Adhesive junctions are involved in a wide range of cellular processes ranging from embryogenesis and tissue formation to cell transformation. They participate in cell-cell recognition, provide tensile strength to epithelial sheets and participate in intracellular signaling that direct cell growth and migration; several component proteins function either as oncogenes or as tumor suppressors. There are two types of adhesive junctions, desmosomes and adherens junction, both of which are well characterized from a biochemical and cell biological perspective and several x-ray crystal structures exist for important domains of several key proteins. Nevertheless, the architectural principles by which these components form a live junction and mediate adhesion remains largely guesswork. This proposal seeks to elucidate this architecture using the method of electron tomography to determine 3D structures of intact junctions in their native cellular environment. In preliminary work, we have studied intact desmosomes from after freeze-substitution and plastic embedding of newborn mouse epidermis. From the resulting tomograms, we have described the organization of desmosomal cadherins within the intercellular space and proposed structural mechanisms for adhesion. For our first aim, frozen-hydrated specimens and methods of cryotomography will be used to eliminate specimen preparation artifacts as a potential factor and to establish this methodology as an alternative to plastic sections. In our second aim, the architecture of adherens junctions will be studied either in epidermis, in cultured keratinocytes, or in lens tissue. Although adherens junctions have an analogous architecture to desmosomes, they have different molecular components, different morphology, and have a more dynamic role in cellular behavior. In our third aim, we will study the structure and function of the dense cytoplasmic plaque, by isolating epidermis from transgenic mice with knockout of several key desmosomal components: plakoglobin, desmoplakin, desmocollin, and keratin 5. In addition, we will explore immunolabelling as a technology for identifying plaque component in tomograms. In our fourth aim, we will study the assembly of both adherens junctions and desmosomes in cultured keratinocytes, using calcium to initiate junction assembly between confluent cell cultures. ? ?
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