TIRF Imaging Research Articles

Cooperative Mechanics of Multi-Motor Axonal Transport Revealed by Novel Nanomanipulation in Live Neurons

Despite remarkable advances in characterizing molecular motors and microtubular transport in vitro, our understanding of intracellular cargo transport is rudimentary. The intracellular mechanochemical properties and cooperative mechanics of multiple motors, sharing load and coordinating cargo direction, are fundamental in this regard. To elucidate these, we developed two novel approaches for manipulating the transport of axonal endosomes, loaded with nanoprobes (<100nm) by receptor-mediated endocytosis, in microfluidic DRG cultures.Firstly, we show that by culturing neurons in a novel microfluidic-magnetic device, with axons aligned along high magnetic gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (MNP∼100nm). TIRF imaging of axonal MNP-endosome transport under external load reveals that mechanical means alone can transiently reverse endosome directionality. This suggests that mechanical force balance between kinesins and dyneins is critical for the regulation of endosome direction.Secondly, we show that laser-absorption by a gold nanoparticle (GNP∼80nm) in the endosome, stochastically results in the endosome being elastically tethered in axon (mechanism will be discussed). Motors driving the tethered endosome gradually come to a stall (like beads held by an optical trap) before detaching from the microtubule. The instantaneous recoil velocity of the endosome following lead-motor(s) detachment is proportional to the multi-motor stall force. We captured the recoil velocity distribution of dynein-driven retrograde and kinesin-driven anterograde endosomes using darkfield imaging at <2ms time, <5nm spatial resolutions. The peak structure of these distributions reflects discrete multi-motor stall forces and the peak-separation represents single-motor detachment velocity. Surprisingly, we find the single-motor detachment velocity for dynein comparable to that for kinesin suggesting comparable single-motor stall forces in this case. Further high-resolution motion analyses and stochastic modeling of GNP-endosome trajectories reveal several key signatures of cooperative mechanics, multi-motor force profiles and detachment kinetics.

Long-Term Live Observation of Membrane Protein Interaction with Lipid Nanodomains Show Dependence on Cell Cycle and Time After Transfection

The study of cholesterol-stabilized nanodomains in the cell membrane, aka lipid rafts, is complicated by differences in techniques, cell type, physiological parameters and possible changes of cell and membrane composition during the cell cycle and after transient transfection. We recently developed a non-destructive and non-perturbing technique combining fluorescence correlation spectroscopy (FCS) with spatially-resolved camera TIRF imaging, bimFCS. This method provides a quantification of the strength of the membrane protein - lipid nanodomain interaction, the membrane protein concentration and its diffusion coefficient. As an optical method it can be combined with other imaging methods such as we record changes in cytoskeleton structure simultaneously. bimFCS data was acquired with hourly intervals for periods longer than one day while keeping the cells growing at physiological temperature on the microscope stage. Transiently transfecting PtK2 or CHO cells with GPI-anchored membrane proteins associated with cholesterol-stabilized nanodomains, we observed a higher association with such domains in earlier stages of protein expression and slower diffusion compared to later times. Using synchronized cell cultures, we also quantify the membrane protein interaction with lipid nanodomains and record the images of membrane cytoskeletal actin bundles as function of the cell cycle. To discriminate between possible membrane ultrastructure models, we combine the experimental results with diffusion simulations of molecules interacting with such domains in the presence and absence of linear actin meshwork acting as pinpoints for the domains.

Real-time investigation of plasma membrane deformation and fusion pore expansion using polarized Total Internal Reflection Fluorescence Microscopy.

Polarized Total Internal Reflection Fluorescence Microscopy (pTIRFM) allows for real-time observation of plasma membrane deformations. The technique provides insights into the dynamics of biological processes requiring rapid and localized changes in membrane shape. Such processes include exocytosis, endocytosis, cytokinesis, and cell motility. In this chapter, we describe how to implement a polarization-based TIRF imaging system to monitor exocytosis in adrenal chromaffin cells.

Aurora B Inhibits MCAK Activity through a Phosphoconformational Switch that Reduces Microtubule Association

Proper spindle assembly and chromosome segregation rely on precise microtubule dynamics, which are governed in part by the kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood. Here, we develop the first Förster resonance energy transfer (FRET)-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) we show that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging, we find that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule. Unlike motile kinesins, which are open when doing work, the high-affinity binding state for microtubule-depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule-depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, our work provides a mechanism by which the robust microtubule depolymerization activity of kinesin-13s can be rapidly modulated to control cellular microtubule dynamics.

