is not understood. To investigate the EF sensor, we studied the migration of keratinocytes in a DC EF of 100 V/m, alternating current (AC) EFs of 40 V/m at either 1.6 or 160 Hz, and combinations of DC and AC EFs. In the AC EFs alone, keratinocytes migrated randomly. The 1.6 Hz AC EF combined with the DC EF suppressed the direction of migration but had no effect on speed. In contrast, the 160 Hz AC EF combined with the DC EF did not affect the direction of migration but increased the migration speed compared to the DC EF alone. These results can be understood in terms of an electromechanical transduction model, but not an electrodiffusion/osmosis or a voltage‐gated channel model. Bioelectromagnetics 34:85–94, 2013. © 2012 Wiley Periodicals, Inc.     

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system^1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans^3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types^5,6, including neural progenitor cells (NPCs)^7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously^5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures^9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.     

磷脂酰肌醇3激酶通过Akt和ERK抑制盘基网柄菌细胞的趋电性

目的 磷脂酰肌醇3激酶（PI3Ks）通过调控肌动蛋白在细胞定向运动中发挥重要作用。然而,PI3Ks的结构和功能很复杂,人们对PI3Ks在细胞趋电性运动中的作用并不完全清楚。因此,本文以模式生物盘基网柄菌细胞为实验材料,探究其中的PI3K1和PI3K2在细胞趋电性运动中的作用。方法 首先利用CRISPR/Cas9系统介导分别构建PI3K1编码基因pikA基因敲除突变株和PI3K2编码基因pikB基因敲除突变株;随后将2个突变株置于强度为12 V/cm的直流电场中,记录并分析两个突变株的趋电性。结果 数据分析显示,野生型细胞在直流电场中的方向指数为（0.86±0.03）,而pikA-和pikB-突变株在直流电场中的运动方向指数分别为（0.95±0.02）和（0.94±0.03）;此外,野生型细胞在电场中的平均轨迹速度（3.34±0.08）μm/min,而pikA-和pikB-突变株的平均轨迹速度分别为（4.85±0.20）μm/min和（5.48±0.15）μm/min,t检验表明突变株和野生型的方向性指数和运动速度都存在极显著的差异。蛋白质印迹实验结果显示,pikA-和pikB-突变株中...     

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic microfluidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.     

Electrotaxis of zoospores of Phytophthora palmivora at physiologically relevant field strengths

Plant roots generate electrical fields in the rhizosphere as a consequence of their ion transport activities. We show here that zoospores of the plant pathogen Phytophthora palmivora exhibit anodal electrotaxis in electrical fields ≥0.5 V m⁻¹ comparable in size to the physiological fields around roots. An experimental protocol for applying weak electrical fields and quantifying electrotaxis is described. In this system, zoospore suspensions are isolated from the electrodes and their products using agarose bridges. Therefore, electrotaxis was not due to movement or trapping of zoospores in chemical, oxygen, pH or inhibitor gradients established by electrolysis. The electrophoretic and electroosmotic mobilities of encysted zoospores were measured. These forces did not influence the distribution of zoospores in electrotactic experiments at physiological field strengths. The electrotactic response saturated at fields above 10 V m⁻¹ was inhibited in media of osmotic strength below 400 Osmol m⁻³, was maximal at pH 7.5 and increased at high zoospore densities. These data suggest that electrotaxis may be a useful adjunct to chemotaxis in root targeting by zoospores.     

Electrotaxis of lung cancer cells in a multiple-electric-field chip

We report a microfluidic cell culture chip that was used for long-term electrotaxis study on a microscope. The cellular response under three different electric field strengths was studied in a single channel microfluidic chip. Electric field (EF) inside the microchamber was numerically simulated and compared to the measured value. Lung cancer cell lines with high and weak metastasis potential, CL1–5 and CL1–0, respectively, were used to demonstrate the function of the multi-field chip (MFC). The two cell lines exhibited greatly different response under the applied EF of E = 74–375 mV/mm. CL1–5 cells migrated toward the anode while CL1–0 cells did not show obvious response. Under the applied EF, cell orientation was observed accompanying the cell migration. Judging from the different temporal responses of the orientation and the migration, it is proposed that the two EF-induced responses may involve different signaling pathways.     

Genetic analysis of the role of G protein-coupled receptor signaling in electrotaxis

Cells display chemotaxis and electrotaxis by migrating directionally in gradients of specific chemicals or electrical potential. Chemotaxis in Dictyostelium discoideum is mediated by G protein–coupled receptors. The unique Gβ is essential for all chemotactic responses, although different chemoattractants use different receptors and Gα subunits. Dictyostelium amoebae show striking electrotaxis in an applied direct current electric field. Perhaps electrotaxis and chemotaxis share similar signaling mechanisms? Null mutation of Gβ and cAMP receptor 1 and Gα2 did not abolish electrotaxis, although Gβ-null mutations showed suppressed electrotaxis. By contrast, G protein signaling plays an essential role in chemotaxis. G protein–coupled receptor signaling was monitored with PHcrac–green fluorescent protein, which translocates to inositol phospholipids at the leading edge of cells during chemotaxis. There was no intracellular gradient of this protein during electrotaxis. However, F-actin was polymerized at the leading edge of cells during electrotaxis. We conclude that reception and transduction of the electrotaxis signal are largely independent of G protein–coupled receptor signaling and that the pathways driving chemotaxis and electrotaxis intersect downstream of heterotrimeric G proteins to invoke cytoskeletal elements.     

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson''s disease (PD), which involves the degeneration of dopaminergic (DA) neurons ¹. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds ^2,3.Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans'' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") ⁴. Reversing the field''s polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response ⁵. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae ⁴. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype ⁶.We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae ⁷. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel ⁸.Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.