Correction: A bioenergetic mechanism for amoeboid-like cell motility profiles tested in a microfluidic electrotaxis assay.

Correction for 'A bioenergetic mechanism for amoeboid-like cell motility profiles tested in a microfluidic electrotaxis assay' by Hagit Peretz-Soroka et al., Integr. Biol., 2017, DOI: .

A bioenergetic mechanism for amoeboid-like cell motility profiles tested in a microfluidic electrotaxis assay.

The amoeboid-like cell motility is known to be driven by the acidic enzymatic hydrolysis of ATP in the actin-myosin system. However, the electro-mechano-chemical coupling, whereby the free energy of ATP hydrolysis is transformed into the power of electrically polarized cell movement, is poorly understood. Previous experimental studies showed that actin filaments motion, cytoplasmic streaming, and muscle contraction can be reconstituted under actin-activated ATP hydrolysis by soluble non-filamentous myosin fragments. Thus, biological motility was demonstrated in the absence of a continuous protein network. These results lead to an integrative conceptual model for cell motility, which advocates an active role played by intracellular proton currents and cytoplasmic streaming (iPC-CS). In this model, we propose that protons and fluid currents develop intracellular electric polarization and pressure gradients, which generate an electro-hydrodynamic mode of amoeboid motion. Such energetic proton currents and active streaming are considered to be mainly driven by stereospecific ATP hydrolysis through myosin heads along oriented actin filaments. Key predictions of this model are supported by microscopy visualization and in-depth sub-population analysis of purified human neutrophils using a microfluidic electrotaxis assay. Three distinct phases in cell motility profiles, morphology, and cytoplasmic streaming in response to physiological ranges of chemoattractant stimulation and electric field application are revealed. Our results support an intrinsic electric dipole formation linked to different patterns of cytoplasmic streaming, which can be explained by the iPC-CS model. Collectively, this alternative biophysical mechanism of cell motility provides new insights into bioenergetics with relevance to potential new biomedical applications.

Photobleaching of fluorescein as a probe for oxidative stress in single cells.

BACKGROUND: ROS are involved in the regulation of many physiological and pathological processes. Apoptosis and necrosis are processes that are induced by changes in concentrations of Reactive Oxygen Species (ROS). This study aims to detect and quantify the cellular response to changing ROS concentrations in the scope of apoptosis and necrosis. METHODS: Photobleaching of the fluorescent substrate fluorescein is used as a probe to detect the response of individual Jurkat-T-lymphocytes and Prostate-Cancer-3(PC-3) cells to oxidative stress, induced by hydrogen peroxide (H2O2). A kinetic model is proposed to describe changes in intracellular dye quantities due to photobleaching, dye hydrolysis, influx and leakage, yielding a single time-dependent decaying exponent+constant. RESULTS: Fluorescein photobleaching is controlled and used to detect intracellular ROS. An increase in the decay time of fluorescence of intracellular fluorescein (slow photobleaching) was measured from cells incubated with H2O2 at 50 µM. At higher H2O2 concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H2O2 in phosphate buffer saline (PBS), where the addition of H2O2 decreases the decay time, regardless of the irradiation dose used. CONCLUSIONS: The anomalous, ROS-concentration dependent reduction of the photobleaching rate in cells, as opposed to solutions, might indicate on the regulation of the activity of intracellular oxidative-stress protective mechanisms, as seen earlier with other methods. SIGNIFICANCE: Assessing photobleaching via the time decay of the fluorescence intensity of an ROS-sensitive fluorophore may be adapted to monitor oxidative stress or ROS-related processes in cells.

Optically-gated self-calibrating nanosensors: monitoring pH and metabolic activity of living cells.

