Embryonic cell motility can be guided by physiological electric fields

Migratory embryonic quail somitic fibroblasts display a striking sensitivity to small, steady electric fields. There are three components to their response. They begin to orient their long axes perpendicular to the field lines within 5 min of current application at the optimal field strength of 600 mV/mm. The threshold field for significant orientation in 90 min is 150 mV/mm (only 3 mV/cell width). The cells migrate toward the cathode with a similar low threshold. At field strengths greater than 400 mV/mm, the cells also elongate beginning about 1 h after field application. The importance of this embryonic cell galvanotaxis and orientation by electric fields lies in the possible utilization of this behavior both by the embryo in the guidance of embryonic cell migration in vivo and by the investigator to control cell morphology and directionality of movement in vitro in order to study mechanisms of motility.     

Embryonic fibroblast motility and orientation can be influenced by physiological electric fields

Epithelial layers in developing embryos are known to drive ion currents through themselves that will, in turn, generate small electric fields within the embryo. We hypothesized that the movement of migratory embryonic cells might be guided by such fields, and report here that embryonic quail somite fibroblast motility can be strongly influenced by small DC electric fields. These cells responded to such fields in three ways: (a) The cells migrated towards the cathodal end of the field by extending lamellipodia in that direction. The threshold field strength for this galvanotaxis was between 1 and 10 mV/mm when the cells were cultured in plasma. (b) The cells oriented their long axes perpendicular to the field lines. The threshold field strength for this response for a 90-min interval in the field was 150 mV/mm in F12 medium and between 50 and 100 mV/mm in plasma. (c) The cells elongated under the influence of field strengths of 400 mV/mm and greater. These fibroblasts were therefore able to detect a voltage gradient at least as low as 0.2 mV across their width. Electric fields of at least 10- fold larger in magnitude than this threshold field have been detected in vivo in at least one vertebrate thus far, so we believe that these field effects encompass a physiological range.     

Cell motility in a new single-cell wound model

Summary  Until now researchers have used a monolayer of cultured cells to investigate cell motility toward an injured cell. However, we suspect that, when using this method, adjacent cells move to the free space due to relief of contact inhibition. The current study was designed to investigate the cell motility nearby an injured cell in varying cell connectivity. A lowpower laser beam was used to damage one cell selectively with the silver coating beads. After injury, we observed the cell motility in three different cell types: (1) those immediately adjacent to the injured cell, 92) those removed from the injured cell by interposition of another cell, and (3) those removed from the injured cell by free space. The cells that are in direct contact with the injured cell moved toward the injured cell within 1.5–3.0 h. Indirectly connected cells and cells with no contact, on the other hand, showed no significant movement toward the injured cell. This suggests that the cell motility toward the cell injury is not only due to relief of contact inhibition but might also be caused by cell-to-cell signaling via cell connection. The current method will provide a tool to create a cell injury without damaging adjacent cells.     

Acting on actin: the electric motility assay

We have developed a novel technique which allows one to direct the two dimensional motion of actin filaments on a myosin coated sheet using a weak electric field parallel to the plane of motion. The filament velocity can be increased or decreased, and even reversed, as a function of orientation and strength of the field. PMMA (poly(methylmethacrylate)) gratings, which act as rails for actin, allow one for the first time to explore three quadrants of the force velocity diagram. We discuss effective friction, duty ratio and stall force at different myosin densities. A discontinuity in the velocity force relationship suggests the existence of dynamical phase transition. Received: 2 March 1998 / Accepted: 23 March 1998     

