Thomas Wilkop

Regenerable Tethered Bilayer Lipid Membrane Arrays for Multiplexed Label-Free Analysis of Lipid−Protein Interactions on Poly(dimethylsiloxane) Microchips Using SPR Imaging

We report a microfabrication approach to generate well-defined, addressable, and regenerable lipid membrane arrays in poly(dimethylsiloxane) (PDMS) microchips for label-free analysis of lipid-protein interactions with surface plasmon resonance imaging (SPRi). The multiplexed detection is demonstrated with a tethered bilayer membrane array built in parallel microchannels. These channels allow multiple measurements to be carried out simultaneously, showing low deviations for element-to-element variation in quantifiable signal. Lipid-conjugated receptors were utilized as model systems for protein binding analysis, and the feasibility of regenerating the tethering sublayer after binding was investigated. The results show that the lipid membrane can be removed effectively by nonionic surfactant Triton X-100. The small variance in SPR signal for the buildup process, i.e., <4% RSD for 3 cycles of detection, removal, and regeneration, indicates the sensing interface is highly reproducible. A calibration curve was obtained for cholera toxin using the monosialoganglioside (GM1) receptor, displaying a linear relationship in the 25 to 175 microg/mL range with a limit of detection of 260 nM. In addition, interaction of a phosphatidylinositol (PIP) with its binding protein and biotin/avidin interactions were employed for array measurements. To further enhance the SPR detection signal, a layer-by-layer amplification strategy was demonstrated that uses biotinylated antibody, NeutrAvidin and biotinylated anti-avidin, and the signal for protein binding on the membrane increased by 400%. The tethered membrane array technology, in combination with SPRi, offers an attractive platform for studies of membrane proteins, and can also find a range of applications for rapid screening of drug candidates interacting with proteins embedded in the near-native environment.

Bi-functionalization of a patterned Prussian blue array for amperometric measurement of glucose via two integrated detection schemes

A novel amperometric sensor that integrates two independent measurement schemes into a single chip for detection of glucose is fabricated. The sensor uses micro-patterned Prussian blue (PB) and ferrocene modified glucose oxidase covered by a thin Nafion membrane. We have developed an amperometric sensor for the detection of glucose that integrates two measurement schemes into a single chip. For fabrication of the sensing interface, micro-contact printing was used to transfer a self-assembled monolayer template onto a gold substrate, allowing selective electrochemical deposition of a PB array. The protective layer of the PB array was subsequently removed and replaced with a layer of redox-functionalized glucose oxidase (GOx), while the entire surface was finally covered with a perm-selective GOx-Nafion membrane. A variety of surface analytical techniques, including atomic force microscopy, surface plasmon resonance imaging and spectroscopic ellipsometry were employed to characterize the composite PB array electrode. The hybrid sensing interface allowed amperometric measurements of glucose to be carried out with two independent schemes at different potentials. The cathodic current was obtained with the PB array functioning as the electrocatalyst, while the anodic current was measured at a higher potential via a mediation mechanism using the ferrocene-modified GOx. For the quantitative detection of glucose, flow-injection analysis was used, and both the operating conditions and the design parameters were optimized. Linear responses were obtained for both anodic and cathodic signals over a concentration range from 0.1 to 50 mM, with a detection limit of 75 microM. The specificity of the sensor was demonstrated with respect to ascorbic and lactic acid. The implementation of integrated detection mechanisms allows the independent measurement of amperometric signals at two separate potentials. This improves the information gathering and opens up new avenues for developing novel methods that potentially eliminate false signal readings.

