Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity.

Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, we find that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification.

Methods for studying planar cell polarity.

Planar cell polarity (PCP) is the polarity of epithelial cells in the plane orthogonal to the apical-basal axis, and is controlled by a partially defined signaling system. PCP related signaling also plays roles in cell migration, tissue re-organization and stem cell differentiation during embryonic development, and later, in regeneration and repair. Aberrant signaling has been linked to a broad range of pathophysiologies including cancer, developmental defects, and neurological disorders. The deepest mechanistic insights have come from studies of PCP in Drosophila. In this chapter we review tools and methods to study PCP signaling in Drosophila epithelia, where it was found to involve asymmetric protein localization that is coordinated between adjacent cells. Such signaling has been most extensively studied in wing, eye, and abdomen, but also in other tissues such as leg and notum. In the adult fly, PCP is manifested in the coordinated direction of hairs and bristles, as well as the organization of ommatidia in the eye. The polarity of these structures is preceded by asymmetric localization of PCP signaling proteins at the apical junctions of epithelial cells. Based on genetic and molecular criteria, the proteins that govern PCP can be divided into distinct modules, including the core module, the Fat/Dachsous/Four-jointed (Fat/Ds/Fj) module (often referred to as the 'global' module) as well as tissue specific effector modules. Different tissues and tissue regions differ in their sensitivity to disturbances in the various modules of the PCP signaling system, leading to controversies about the interactions among the modules, and emphasizing the value of studying PCP in multiple contexts. Here, we review methods including those generally applicable, as well as some that are selectively useful for analyses of PCP in eye (including eye discs), wing (including wing discs), pupal and adult abdomen, and the cuticle of larvae and embryos.

Probing enzymatic activity inside single cells.

We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 µM ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.

Microfluidic flow cell for sequential digestion of immobilized proteoliposomes.

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 µg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 µg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.

Single-cell electroporation.

Single-cell electroporation (SCEP) is a relatively new technique that has emerged in the last decade or so for single-cell studies. When a large enough electric field is applied to a single cell, transient nano-pores form in the cell membrane allowing molecules to be transported into and out of the cell. Unlike bulk electroporation, in which a homogenous electric field is applied to a suspension of cells, in SCEP an electric field is created locally near a single cell. Today, single-cell-level studies are at the frontier of biochemical research, and SCEP is a promising tool in such studies. In this review, we discuss pore formation based on theoretical and experimental approaches. Current SCEP techniques using microelectrodes, micropipettes, electrolyte-filled capillaries, and microfabricated devices are all thoroughly discussed for adherent and suspended cells. SCEP has been applied in in-vivo and in-vitro studies for delivery of cell-impermeant molecules such as drugs, DNA, and siRNA, and for morphological observations.

Control of the release of freely diffusing molecules in single-cell electroporation.

Single-cell electroporation using an electrolyte-filled capillary is an emerging technique for transient pore formation in adherent cells. Because adherent cells do not have a simple and consistent shape and because the electric field emanating from the tip of the capillary is inhomogeneous, the Schwan equation based on spherical cells in homogeneous electrical fields does not apply. We sought to determine experimental and cell parameters that influence the outcome of a single-cell electroporation experiment. A549 cells were exposed to the thiol-reactive dye Thioglo-1, leading to green fluorescence from intracellular thiol adducts. Electroporation causes a decrease with time of the intracellular fluorescence intensity of Thioglo-1-loaded cells from diffusive loss of thiol adducts. The transient curves thus obtained are well-described by a simple model originally developed by Puc et al. We find that the final fluorescence following electroporation is related to the capillary tip-to-cell distance and cell size (specifically, 2(A/pi)(1/2) where A is the area of the cell's image in pixels. This quantity is the diameter if the image is a circle). In separate experiments, the relationship obtained can be used to control the final fluorescence following electroporation by adjusting the tip-to-cell distance based on cell size. The relationship was applied successfully to A549 as well as DU 145 and PC-3 cells. Finally, F-tests show that the variability in the final fluorescence (following electroporation) is decreased when the tip-to-cell distance is controlled according to the derived relationship in comparison to experiments in which the tip-cell distance is a constant irrespective of cell size.

