Repair of damaged or incorrectly matched DNA is essential to the survival of all organisms. Consequently cells have devised a plentitude of pathways for repair. We have been investigating the mechanisms of mismatch repair and base excision repair. Both of these repair processes involve a large number of proteins that interact with one another as well as with DNA. Our long-term goal is to assemble complexes that are fully functional for DNA repair and to image the process of DNA repair. In addition, we wish to i) determine the stoicheometry of binding of the protein complexes to each other and to DNA, ii) monitor conformational changes due to substrate binding, iii) measure physical properties of DNA and the complexes. To accomplish this end, we have endeavored to improve techniques for solution imaging as well as those for data analysis. In this presentation I will discuss data on the stoicheometry of binding in several protein complexes and data on the physical properties of DNA.

To measure the physical properties of DNA, we utilize a nanoManipulator, a modified Scanning Force Microscope with a novel, user-friendly interface that allows easy and controlled manipulation of nanometer-sized samples.

Microtubules are long, hollow, stiff polymers that extend throughout the cytoplasm. They are involved in such diverse functions as governing the location of membrane-bounded organelles and chromosome separation during mitosis. The basic structural unit is tubulin, which is a heterodimer consisting of two closely related and tightly linked globular polypeptides called α and β-tubulin. Alternating α and β tubulin subunits form protofilaments, 13 of which bundle around a central core to form microtubule. The detailed structures of cytoplasmic microtubules have been studied extensively using various electron microscopic techniques. As microtubules are dynamic structures in constant transitions between growing and shrinking phases, it would be extremely interesting to investigate the structural organization of the subunit tubulin molecules in a buffer close to physiological conditions. With its high resolution and ability to image in fluid, atomic force microscope (AFM) makes it possible to study the biological structures in a native environment.

A major advantage of atomic force microscopy (AFM) is its intrinsic high contrast, owing to its extremely high sensitivity to height variations. For clean, flat specimens, high-resolution images of a variety of bio-molecules have been obtained, demonstrating the usefulness of AFM in structural biology. However, for large molecular complexes, the effectiveness of AFM is seriously limited, partly owing to the excessive deformation induced by the probe force exerted on the specimen. To overcome this difficulty, we have developed a cryo-AFM, operated under liquid nitrogen vapor at a temperature only a few degrees above 77K. In this system, specimen or tip contamination is significantly reduced, and the resolution achieved with isolated macromolecules is generally higher than that obtainable at room temperature with AFM. Therefore, cryo-AFM has been proven to be a worthy alternative for conventional electron microscopy (EM) with the potential of higher resolution without image averaging.

A particularly fruitful application of cryo-AFM is the study of the spatial structure of several immuno-proteins, where the 3D surface topology of the molecule has been obtained.

Enamel, which covers the anatomical crown of the tooth, is the hardest tissue in human body. Supported by a soft but tough dentin structure, the tooth is an advanced nanocomposite that can endure mastication stresses throughout a lifetime. A detail understanding the structure of the tooth, and sepecifically detin-enamel junction (DEJ), not only provides a sound basis for a model for synthetic dental restoration, but also provides lessons from nature on biomimetic regeneration with mechanical integrity. Enamel is a non-growing mineralized tissue and is subjected to most mechanical abuse. Dentin-enamel junction plays a critical role of distributing load between two very dissimilar materials - enamel and dentin. Bulk scale mechanical tests have shown that induced cracks on enamel tend to be arrested DEJ. Furthermore, nanoindentation measurements have also shown that there is a gradual decrease in hardness from enamel to dentin in the DEJ zone suggests a strong mechanical coupling in both deciduous and adult incisors. The objective of this investigation, through microscopical study, is to understand how these two dental hard tissues structurally couple through their junction zone.

Recent AFM research in our laboratory has covered such diverse biomaterials as laminin and other macromolecules from basement membranes (Fig. 1) (1), DNA condensed for gene therapy (Fig. 2) (2), DNA-protein complexes in the yeast kinetochore (Fig. 3) (3), and biofilms of the bacterium Pseudomonas putida (Fig. 4) (4).

Laminin is a major protein of basement membranes. When analyzed by AFM in air, it shows a variety of conformations of its cruciform structure (Fig. 1A). Time-lapse images of a single laminin molecule in aqueous solution show the flexibility of the laminin arms as they move and bend (Fig. 1B)(1).

