Chromophore-assisted light inactivation and self-organization of microtubules and motors
Chromophore assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use fluorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of CALI to an in vine system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without influencing structure formation elsewhere.
Additional Information© 1998 by the National Academy of Sciences. Communicated by Kai Simons, European Molecular Biology Laboratory, Heidelberg, Germany, December 29, 1997 (received for review December 9, 1997). We thank Jeff Gelles for plasmid pEY4, Mark Rose for plasmid pGTEPI, Brendan Cormack for GFP mutants, and Jill Johnson for taxol. We also thank Joseph Forkey for running the pulsed laser apparatus and Jerome Zawadsky and Bob Austin for help in the cw laser experiments. We acknowledge Martha Fonseca for preparing C12A5 cell culture supernatants. The partial support of the National Institutes of Health, the National Science Foundation, and the Human Frontier Science Program Organization is gratefully acknowledged. T.S.'s research was also supported by the Deutsche Forschungsgemeinschaft. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Published - SURpnas98.pdf