Electron microscopic tomography is proving to be a valuable tool for investigating the 3D structure and organization of cellular organelles. Important progress is being made in the application of the technique to frozen-hydrated material, but it is likely that success with thick specimens will be limited by the low contrast and beam sensitivity of naked biological material. Thus, optimizing procedures for fixing, embedding, staining, and selectively labelling cells for 3D electron microscopy remains a priority.

Tomography of chemically fixed and plastic-embedded rat-liver tissue and isolated mitochondria has shown that the cristae (the invaginations of the mitochondrial inner membrane) are pleiomorphic and connected to each other and to the surface of the inner membrane by tubular regions 30-40 nm in diameter. This basic design feature has important implications for the microcompartmentation of ions and molecules within this organelle.

The mitochondrial inner membrane contains the machinery of oxidative phosphorylation. This membrane has invaginations called cristae which vary widely in shape between organisms and between tissues in the same organism. Electron microscopic tomography indicates that, despite this pleiomorphism, there is a common design feature, namely, the cristal membranes connect to each other and to the periphery of the inner membrane by tubular regions 30-40 nm in diameter. This finding has important implications for the internal diffusion of ions, metabolites and macromolecules within mitochondria.

In some types of mitochondria, the cristae exhibit periodicity.1 In the case of the amoeba Chaos carolinensis, detailed analysis and modeling of thin-section images of mitochondria in starved cells indicate that the highly curved cristae correspond to periodic cubic surfaces. We are undertaking electron microscopic tomographic and crystallographic approaches to more thoroughly characterize these membrane phases and, in particular, establish the continuity of the internal compartments which they define.

Advances in both electron cryomicroscopy and tomographic reconstruction methods have made it possible to collect and analyze data from large macromolecular assemblies frozen without fixation, sectioning, or staining. The Spindle Pole Body (SPB) is the microtubule organizing center in the yeast Saccharomyces, and is involved in mitosis, nuclear movement, budding, and mating. We have used electron cryomicroscopy and tomographic reconstruction to investigate the 1500Å diameter, 600Å high cylindrical core structure produced by limited heparin treatment of isolated SPBs.

Tilt series' of SPB cores preserved in vitreous ice were taken under low electron dose conditions. These images have been analyzed using iterative correlation alignment and computed back projections for tilt data without fiducial markers. In these studies, electron micrographs were digitized, and translationally aligned using correlation methods.

The organization of the microtubule network of eukariotic cells is controlled by the centrosome. This organelle which duplicates once during the cell cycle comprises two centrioles and a dense filamentous material called pericentriolar material. The centrioles are cylinders of ≈ 0.25 μm in diameter and ≈ 0.5μm in length and are composed of 9 microtubule triplets at the proximal end and 9 doublets at the distal end. For the last 50 years the centrosome and the centrioles in particular have been the object of many optical and electron microscopy studies. But, in spite of their highly symmetric organization, their fine structure is still not well understood. The reconstruction presented here is a substantial step forward towards a more detailed understanding of their structure.

Centrosomes were isolated from a human lymphoblastoma cell line (1). 10 nm gold particles were applied to formvar-carbon coated grids to provide markers for the alignment procedure (2,3). Centrosomes were centrifuged onto the grids and negatively stained.

Smooth muscle is a machine consisting of working and supporting elements whose structure and 3D organization must be elucidated for the mechanics of shortening and tension generation to be understood. Based on longitudinal and serial transverse sections of rabbit portal vein it was suggested that the contractile elements of smooth muscle formed “mini-sarcomeres”, analogous to skeletal muscle, containing parallel arrays of 3-5 myosin filaments 1.6-2.2 um long. Observations at the light microscopic level were consistent with this idea. The past decade has seen little further investigation into the in situ ultrastructure of this or other smooth muscles, and the general applicability of these findings remains unknown. We have taken advantage of recent methodological advances, which can provide full 3D computer representations of cellular organization based on EM data, using guinea pig taenia coli muscle as a model system.

Serial transverse sections (Fig 1) were used to generate 3D reconstructions of the organization of the myosin filaments and their relation to dense bodies, actin bundles, mitochondria and other organelles.