Direct access and control of the intracellular solution environment in single cells.

Methods that can control and vary the solution environment around single cells are abundant. In contrast, methods that offer direct access to the intracellular proteome and genome in single cells with the control, flexibility, and convenience given by microfluidic methods are both scarce and in great demand. Here, we present such a method based on using a microfluidic device mounted on a programmable scanning stage and cells on-chip permeabilized by the pore-forming glycoside digitonin. We characterized the on-chip digitonin poration, as well as the solution exchange within cells. Intracellular solution exchange times vary with the dose of exposure to digitonin from less than a second to tens of seconds. Also, the degree of permeabilization obtained for cells treated with the same dose varies considerably, especially for low doses of digitonin exposure and low permeabilities. With the use of the presented setup, the degree of permeabilization can be measured during the permeabilization process, which allows for "on-line" optimization of the digitonin exposure time. Using this calibrated permeabilization method, we demonstrate the generation of intracellular oscillations, intracellular gradients, and the delivery of substrate to initiate enzymatic reactions in situ. This method holds the potential to screen and titrate intracellular receptors or enzymes or to generate intracellular oscillations, useful in the study of signaling pathways and oscillation decoding among other applications.

Automated control of local solution environments in open-volume microfluidics.

We present an open-volume microfluidic system capable of on-line modification of a patterned laminar flow by using programmable inlet valves. Each separate solution environment in the flow pattern can be independently exchanged between different preloaded input solutions where each exchange requires 20 s. The number of flow patterns that can be generated by one device is N(n), where N represents the number of valve inlets and n the number of microchannels in the microfluidic system. Furthermore, the system can be operated as a combinatorial mixer, in which mixture of the different input solutions can be obtained independently in each channel. Since the flow patterns are generated in an open volume, they are accessible to many different detection methods and types of probes, e.g., microelectrodes, cells, or cell fragments. This technology offers the possibility to adjust the flow pattern composition in response to an output from a probe. This is the first step toward creating an automated feedback device controlled by, for example, biological cells.

Scanning electroporation of selected areas of adherent cell cultures.

We present a computer-controlled scanning electroporation method. Adherent cells are electroporated using an electrolyte-filled capillary in contact with an electrode. The capillary can be scanned over a cell culture and locally deliver both an electric field and an electroporation agent to the target area without affecting surrounding cells. The instantaneous size of the targeted area is determined by the dimensions of the capillary. The size and shape of the total electroporated area are defined by these dimensions in combination with the scanning pattern. For example, striped and serpentine patterns of electroporated cells in confluent cultures can be formed. As it is easy to switch between different electroporation agents, the method is suitable for design of cell cultures with complex composition. Finite element method simulations were used to study the spatial distributions of the electric field and the concentration of an electroporation agent, as well as the fluid dynamics related to scanning and flow of electroporation agent from the capillary. The method was validated for transfection by introduction of a 9-base-pair-long randomized oligonucleotide into PC12 cells and a pmaxGFP plasmid coding for green fluorescent protein into CHO and WSS cells.

Effect of cell size and shape on single-cell electroporation.

Single-cell electroporation was performed using electrolyte-filled capillaries on fluorescently labeled A549 cells. Cells were exposed to brief pulses (50-300 ms) at various cell-capillary tip distances. Cell viability and electroporation success were measured. In order to understand the variability in single-cell electroporation, logistic regression was used to determine whether the probabilities of cell survival and electroporation depend on experimental conditions and cell properties. Both experimental conditions and cell properties (size and shape) have a significant effect on the outcome. Finite element simulations were used to compare bulk electroporation to single-cell electroporation in terms of cell size and shape. Cells are more readily permeabilized and are more likely to survive if they are large and hemispherical as opposed to small and ellipsoidal with a high aspect ratio. The dependence of the maximum transmembrane potential across the cell membrane on cell size is much weaker than it is for bulk electroporation. Observed survival probabilities are related to the calculated fraction of the cell's surface area that is electroporated. Observed success of electroporation is related to the maximum transmembrane potential achieved.

