Wild type and tailless CD8 display similar interaction with microfilaments during capping PASCALE ANDRE 1 , JEAN GABERT 2 '*, ANNE MARIE BENOLIEL 1 , CHRISTIAN CAPO 1 , CLAUDE BOYER 2 , ANNE MARIE SCHMITT-VERHULST 2 , BERNARD MALISSEN 2 and PIERRE BONGRAND 1 1 Laboratoire d'Immunologie, Hdpital de Sainte-Marguerite, BP 29, 13277 Marseille Cedex 09, France 2 Centre d'Immunologie de Marseille Luminy, Case 906, 13288 Marseille Cedex 9, France * Present address: Institut Paoli-Calmettes, BP 156, 13273 Marseille Cedex 9, France Summary We examined the influence of the intracytoplasmic region of CD8a on capping and interaction with microfilaments. We used cell clones obtained by transfecting a CD4+ T-cell hybridoma with (a) T-cell receptor (TCR) a and fi chains from a cytolytic clone and (b) CD8« genes that were either native or modified by extensive deletion of the intracytoplas- mic region or replacement of the transmembrane and intracytoplasmic domains with those of a class I major histocompatibility complex gene (Letourneur et al. (1990). Proc. natn. Acad. Sci. U.S.A. 87, 2339—2343). Different cell surface structures were cross-linked with anti-T-cell receptor, anti-CD8 or anti-class I monoclonal antibodies and anti-immuno- globulin (Fab') 2 . Double labeling and quantitative image analysis were combined to monitor fluor- escence anisotropy and correlation between differ- ent markers. Microfilaments displayed maximal polarization within two minutes. The correlation between these structures and surface markers was then maximal and started decreasing, whereas the redistribution of surface markers remained stable or continued. Furthermore, wild type and altered CD8« exhibited similar ability to be capped and to induce co-capping of TCR and MHC (major histocompati- bility complex) class I: the fraction of cell surface label redistributed into a localized cap ranged between 40% and 80%. Finally, cytochalasin D dramatically decreased CD8 capping in all tested clones. It is concluded that the transmembrane and/or intracellular domains of CD8 molecules are able to drive the extensive redistributions of membrane structures and cytoskeletal elements that are trig- gered by CD8 cross-linking. Key words: membrane, molecular motion, cytoplasmic domain, cytoskeleton, CD8. Introduction Since the elaboration of the fluid mosaic model of the plasma membrane by Singer and Nicolson (1972), it has become clear that many cell functions are affected by lateral movements of membrane molecules. Cell adhesion is thus highly dependent on the lateral mobility of ligand molecules (Rutishauser and Sachs, 1974). Clearly, the formation of strong intercellular bonds may require that ligand molecules be concentrated in adhesion areas (Bell et al. 1984), and this concentration was indeed observed in several experimental studies using different method- ologies such as electron microscopy (Singer, 1975), micro- fluorometry (Poo and McCloskey, 1986) and conventional (Kupfer and Singer, 1989) or computer-assisted (Andre et al. 1990a,b)fluorescencemicroscopy. Also, the microaggre- gation of cell surface receptors was found to enhance their adhesive capacity (Detmers et al. 1987). However, the mechanisms of molecular motion in the plasma membrane remain incompletely understood. When the mobility of cell surface proteins was studied with the fluorescence photobleaching recovery (Schless- inger et al. 1976) or post electric field relaxation (Poo et al. 1978) method, the diffusion coefficient of concanavalin A Journal of Cell Science 100, 329-337 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 'receptors' (Schlessinger et al. 1976; Poo et al. 1978; Smith et al. 1979), class I histocompatibility molecules (Edidin and Zuniga, 1984; Bierer et al. 1987; Wier and Edidin, 1986) or lymphocyte surface immunoglobulins (Dragsten et al. 1979; Barisas, 1984) varied between less than 10~ 12 and about 2xlO~ 9 cm 2 s~ 1 . The latter value was roughly consistent with theoretical estimates for free diffusion in a thick viscous sheet (Wiegel, 1984). Thus, it appeared reasonable to view the motion of cell surface proteins as a hindered-diffusion process. In addition, these proteins may undergo active displacements as exemplified by the capping process (Taylor et al. 1971; Bourguignon and Bourguignon, 1984). Strong experimental evidence suggests that interac- tions between cytoskeletal elements and membrane intrinsic proteins are dynamic events (Flanagan and Koch, 1978; Burn et al. 1988) that may hinder random diffusion (Koppel et al. 1981; Tank et al. 1982). Further, active movements may be affected by cytoskeletal inhibi- tors (Taylor et al. 1971; Bourguignon and Bourguignon, 1984). However, the precise molecular interactions in- volved in these phenomena remain incompletely under- stood and it is not well known whether cytoskeletal elements are bound to many membrane proteins or 329