The Formin Daam1 and Fascin Directly Collaborate to Promote Filopodia Formation

Filopodia are slender cellular protrusions that dynamically extend and retract to facilitate directional cell migration, pathogen sensing, and cell-cell adhesion. Each filopodium contains a rigid and organized bundle of parallel actin filaments, which are elongated at filopodial tips by formins and Ena/VASP proteins. However, relatively little is known about how the actin filaments in the filopodial shaft are spatially organized to form a bundle with appropriate dimensions and mechanical properties. Here, we report that the mammalian formin Daam1 (Disheveled-associated activator of morphogenesis 1) is a potent actin-bundling protein and localizes all along the filopodial shaft, which differs from other formins that localize specifically to the tips. Silencing of Daam1 led to severe defects in filopodial number, integrity, and architecture, similar to silencing of the bundling protein fascin. This led us to investigate the potential relationship between Daam1 and fascin. Fascin and Daam1 coimmunoprecipitated from cell extracts, and silencing of fascin led to a striking loss of Daam1 localization to filopodial shafts, but not tips. Furthermore, purified fascin bound directly to Daam1, and multicolor single-molecule TIRF imaging revealed that fascin recruited Daam1 to and stabilized Daam1 on actin bundles in vitro. Our results reveal an unanticipated and direct collaboration between Daam1 and fascin in bundling actin, which is required for proper filopodial formation.

Mapping the Evolution of Molecular Flow Fields in Migrating Cells with Time-Resolved STICCS

Cell migration is a complex process that involves an intricate choreography between cytoskeleton and adhesion molecules with precise regulation of their transport and interactions in space and time in living cells. Although there have been many advances recently in improving spatial resolution of optical microscopy methods, temporal resolution remains a bottleneck for many methods. Previously we have reported spatio-temporal image cross-correlation spectroscopy (STICCS) in combination with TIRF microscopy to provide snap shots of flow fields of adhesion proteins and the cyctoskeleton in migrating cells. However, the temporal resolution of the STICCS transport maps was low compared to the frame time resolution of the TIRF imaging. Here we report the extension of STICCS to its absolute temporal resolution limit as set by the imaging frame rate by applying a short correlation window of 10 or fewer frames with the analysis window iterated sequentially by single frame steps. Although computationally more intensive, this enables us to capture the time evolution of the flow transport and interactions of adhesion components in living cells with a STICCS vector map for every frame of the TIRF movie. We illustrate the method with measurements of time evolving transport maps of the adhesion related macromolecules alpha5, alpha6 and alphaL integrins with paxillin, and actin within, or associated with the basal membrane in adherent U2OS and CHO.B2 cells plated on extracellular matrix components fibronectin, laminin, or ICAM-1. The time resolved cross-correlation vector maps clearly show that the dynamic interactions between alpha6 or alphaL integrins with paxillin evolve in space and time only at active adhesions in protruding and retracting regions of the cells.

A New Approach to Evaluation of Mitochondrial Ca-Signaling in Cardiomyocytes

Cardiac EC-coupling is mainly controlled by Ica-gated Ca-release, but the extent to which mitochondria contribute to the overall Ca-signaling remains controversial. To examine the possible role of mitochondria in Ca-signaling, we used a lower Ca-affinity mitochondrial probe, mitycamE31Q (300MOI, 48-72h) in conjunction with TMRE and Fura-2AM in cultured neonatal rat and adult feline cardiomyocytes using a dual wavelength TIRF imaging system (< 150nm penetration, 10Hz). Calcium was simultaneously measured in cytosol and mitochondria using mitycamE31Q and Fura-2. TMRE was used to monitor mitochondrial membrane potential. MitycamE31Q and TMRE staining of feline cardiomyocytes showed fluorescent longitudinal bands. Caffeine application caused a rapid decline in Fura-2 signal (increased Ca2+) that recovered slowly while mitycamE31Q signal initially increased (loss of [Ca2+]m or [H+]m) before decaying back to or below baseline ([Ca2+]m or [H+]m uptake). Simultaneously measured TMRE signal showed mitochondrial depolarization and repolarization. Low Na+ slowly decreases both Fura-2AM and mitycamE31Q signals, consistent with rise of cytosolic and mitochondrial Ca2+. FCCP produced little changes in the Fura-2AM signal, but increased the rate of spontaneous Ca-waves as it increased mitochondrial Ca2+ and depolarized mitochondrial membrane potential (increase TMRE signal). In contrast, single mitochondrial signals demonstrated heterogeneous populations of mitochondrial activity. Our findings suggest that Ca2+ in the mitochondria not only shows transients that are generally delayed and attenuated compared to the cytosolic Ca2+ signals, but at intervals can also be released locally and spontaneously. When synchronized, the local mitochondrial Ca2+ releases generate large cellular cytosolic Ca2+ releases that may interfere with normal Ca2+ signaling. (Support: NIH, RO1-HL16152, RO1-HL107600).

