Liao J C, Berg L J, Jay D G
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Photochem Photobiol. 1995 Nov;62(5):923-9. doi: 10.1111/j.1751-1097.1995.tb09157.x.
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-alpha, TCR-beta and CD3-epsilon. The CALI of TCR-beta resulted in the disruption of the beta subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR-alpha but caused no effect on another subunit, CD3-epsilon. Reciprocal experiments directing CALI to TCR-alpha and CD3-epsilon 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.
生色团辅助激光失活（CALI）是一种分子光消融技术，已被用于阐明特定蛋白质在神经发育中的体内作用。对其在活细胞中对蛋白质影响的解释依赖于了解CALI诱导的损伤在体内的空间限制程度。为了确定CALI在活细胞中的空间特异性，我们将CALI应用于培养的2B4杂交瘤细胞表面的T细胞受体（TCR）复合物的各个亚基，并检测了TCR-α、TCR-β和CD3-ε随后的结构和功能完整性。TCR-β的CALI导致β亚基的破坏，也对单独与相邻TCR-α结合的抗体产生了微小影响，但对另一个亚基CD3-ε没有影响。将CALI应用于TCR-α和CD3-ε的反向实验得到了一致的结果。对于这些CALI实验中的任何一个，除了简单的功能丧失外，没有观察到其他影响。这些数据证明了活细胞中多亚基复合物内CALI诱导损伤的程度，并为该技术未来应用于理解复杂细胞过程中蛋白质的体内功能提供了更大的信心。
