Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts

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We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocynanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion of DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 ± 0.12) · 10 −10 cm 2 s −1, was approximately two times faster than that observed in normal CEF, (0.61 ± 0.06) · 10 −10 cm 2 s −1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 ± 0.13) · 10 −10 cm 2 s −1 to a value comparable to the RSV-CEF transformed cells, (1.74 ± 0.20) · 10 −10 cm 2 s −1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35°C) unless stated otherwise. The changes in the lateral diffusion of TRICT-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.