18 research outputs found
Parallel Separations on Microfluidic Chips for High Throughput Monitoring of Insulin Secretion from Single Islets of Langerhans.
Microfluidic devices for the simultaneous characterization of insulin release from four and 15 isolated pancreatic islets were developed. Quantification of released insulin from islet samples was performed using parallel immunoassays coupled to capillary electrophoresis with fluorescence detection. Assays for insulin were completed in a serial fashion on each channel every 6 – 10 s, giving fast temporal resolution used for investigations into insulin secretion dynamics. Assay limits of detection were between 0.5 – 10 nM insulin.
Individual islets were housed on the chips while perfusion streams carrying glucose or other secretagogues were used to stimulate insulin release. Secreted insulin was then mixed with fluorescently-labeled insulin and anti-insulin antibody in reaction channels for a competitive immunoassay. Portions of the continuously flowing reaction streams were injected onto separation channels where bound fluorescent insulin:antibody and free fluorescent insulin complexes were separated electrophoretically and detected via fluorescence. Relative amounts of these products were used to determine the amount of released insulin.
The 15-islet microchip was used to investigate possible roles of leptin signaling on insulin secretion. Through a collaborative effort, islets from mice lacking leptin receptors only in the pancreas were compared to control islets through insulin release studies. Specifically, the effects of leptin, glibenclamide, glucagon-like peptide-1, and palmitic acid on glucose-stimulated insulin secretion were investigated. It was observed that leptin produces an inhibitory effect on insulin release and that lack of leptin signaling in islets enhances insulin release stimulated with glucose.
Modifications were made to the 15-islet chip to ensure more uniform sampling of insulin from islets. The new islet sampling method was used to characterize oscillatory insulin release under various conditions. It was found that when treated appropriately, islets from individual mice displayed similar insulin secretion and Ca2+ flux oscillation frequencies. These frequencies were shown to be different from mouse to mouse, complementing previous studies. Additionally, the effects of free fatty acid-induced liptoxicity on pulsatile insulin release were investigated. Results from these experiments demonstrate the usefulness of single islet data not previously available at this level of throughput.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61556/1/johndish_1.pd
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 KATP-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
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 motor1,2. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. 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 (NLS) suggests that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a Ran-GTP gradient across the ciliary base. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with importin-β2 in a manner dependent on the CLS and inhibited by Ran-GTP. We propose that Ran plays a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments
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
Serial Immunoassays in Parallel on a Microfluidic Chip for Monitoring Hormone Secretion from Living Cells
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