Simultaneous Imaging of Vesicle Trafficking and Calcium-Mediated Exocytosis in Pancreatic Beta-Cells

It has long been known that only a small fraction (<10%) of the insulin in pancreatic beta-cells can be released. Clinical treatments of type 2 diabetes focus on increasing insulin release, so an understanding of insulin vesicle trafficking and release may lead to novel therapeutic strategies. Towards this understanding, we have utilized a novel quantitative imaging assay, based on a double fusion to the integral vesicle protein phogrin. In this construct, a pH-dependent ecliptic pHluorinFP is inside the lumen of the granule and a mOrangeFP is outside the granule in the cytoplasm. The pHluorin fluorescence is quenched by the low pH inside the granule until exocytosis when the lumen of the granule mixes with the extracellular media. This increases the local pH and the green fluorescence can then be measured as a readout of exocytosis. Experiments were done on the multicolor TIRF system with a photoactivation module. We photoconverted the mOrange-phogrin to a deep red protein in the perinuclear region, and then watched where that population of granules went as we stimulated the cells with glucose. Having only a small subset of vesicles labeled greatly facilitates tracking. We used this approach with TIRF microscopy to measure secreted granules and determine if they came from previously docked granules or from regions beyond the TIRF imaging field. In beta-cells, we find that secreted vesicles do not come from a docked pool, with secretion happening within about 1 second of the vesicle's arrival at the membrane. This time is decreased even further with the addition of glucose. These data suggest that the releasable insulin pool in beta-cells may not be docked at the membrane as are synaptic vesicles, but rather are maintained deeper with in the cell.

The First Five Seconds in the Life of a Clathrin-Coated Pit

Coated pits assemble by growth of a clathrin lattice, which is linked by adaptors to the underlying membrane. How does this process start? We used live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution to detect arrival of the clathrin triskelions and AP2 adaptors that initiate coat assembly. Unbiased object identification and trajectory tracking, together with a statistical model, yield the arrival times and numbers of individual proteins, as well as experimentally confirmed estimates of the extent of substitution of endogenous by expressed, fluorescently tagged proteins. Pits initiate by coordinated arrival of clathrin and AP2, which is usually detected as two sequential steps, each of one triskelion with two adaptors. PI-4,5-P2 is essential for initiation. The accessory proteins FCHo1/2 are not; instead, they are required for sustained growth. This objective picture of coated pit initiation also shows that methods outlined here will be broadly useful for studies of dynamic assemblies in living cells.

High cellular organization of pyoverdine biosynthesis in Pseudomonas aeruginosa: clustering of PvdA at the old cell pole

Pyoverdine I (PVDI) is the major siderophore produced by Pseudomonas aeruginosa PAO1 to import iron. Its biosynthesis requires the coordinated action of cytoplasmic, periplasmic and membrane proteins. The individual enzymatic activities of these proteins are well known. However, their subcellular distribution in particular areas of the cytoplasm, periplasm, or within the membrane has never been investigated. We used chromosomal replacement to generate P.aeruginosa strains producing fluorescent fusions with PvdA, one of the initial enzymes in the biosynthetic pathway of PVDI in the cytoplasm, and PvdQ, involved in the maturation of PVDI in the periplasm. Cellular fractionation indicated that a substantial amount of PvdA-YFP was located in the membrane fraction. Epifluorescence microscopy imaging showed that PvdA-YFP was mainly clustered at the old cell pole of bacteria, indicating a polar segregation of the protein. Epifluorescence and TIRF imaging on cells expressing labelled PvdQ showed that this enzyme was uniformly distributed in the periplasm, in contrast with PvdA-YFP. The description of the intracellular distribution of these enzymes contributes to the understanding of the PVDI biosynthetic pathway.