Endosidin 7 Specifically Arrests Late Cytokinesis and Inhibits Callose Biosynthesis, Revealing Distinct Trafficking Events during Cell Plate Maturation

Fluorescent protein markers and a newly discovered specific inhibitor of callose synthesis identify discrete trafficking events during late cytokinesis. Although cytokinesis is vital for plant growth and development, our mechanistic understanding of the highly regulated membrane and cargo transport mechanisms in relation to polysaccharide deposition during this process is limited. Here, we present an in-depth characterization of the small molecule endosidin 7 (ES7) inhibiting callose synthase activity and arresting late cytokinesis both in vitro and in vivo in Arabidopsis (Arabidopsis thaliana). ES7 is a specific inhibitor for plant callose deposition during cytokinesis that does not affect endomembrane trafficking during interphase or cytoskeletal organization. The specificity of ES7 was demonstrated (1) by comparing its action with that of known inhibitors such as caffeine, flufenacet, and concanamycin A and (2) across kingdoms with a comparison in yeast. The interplay between cell plate-specific post-Golgi vesicle traffic and callose accumulation was analyzed using ES7, and it revealed unique and temporal contributions of secretory and endosomal vesicles in cell plate maturation. While RABA2A-labeled vesicles, which accumulate at the early stage of cell plate formation, were not affected by ES7, KNOLLE was differentially altered by the small molecule. In addition, the presence of clathrin-coated vesicles in cells containing elevated levels of callose and their reduction under ES7 treatment further support the role of endocytic membrane remodeling in the maturing cell plate while the plate is stabilized by callose. Taken together, these data show the essential role of callose during the late stages of cell plate maturation and establish the temporal relationship between vesicles and regulatory proteins at the cell plate assembly matrix during polysaccharide deposition.

Development of air-stable, supported membrane arrays with photolithography for study of phosphoinositide-protein interactions using surface plasmon resonance imaging.

We report the development of an air-stable, supported membrane array by use of photolithography for label-free detection of lipid-protein interactions. Phosphoinositides and their phosphorylated derivatives (PIPs) were studied for their binding properties to proteins with lipid microarray in combination with surface plasmon resonance imaging (SPRi). We have demonstrated a simple method to fabricate lipid arrays using photoresist and carried out a series of surface characterizations with SPRi, ac impedance, cyclic voltammetry, and fluorescence microscopy to validate the array quality and lipid bilayer formation. A number of lipid compositions have been tested for the robustness of resulting membranes when undergoing dehydration and rehydration procedures, and the 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine/poly(ethylene glycol)-phosphatidylethanolamine (DOPC+/PEG-PE) system stands out as the best performer that yields the recovery to within 2% of the original state according to SPR sensorgrams. Limits of detection on the dehydrated/rehydrated DOPC+/PEG-PE membranes were determined to be 33 nM for avidin binding to biotinylated lipids, 73.5 nM for cholera toxin to GM1, and 25 nM for PtdIns(4,5)P2-binding protein (P(4,5)BP) to PtdIns(4,5)P2 lipid, respectively. These results demonstrate the suitability and sensitivity of this membrane for constructing membrane arrays for SPRi analysis under ambient conditions. With the use of this addressable and functional lipid membrane array, the screening of specific lipid-protein interactions has been conducted. Strong and specific interactions between P(4,5)BP and PtdIns(4,5)P2/DOPC+/PEG-PE membrane were observed as expected, while cross reactions were spotted for P(4,5)BP/PtdIns(4)P and avidin/GM1 at varied degrees. The air-stable membrane array demonstrated here presents a simple, effective approach for constructing functional membrane surfaces for screening applications, which opens a new avenue for the label-free study of membrane proteins and other forms of lipid-membrane interactions.

CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1

The compound CESTRIN reduces cellulose content and inhibits the trafficking of CESA in Arabidopsis hypocotyls. Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.

Electrochemical characterization of pore formation by bacterial protein toxins on hybrid supported membranes.

