Neurodegenerative disorders including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, and stroke are rapidly increasing as population ages. The field of nanomedicine is rapidly expanding and promises revolutionary advances to the diagnosis and treatment of devastating human diseases. This paper provides an overview of novel nanomaterials that have potential to improve diagnosis and therapy of neurodegenerative disorders. Examples include liposomes, nanoparticles, polymeric micelles, block ionomer complexes, nanogels, and dendrimers that have been tested clinically or in experimental models for delivery of drugs, genes, and imaging agents. More recently discovered nanotubes and nanofibers are evaluated as promising scaffolds for neuroregeneration. Novel experimental neuroprotective strategies also include nanomaterials, such as fullerenes, which have antioxidant properties to eliminate reactive oxygen species in the brain to mitigate oxidative stress. Novel technologies to enable these materials to cross the blood brain barrier will allow efficient systemic delivery of therapeutic and diagnostic agents to the brain. Furthermore, by combining such nanomaterials with cell-based delivery strategies, the outcomes of neurodegenerative disorders can be greatly improved.

The study of protein interactions with DNA is important to gain a fundamental understanding of how numerous biological processes occur, including recombination, transcription, repair, etc. In this study, we use the EcoRII restriction enzyme, which employs a three-site binding mechanism in order to catalyze cleavage of a single recognition site. Using high-speed atomic force microscopy (HS-AFM) to image single-molecule interactions in real time, we were able to observe binding, translocation, and dissociation mechanisms of the EcoRII protein. The results show that the protein can translocate along DNA to search for the specific binding site. Also, once specifically bound at a single site, the protein is capable of translocating along the DNA to locate the second specific binding site. Furthermore, two alternative modes of dissociation of the EcoRII protein from the loop structure were observed, which result in the protein stably bound as monomers to two sites or bound to a single site as a dimer. From these observations, we propose a model in which this pathway is involved in the formation and dynamics of a catalytically active three-site complex.The formation of synaptic protein-DNA complexes is central to many biological processes which require communication between two or more DNA regions, including recombination (1,2), replication (3), transcriptional regulation (4), repair (5), transposition (6), and restriction (7,8). Restriction enzymes serve as useful models to study mechanisms by which the intracellular protein machinery functions on DNA, including synapsis. Restriction enzymes (REases), which require binding to two or more cognate recognition sites in order to be catalytically active are widely spread (9). A multi-site mechanism suggests that restriction enzymes serve as evolutionary precursors to many DNA regulatory factors in the cell (10,11). Such a mechanism could also serve an inhibitory function to prevent rare unmethylated recognition sites in the host genome from undergoing restriction (12). In addition to systems involving interactions of two DNA helices, interactions of three or more DNA molecules may occur (13-17).EcoRII is a dimer which recognizes the sequence 5′-CCWGG-3′. It is generally known as a type IIE restriction enzyme. In general, the definition of typeIIE REases is that they bind two DNA recognition sites in order to cleave one of the sites (18). However, recent evidence suggests that the EcoRII protein actually requires three sites to concertedly cleave both strands For this study, we used high speed atomic force microscopy (HS-AFM) to directly image single molecule dynamics of the protein-DNA complexes formed by EcoRII restriction enzyme. It has been used previously to visualize looping and translocation mechanisms of the type III restriction enzyme EcoP15I (31). This HS-AFM relies on a small cantilever design based on the original design by T. Ando (32). This technique has the ability to observe molecular dynamics on a timescale that is 100 times faster than conventional...

Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein-DNA complexes. This is a ubiquitous phenomenon which is critical in various genetic processes. Although such interactions typically occur between two sites, interactions among three specific DNA regions have been identified, and a corresponding model has been proposed. Atomic force microscopy was used to test this model for the EcoRII restriction enzyme and provide direct visualization and characterization of synaptic protein-DNA complexes involving three DNA binding sites. The complex appeared in the images as a two-loop structure, and the length measurements proved the site specificity of the protein in the complex. The protein volume measurements showed that an EcoRII dimer is the core of the three-site synaptosome. Other complexes were identified and analyzed. The protein volume data showed that the dimeric form of the protein is responsible for the formation of other types of synaptic complexes as well. The applications of these results to the mechanisms of the protein-DNA interactions are discussed.

