We continued the development and application of fracture-flip, a method that we discovered last year. This new method provides macromolecular resolution views of the membrane surfaces of cell membranes. We combined fracture-flip with post-fracture extraction of unfractured membranes by Triton X-100. This new version of fracture-flip allows exposure and imaging of the inner (cytoplasmic) surfaces of plasma membranes and, also, of the exoplasmic surfaces in double-membrane organelles. We combined this method with immunogold cytochemistry to study the ultrastructure of the inner surface of the red cell membrane as well as the planar distribution of Band 3 and spectrin (K. Fujimoto). Fracture-flip Triton X was also used to investigate the ultrastructure of nuclear pores in isolated rat liver nuclei (K. Fujimoto). In other studies, we were able to show that the presence of a surface meshwork of 44 nm fusiform of filaments characterizes the infective stage of Leishmania major. We proposed that these filaments represent metacyclic developmental lipophosphoglycan (P. Pimenta; D. Sacks; R.P. da Silva). We investigated and were able to differentiate the ultrastructure and lectin cytochemistry of the main developmental stages of Trypanosoma cruzi (P. Pimenta, W. de Sousa, T. SoutoPadron). We continued the investigation of the surface ultrastructure of developing nerve cells in culture (C. Andersson-Forsman). Studies now in progress address: 1 the surface ultrastructure of H9 cells infected by HTLV-II(B) (S. Ru-Long); 2) the surface ultrastructure and distribution of specific proteins in sea urchin spermatozoa at different stages of development (S. Ru-Long, D. Nishioka); 3) surface ultrastructure of Giardia lambia (P. Pimenta, W. de Sousa); 4) surface ultrastructure and molecular cytochemistry of lymphocyte capping of CD3, CD4 and HL-A antigens (M.R. Torrist, A. Pavan); 5) surface ultrastructure of Mycobacterium leprae and M. avium (A. Aguas, M. Teixeira da Silva; see Lepra, new project); and 6) surface microanatomy of Schistosoma mansoni collected during its life cycle (P. Pimenta, A. Sher).