AFM analysis of DNA condensed for receptor-mediated gene therapy poses a puzzle (Fig. 2)(2). Polylysine condenses DNA poorly (Fig. 2A), while AsOR-polylysine condenses DNA into compact toroids and short rods (Fig. 2B). AsOR (asialoorosomucoid) is a 38-kDa glycoprotein with a net negative charge of 5 carboxylic acid groups per molecule. The puzzle is why this negative glycoprotein, covalently attached to polylysine, can enhance the condensation of DNA so much over the condensation caused by the polycation, polylysine, alone.

The Atomic Force Microscope is a simple instrument capable of imaging single macromolecules with submolecular resolution and measure the forces required to unfold a single protein domain (Fig. 1).

The AFM method of unfolding a tandem modular protein generates a characteristic sawtooth pattern in the force-extension curves (Fig. 2). The measured spaces between the peaks are thought to correspond to the increase in contour length (ΔΔLC) of the protein caused by the unfolding of one of its modules. In order to demonstrate the validity of this model and measure the sensitivity of the AFM we have designed an experiment where we have inserted a cluster of 5 Gly into the loop joining the F and G b-strands of the I27 module of the I band from human cardiac titin. This loop is clearly within the folded structure of the module and hence, if our predictions are correct, unfolding of the I27-5Gly module should reveal an expanded ΔLC. We expect to measure an increase in the contour length increment of ΔΔLC = 5 ✗ 0.38 nm =1.9 nm.

The high spatial resolution and the force sensitivity of the Atomic Force Microscope have made it possible to perform mechanical experiments with single molecules. These experiments give direct access to the forces that stabilize biomolecule structure. In a variety of examples we show how different molecular interactions determine the mechanical stability of polysaccharides, proteins, DNA and other biomolecules.

Under the influence of a stretching force of around 750 pN the polysaccharide dextran undergoes a transition into a stretched conformation during which bond angles flip into a new conformation. Molecular dynamics simulations corroborated this conformational change.

Proteins fold into compact domains. Especially for structural proteins the mechanical stability of these domains is of importance. We show that the immunoglobulin and fibronectin III domains of the muscle protein titin exhibit exceptionally high unfolding forces (150-250 pN) when stretched at speeds of normal muscle operation (Fig. 1).

Recently, a quantum leap has been achieved in the analysis of DNA and proteins through the advent of the biochip technology. This technology is a product of a broad interdisciplinary approach combining biochemical analysis, semiconductor manufacturing and computer software. Biochips can be defined as miniaturized ordered arrays of macro molecules or pieces thereof that are immobilized in a precise spatial manner on support media and can be used in highly automated, large-scale and high-throughput fashion to analyze biological material. The biochip can be used in a wide variety of areas related to basic research and can find versatile applications in almost all areas of human activities connected to biotechnology, medicine, agriculture, and environment monitoring and bioremediation.

The power of the technology has already been demonstrated in areas like gene sequencing and proofreading, detection of single-nucleotide mutation and polymorphism, identification of genes, identification of viruses and microorganisms, gene expression analysis, analysis of sequencespecific ligands and proteins, and others.

Connexins oligomerize into hemichannels, traffic to the cell surface and dock with hemichannels from an adjacent cell to form intercellular gap junction channels. Pulsechase studies have revealed that connexins are subject to a short half-life ranging from 1- 5 hrs. To examine the mechanisms involved in connexin trafficking, gap junction assembly and internalization in living cells we fused green fluorescent protein (GFP) to the amino or carboxyl terminus of full length connexins (Cx). When cDNA encoding Cx43-GFP was transfected into communication-competent NRK cells, Cx43-negative MDCK cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into functional gap junctions (Figure 1). Most notably, the fusion of GFP to the amino terminal of Cx43 (GFP-Cx43) or Cx32 (GFP-Cx32) did not inhibit the co-translational insertion of these polytopic type IV connexins into the membrane and as a result gap junction plaques assembled at cellcell interfaces.

Solid tumors frequently contain cells that are heterogeneous in the copy number of DNA loci. This fact implies the existence of genetic instability, which may be associated with disease aggressiveness. Accurate measurement of this phenomenon requires analysis of intact nuclei within their natural tissue context. We perform these measurements by preparing >30μm thick tissue sections, labeling them with fluorescent labels for total DNA and for specific DNA loci using fluorescence in situ hybridization (FISH) which retain the transparency of the tissue and acquiring 3D images of the tissue using confocal microscopy (figure 1). In this study, we combined automated 3D image analysis (IA) algorithms for segmenting individual nuclei based on the total DNA stain1 and for segmenting the punctuate FISH signals of DNA loci. This enables us to efficiently enumerate the copy number of specific DNA loci in individual cells and as a function of the cell's location in the tissue.