The interaction of pore-forming streptolysin O (SLO) with biomimetic lipid membranes has been studied by electrochemical methods. Phosphatidylcholine lipid vesicles were deposited onto gold electrodes modified with supporting layers of hexyl thioctate (HT) or thioctic acid tri(ethylene glycol) ester (TA-TEGE), and integrity and permeability of the resulting membranes were characterized by cyclic voltammetry and impedance spectroscopy. Both positively and negatively charged electrochemical probes, potassium ferrocyanide, hexaammineruthenium(III) chloride, and ferrocene carboxylic acid (FCA), were employed to evaluate their suitability to probe the membrane permeability properties, with FCA exhibiting ideal behavior and thus employed throughout the work. Fusion of vesicles incubated with SLO on the electrodes yielded membranes that showed a distinctive response pattern for FCA as a function of SLO concentration. A direct dependence of both the currents and peak separation of FCA in the cyclic voltammograms was observed over a concentration range of 0-10 hemolytic units (HU)/microL of the toxin. The interaction of SLO with preformed supported lipid membranes was also investigated, and much lower response was observed, suggesting a different extent of membrane-toxin interactions on such an interface. Nonionic surfactant Triton was found to disrupt the vesicle structure but could not completely remove a preformed membrane to fully restore the electrode response. The information reported here offers some unique insight into toxin-surface interactions on a hybrid membrane, facilitating the development of electrochemically based sensing platforms for detecting trace amounts of bacterial toxins via the perforation process.

On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging

The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein-ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for high-throughput analysis of their properties and constituents.

On-Demand Self-Assembly of Supported Membranes Using Sacrificial, Anhydrobiotic Sugar Coats

Borrowing principles of anhydrobiosis, we have developed a technique for self-assembling proteolipid-supported membranes on demand--simply by adding water. Intact lipid- and proteolipid vesicles dispersed in aqueous solutions of anhydrobiotic trehalose are vitrified on arbitrary substrates, producing glassy coats encapsulating biomolecules. Previous efforts establish that these carbohydrate coats arrest molecular mobilities and preserve native conformations and aggregative states of the embedded biomolecules, thereby enabling long-term storage. Subsequent rehydration, even after an extended period of time (e.g., weeks), devitrifies sugar--releasing the cargo and unmasking the substrate surface--thus triggering substrate-mediated vesicle fusion in real time, producing supported membranes. Using this method, arrays of membranes, including those functionalized with membrane proteins, can be readily produced in situ by spatially addressing vitrification using common patterning tools--useful for multiplexed or stochastic sensing and assaying of target interactions with the fluid and functional membrane surface.

The RAB GTPase RABA1e localizes to the cell plate and shows distinct subcellular behavior from RABA2a under Endosidin 7 treatment.

Cytokinesis in plants requires the activity of RAB GTPases to regulate vesicle-mediated contribution of material to the developing cell plate. While some plant RAB GTPases have been shown to be involved in cell plate formation, many still await functional assignment. Here, we report cell plate localization for YFP-RABA1e in Arabidopsis thaliana and use the cytokinesis inhibitor Endosidin 7 to provide a detailed description of its localization compared to YFP-RABA2a. Differences between YFP-RABA2a and YFP-RABA1e were observed in late-stage cell plates under DMSO control treatment, and became more apparent under Endosidin 7 treatment. Taken together, our results suggest that individual RAB GTPases might make different contributions to cell plate formation and further demonstrates the utility of ES7 probe to dissect them.

Unconventional Protein Secretion in Plants

Unconventional protein secretion (UPS) describes secretion pathways that bypass one or several of the canonical secretion pit-stops on the way to the plasma membrane, and/or involve the secretion of leaderless proteins. So far, alternatives to conventional secretion were primarily observed and studied in yeast and animal cells. The sessile lifestyle of plants brings with it unique restraints on how they adapt to adverse conditions and environmental challenges. Recently, attention towards unconventional secretion pathways in plant cells has substantially increased, with the large number of leaderless proteins identified through proteomic studies. While UPS pathways in plants are certainly not yet exhaustively researched, an emerging notion is that induction of UPS pathways is correlated with pathogenesis and stress responses. Given the multitude UPS events observed, comprehensively organizing the routes proteins take to the apoplast in defined UPS categories is challenging. With the establishment of a larger collection of studied plant proteins taking these UPS pathways, a clearer picture of endomembrane trafficking as a whole will emerge. There are several novel enabling technologies, such as vesicle proteomics and chemical genomics, with great potential for dissecting secretion pathways, providing information about the cargo that travels along them and the conditions that induce them.