Steven Lenhert

Multiplexed Lipid Dip-Pen Nanolithography on Subcellular Scales for the Templating of Functional Proteins and Cell Culture

Molecular patterning processes taking place in biological systems are challenging to study in vivo because of their dynamic behavior, subcellular size, and high degree of complexity. In vitro patterning of biomolecules using nanolithography allows simplification of the processes and detailed study of the dynamic interactions. Parallel dip-pen nanolithography (DPN) is uniquely capable of integrating functional biomolecules on subcellular length scales due to its constructive nature, high resolution, and high throughput. Phospholipids are particularly well suited as inks for DPN since a variety of different functional lipids can be readily patterned in parallel. Here DPN is used to spatially pattern multicomponent micro- and nanostructured supported lipid membranes and multilayers that are fluid and contain various amounts of biotin and/or nitrilotriacetic acid functional groups. The patterns are characterized by fluorescence microscopy and photoemission electron microscopy. Selective adsorption of functionalized or recombinant proteins based on streptavidin or histidine-tag coupling enables the semisynthetic fabrication of model peripheral membrane bound proteins. The biomimetic membrane patterns formed in this way are then used as substrates for cell culture, as demonstrated by the selective adhesion and activation of T-cells.

Lipid multilayer gratings

The interaction of electromagnetic waves with matter can be controlled by structuring the matter on the scale of the wavelength of light, and various photonic components have been made by structuring materials using top-down or bottom-up approaches. Dip-pen nanolithography is a scanning-probe-based fabrication technique that can be used to deposit materials on surfaces with high resolution and, when carried out in parallel, with high throughput. Here, we show that lyotropic optical diffraction gratings--composed of biofunctional lipid multilayers with controllable heights between approximately 5 and 100 nm--can be fabricated by lipid dip-pen nanolithography. Multiple materials can be simultaneously written into arbitrary patterns on pre-structured surfaces to generate complex structures and devices, allowing nanostructures to be interfaced by combinations of top-down and bottom-up fabrication methods. We also show that fluid and biocompatible lipid multilayer gratings allow label-free and specific detection of lipid-protein interactions in solution. This biosensing capability takes advantage of the adhesion properties of the phospholipid superstructures and the changes in the size and shape of the grating elements that take place in response to analyte binding.

Nucleic acid supercoiling as a means for ionic switching of DNA--nanoparticle networks.

Oligomeric nanoparticle networks, generated by the self‐assembly of bis‐biotinylated double‐stranded DNA fragments and streptavidin, have been studied by scanning force microscopy (SFM). SFM imaging revealed the presence within the networks of irregular thick DNA molecules, which were often associated with distinct, Y‐shaped structural elements. Closer analysis revealed that the Y structures are formed by condensation (thickening and shortening) of two DNA fragments, most likely through the supercoiling of two DNA molecules bound to adjacent binding sites of the streptavidin particle. The frequency of supercoiling was found to be dependent on the ionic strength applied during the immobilization of the oligomeric networks on mica surfaces. Potential applications of the structural changes as a means for constructing ion‐dependent molecular switches in nanomaterials are discussed.

Common Genetic Pathways Regulate Organ-Specific Infection-Related Development in the Rice Blast Fungus

This study describes fungal infection–related development of Magnaporthe oryzae induced on rice roots and on hydrophilic polystyrene. A fungal mutant with abnormal preinfection hyphae and lacking the ortholog of the karyopherin exportin-5 had defects in full disease symptom production on leaves and roots, showing that this fungal karyopherin plays an important role during plant colonisation. Magnaporthe oryzae is the most important fungal pathogen of rice (Oryza sativa). Under laboratory conditions, it is able to colonize both aerial and underground plant organs using different mechanisms. Here, we characterize an infection-related development in M. oryzae produced on hydrophilic polystyrene (PHIL-PS) and on roots. We show that fungal spores develop preinvasive hyphae (pre-IH) from hyphopodia (root penetration structures) or germ tubes and that pre-IH also enter root cells. Changes in fungal cell wall structure accompanying pre-IH are seen on both artificial and root surfaces. Using characterized mutants, we show that the PMK1 (for pathogenicity mitogen-activated protein kinase 1) pathway is required for pre-IH development. Twenty mutants with altered pre-IH differentiation on PHIL-PS identified from an insertional library of 2885 M. oryzae T-DNA transformants were found to be defective in pathogenicity. The phenotypic analysis of these mutants revealed that appressorium, hyphopodium, and pre-IH formation are genetically linked fungal developmental processes. We further characterized one of these mutants, M1373, which lacked the M. oryzae ortholog of exportin-5/Msn5p (EXP5). Mutants lacking EXP5 were much less virulent on roots, suggesting an important involvement of proteins and/or RNAs transported by EXP5 during M. oryzae root infection.