Dynamin is a 100 kD GTPase that plays an essential role in clathrin-coated vesicle formation during receptor mediated endocytosis, and in caveolae internalization and may play a role in intracellular membrane trafficking (1). It shares an extensive sequence homology (70% identity) to shibiregene product in Drosophila(2,3). The shibiretsmutants exhibit a rapid and reversible paralysis at non-permissive temperature due to a depletion of synaptic vesicles in their nerve termini which is believed to be caused by a block in endocytosis since there is an accumulation of “collared” clathrin-coated pits at the plasma membrane (4). Synaptosomes treated with GTPγs produces elongated necks surrounded by dynamin (6). Purified recombinant dynamin itself can assemble to form spirals and bind to lipid vesicles to form tubes, which resemble the “collar” at the necks of coated pits (5). These dynamin tubes vesiculate upon GTP treatment (7), suggesting a unique role of dynamin acting as a mechanoenzyme which causes clathrin-coated vesicles to be pinched off plasma membrane.

The chemical synapse is a key structure used by neurons to communicate with neighboring cells. This is an asymmetric structure composed of two elements. The presynaptic part contains small synaptic vesicles filled with neurotransmitter. In an active synapse synaptic vesicles fuse with the presynaptic membrane and release neurotransmitter into the synaptic cleft. This results in activation of receptor molecules and respective ion channels on the postsynaptic cell. After a fusion event, the vesicle membrane is retrieved to form a new vesicle. Clathrin-mediated endocytosis is one of the main pathways for synaptic vesicle recycling. It involves a series of steps that starts with a flat plasma membrane and ends with an internalized coated vesicle. Four distinct stages have been defined: (1) the assembly of the clathrin coat, (2) the invagination of the coated membrane into a pit, (3) the constriction of the coated pit, and (4) the fission reaction that liberates the coated vesicle (e.g. De Camilli and Takei, 1996; Brodin et al., 1997). Analysis of mutant yeast and animal cells have linked a number of proteins to each of these stages. Taken together with data from in vitro binding studies, these observations indicate the existence of a complex molecular network that regulate the endocytic reactions.

Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane proteins that function as cation selective, ligand-gated ion channels and are widely distributed throughout the vertebrate nervous system. One of the most abundant nAChRs is a species that contains the α7 gene product (α7-AChRs), binds the snake venom oc-bungarotoxin, and rapidly desensitizes. These receptors have been shown to function at presynaptic sites to modulate transmitter release, as well as on postsynaptic cells where they mediate transmission. Interestingly, these receptors have an exceptionally high relative permeability to calcium.

In the chick ciliary ganglion, the α 7-AChRs play a prominent role, by generating large synaptic currents, but the receptors appear to be excluded from postysnaptic densities on the cell. Immunohistochemical studies have shown that the receptors form large clusters on the surface of the ciliary ganglion neurons. We have recently shown that the α 7-containing receptors are concentrated on mats of somatic spines in close proximity to putative sites of presynaptic transmitter release.

Observations that polyribosomes are localized near dendritic spines and beneath synapses has led to a hypothesis of synapse-specific gene expression in which local synthesis would provide a mechanism to influence synaptic structure and strength (l).The active transport of specific mRNAs into dendrites and spines may be a regulated mechanism to target synaptic and regulatory proteins to postsynaptic locations and influence synaptic activity. RNA granules labeled with the vital dye, SYT014, were observed to localize into developing neurites in response to the neurotrophin, NT-3 (2). Neurotrophins have been shown to enhance synaptic activity by a process which requires new protein synthesis (3). The identity and source of newly synthesized proteins which are required to enhance synaptic strength in response to NT-3 are unknown. CaMKIIαRNA localization into dendrites has been shown to occur during long term potentiation (4).

The transmembrane receptor for aspartate, Tar, in Escherichia coliand Salmonellais representative of a large class of receptors that generates chemotaxis responses by regulating the activity of an associated histidine kinase, CheA. Tar is composed of an Nterminal extracellular ligand-binding domain linked through a transmembrane sequence to a C-terminal signaling domain in the cytoplasm. The isolated cytoplasmic domain of Tar fused to a leucine zipper sequence forms soluble ternary complexes with CheA and an adapter protein, CheW. The isolated complex is biochemically active, has a molecular weight of 1,400,000 Daltons, and includes approximately 28 receptor signaling domains for 2 CheA dimers.

Electron microscopy of the complexes indicates well-defined bundles, presumably composed of numerous receptor filaments surrounding a core of CheA dimers and CheW. CheA also interacts with CheY, the response regulator of the bacterial flagellar motor. Immunoelectron microscopy has provided a general picture of the domain organization of the complexes.