The developmental regulation of single-cell motility in Dictyostelium discoideum

The velocity of single amebae in the absence of a chemotactic signal has been analyzed during growth, development, rapid recapitulation, and dedifferentiation in the cellular slime mold Dictyostelium discoideum. It is demonstrated that (1) the velocity of axenically grown cells in half that of bacterially grown cells, (2) the velocity of bacterially grown cells decreased to roughly the same low level as axenically grown cells approximately 5 hr after the removal of exogeneous bacteria, (3) the velocity remains low for a 7-hr period preceding the onset of aggregation in both axenically and bacterially grown cells, (4) the velocity increases transiently at the onset of aggregation for both axenically and bacterially grown cells, (5) the velocity decreases to a very low level after the formation of loose aggregates and remains at that level at least through the early culminate I stage, (6) the velocity is not stimulated in 13-hr developing cells (finger stage) by inducing rapid recapitulation, (7) the velocity decreases after the erasure event in cultures of 7-hr developing cells (ripple stage) stimulated to undergo dedifferentiation, but the inhibition of the erasure event by the addition of 10(-4) M cAMP does not block this decrease. These results demonstrate that the basal level of single-cell motility in growing cultures is significantly influenced by the nutrient composition of the supporting medium, and that the transient increase in single-cell motility at the onset of aggregation is under the rigid control of the initial developmental program. Both rapid recapitulation and the program of dedifferentiation appear to have no influence on the basal level of single-cell motility.     

Quantifying the impact of membrane microtopology on effective two-dimensional affinity

Just as interactions of soluble proteins are affected by the solvent, membrane protein binding is influenced by the surface environment. This is particularly true for adhesion receptors because their function requires tightly apposed membranes. We sought to demonstrate, and further, to quantify the possible scale of this phenomenon by comparing the effective affinity and kinetic rates of an adhesion receptor (CD16b) placed in three distinct environments: red blood cells (RBCs), detached Chinese hamster ovary (CHO) cells, and K562 cells. Effective affinity reflects both the intrinsic receptor-ligand kinetics and the effectiveness of their presentation by the host membranes. Expression of CD16b, a low affinity Fcgamma receptor, was established by either transfection or spontaneous insertion via its glycosylphosphatidylinositol anchor. Binding to IgG-coated RBCs, measured using a micropipette method, indicated a 50-fold increase in effective affinity for receptors on RBCs over CHO and K562 cells, whereas the off rates were similar for all three. Electron microscopy confirmed that specific tight contacts were broad in RBC-RBC conjugates but sparse in CHO-RBC conjugates. We suggest that through modulation of surface roughness the cytoskeleton can greatly impact the effectiveness of adhesion molecules, even those with no cytoplasmic structures. Implications for locomotion and static adhesion are discussed.     

Quantifying the impact of protein tertiary structure on molecular evolution

To investigate the evolutionary impact of protein structure, the experimentally determined tertiary structure and the protein-coding DNA sequence were collected for each of 1,195 genes. These genes were studied via a model of sequence change that explicitly incorporates effects on evolutionary rates due to protein tertiary structure. In the model, these effects act via the solvent accessibility environments and pairwise amino acid interactions that are induced by tertiary structure. To compare the hypotheses that structure does and does not have a strong influence on evolution, Bayes factors were estimated for each of the 1,195 sequences. Most of the Bayes factors strongly support the hypothesis that protein structure affects protein evolution. Furthermore, both solvent accessibility and pairwise interactions among amino acids are inferred to have important roles in protein evolution. Our results also indicate that the strength of the relationship between tertiary structure and evolution has a weak but real correlation to the annotation information in the Gene Ontology database. Although their influences on rates of evolution vary among protein families, we find that the mean impacts of solvent accessibility and pairwise interactions are about the same.     

Quantifying the impact and relevance of scientific research

Qualitative and quantitative methods are being developed to measure the impacts of research on society, but they suffer from serious drawbacks associated with linking a piece of research to its subsequent impacts. We have developed a method to derive impact scores for individual research publications according to their contribution to answering questions of quantified importance to end users of research. To demonstrate the approach, here we evaluate the impacts of research into means of conserving wild bee populations in the UK. For published papers, there is a weak positive correlation between our impact score and the impact factor of the journal. The process identifies publications that provide high quality evidence relating to issues of strong concern. It can also be used to set future research agendas.     

Quantifying the impact of periphytic algae on nutrient availability for phytoplankton

SUMMARY.

