This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT Desmosomes and adherens junctions represent the two different kinds of intercellular junctions that are responsible for cell-cell adhesion (Eelkema and Cowin 2001). Cadherins are proteins that directly mediate adhesion by forming protein-protein contacts across the intercellular gap. Cadherins cross the membrane and interact with a plaque of intracellular proteins that serve to cluster constituents into a defined junction and to link the cadherins to the the cytoskeleton. A number of contradictory molecular models for cadherin interactions have arisen from x-ray crystal structures coupled with biophysical and biochemical studies(Koch et al. 1999). Initial tomographic reconstructions with plastic sections supported one particular mode of cadherin interaction and suggested a unique mechanism for propagating this interaction across the junction. It is now necessary to confirm these observations with a specimen that has not undergone the rigorous preparative procedures required for making plastic sections. There is limited structural evidence for interactions amongst constituents of the cytoplasmic plaque and this remains a goal for future studies of electron tomography. Determination of 3D tomograms from plastic-embedded material is relatively rare and the fitting of molecular structures to such tomograms is even rarer. Despite documented preservation of muscle, viruses, and 3D crystals in plastic resin, the general community remains unconvinced about the potential for molecular preservation (Baumeister et al. 1999). On the other hand, collecting the requisite number of images with sufficient signal-to-noise ratio from an unstained sample is daunting. Thus, as tomography struggles to become an accepted technique, it is important to establish the limitations imposed by sample preparation and the imaging requirements, respectively. The desmosome is well-suited to this role for several reasons. The low water content in skin makes it easy to freeze and to section with minimal artifacts. Also, the high density of protein-protein contacts that characterize this junction appears to offer mechanical resistance to sectioning compression, thus maximizing the potential for a high-resolution tomographic reconstruction. Cell-cell adhesion is a fundamentally structural phenomenon, with one group of molecules forming a mechanically rigid scaffold and a second group of proteins associating to form intermolecular bonds. To date, investigators have used methods of x-ray crystallography, molecular and cellular biology, and biophysics in an attempt to deduce the roles of proteins within the different types of junctions. These investigations have failed to reach a solid consensus for the architecture of the junctions, primarily because they rely on isolated proteins in artificial, in vitro environments (e.g., 3D crystals, mica surfaces, or genetically engineered cultured cells). Electron tomography has the very great advantage of visualizing the 3D organization of molecules in their native cellular environment and stands an excellent chance of resolving some of the controversies that have arisen. Mouse epidermis represents a ready source of desmosomes that has previously been used for preparing plastic sections. This tissue has been frozen in the vitreous state using the high-pressure freezer at the Wadsworth Center. In initial studies, this frozen-tissue was used for freeze-substitution and the same freezing method is ideally suited to cutting frozen-hydrated sections. However, whereas hexadecene or polyvinylpropylene are most suitable as cryoprotectants for freeze-substitution, dextran appears to be the best for obtaining frozen-hydrated sections. Frozen-hydrated sections will be cut with a Leica Ultracut UCT/EM-FCS cryo-ultramicrotome, and pressed onto R2/1 Quantifoil grids. Before applying the sections, a 10nm thick carbon film carrying 10 nm colloidal gold particles will be deposited on the grids. Tomographic tilt series will be collected at 1 or 2 intervals over a 120 angular range, using a JEOL JEM-4000FX operated at 400 kV. Zero-loss imaging will be employed using a Gatan GIF 2002 with a 15 eV slit width. Imaging of the tilt series will be accomplished with a semi-automatic data collection strategy and a total electron dose of 7000-8000 e-/nm2. Alignment and reconstruction will be done using SPIDER or IMOD. As in previous work, segmentation and visualization will be done with Amira. The primary goal is to reveal the organization of molecules responsible for forming adhesive junctions between cells. Initial studies have employed tissue preserved within plastic sections and have demonstrated the potential of tomography for not only delineating cadherin molecules in the intercellular gap, but in defining the molecular interface responsible for adhesion (He et al. 2003). This structure suggested a new paradigm in the associations between cadherin molecules, but has raised doubts due to the harsh procedures required for embedding tissue within plastic resin, such as dehydration and staining. We now seek confirmation of this structure from tissue preserved in a more native state, i.e., fully hydrated and unstained. Not only will this work address the physiological mechanisms responsible for adhesion, but will also serve as a guide to others pursuing tomographic studies of cellular assemblies. In particular, the methods of plastic embedding are widely employed, but the degree of structural preservation within the resulting sections is poorly established. On the other hand, there are serious technical challenges to preparing and imaging frozen-hydrated sections with fundamental limits to resolution presented by the innate radiation sensitivity of native protein samples. By conducting a comparative study of junctions, we hope to establish the use of plastic sections for studying molecular structure and to push the that can be obtained from frozen-hydrated sections. Our current challenge is to optimize specimen preparation and imaging methodologies of electron tomography to enable visualization of constituent molecules and their interactions. The resources at RVBC are uniquely suited to this task. Firstly, the high-pressure freezer is a prerequisite to obtaining vitrified tissue. The development of cryoultramicrotomy offers the least invasive procedure for imaging undisturbed junctions. The use of high voltage and energy filtration is optimal for imaging the resulting thick sections with a maximal signal-to-noise ratio. The routine functioning of all this advanced instrumentation at RVBC together with dedicated, expert staff makes a collaborative effort the most efficient way to pursue this project. References 1. Baumeister W, Grimm R, Walz J. 1999. Electron tomography of molecules and cells. Trends in Cell Biology 9:81-85. 2. Eelkema R, Cowin P. 2001. General themes in cell-cell junctions and adhesion. In: Cereijido M, Anderson J, editors. Tight Junctions. Boca Raton: CRC Press. p 121-145. 3. He W, Cowin P, Stokes DL. 2003. Untangling desmosomal knots with electron tomography. Science 302(5642):109-113. 4. Koch AW, Bozic D, Pertz O, Engel J. 1999. Homophilic adhesion by cadherins. Curr. Opin. Struct. Biol. 9(2):275-281.
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