Experimentally determining the iR drop in solution at carbon fiber microelectrodes with current interruption and application to single-cell electroporation.

Single-cell electroporation uses microelectrodes, capillaries, or micropipets positioned near single, adherent cells to increase transiently the membrane permeability of the cell. The increased permeability permits, for example, transfection without chemical reagents. When using microelectrodes to apply an electric field to the cell, there is a question of how much voltage to apply. Unlike in bulk electroporation, where hundreds of volts may be applied between electrodes, a rather small voltage is applied to a microelectrode in single-cell electroporation. In the single-cell experiment with microelectrodes, a substantial fraction of the voltage is lost at the interface and does not therefore exist in solution. This problem is the same as the classical electrochemist's problem of knowing the "iR" drop in solution and correcting for it to obtain true interfacial potential differences. Therefore, we have used current interruption to determine the iR drop in solution near microcylinder electrodes. As the field is inhomogeneous, calculations are required to understand the field distribution. Results of the current interruption are validated by comparing two independent measurements of the resistance in solution: one value results from the measured iR drop in conjunction with the known applied current. The other value results from a measured solution conductivity and a computed cell constant. We find substantial agreement in the range of resistances from about 2 to 50 kOmega, but not at higher resistances. We propose a simple, four-step plan that takes a few minutes to calculate the approximate current required to electroporate a cell with an electrode of a particular size, shape, and distance from the cell. We validate the approach with electroporation of single A549 cells.

Microfluidic gradient-generating device for pharmacological profiling.

We describe an on-chip microfluidic gradient-generating device that generates concentration gradients spanning nearly 5 orders of magnitude starting from a single concentration. The exiting stream of drugs held at different concentrations remains laminar in a recording chamber and can be presented as 24 discrete solutions to a cell-based sensor. The high-performance characteristics of the device are demonstrated by pharmacological screening of voltage-gated K+ channels (hERG) and ligand-gated GABA(A) receptors using scanning-probe patch-clamp measurements. Multiple data point dose-response curves and IC50 and EC50 values were rapidly obtained, typically in less than 30 min, through its combined functionality of gradient generation and open-volume laminar flow. The device facilitates rapid pharmacological profiling of ion channel and GPCR effectors and enables the acquisition of large numbers of data points with minute sample consumption and handling.

Generation of focused electric field patterns at dielectric surfaces.

We here report on a concept for creating well-defined electric field gradients between the boundaries of capillary electrode (a capillary of a nonconducting material equipped with an interior metal electrode) outlets, and dielectric surfaces. By keeping a capillary electrode opening close to a boundary between a conducting solution and a nonconducting medium, a high electric field can be created close to the interface by field focusing effects. By varying the inner and outer diameters of the capillary, the span of electric field strengths and the field gradient obtained can be controlled, and by varying the slit height between the capillary rim and the surface, or the applied current, the average field strength and gradient can be varied. Field focusing effects and generation of electric field patterns were analyzed using finite element method simulations. We experimentally verified the method by electroporation of a fluorescent dye (fluorescein diphosphate) into adherent, monolayered cells (PC-12 and WSS-1) and obtained a pattern of fluorescent cells corresponding to the focused electric field.

A chemical waveform synthesizer.

Algorithms and methods were developed to synthesize complex chemical waveforms in open volumes by using a scanning-probe microfluidic platform. Time-dependent variations and oscillations of one or several chemical species around the scanning probe, such as formation of sine waves, damped oscillations, and generation of more complex patterns, are demonstrated. Furthermore, we show that intricate bursting and chaotic calcium oscillations found in biological microdomains can be reproduced and that a biological cell can be used as a probe to study receptor functionalities as a function of exposure to time-dependent variations of receptor activators and inhibitors. Thus, the method allows for studies of biologically important oscillatory reactions. More generally, the system allows for detailed studies of complex time-varying chemical and physical phenomena in solution or at solution/surface interfaces.

Single-file electrophoretic transport and counting of individual DNA molecules in surfactant nanotubes.

We demonstrate a complete nanotube electrophoresis system (nanotube radii in the range of 50 to 150 nm) based on lipid membranes, comprising DNA injection, single-molecule transport, and single-molecule detection. Using gel-capped electrodes, electrophoretic single-file transport of fluorescently labeled dsDNA molecules is observed inside nanotubes. The strong confinement to a channel of molecular dimensions ensures a detection efficiency close to unity and identification of DNA size from its linear relation to the integrated peak intensity. In addition to constituting a nanotechnological device for identification and quantification of single macromolecules or biopolymers, this system provides a method to study their conformational dynamics, reaction kinetics, and transport in cell-like environments.

A microfluidics approach to the problem of creating separate solution environments accessible from macroscopic volumes.

We report on a microfluidic device that generates separate solution environments in macroscopic volumes. Spatially distinct patterns are created by emitting fluids from 16 different sources (closely spaced microchannels) into a solution-filled macroscopic chamber. The fluid in neighboring microchannels couples viscously in the macroscopic container, generating one single interdigitated stream. Scanning nanoelectrode amperometry was used for characterizing the concentration landscape and the diffusion zones between solutions running in parallel at different coordinates in the stream. These experiments were complemented by finite element simulations of the Navier-Stokes and mass transport equations to describe the velocity distributions and the diffusion behavior. For in channel flow velocities of 50 mm.s(-1), patterns could persist on the order of millimeters to centimeters in the open volume. The most narrow diffusion zones with widths less than 10 microm (5-95% concentration change) were found some tens of micrometers out in the macroscopic container. We demonstrate that a 14-microm-diameter nearly spherical object (biological cell) attached to a micropipet can be moved from one solution environment to another by a lateral displacement of only 8 microm. The device is suitable for applications where the solution environment around a microscopic or nanoscopic sensor needs to be changed multiple times, i.e., in order to build layered structures, for obtaining binding isotherms, and kinetic information, for example, on ion channels, enzymes, and receptors as well as in applications where different loci on an object need to be exposed to different environments or where complex solution environments need to be created for studies of interfacial chemistry between two streaming layers.

Single-cell electroporation.

Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.

Stabilization of high-resistance seals in patch-clamp recordings by laminar flow.

The formation of a high-resistance electrical seal between a cell membrane and a glass micropipet tip is essential in patch-clamp experiments. We have studied the electrical properties and the mechanical stability of the seal using a microfluidic chip generating laminar flow in open volumes. We show that, by using fluid flow (1-10 mm/s) acting along the symmetry axis of the cell-pipet, seals of a higher mechanical stability with increased resistances can be achieved, allowing up to 100% longer recording times and over 40% decreased noise levels (Irms). These improved properties are beneficial for high-sensitivity patch-clamp recordings, in particular, in longtime studies of ion channel receptor systems that are relevant in biosensor applications of the technique. Furthermore, these observations support the combination of patch-clamp with microfluidic devices, for example, for rapid solution exchange around a single cell sensor for high-throughput electrophysiology and for highly resolved kinetic studies.

A cell-based bar code reader for high-throughput screening of ion channel-ligand interactions.

This paper presents a microfluidics-patch clamp platform for performing high-throughput screening and rapid characterization of weak-affinity ion channel-ligand interactions. This platform integrates a microfluidic chip consisting of multiple channels entering an open volume with standard patch clamp equipment. The microfluidic chip is placed on a motorized scanning stage and the method relies on the ability to scan rapidly, on the order of milliseconds, a patch-clamped cell across discrete zones of different solutions created in the open volume. Under ideal conditions, this method has the capacity to obtain kinetically resolved patch clamp measurements and dose-response curves of up to 10(3) ligand solutions in a single day.