Troy W. Lowry

Compartmentalization of the protein repair machinery in photosynthetic membranes

Significance The fitness and robustness of plants crucially depend on the molecular repair of the vulnerable photosystem II (PS II) supercomplex, embedded in photosynthetic thylakoid membranes. To maintain photosynthetic performance, plants evolved an efficient multistep PS II repair cycle. The PS II repair cycle relies on a well-defined order of reactions and partial separation of individual repair steps. By combining biochemical, spectroscopic, and ultrastructural techniques, we discover that plants establish reaction order and separation by confinement of the enzymes that catalyze the individual steps to spatially separated thylakoid subcompartments—grana, grana margins, and stroma lamellae—formed by the stacked membranes. Structural flexibility of the thylakoid architecture allows controlled access of the damaged PS II by the repair machinery. 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.

Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes

Background: The functional significance of semicrystalline protein states in photosynthetic membranes is unknown. Results: A mutant with high levels of semicrystalline PSII arrays shows facilitated diffusion of small lipophilic molecules but restricted mobility of large supercomplexes. Conclusion: The results indicate that supramolecular protein organizations control photoprotection, electron transport, and protein repair. Significance: Changes in supramolecular organization of thylakoid membranes seem to underlie acclimation processes. 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

Abstract: 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.

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.

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.