High‐Quality Mapping of DNA–Protein Complexes by Dynamic Scanning Force Microscopy

Biomolecular complexes, nanocircles and aggregates of DNA and streptavidin (STV) are studied by dynamic scanning force microscopy. More structural details are observed with an improved dynamic mode assisted with a special feedback circuit (Q-control), as shown in the picture. Under otherwise identical conditions, these improvements indicate that the well cited models relating enlarged lateral size to the finite geometry of the SFM tip have to be modified for soft samples susceptible to tip-sample interactions.

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.

Lipid multilayer microarrays for in vitro liposomal drug delivery and screening

Screening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed. Small-molecule arrays and microfluidics are promising approaches. Here we introduce a simple method of surface-mediated delivery of drugs to cells from a microarray of phospholipid multilayers (layers thicker than a bilayer) encapsulating small molecules. The multilayer patterns are of sub-cellular dimensions and controllable thickness and were formed by dip-pen nanolithography. The patterns successfully delivered a rhodamine-tagged lipid and drugs only to the cells directly over them, indicating successful encapsulation and no cross-contamination to cells grown next to the patterns. We also demonstrated multilayer thickness-dependant uptake of the lipids from spots with sub-cellular lateral dimensions, and therefore the possibility of delivering different dosages from different areas of the array. The efficacies of two drugs were assayed on the same surface, and we were able to deliver dosages comparable to those of solution based delivery (up to the equivalent of 30 μg/mL). We expect our method to be a promising first step toward producing a single high-throughput liposome-based screening microarray plate that can be used in the same way as a standard well plate.

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.

A Self‐Correcting Inking Strategy for Cantilever Arrays Addressed by an Inkjet Printer and Used for Dip‐Pen Nanolithography

Dip-pen nanolithography (DPN) allows one to directly print a wide variety of biomaterials including DNA, phospholipids, and proteins on a surface with high registry and sub-50-nm resolution. The recent development of massively parallel DPN has substantially increased the throughput of DPN through the use of 2D pen arrays consisting of as many as 55 000 atomic force microscopy (AFM) cantilevers per square centimeter. Multiplexing, or the ability to simultaneously generate structures made of different materials, is the next step in developing a suite of DPN-based nanofabrication tools. Such capabilities will allow researchers to: 1) fabricate nanoarrays with unprecedented chemical and biochemical complexity; 2) control materials assembly through the use of affinity templates, such that each patterned feature controls the placement of different building blocks for fabricating higher-ordered architectures; and 3) develop an understanding of multivalent interactions between patterned surfaces and proteins, viruses, spores, and cells on a length scale that is biologically meaningful. Methods for multiplexing in the context of a DPN experiment thus far have been limited owing to the challenges associated with

In situ lipid dip‐pen nanolithography under water

Lipids form the structural and functional basis of biological membranes, and methods for studying their self-organization in well-defined nano- and micro-scale model systems can provide insights into biology. Using lipids as an ink for dip-pen nanolithography (lipid DPN) permits the rapid nanostructuring of multicomponent model lipid membrane systems, but this technique has so far been limited to air. Here we demonstrate that lipid DPN can be carried out under water with single tips or parallel arrays. Using the same tip for deposition and imaging in aqueous solution permits imaging of self-spreading lipid bilayer spots in situ and quantification of the nanoscale spreading kinetics in real time by means of lateral-force microscopy. Furthermore, using fluorophore-labeled phospholipids, we directly observed, by confocal laser scanning microscopy, a two-phase (oil in water) meniscus formed around the contact point between the DPN tip and surface, gaining insights into the mechanisms of the ink transport. The methods described here provide a new tool and environment for high-resolution studies of lipid nanodynamics and molecular printing processes in general.