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 vitro 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.
Surrey
T
T
Department of Physics, Princeton University, Princeton, NJ 08544, USA.
Elowitz
M B
MB
Wolf
P E
PE
Yang
F
F
Nédélec
F
F
Shokat
K
K
Leibler
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001703:Biophysics / Q000379:methods; D004330:Drosophila; D004926:Escherichia coli; D049452:Green Fluorescent Proteins; D006388:Hemagglutinins / Q000737:chemistry / Q000528:radiation effects; D016547:Kinesins / Q000737:chemistry* / Q000528:radiation effects / Q000648:ultrastructure; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D008870:Microtubules / Q000528:radiation effects / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000528:radiation effects; D016324:Sequence Tagged Sites; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism
D006388:Hemagglutinins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D016547:Kinesins