Odor Discrimination by Lipid Membranes.

Odor detection and discrimination in mammals is known to be initiated by membrane-bound G-protein-coupled receptors (GPCRs). The role that the lipid membrane may play in odor discrimination, however, is less well understood. Here, we used model membrane systems to test the hypothesis that phospholipid bilayer membranes may be capable of odor discrimination. The effect of S-carvone, R-carvone, and racemic lilial on the model membrane systems was investigated. The odorants were found to affect the fluidity of supported lipid bilayers as measured by fluorescence recovery after photobleaching (FRAP). The effect of odorants on surface-supported lipid multilayer microarrays of different dimensions was also investigated. The lipid multilayer micro- and nanostructure was highly sensitive to exposure to these odorants. Fluorescently-labeled lipid multilayer droplets of 5-micron diameter were more responsive to these odorants than ethanol controls. Arrays of lipid multilayer diffraction gratings distinguished S-carvone from R-carvone in an artificial nose assay. Our results suggest that lipid bilayer membranes may play a role in odorant discrimination and molecular recognition in general.

Modulating the Excitability of Olfactory Output Neurons Affects Whole-Body Metabolism.

Metabolic state can alter olfactory sensitivity, but it is unknown whether the activity of the olfactory bulb (OB) may fine tune metabolic homeostasis. Our objective was to use CRISPR gene editing in male and female mice to enhance the excitability of mitral/tufted projection neurons (M/TCs) of the OB to test for improved metabolic health. Ex vivo slice recordings of MCs in CRISPR mice confirmed increased excitability due the targeted loss of Kv1.3 channels, which resulted in a less negative resting membrane potential (RMP), enhanced action potential (AP) firing, and insensitivity to the selective channel blocker margatoxin (MgTx). CRISPR mice exhibited enhanced odor discrimination using a habituation/dishabituation paradigm. CRISPR mice were challenged for 25 weeks with a moderately high-fat (MHF) diet, and compared with littermate controls, male mice were resistance to diet-induced obesity (DIO). Female mice did not exhibit DIO. CRISPR male mice gained less body weight, accumulated less white adipose tissue, cleared a glucose challenge more quickly, and had less serum leptin and liver triglycerides. CRISPR male mice consumed equivalent calories as control littermates, and had unaltered energy expenditure (EE) and locomotor activity, but used more fats for metabolic substrate over that of carbohydrates. Counter to CRISPR-engineered mice, by using chemogenetics to decrease M/TC excitability in male mice, activation of inhibitory designer receptors exclusively activated by designer drugs (DREADDs) caused a decrease in odor discrimination, and resulted in a metabolic profile that was obesogenic, mice had reduced EE and oxygen consumption (VO2). We conclude that the activity of M/TC projection neurons canonically carries olfactory information and simultaneously can regulate whole-body metabolism.SIGNIFICANCE STATEMENT The olfactory system drives food choice, and olfactory sensitivity is strongly correlated to hunger and fullness. Olfactory function thereby influences nutritional balance and obesity outcomes. Obesity has become a health and financial crisis in America, shortening life expectancy and increasing the severity of associated illnesses. It is expected that 51% of Americans will be obese by the year 2030. Using CRISPR gene editing and chemogenetic approaches, we discovered that changing the excitability of output neurons in the olfactory bulb (OB) affects metabolism and body weight stabilization in mice. Our results suggest that long-term therapeutic targeting of OB activity to higher processing centers may be a future clinical treatment of obesity or type II Diabetes.

Aptamer Functionalized Lipid Multilayer Gratings for Label-Free Analyte Detection.

Lipid multilayer gratings are promising optical biosensor elements that are capable of transducing analyte binding events into changes in an optical signal. Unlike solid state transducers, reagents related to molecular recognition and signal amplification can be incorporated into the lipid grating ink volume prior to fabrication. Here we describe a strategy for functionalizing lipid multilayer gratings with a DNA aptamer for the protein thrombin that allows label-free analyte detection. A double cholesterol-tagged, double-stranded DNA linker was used to attach the aptamer to the lipid gratings. This approach was found to be sufficient for binding fluorescently labeled thrombin to lipid multilayers with micrometer-scale thickness. In order to achieve label-free detection with the sub-100 nm-thick lipid multilayer grating lines, the binding affinity was improved by varying the lipid composition. A colorimetric image analysis of the light diffracted from the gratings using a color camera was then used to identify the grating nanostructures that lead to an optimal signal. Lipid composition and multilayer thickness were found to be critical parameters for the signal transduction from the aptamer functionalized lipid multilayer gratings.

Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings.