N‐3 PUFAs reorganize B cell membrane rafts and molecular order accompanied by differential effects on B cell function

n‐3 PUFAs manipulate lipid raft organization to suppress T cell and macrophage function; however, little is known about this relationship with B cells, especially at the animal level. We previously established a high n‐3 PUFA dose remodeled mouse B cell raft clustering. To establish relevance, we first tested a n‐3 PUFA dose modeling human intake on B cell raft organization, relative to a control, using biochemical and quantitative imaging methods. Biochemical analysis revealed incorporation of docosahexaenoic acid into phosphatidylcholines of raft‐like domains with no change in cholesterol distribution. TIRF and fluorescence polarization imaging showed n‐3 PUFAs manipulated B cell raft size, increased GM1 expression, and increased membrane order upon cross‐linking GM1 molecules. Finally, we tested the hypothesis that modifying rafts would suppress B cell responses ex vivo. Contrary to expectations, n‐3 PUFAs enhanced LPS‐induced B cell activation, yet suppressed B cell stimulation of naïve CD4+ T cells. Overall, the results demonstrated a relevant n‐3 PUFA dose remodeled rafts; furthermore, the data showed raft remodeling was accompanied by unique changes in B cell function.Grant Funding Source: NIH R15AT006122

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)1. Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)1. Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways. One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

Axonal Traffic Control in Live Neurons by Tailor-Designed Magnetic Forces

The axon acts as a conduit for organized transport of material, between the cell body and the synapse, which is essential for the function and survival of neurons. Axonal traffic jams caused by local accumulation of cargo have been implicated in many neurodegenerative diseases. In order to study the neuronal response to axonal traffic jams we need new noninvasive assays capable of A) slowing/stalling axonal cargo by external forces to induce controlled traffic jams B) monitoring the perturbed transport and the ensuing neuronal response in real time. Here, we present an integrated methodology based on microfluidic neuron culture, high-gradient magnetic trapping and multi-color TIRF imaging that permits external control of axonal traffic in live neurons via magnetic forces. We fabricated a novel microfluidic device for neuron culture by patterned electrodeposition of soft micromagnets (permalloy) on glass coverslips. In the presence of an external magnetizing field, the soft micromagnetic pattern gives rise to local zones of high magnetic gradients. By culturing neurons in this device, with axons aligned along these high gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (MNPs, 50 nm). The magnetic forces counter the molecular motor forces to physically stall the endosomes, which leads to axonal traffic jams. The axonal growth and the delivery of MNP-loaded axonal endosomes along the high gradient zones are achieved by microfluidic compartmentalization of neuron culture. We have successfully A) compartmentalized DRG neurons in prototype magnetic devices B) characterized lectin-mediated axonal transport of 50 nm MNPs by pseudo-TIRF imaging, with/without external magnetic forces C) demonstrated the magnetic induction of controlled axonal traffic jams. These advances can potentially unravel the cooperative mechanics of multi-motor axonal transport and also elucidate the generic links between traffic jams, axonal swellings and neurodegeneration.

Membrane Protein Cluster Titration in Early Lymphocyte Signaling

T cells are known to be sensitive to extremely small numbers of agonist peptides. Early T cell signaling originates in and is sustained by T Cell Receptor (TCR) microclusters. Recent reports indicate that T cell triggering thresholds are determined by the number of agonist peptide in individual TCR microclusters, a conclusion supported by the observation that 2D peptide-MHC kinetics are characterized by unexpectedly fast on-rates. Yet, questions remain regarding the interplay, if any, between signaling microclusters associated with the plasma membrane. We have studied the relationship between peptide number and protein cluster formation in early T cell signaling by direct, simultaneous observation of fluorescently labeled agonist peptides and the early T cell signaling molecule Zap70 genetically fused to EGFP in primary murine T cells. Our experiments replace the antigen-presenting cell with a supported lipid bilayer (SLB) chemically functionalized with peptide-MHC and ICAM-1. This approach allows us to titrate the density of triggering agonist peptide, while also providing a flat interface suitable for high-resolution TIRF imaging. By integrating micro-patterned chromium supports onto a cover glass we can also segregate the SLB into an array of spatially distinct micron-scale reaction centers. Using this method we measure antigen input vs. TCR triggering output in living primary T cells. These results have important implications concerning the role of cooperativity between adjacent TCR clusters in tuning the sensitivity of T cell triggering by exogenous agonist peptides.

Multicolor Super-Resolution Imaging using a Laser Line Scanning Hyperspectral Microscope

Single-molecule super-resolution imaging techniques have proven to be successful in observing cellular structures with a resolution below the diffraction limit. However, these experiments often restrict observations to a region of shallow z-depth near the cover slip due to TIRF imaging or else suffer from high backgrounds due to wide field excitation. Confocal microscopy, which offers good background rejection, is prohibitively slow and cannot be used effectively for single-molecule localization based super-resolution. Here we introduce a new method that uses a fast, laser line scanning system that can be used to image a z-section anywhere within a cell and is not restricted to regions near the cover slip. The line scanning system allows a trade-off between acquisition speed and background rejection. Emission light is passed through a prism spectrometer, and a complete spectrum is collected for each spatial pixel. We exploit this hyperspectral emission path to make simultaneous measurements of multiple fluorophore species in the same sample.