Quantitative detection of biological and chemical species is critical to numerous areas of medical and life sciences. In this context, information regarding pH is of central importance in multiple areas, from chemical analysis, through biomedical basic studies and medicine, to industry. Therefore, a continuous interest exists in developing new, rapid, miniature, biocompatible and highly sensitive pH sensors for minute fluid volumes. Here, we present a new paradigm in the development of optoelectrical sensing nanodevices with built-in self-calibrating capabilities. The proposed electrical devices, modified with a photoactive switchable molecular recognition layer, can be optically switched between two chemically different states, each having different chemical binding constants and as a consequence affecting the device surface potential at different extents, thus allowing the ratiometric internal calibration of the sensing event. At each point in time, the ratio of the electrical signals measured in the ground and excited states, respectively, allows for the absolute concentration measurement of the molecular species under interest, without the need for electrical calibration of individual devices. Furthermore, we applied these devices for the real-time monitoring of cellular metabolic activity, extra- and intracellularly, as a potential future tool for the performance of basic cell biology studies and high-throughput personalized medicine-oriented research, involving single cells and tissues. This new concept can be readily expanded to the sensing of additional chemical and biological species by the use of additional photoactive switchable receptors. Moreover, this newly demonstrated coupling between surface-confined photoactive molecular species and nanosensing devices could be utilized in the near future in the development of devices of higher complexity for both the simultaneous control and monitoring of chemical and biological processes with nanoscale resolution control.

Functional analysis of individual cells and microenvironment of breast cancer-draining lymph nodes.

The development of distant metastases is the major cause of death in breast cancer (BC). In many BC cases, metastases are present in patients with no metastasis-positive lymph nodes (LN). Hence, there is a need to improve prognosis by a better prediction of the nodal status and tumor spread. The current study is designed to develop and utilize new functional characteristics of the cells and microenvironment of BC-draining LN, which may help to improve the estimation of LN metastatic involvement. Innovative devices and methodologies were developed for collecting, transferring, and analyzing LN at an individual-cell resolution. Using these devices, a suspension of living cells were prepared from the LN and processed for various assays, including immunophenotypic analysis, activation status, and invasion activity. The functional profile of tumor-activated LN cells showed an increase in the intracellular enzymatic reaction rate, accompanied by a homogeneous distribution of transferrin receptor as well as by a significant increase in matrix metalloproteinase proteolytic activity. Moreover, the proportion of cells exhibiting such a profile was significantly higher in tumor-containing LN than in tumor-free LN. Thus, the live and postfixation features of LN cells and their microenvironment, correlated with the functional status of the LN, may serve to improve their predictive value in breast cancer examination.

Tracing apoptosis and stimulation in individual cells by fluorescence intensity and anisotropy decay.

Presented is the use of fluorescence lifetime (FLT), anisotropy decay, and associated parameters as differential indicators of cellular activity. A specially designed combination of a frequency mode based time resolved microscope and a picoliter well-per-cell array have been used to perform temporal measurements in individual cells under various biological conditions. Two biological models have been examined: mitogenic activation of peripheral blood mononuclear cells (PBMC) and induction of programmed cell death (apoptosis) in Jurkat T cells (JTC). The FLT of fluorescein stained PBMC was found to increase from 4+/-0.02 to 4.5+/-0.025 ns due to mitogenic activation, whereas during apoptosis in fluorescein stained JTC, the FLT remained constant. Notably, the rotational correlation times changed in both models: decreased in PBMC from 2.5+/-0.08 to 2+/-0.1 ns, and increased in JTC from 2.1+/-0.07 to 3.3+/-0.09 ns. FLT and rotational correlation time were used to calculate the steady state fluorescence anisotropy (FA) which was compared to directly measured FA values. The present study suggests that in addition to bioindication, the said parameters can provide valuable information about cellular mechanisms that may involve complex molecular diffusion dynamics, as well as information about structural changes that a cellular fluorophore undergoes in the course of cell activation.

Fluorescence polarization: a novel indicator of cardiomyocyte contraction.

The changes measured in intracellular fluorescein fluorescence polarization (IFFP) are used as a new tool for tracing cytoplasmic effects during contractile cycles of cardiac myocytes (1-2-day-old rat hearts), in addition to the established Ca(2+) monitoring and/or videometric methods of tracking cell-shortening. This novel method was found to be non-intrusive to the contraction cycles. The decay of the transient IFFP signal (from 0.220+/-0.01 to 0.170+/-0.013) seems to be closely related to the extended phase of contractile activation. This fact was further supported when Ca(2+) exchanger inhibitor was introduced and significantly decreased (90%) the rate of beats of contraction and IFFP, but not the Ca(2+) beat rate changes. This result suggests that the IFFP indicator is probably associated with the physiological activation, rather than with Ca(2+) alterations. The IFFP measure monitors the average of effective changes in the micro-viscosity of the cytoplasm protein matrix, associated with cellular activation.