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a sideby-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg 2؉ cations with Ca 2؉ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments (1). This DNA rearrangement process, known as V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called RAG1 and RAG2, assemble a multiprotein synaptic complex with a pair of antigen receptor gene segments and subsequently introduce a DNA double strand break at the end of each gene segment (2). A recombination signal sequence (RSS) 3 that abuts each participating gene segment serves as the binding site of the RAG proteins and directs the location of DNA cleavage. Each RSS contains conserved heptamer and nonamer sequences that are separated by either 12 or 23 bp of DNA of more varied sequence (12RSS and 23RSS, respectively); efficient V(D)J recombination generally only occurs between two RSSs in which the lengths of DNA separating the heptamer and nonamer differ (the 12/23 rule). The RAG proteins mediate DNA cleavage via a nick-hairpin mechanism, breaking the DNA between the RSS heptamer and the coding segment; these reaction products are subsequently processed and repaired by the non-homologous end-joining pathway (1, 3).Previous studies suggest that RAG synaptic complexes are assembled through the stepwise binding of a 12RSS followed by the capture of a 23RSS (4 -6). In vitro biochemical studies suggest synapsis is mediated by a RAG1/2 heterotetramer, but there remains disagreement over the stoichiometry of RAG1 in these complexes (7). In addition, fluorescence resonance energy transfer techniques have recently been applied to examine the orientat...

The dynamics of the cell membrane and submembrane structures are closely linked, facilitating various cellular activities. Although cell surface research and cortical actin studies have shown independent mechanisms for the cell membrane and the actin network, it has been difficult to obtain a comprehensive understanding of the dynamics of these structures in live cells. Here, we used a combined atomic force/optical microscope system to analyze membrane-based cellular events at nanometer-scale resolution in live cells. Imaging the COS-7 cell surface showed detailed structural properties of membrane invagination events corresponding to endocytosis and exocytosis. In addition, the movement of mitochondria and the spatiotemporal dynamics of the cortical F-actin network were directly visualized in vivo. Cortical actin microdomains with sizes ranging from 1.7 3 10 4 to 1.4 3 10 5 nm 2 were dynamically rearranged by newly appearing actin filaments, which sometimes accompanied membrane invaginations, suggesting that these events are integrated with the dynamic regulation of submembrane organizations maintained by actin turnovers. These results provide novel insights into the structural aspects of the entire cell membrane machinery which can be visualized with high temporal and spatial resolution.

Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg²⁺ or Ca²⁺ at a scan rate of 1-2 fps. The increased time resolution allowed us to visualize the concerted cleavage of the protein at two cognate sites. The four termini generated by the cleavage were released in a multistep manner. The high temporal resolution enabled us to visualize the translocation of a DNA strand on a looped complex and intersegmental transfer of the SfiI protein in which swapping of the site is performed without protein dissociation. On the basis of our results, we propose that the SfiI tetramer can remain bound to one of the sites even after cleavage, allowing the other site on the DNA molecule to fill the empty DNA-binding cleft by combining a one-dimensional diffusion-mediated sliding and a segment transfer mechanism.

Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (ΔrecA) mutation suppresses the plasmid transformation defect of competent ΔrecU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ΔrecX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA–DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth.

Caprice [C19orf21 actin-bundling protein in characteristic epithelial cells, also called mitotic interactor and substrate of Plk1 (MISP)] is a novel actin-related protein identified in the highly-insoluble subcellular scaffold proteins. This protein contains multiple actin-binding sites, forms characteristic mesh-like F-actin bundles in vitro, and exhibits capricious localization and expression patterns in vivo. Overexpression or knock-down of Caprice resulted in a dramatic effect on cellular morphology by inducing stress fiber-like thick filaments or filopodial formations, respectively. Caprice is expressed and localized in distinct cells and tissues with specialized actin-based structures, such as growth cones of migrating neurons and stereocilia of inner ear hair cells. However, Caprice gene expression is varied among different cell types; especially enriched in several epithelial cells whereas relatively suppressed in a subset of epithelial cells, fibroblasts, and neuroblastoma cells at the transcriptional level. Thus, this protein is expected to be an effector for cell type-specific actin reorganization with its direct actin-binding properties and provides a novel model of cell morphology regulation by a nonubiquitous single actin-bundling protein.