Light microscopic imaging has experienced a renaissance in the past decade or so, as new techniques for high resolution 3D light microscopy have become readily available. Light microscopic (LM) analysis of cellular details is desirable in many cases because of the flexibility of staining protocols, the ease of specimen preparation and the relatively large sample size that can be obtained compared to electron microscopic (EM) analysis. Despite these advantages, many light microscopic investigations require additional analysis at the electron microscopic level to resolve fine structural features.

High voltage electron microscopy allows the use of relatively thick sections compared to conventional EM and provides the basis for excellent new methods to bridge the gap between microanatomical details revealed by LM and EM methods. When combined with electron tomography, investigators can derive accurate 3D data from these thicker specimens. Through the use of correlated light and electron microscopy, 3D reconstructions of large cellular or subcellular structures can be obtained with the confocal microscope,

Three-dimensional reconstructions from projections are usually ridden by noise from different sources. A common problem among many three-dimensional reconstruction techniques is the systematic absence of certain projections, but also the accidental absence of spurious projections. In these three-dimensional reconstructions such absences are visible as directional smearing due to convolution. Other convolution effects such as those due to the optics of the instrument used to record the data usually cause severe damping of high frequencies and even contrast reversal (common in images from electron microscopes).

Several approaches to overcome these ‘noise’ effects in three-dimensional reconstructions have been developed, but they usually suffer from the very small radius of convergence. Very easily and commonly, the refinement iterations end up stuck in a premature choice of a minimum. We have developed another algorithm, constrained maximum entropy tomography (COMET), that in practice has been shown to overcome these problems.

Microscope specimens are 3-dimensional objects, but the images from conventional light microscopes are flat - they only show two dimensions. Multiple oblique illumination is a novel lighting technique for transmitted light microscopes that produces multiple (i.e. two or more) high definition 3-dimensional images using conventional microscope objective lenses. We have previously described its use in transmitted light. The same optical theory has now been expanded to include reflection microscopy. In the present paper, we describe a new development which will make this approach more widely available. It is a retrofit illumination system that will produce true 3-dimensional images directly through the eyepieces of conventional microscopes.

By seeing z-axis information in real-time and in the context of a specimen's entire thickness, researchers can gain additional, unambiguous information about the interrelationships between structures, whereas critical information about 3-dimensional structures can be obscured, lost or misinterpreted when using 2-dimensional instruments. The importance of accurate z-axis information has popularized methods such as deconvolution and confocal microscopy.

Cytosolic and organellar free Ca2+ concentrations are very dynamic; they are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations.

Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jelly fish Aequorea victoria. Its cDNA can be concatenated with those encoding many other proteins, and the resulting fusion proteins are usually fluorescent and often preserve the biochemical functions and cellular localizations of the partner proteins. Mutagenesis has produced GFP mutants with shifted wavelengths of excitation or emission that can serve as donors and acceptors for fluorescence resonance energy transfer (FRET).

Our new indicators consist of tandem fusions of a blue- or cyan-emitting mutant of GFP, calmodulin (CaM), the calmodulin-binding peptide Ml3, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the Ml3 domain, increasing the FRET between the flanking GFPs (Fig. 1).

Previously, we showed rapid mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during the contractile cycle. Ruthenium red, an inhibitor of mitochondrial Ca2+ uptake by the Ca2+ uniporter, inhibits mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during electrical stimulation. Here, we extend this finding to show that ruthenium red inhibition is specific for mitochondrial Ca2+ transients and not cytosolic Ca2+ transients.

Ca 2+-tolerant adult rabbit cardiac myocytes were isolated by collagenase and hyaluronidase digestion and loaded in the cold with 2.5 μM Rhod 2-AM for 30 minutes. Subsequently, the cells were incubated in nutrient medium at 37°C for 4-6 hours during which time cytosolic but not mitochondrial Rhod 2 was lost. Subsequently, the myocytes were loaded with 10 μM Fluo 3-AM for 15 minutes at 37°C, conditions that lead to predominantly cytosolic localization of Fluo 3. In sequential scans, the red fluorescence of Rhod 2 and the green fluorescence of Fluo 3 were imaged using a Zeiss LSM 410 laser scanning confocal microscope.