• 1 Recent laboratory studies demonstrate that periphytic algae growing on the sediment surface reduce nutrient availability in the overlying water. Consequently, periphytic algae may competitively reduce growth of phytoplankton.
• 2 The aim of this study was to quantify the competitive impact of sediment-attached periphytic algae on phytoplankton in the presence of all other factors simultaneously affecting nutrient dynamics in natural systems.
• 3 In enclosure experiments, performed in three lakes of different productivity, the periphytic algal biomass was manipulated. When compared to enclosures with high biomass of periphytic algae, those with reduced biomass showed an increase in total phosphorus concentration in the water of 32–44%. Extrapolation of the experimental results to whole lakes predicts an increase in original total phosphorus concentration of between 1.5% and 8.0%. According to existing regressions between total phosphorus and phytoplankton chlorophyll, the potential increase in original phytoplankton biomass will be between 2.5% and 12.6%.
• 4 With respect to the shallow parts of lakes, my results support the conclusions revealed from laboratory studies that periphytic algae have a significant impact on the phosphorus concentration in the overlying water. However, when considering whole-lake dynamics, the competitive impact of periphytic algae on phytoplankton biomass development is probably of minor importance.
• 5 Rather, the main competitive advantage of growing on the sediment surface, compared to in the water, may be the exclusive access to nutrients in the sediment.

     

Quantifying the impact of ecological memory on the dynamics of interacting communities

Ecological memory refers to the influence of past events on the response of an ecosystem to exogenous or endogenous changes. Memory has been widely recognized as a key contributor to the dynamics of ecosystems and other complex systems, yet quantitative community models often ignore memory and its implications.Recent modeling studies have shown how interactions between community members can lead to the emergence of resilience and multistability under environmental perturbations. We demonstrate how memory can be introduced in such models using the framework of fractional calculus. We study how the dynamics of a well-characterized interaction model is affected by gradual increases in ecological memory under varying initial conditions, perturbations, and stochasticity.Our results highlight the implications of memory on several key aspects of community dynamics. In general, memory introduces inertia into the dynamics. This favors species coexistence under perturbation, enhances system resistance to state shifts, mitigates hysteresis, and can affect system resilience both ways depending on the time scale considered. Memory also promotes long transient dynamics, such as long-standing oscillations and delayed regime shifts, and contributes to the emergence and persistence of alternative stable states. Our study highlights the fundamental role of memory in communities, and provides quantitative tools to introduce it in ecological models and analyse its impact under varying conditions.     

Flagella and pili-mediated near-surface single-cell motility mechanisms in P. aeruginosa

Bacterial biofilms are structured multicellular communities that are responsible for a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near a surface is crucial for understanding the transition from the planktonic to the biofilm phenotype. By translating microscopy movies into searchable databases of bacterial behavior and developing image-based search engines, we were able to identify fundamental appendage-specific mechanisms for the surface motility of Pseudomonas aeruginosa. Type IV pili mediate two surface motility mechanisms: horizontally oriented crawling, by which the bacterium moves lengthwise with high directional persistence, and vertically oriented walking, by which the bacterium moves with low directional persistence and high instantaneous velocity, allowing it to rapidly explore microenvironments. The flagellum mediates two additional motility mechanisms: near-surface swimming and surface-anchored spinning, which often precedes detachment from a surface. Flagella and pili interact cooperatively in a launch sequence whereby bacteria change orientation from horizontal to vertical and then detach. Vertical orientation facilitates detachment from surfaces and thereby influences biofilm morphology.     

The influence of electric fields on bacterial growth

Growth of S.MARCESCENS (on TS agar) and of E.COLI (on EMB agar) was examined at various constant field strengths (growth being expressed by the diameter of colonies after incubation at 37°C for 46 hr). In the case of S. MARCESCENS the field strengths were calculated at 0, + or ÷ 940, and + or ÷ 3,125 v/cm;in the case of E.COLI at 0 and + or ÷ 3,125 v/cm. In experiments using S. MARCESCENS the mean diameters of colonies were found to be significantly reduced only at afield strength of + 940 v/cm as compared with diameters seen under field-free conditions. In experiments using E.COLI the mean diameters of the colonies were found to be significantly reduced at field strengths of + and ÷ 3,125 v/cm as compared with the diameters seen under field-free conditions.

