High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip ANTONIO PAVAN, PATKIZIA MANCINI, GIUSEPPE LUCANIA, LUIGI FRATI, MARIA ROSARIA TORRISI* Dipartimento di Medicina Sperimentale, Universita di Roma "La Sapienza", 324 Viaie Regina Elena, 00161, Roma, Italy and PEDRO PINTO da SILVA Membrane Biology Section/Laboratory of Mathematical Biology, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701, USA * Author for correspondence Summary The surface ultrastructure of lymphocytes during capping of two transmembrane proteins is shown. As seen by fracture-flip the plasma membranes of hu- man lymphocytes are covered by a high density of surface particles. Incubation in 30 % glycerol leads to aggregation of these surface particles. Immunogold labelling shows that the transmembrane proteins bearing HLA class I and CD4 antigens are confined to the particle aggregates. These results indicate that surface particles revealed by fracture-flip represent surface protrusions of integral membrane proteins seen as intramembrane particles in freeze-fractured lymphocytes. During capping HLA or CD4 antigens aggregate into progressively larger patches and, finally, into single caps. As revealed by fracture-flip the patches/ caps are seen as clearly differentiated raised plat- forms that are clearly and sharply demarcated rela- tive to contiguous areas of the surface. In non- patched (non-capped) regions, the pattern of distri- bution and apparent density of surface particles remain unaltered. Immunogold labelling clearly demarcates patches and caps, and shows that vir- tually no antigen molecules remain dispersed over the non-patched (non-capped) regions. Estimates of the surface density of either HLA or CD4 antigens over the capped areas point to high planar concen- trations of the transmembrane proteins that bear these antigens. Key words: fteeze-fracture, fracture-flip, lymphocytes. Introduction Interest in the phenomenon of capping in lymphocytes goes beyond the immunological significance of capping. Few phenomena that can be analyzed ultrastructurally can so well illustrate dynamic events in biological mem- branes (Nelson et al. 1983; Bourguignon and Bourguignon, 1984; Turner et al. 1988). The recent introduction of the fracture-flip method made it possible to observe high-resolution Pt/C replicas of cell surfaces (Andersson Foreman and Pinto da Silva, 1988a; Fujimoto and Pinto da Silva, 1988; Pinto da Silva et al. 1989). In fracture-flip the exoplasmic halves of membranes are stabilized by, and remain attached to, carbon replicas of freeze-fractured specimens. Inversion ('flipping*) of these carbon replicas exposes the actual outer surfaces of plasma membranes, which are then shadowed by Pt/C evaporation. The method is simple and can be easily combined with immunogold labelling to produce images of the planar distribution of surface antigens and receptors (Andersson Forsman and Pinto da Silva, 1988a; Pimenta et al. 1989). Immunogold labelling can be combined with fracture-flip by labelling the specimens either before Journal of Cell Science 96, 151-157 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 (Andersson Foreman and Pinto da Silva, 1988a) or after freeze-fracture (Pimenta et al. 1989). In previous papers we used label-fracture (Pinto da Silva and Kan, 1984; Kan and Pinto da Silva, 1987; Andersson Foreman and Pinto da Silva, 19886) to address the dynamics and relationships between intramembrane particles (revealed by freeze-fracture) and the surface distribution and redistribution of surface antigens (Pavan et al. 1989a,6). Here, we use fracture-flip and immunogold fracture-flip to re-approach the investigation of capping phenomena from the ultrastructural/cytochemical point of view. Materials and methods Human peripheral lymphocytes were isolated from fresh heparin- ized blood of healthy donors by Ficoll/Hypaque density gradient centrifugation, and were washed three times in phosphate- buffered saline (PBS), pH7.4. The native distribution of HLA class I and CD4 antigens was assessed by treating lymphocytes unfixed or chemically fixed (1 % glutaraldehyde in PBS, 1 h, 25 °C) in anti-HLA A,B,C (class I) monoclonal antibody (1:100, in PBS, lh, 4°C), (a generous gift from Dr M. C. Mazzilli), or 0KT4 151