Connexin43 Modulates Cell Polarity and Directional Cell Migration by Regulating Microtubule Dynamics

Knockout mice deficient in the gap junction gene connexin43 exhibit developmental anomalies associated with abnormal neural crest, primordial germ cell, and proepicardial cell migration. These migration defects are due to a loss of directional cell movement, and are associated with abnormal actin stress fiber organization and a loss of polarized cell morphology. To elucidate the mechanism by which Cx43 regulates cell polarity, we used a wound closure assays with mouse embryonic fibroblasts (MEFs) to examine polarized cell morphology and directional cell movement. Studies using embryonic fibroblasts from Cx43 knockout (Cx43KO) mice showed Cx43 deficiency caused cell polarity defects as characterized by a failure of the Golgi apparatus and the microtubule organizing center to reorient with the direction of wound closure. Actin stress fibers at the wound edge also failed to appropriately align, and stabilized microtubule (Glu-tubulin) levels were markedly reduced. Forced expression of Cx43 with deletion of its tubulin-binding domain (Cx43dT) in both wildtype MEFs and neural crest cell explants recapitulated the cell migration defects seen in Cx43KO cells. However, forced expression of Cx43 with point mutation causing gap junction channel closure had no effect on cell motility. TIRF imaging revealed increased microtubule instability in Cx43KO cells, and microtubule targeting of membrane localized Cx43 was reduced with expression of Cx43dT construct in wildtype cells. Together, these findings suggest the essential role of Cx43 gap junctions in development is mediated by regulation of the tubulin cytoskeleton and cell polarity by Cx43 via a nonchannel function.

Subcellular and single-molecule imaging of plant fluorescent proteins using total internal reflection fluorescence microscopy (TIRFM)

Total internal reflection fluorescence microscopy (TIRFM) has been proven to be an extremely powerful technique in animal cell research for generating high contrast images and dynamic protein conformation information. However, there has long been a perception that TIRFM is not feasible in plant cells because the cell wall would restrict the penetration of the evanescent field and lead to scattering of illumination. By comparative analysis of epifluorescence and TIRF in root cells, it is demonstrated that TIRFM can generate high contrast images, superior to other approaches, from intact plant cells. It is also shown that TIRF imaging is possible not only at the plasma membrane level, but also in organelles, for example the nucleus, due to the presence of the central vacuole. Importantly, it is demonstrated for the first time that this is TIRF excitation, and not TIRF-like excitation described as variable-angle epifluorescence microscopy (VAEM), and it is shown how to distinguish the two techniques in practical microscopy. These TIRF images show the highest signal-to-background ratio, and it is demonstrated that they can be used for single-molecule microscopy. Rare protein events, which would otherwise be masked by the average molecular behaviour, can therefore be detected, including the conformations and oligomerization states of interacting proteins and signalling networks in vivo. The demonstration of the application of TIRFM and single-molecule analysis to plant cells therefore opens up a new range of possibilities for plant cell imaging.

DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice β cells

Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single β-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2weeks. Perfusion studies showed that 2weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.

TIRF Imaging of Lipid Bilayer Arrays

A TIRF imaging platform for the simultaneous interrogation of multiple lipid bilayers in an arrayed format has been developed. Here, a proof of principle demonstration is made with the quantification of Calcium flux through the trans membrane pore, alpha-hemolysin. The effect of pore blocking by cyclodextrins is also demonstrated. Utilising a droplet on hydrogel method of lipid bilayer formation, an array of multiple lipid bilayers are illuminated simultaneously by the evanescent field of laser light undergoing total internal reflection at the surface of the glass hydrogel support layer. The droplet based method of bilayer production is ideally suited to an array platform, with the segregated contents of each droplet affording the opportunity to incorporate various membrane proteins, or simultaneously screen a variety of molecular candidates for their interaction with incorporated membrane components. The system offers the opportunity of asymmetric bilayer generation and the option of rapidly screening environmental dependencies for protein function. The scalable and automatable nature of the approach, together with the ability to tailor droplet contents, creates many exciting opportunities for characterisation of membrane protein behaviour and ligand interactions, the focus of future studies with the platform.