Synthesis and assembly of gap junction proteins into intercellular channels is an important biological problem with direct bearing on embryonic development. In particular, transgenic mice Cx43 gap junction protein knock-outs 1 and mutations in Cx43 sequence in human Viscera Hyper Taxia patients result in severely deformed hearts.

NRK cells are one of the best studied systems for gap junction synthesis, transport and assembly. Gap junctions contain numerous cell-cell channels which are composed of two paired oligomers (called a connexon or hemichannel) each contributed from the plasma membrane of apposing cells. Although biochemical studies have shown indirectly that unpaired Cx43 connexons are in the plasma membrane, direct hemichannel visualization has not been demonstrated. Musil and Goodenough have shown that oligomerization occurs in the trans-Golgi network and the connexon is transported to the plasma membrane.

The use of animation can significantly enhance the visualization of three-dimensional (3D) structures. It can present a focused train of thought, or it can be used to systematically scan through previously unfathomable quantities of data to examine for unknown features and consistencies. In order to establish a modern animation facility requires a variety of technical, psychological and artistic skills, in addition to advanced computer graphics and related equipment. 3D graphics combined with animation has proven to be a very effective tool in storytelling. The dynamic visual image is uniquely suited for thought experiments, simulations and traversing vast quantities of data otherwise incomprehensible to the human mind. Sometimes animation is the only method that allows a clear presentation of complex empirical or theoretical information in many dimensions. Scientific visualization is by its nature an exploratory process. And frequently animations are iteratively refined and polished to enhance comprehensibility for the researchers and their peers.

The structure of tubulin, the major protein component of microtubules, has been obtained by electron crystallography of zinc-induced 2-D crystals. The atomic model of the tubulin dimer, built into a 3.7 Å density map, shows that α- and β-tubulin have basically identical structures. Each monomer is very compact and is formed by two interacting beta sheets surrounded by helices. The structure can be divided into three sequential domains that are functionally distinctive. The N-terminal domain forms a Rossmann fold that binds the nucleotide. Connected to the nucleotide-binding domain by the core helix is a second domain that in β-tubulin contains the binding site of taxol. The C-terminal region is formed by two long helices that constitute most of the interactive, outside surface of the microtubule. The nucleotide-binding and second domains are common with FtsZ, a bacterial homologue of tubulin essential for cell division. The bacterial protein, which lacks the C-terminal helices of tubulin, has generally shorter loops and contains an extra helix at the N-terminus.

Molecular motors belonging to the myosin and kinesin superfamilies utilize ATP to move along their respective F-actin and microtubule tracks. The track-motor complexes have not been amenable to crystallization, so x-ray crystallographic investigations have focused on structure determinations of the individual proteins. Although providing detailed descriptions of the structure of each protein, this approach cannot reveal the geometry of interaction of the proteins nor the conformational changes which occur during the mechanochemical cycle. To obtain this information, we use cryoelectron microscopy and image analysis to calculate three dimensional maps of the track-motor complexes at moderate resolution (15-30A) and combine these data with the high resolution x-ray structures to provide near-atomic models of the working assemblies.

We have so far built models of the rigor (nucleotide-free) and ADP actomyosin complexes. In smooth muscle myosin II (a collaboration with H.L. Sweeney, U. Perm.) and brush border myosin I (BBMI), the motor domain of the myosin head is similar in both biochemical states.

A superfamily of actin crosslinking proteins, the calponin-homology domain (CH) family is implicated in directing the assembly and controlling the organization of the cytoskeleton through a highly conserved actin binding domain (ABD). This domain, composed of a tandem pair of CH motifs, is found in actin crosslinking proteins such as fimbrin, alpha-actinin, spectrin, and dystrophin. In addition to its role in crosslinking actin filaments, a putative homologous ABD in intermediate filament-binding proteins, plectin, dystonin, and BPAGlnl, suggests a possible role in crosslinks between desmosomal and vimentin intermediate filaments and actin. Thus, the CH domain could integrate the functions of the actin and IF cytoskeletons by physically linking these structural systems. We present an atomic model for the complex of the CH domain and actin.

In crosslinking proteins that organize actin into two and three dimensional networks or gels the ABDs lie at the ends of long, flexible molecules.