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid-protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (K D ) and kinetics (kon and koff ). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached.

Lipid Multilayer Grating Arrays Integrated by Nanointaglio for Vapor Sensing by an Optical Nose.

Lipid multilayer gratings are recently invented nanomechanical sensor elements that are capable of transducing molecular binding to fluid lipid multilayers into optical signals in a label free manner due to shape changes in the lipid nanostructures. Here, we show that nanointaglio is suitable for the integration of chemically different lipid multilayer gratings into a sensor array capable of distinguishing vapors by means of an optical nose. Sensor arrays composed of six different lipid formulations are integrated onto a surface and their optical response to three different vapors (water, ethanol and acetone) in air as well as pH under water is monitored as a function of time. Principal component analysis of the array response results in distinct clustering indicating the suitability of the arrays for distinguishing these analytes. Importantly, the nanointaglio process used here is capable of producing lipid gratings out of different materials with sufficiently uniform heights for the fabrication of an optical nose.

Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes.

The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion.

Evaporative edge lithography of a liposomal drug microarray for cell migration assays.

Lipid multilayer microarrays are a promising approach to miniaturize laboratory procedures by taking advantage of the microscopic compartmentalization capabilities of lipids. Here, we demonstrate a new method to pattern lipid multilayers on surfaces based on solvent evaporation along the edge where a stencil contacts a surface called evaporative edge lithography (EEL). As an example of an application of this process, we use EEL to make microarrays suitable for a cell-based migration assay. Currently existing cell migration assays require a separate compartment for each drug which is dissolved at a single concentration in solution. An advantage of the lipid multilayer microarray assay is that multiple compounds can be tested on the same surface. We demonstrate this by testing the effect of two different lipophilic drugs, Taxol and Brefeldin A, on collective cell migration into an unpopulated area. This particular assay should be scalable to test of 2000 different lipophilic compounds or dosages on a standard microtiter plate area, or if adapted for individual cell migration, it would allow for high-throughput screening of more than 50,000 compounds per plate.

Compartmentalization of the protein repair machinery in photosynthetic membranes.

A crucial component of protein homeostasis in cells is the repair of damaged proteins. The repair of oxygen-evolving photosystem II (PS II) supercomplexes in plant chloroplasts is a prime example of a very efficient repair process that evolved in response to the high vulnerability of PS II to photooxidative damage, exacerbated by high-light (HL) stress. Significant progress in recent years has unraveled individual components and steps that constitute the PS II repair machinery, which is embedded in the thylakoid membrane system inside chloroplasts. However, an open question is how a certain order of these repair steps is established and how unwanted back-reactions that jeopardize the repair efficiency are avoided. Here, we report that spatial separation of key enzymes involved in PS II repair is realized by subcompartmentalization of the thylakoid membrane, accomplished by the formation of stacked grana membranes. The spatial segregation of kinases, phosphatases, proteases, and ribosomes ensures a certain order of events with minimal mutual interference. The margins of the grana turn out to be the site of protein degradation, well separated from active PS II in grana core and de novo protein synthesis in unstacked stroma lamellae. Furthermore, HL induces a partial conversion of stacked grana core to grana margin, which leads to a controlled access of proteases to PS II. Our study suggests that the origin of grana in evolution ensures high repair efficiency, which is essential for PS II homeostasis.

Multifunctional lipid multilayer stamping.

Nanostructured lipid multilayers on surfaces are a promising biofunctional nanomaterial. For example, surface-supported lipid multilayer diffraction gratings with optical properties that depend on the microscale spacing of the grating lines and the nanometer thickness of the lipid multilayers have been fabricated previously by dip-pen nanolithography (DPN), with immediate applications as label-free biosensors. The innate biocompatibility of such gratings makes them promising as biological sensor elements, model cellular systems, and construction materials for nanotechnology. Here a method is described that combines the lateral patterning capabilities and scalability of microcontact printing with the topographical control of nanoimprint lithography and the multimaterial integration aspects of dip-pen nanolithography in order to create nanostructured lipid multilayer arrays. This approach is denoted multilayer stamping. The distinguishing characteristic of this method is that it allows control of the lipid multilayer thickness, which is a crucial nanoscale dimension that determines the optical properties of lipid multilayer nanostructures. The ability to integrate multiple lipid materials on the same surface is also demonstrated by multi-ink spotting onto a polydimethoxysilane stamp, as well as higher-throughput patterning (on the order of 2 cm(2) s(-1) for grating fabrication) and the ability to pattern lipid materials that could not previously be patterned with high resolution by lipid DPN, for example, the gel-phase phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or the steroid cholesterol.