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Zusammenfassung Das Wachstum von S.MARCESCENS (auf TG agar) und E.COLI (auf EBM agar) wurde bei verschiedenen konstanten Feldstärken untersucht. Für S. MARCESCENS wurden die Feldstärken berechnet bei 0, + oder 940 und + oder ÷ 3.125 v/cm;für E. COLI bei 0 und + oder ÷ 3.125 v/cm. Der mittlere Durchmesser der S.MARCESCENS Kolonien war nur bei der Feldstärke + 940 v/cm signifikant vermindert;der mittlere Durchmesser der E.COLI Kolonien war bei den Feldstärken von + und ÷ 3.125 v/cm signifikant vermindert, beide bezogen auf das Wachstum unter feldfreien Bedingungen.

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Resume On a observé la vitesse de croissance de S.MARCESCENS (sur Agar TS) et de E.COLI (sur Agar EBM) et cela dans divers champs électriques constants(la croissance était déterminée par le diamètre des colonies après une incubation de 46 heures à 37 °C). Pour S.MARCESCENS on a utilisé des champs de 0, + ou + 940 et + ou ÷ 3.125 v/cm. Pour E.COLI, les champs furent de 0 et de + ou ÷ 3.125 v/cm.Le diamètre moyen des colonies de S.MARCESCENS n'a été réduit de façon significative que pour un champ de + 940 v/cm. Le diamètre moyen des colonies de E.COLI a diminué de façon significative pour des champs de + ou ÷ 3.125 v/cm. Dans les deux cas, la diminution se rapporte aux conditions de croissances déterminées en l'absence de champ électrique.

     

Migration of isogenic cell lines quantified by dynamic multivariate analysis of single-cell motility

Cell migration is essential in many physiological and pathological processes. To understand this complex behavior, researchers have turned to quantitative, in vitro, image-based measurements to dissect the steps of cellular motility. With the rise of automated microscopy, the bottleneck in these approaches is no longer data acquisition, but data analysis. Using time-lapse microscopy and computer-assisted image analysis, we have developed a novel, quantitative assay that extracts a multivariate profile for cellular mo-tility. This technique measures three dynamic parameters per single cell: speed, surface area, and an in-dex of cell expansion/contraction activity (DECCA). Our assay can be used in combination with a variety of extracellular matrix components, or other soluble agents, to analyze the effects of the microenviron-ment on cellular migration dynamics in vitro. Our application was developed and tested using A431 and HT-1080 cell lines plated on laminin-332 or fibronectin substrates. Our results indicate that HT-1080 cells migrate faster, have a greater surface area, and have a higher DECCA index than A431 cells on both matrices (for all parameters, p < 0.05). Spearman’s correlation coefficients suggest that for these cell lines and matrices, various combinations of the three measurements display low to medium-high levels of correlation. These findings compare well with previous literature. Our approach provides new tools to measure cellular migration dynamics and address questions on the relationship between cell motility and the microenvironment, using only common microscopy techniques, accessible image analysis applica-tions, and a basic desktop computer for image processing.     

Residential magnetic and electric fields

A magnetic flux density (MFD) and electric-field (E-field) data-acquisition system was built for characterizing extremely low-frequency fields in residences. Every 2 min during 24-h periods, MFD and E-field measurements were made in 43 homes in King, Pierce, and Snohomish counties of Washington State. The total electrical energy used in each residence during the 24-h measurement period was also recorded, and maps were drawn to scale of the distribution wiring within 43 m (140 ft) of these homes. Finally, on a separate date, field measurements were made in each home during an epidemiological interview. The results of this study can be summarized as follows: 1) 24-h-average MFD measured at two separate points in the family room were correlated, as were a 24-h-average bedroom measurement and the mean of the two family-room measurements. 2) The 24-h-average family-room MFD and E-field measurements were uncorrelated. 3) The 24-h-average total harmonic distortions of family-room MFD and E-fields were less than about 24% and 7%, respectively. 4) Residential MFD exhibited a definite 24-h (diurnal) cycle. 5) The 24-h-average and interviewer-measured MFD were correlated. 6) Residential 24-h-average MFD were correlated with the wiring code developed by Wertheimer and Leeper. 7) An improved prediction of 24-h-average residential MFD was obtained using the total number of service drops, the distance to neighboring transmission lines, and the number of primary phase conductors.     

Cryosurgery with pulsed electric fields

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion.