Aqueous Two-Phase System Rehydration of Antibody-Polymer Microarrays Enables Convenient Compartmentalized Multiplex Immunoassays.

Multiplex immunoassays are rapidly increasing in popularity due to the offered advantages of increased throughput and decreased sample volume requirements. However, a major weakness inherent to multiplex enzyme-linked immunosorbent assays (ELISA) is generation of false signals through reagent-driven cross-talk. Typically, multiplex platforms necessitate bath application of antibody cocktails, increasing probability of nonspecific antibody binding, especially when multiplexing large numbers of analytes. Aqueous two-phase systems (ATPS) exploiting the phase-separating polymers poly(ethylene) glycol (PEG) and dextran (DEX) have been used to compartmentalize antibodies and prevent cross-talk in multliplex, plate-based ELISA. However, the resulting protocol is tedious and lengthy, and requires too many user steps to be practical for widespread use. Here, we report an improved, user-friendly, cross-talk-free multiplex ELISA method in which dehydrated arrays of colocalized capture and detection antibodies in DEX are prepared on multiwell plates. Addition of a PEG-based sample buffer rehydrates antibody/DEX droplets for analysis. In this report, we demonstrate rehydrated ATPS components for multiplex ELISA retain the ability to compartmentalize antibodies and prevent cross-talk, while analytes in sample buffer partition into rehydrated DEX droplets for analysis. Utility of this method was demonstrated through successful quantitative analysis of five inflammatory cytokines in lipopolysaccharide-stimulated ThP-1 cell culture supernatant.

Disruption of leptin receptor expression in the pancreas directly affects beta cell growth and function in mice.

Obesity is characterized by hyperinsulinemia, hyperleptinemia, and an increase in islet volume. While the mechanisms that hasten the onset of diabetes in obese individuals are not known, it is possible that the adipose-derived hormone leptin plays a role. In addition to its central actions, leptin exerts biological effects by acting in peripheral tissues including the endocrine pancreas. To explore the impact of disrupting leptin signaling in the pancreas on beta cell growth and/or function, we created pancreas-specific leptin receptor (ObR) KOs using mice expressing Cre recombinase under the control of the pancreatic and duodenal homeobox 1 (Pdx1) promoter. The KOs exhibited improved glucose tolerance due to enhanced early-phase insulin secretion, and a greater beta cell mass secondary to increased beta cell size and enhanced expression and phosphorylation of p70S6K. Similar effects on p70S6K were observed in MIN6 beta cells with knockdown of the ObR gene, suggesting crosstalk between leptin and insulin signaling pathways. Surprisingly, challenging the KOs with a high-fat diet led to attenuated acute insulin secretory response to glucose, poor compensatory islet growth, and glucose intolerance. Together, these data provide direct genetic evidence, from a unique mouse model lacking ObRs only in the pancreas, for a critical role for leptin signaling in islet biology and suggest that altered leptin action in islets is one factor that contributes to obesity-associated diabetes.

Quantitative monitoring of insulin secretion from single islets of Langerhans in parallel on a microfluidic chip.

Quantification of insulin release from pancreatic islets of Langerhans is of interest for diabetes research. Typical insulin secretion experiments are performed using offline techniques that are expensive, slow, have low-throughput, and require multiple islets. We have developed a microfluidic device for high-throughput, automated, and online monitoring of insulin secretion from individual islets in parallel. This chip consists of 15 channel networks each capable of superfusing a single islet and mixing superfusate from each islet online with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for a competitive immunoassay. The resulting continuous reaction streams are periodically injected onto parallel electrophoresis channels where the mixtures are separated. The resulting traces are used to quantify relative insulin released from islets. Serial immunoassays were performed at 10 s intervals on all 15 channels, corresponding to 5400 immunoassays per hour, to create temporally resolved insulin release profiles that captured single islet secretion dynamics. The chip was used to demonstrate that free fatty acid induced lipotoxicity in islets eliminates pulsatile insulin secretion.

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system.

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.

An assay for clogging the ciliary pore complex distinguishes mechanisms of cytosolic and membrane protein entry.

As a cellular organelle, the cilium contains a unique protein composition. Entry of both membrane and cytosolic components is tightly regulated by gating mechanisms at the cilium base; however, the mechanistic details of ciliary gating are largely unknown. We previously proposed that entry of cytosolic components is regulated by mechanisms similar to those of nuclear transport and is dependent on nucleoporins (NUPs), which comprise a ciliary pore complex (CPC). To investigate ciliary gating mechanisms, we developed a system to clog the pore by inhibiting NUP function via forced dimerization. We targeted NUP62, a component of the central channel of the nuclear pore complex (NPC), for forced dimerization by tagging it with the homodimerizing Fv domain. As proof of principle, we show that forced dimerization of NUP62-Fv attenuated (1) active transport of BSA into the nuclear compartment and (2) the kinesin-2 motor KIF17 into the ciliary compartment. Using the pore-clogging technique, we find that forced dimerization of NUP62 attenuated the gated entry of cytosolic proteins but did not affect entry of membrane proteins or diffusional entry of small cytosolic proteins. We propose a model in which active transport of cytosolic proteins into both nuclear and ciliary compartments requires functional NUPs of the central pore, whereas lateral entry of membrane proteins utilizes a different mechanism that is likely specific to each organelle's limiting membrane.

Analysis of ciliary import.

Cilia and flagella are microtubule-based organelles that play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in cilia formation and function cause a broad class of human genetic diseases called ciliopathies. To carry out their specialized functions, cilia contain a unique complement of proteins that must be imported into the ciliary compartment. In this chapter, we describe methods to measure the permeability barrier of the ciliary gate by microinjection of fluorescent proteins and dextrans of different sizes into ciliated cells. We also describe a fluorescence recovery after photobleaching assay to measure the entry of ciliary proteins into the ciliary compartment. These assays can be used to determine the molecular mechanisms that regulate the formation and function of cilia in mammalian cells.

A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia.

The cilium is a microtubule-based organelle that contains a unique complement of proteins for cell motility and signalling functions. Entry into the ciliary compartment is proposed to be regulated at the base of the cilium. Recent work demonstrated that components of the nuclear import machinery, including the Ran GTPase and importins, regulate ciliary entry. We hypothesized that the ciliary base contains a ciliary pore complex whose molecular nature and selective mechanism are similar to those of the nuclear pore complex. By microinjecting fluorescently labelled dextrans and recombinant proteins of various sizes, we characterize a size-dependent diffusion barrier for the entry of cytoplasmic molecules into primary cilia in mammalian cells. We demonstrate that nucleoporins localize to the base of primary and motile cilia and that microinjection of nucleoporin-function-blocking reagents blocks the ciliary entry of kinesin-2 KIF17 motors. Together, this work demonstrates that the physical and molecular nature of the ciliary pore complex is similar to that of the nuclear pore complex, and further extends functional parallels between nuclear and ciliary import.

Enhanced GLP-1- and sulfonylurea-induced insulin secretion in islets lacking leptin signaling.

We have previously reported that the absence of leptin signaling in ß-cells enhances glucose-stimulated insulin secretion and improves glucose tolerance in vivo. To investigate the relevance of ß-cell leptin signaling in the context of postprandial or therapeutic insulin secretion, we examined the cross talk between leptin and glucagon-like peptide (GLP)-1 and sulfonylurea actions. Single and size-matched islets isolated from control or pancreas-specific leptin receptor knockout (pancreas-ObR-KO) mice were treated either with GLP-1 or with glibenclamide. Leptin suppressed GLP-1-stimulated intracellular Ca(2+) concentrations ([Ca(2+)](i)) increase that paralleled the decrease in insulin secretion in controls. In contrast, and as expected, the ObR-KO islets were nonresponsive to leptin, and instead, showed a 2.8-fold greater GLP-1-stimulated [Ca(2+)](i) increase and a 1.7-fold greater insulin secretion. Phosphorylation of cAMP-responsive element binding protein was enhanced, and phosphodiesterase enzymatic activity was suppressed in MIN6 ß-cells with ObR knockdown compared with controls. The ObR-KO islets also showed significantly higher glibenclamide-induced insulin secretion compared with control islets, whereas [Ca(2+)](i) was similar to the controls. These data support enhanced insulinotropic effects of glucose, GLP-1, and sulfonylureas in the islets lacking leptin signaling with potential therapeutic implications.

Induction of Ran GTP drives ciliogenesis.

The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin-Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport.

Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP.

The biogenesis, maintenance and function of primary cilia are controlled through intraflagellar transport (IFT) driven by two kinesin-2 family members, the heterotrimeric KIF3A/KIF3B/KAP complex and the homodimeric KIF17 motor. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. Here, we identify a ciliary localization signal (CLS) in the KIF17 tail domain that is necessary and sufficient for ciliary targeting. Similarities between the CLS and classic nuclear localization signals (NLSs) suggest that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a ciliary-cytoplasmic gradient of the small GTPase Ran, with high levels of GTP-bound Ran (RanGTP) in the cilium. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with the nuclear import protein importin-beta2 in a manner dependent on the CLS and inhibited by RanGTP. We propose that Ran has a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments.

Glucose metabolism, islet architecture, and genetic homogeneity in imprinting of [Ca2+](i) and insulin rhythms in mouse islets.

We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+](i)) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed "islet imprinting." We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+](i) oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K(ATP)-channel opener that blocks [Ca2+](i) influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+](i) oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+](i) oscillations. Lastly, to test whether the imprinted [Ca2+](i) patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+](i) oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.

Multiplexed detection and applications for separations on parallel microchips.

Much work has been performed since the development of the lab-on-a-chip concept that has brought microfabricated systems to the forefront of bioanalytical research. The success of using these microchips for performing complicated biological assays faster and cheaper than conventional methods has facilitated their emerging popularity among researchers. A recently exploited advantage of microfabricated technology has led to the creation of single wafers with multiple channel manifolds for high-throughput experiments. Efforts toward parallel microchip development have yielded fascinating new devices for chemical separations showing the potential for replacing conventional multiplexing techniques. This review will focus on recent work toward multiplexed separations on microdevices and complementary detection instrumentation.

Serial immunoassays in parallel on a microfluidic chip for monitoring hormone secretion from living cells.

A microfluidic chip that allows for the continuous monitoring of cellular secretions from multiple independent living samples was developed. Performance of the device was characterized through the analysis of insulin secretion from islets of Langerhans. The chip contained four individual channel networks, each capable of performing electrophoresis-based immunoassays of the perfusate from islets. In the networks, islets were housed in a chamber that was continuously perfused with pressure-driven biological media at 0.6 microL min-1. Electroosmosis was used to pull perfusate containing secreted insulin into 4-cm-long reaction channels where it mixed with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for 60 s. The reaction streams were sampled at 6.25-s intervals and analyzed in parallel using an on-chip capillary electrophoresis separation with laser-induced fluorescence detection by a scanning confocal microscope. The limit of detection for insulin was 10 nM. The device was used to complete over 1450 immunoassays of biological samples in less than 40 min, allowing the parallel monitoring of insulin release from four islets every 6.25 s.