CHROMOPHORE‐ASSISTED LASER INACTIVATION OF SUBUNITS OF THE T‐CELL RECEPTOR IN LIVING CELLS IS SPATIALLY RESTRICTED

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Abstract
— Chromophore‐assisted laser inactivation (CALI) is a molecular photoablation technique that has been used to elucidate the in vivo roles of specific proteins in neural development. The interpretation of its effects on proteins in living cells relies on knowing how spatially restricted the CALI‐induced damage is in vivo. To determine the spatial specificity of CALI in living cells, we have applied CALI to individual subunits of the T‐cell receptor (TCR) complex on the surface of 2B4 hybridoma cells in culture and have examined the consequent structural and functional integrity of the TCR‐α, TCR‐β and CD3‐α. The CALI of TCR‐β resulted in the disruption of the β subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR‐α but caused no effect on another subunit, CD3‐ε. Reciprocal experiments directing CALI to TCR‐α and CD3‐ε gave consistent results. No effects other than a simple loss of function were observed for any of these CALI experiments. These data demonstrate the extent of CALI‐induced damage within a multisubunit complex in living cells and provide greater confidence for the future application of this technique to understanding in vivo function of proteins during complex cellular processes.