The acrosomal process is an intracellular quasi-crystalline organelle in the head of the sperm of the horseshoe crab Limulus polyphemus. It consists of 100 - 130 actin-scruin filaments packed together in a pseudo-hexagonal lattice2 and is up to 60 (im long with a diameter of 0.1 μm. Scruin-scruin interactions are responsible for cross-linking the actin filaments together in the bundle. The goal of the current study is to reveal interfilament interactions in the bundle. We have taken tilt series images in the electron microscope3 to reconstruct its three-dimensional structure without assuming helical symmetry.

The acrosomal process was purified as described.34 Bundles were embedded in vitreous ice over holes on a holey carbon film on copper grids. The specimen was kept at -167°C in a JEOL 4000EX electron microscope operating at 400 kV. Straight 6-10 (im long bundles were found using a TV-rate CCD camera in defocused diffraction mode.

The calcium pump (Ca2+-ATPase) from sarcoplasmic reticulum (SR) is a prominent member of the large family of ATP-dependent cation pumps, which include Na+ /K+-ATPase, H+/K+-ATPase from the stomach, H+-ATPase from yeast and Neurospora, and various detoxifying pumps for Cd+, Cu+ and other metals. In muscle, calcium is stored inside the SR and contraction is initiated by regulated release through specific calcium channels; Ca2+ -ATPase is responsible for relaxation by pumping calcium back into the SR lumen. Many techniques (chemical modification, site mutagenesis, reaction kinetics) have been used to correlate Ca2+-ATPase sequence with function, but no high resolution three-dimensional structure of Ca2+-ATPase, or any P-type pump, has yet been determined. In the current work, we have determined the structure from helical crystals at 8 A resolution and thus revealed the alpha-helical architecture of the transmembrane domain. In addition, a specific inhibitor of Ca2+-ATPase, thapsigargin, was used to promote crystallization and we have characterized the structural consequences of its inhibition.

Gap junction membrane channels mediate the electrical and metabolic coupling between cells. The channels are formed by the end-to-end docking of two hemichannels (connexons), each of which is formed by a hexameric cluster of protein subunits (connexins). The principal gap junction protein in the heart, α1 connexin (also designated Cx43), mediates action potential propagation between cells in order to synchronize cardiac contraction.

We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated two-dimensional (2D) crystals of a recombinant, truncated α1- connexin (α1Cx263T). The projection map at 7Å resolution revealed that each 30kD connexin subunit has a transmembrane α-helix that lines the aqueous pore and a second α-helix in close contact with the membrane lipids [Nature Struct. Biol. 4: 39-43 (1997)]. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by ∼30°. Furthermore, apparent non-crystallographic twofold axes predicted that the two apposing connexons adopt identical conformations.

Potassium channels are ubiquitous ion channel proteins which play a crucial role in a broad spectrum of important cell functions. KcsA is a potassium channel found in the bacterium, Streptomyces lividan, and is believed to have only two transmembrane helices.

The KcsA protein has been cloned, overexpressed and purified to homogeneity, and reconstituted with phospholipids to form two-dimensional (2-D) crystals. Crystal samples were embedded in trehalose and examined in a cryo-holder (-170 °C) using a JEOL 4000EX electron microscope operated at 400kV in low dose mode. Images were recorded in spot scan-mode at a magnification of 60,000X, then digitized on a Perkin Elmer PDS flat bed microdensitometer and processed by using the MRC program suite. Processed images were brought to a common phase origin, and the amplitudes and phases from individual images were vectorially combined.

New state-of-the-art analytical equipment is becoming more specialized and more expensive. With the availability of federal funding decreasing over the years, it has become increasingly difficult for University laboratories to maintain and upgrade existing analytical equipment, or purchase new analytical equipment. There are also instances where large industrial laboratories may only need a particular piece of analytical equipment on a part-time basis, or where small companies cannot afford to purchase a particular technique. An obvious solution to these problems is to merge industry and university into a viable working collaboration whereby resources are shared among partners.

The University of Central Florida and Cirent Semiconductor (a joint venture of Lucent Technologies and Cirrus Logic) have entered into such a collaboration with the development of the UCF/Cirent Materials Characterization facility (MCF). The MCF is a shared user facility that is available to (i) UCF and other State University System (SUS) Florida University students, faculty, and staff, and (ii) MCF industrial affiliates and others requiring specialized instrumentation and expertise.