Chromophore-assisted light inactivation and self-organization of microtubules and motors

Abstract

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.

Figure 1

Inactivation of β-galactosidase activity by fluorescein-labeled antibodies against β-galactosidase after being exposed to 2 mW (64 mW/cm²) of light at 488 nm. Controls with the same level of fluorescein fluorescence were also performed: in one case fluorescein was bound to anti-HA antibodies; in the other case, it was attached to BSA. Each point is the average of at least three independent activity determinations using an orthonitrophenyl galactoside colorimetric assay.

Figure 2

CALI of HAkinesin using fluorescein-labeled anti-HA Fabs in motility assays. (A) An area with a diameter of 75 μm (indicated by a circle) was illuminated through the fluorescein filter set in the absence of microtubules (only immobilized HAkinesin and HA-kinesin-bound, fluorescein-labeled Fab fragments were present). Ten minutes after adding the microtubules and washing the flow cell (see Materials and Methods), microtubules were observed. (B) As in A, but the area was illuminated after adding the microtubules. (Because some gliding microtubules detach from the surface after washing the flow cell, but hardly any immobilized ones do so, the density of microtubules is higher in the illuminated area of B. In experiment B a lower concentration of microtubules was used than in A. Typical microtubule densities are in Table 2.)

Figure 3

Perturbation of aster formation by CALI of HAkinesin using fluorescein-labeled anti-HA Fabs (A), fluorescein-labeled BSA (B), or fluorescein-labeled streptavidin (C). A central area with a diameter of 460 μm (indicated by a circle) was illuminated through the fluorescein filter set. The temperature was immediately shifted to 35°C (t = 0) to start microtubule polymerization and organization of microtubules by motors. (Under these conditions, motors should immediately bind to polymerizing microtubules and therfore no longer diffuse freely.) In the presence of fluorescein-labeled anti-HA Fab fragments, perturbed aster formation is shown 18 min (A1), 22 min (A2), and 27 min (A3) after an illumination period of 0.2 sec. Unperturbed aster formation in the control experiment (labeled BSA instead of labeled Fabs) is shown 4 min (B1), 6 min (B2), and 10 min (B3) after 0.4 sec of illumination. Because the presence of Fabs slows down the speed of kinesin on microtubules, aster formation in the presence of Fabs is comparatively slow. When the motor constructs contained fluorescein-labeled streptavidin (no addition of labeled Fabs), aster formation was locally inhibited by 12 sec of illumination, as shown by the evolving structures after 4 min (C1), 6 min (C2), and 10 min (C3). Each picture was assembled from nine individual, overlapping photographs.

Figure 4

Different perturbations of aster formation generated by varying the timing and extent of CALI illumination. In A, illumination proceeded for 12 sec. In B, 6 sec of illumination was used. In C, 12 sec of illumination was used, as in A, but only after microtubule polymerization had proceeded for 4 min. D shows a control experiment with fluorescein-labeled BSA (12 sec of illumination, as in A). In each case, end states 10 min after the start of microtubule polymerization are shown. Each picture is composed of nine overlapping photographs.