Bacterial flagellum is a helical filament by means of which bacteria swim. Each filament rotated by the motor at its base works as a screw that propels the cell, but it is not simply a rigid propeller. The filament is normally in a left-handed supercoiled form and several of them form a bundle when bacteria swim. Upon quick reversal of the motor rotation, which occurs every few seconds, the filament switches into a right-handed supercoil, making the filament bundle fall apart and enabling the cell to tumble for its tactic behavior. The filament is a tubular structure formed by helical assembly of single protein, flagellin, whose molecular mass is 51.5 kDa in the case of Salmonella typhimurium, which we study. The supercoiling of the filament is thought to involve two distinct subunit conformations and/or packing, whose mechanism is interesting in terms of conformational distinctness and adaptability of flagellin.

To understand the mechanisms of self-assembly and polymorphism of the filament, electron cryomicroscopy (EM) and X-ray fiber diffraction have been used to analyze the structures of two straight filaments with distinct helical symmetries.

Interactions between proteins or protein domains can be imaged by fusing them to cyan (CFP) and yellow (YFP) mutants of Green Fluorescent Protein and observing fluorescence resonance energy transfer (FRET). For example, fusions of CFP, calmodulin, a calmodulin-binding peptide, and YFP are transfectable emission-ratioing Ca2+ indicators with many uses. They are highly suitable for twophoton excitation at 770-810 nm, even at video rates. Applications not possible with previous indicators include detection of submicroscopic domains of Ca2+ by fusion of the indicators to key proteins, and dynamic imaging of Ca2+ in transgenic animals. YFPs have been improved as FRET acceptors by reducing their sensitivity to pH changes. Many other applications of GFP mutants to detect fluctuating protein-protein interactions are underway.

A synthetic alternative to GFPs for protein tagging arises from the ability of membrane-permeant biarsenical dyes to seek out and light up alpha-helical Cys-Cys-X-X-Cys-Cys motifs placed in recombinant proteins in live cells. The new system is much smaller than GFP (6 residues vs. 238), can label internal domains not just N- and C-terminii, and offers novel readouts (e.g. red emission peaking > 600 nm) and better temporal control of the labeling.

Calmodulin is thought to be the most important calcium sensor in signal transduction. When calcium binds to its 4 calcium binding sites, CaMCa4 becomes a multifunctional activator that regulates a large number of critical cell functions. In the nucleus, CaMCa4 controls gene transcription and other important nuclear processes. Here, using confocal microscopy to measure changes in fluorescence resonance energy transfer (FRET) of an indicator composed of two GFP-variants (CFP and YFP) linked by a calmodulin-binding sequence, we show that free cytosolic CaMCa4 concentration increased rapidly after a cell-wide increase in calcium concentration while the initial free CaMCa4 in the nucleus remained much lower and only equilibrated to the cytosolic CaMCa4 concentration after a delay of 1-2 minutes. Thus, immediately after a cell-wide calcium increase, a significant gradient in free CaMCa4 concentration exists between the cytosol and nucleus.

Apoptosis is an actively regulated process of cell death necessary for proper control of tissue growth. The phenomenon is characterized by plasma membrane blebbing, cell shrinkage, chromatin condensation, and degradation of DNA. Apoptosis is distinctly different from necrosis. Unlike necrosis, which is a passive form of cell death, apoptosis appears to be an active physiological process using a controlled genetic “program” of gene expression. Furthermore, apoptosis does not involve severe tissue damage or inflammation. Membrane integrity is preserved, and there is no loss of cellular contents before phagocytosis. Apoptosis is also distinct from necrosis in that it can be triggered or suppressed by tissue-specific hormones and growth factors.

Apoptosis plays a critical role in tissue homeostasis by counterbalancing mitosis. It is an essential component of many physiological processes, including embryonic development, clonal selection in thymocytes, and in protection against disease.

We employed force-distance measurements, carried out by Atomic Force Microscopy (AFM), to explore the local elastic properties of the membrane-skeleton of human erythrocytes. To calculate the elastic constant of the membrane-skeleton we chose that part of the withdraw region in the force-distance curve which corresponds to the stretching of the membrane-skeleton complex. In order to achieve efficient stretching of the membrane we increased the adhesion of the tip to the membrane by polysine coating of the tip.

Fig. 1 is presents the method of stretching AFM on which our measurements are based, and in Fig.2 one can see the theoretical model of the deformation of a circular plate with fixed boundaries by an external force (AFM probe ) used for recalculating the membrane's bending modulus from the experimental data.

Since we obtained different detachment patterns of tip from the membrane, we set selection criteria for the choice of appropriate curves corresponding to membrane stretching.