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 f luorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use f luorescein-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 inf luencing structure formation elsewhere.

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

“…In this work, a standard epifluorescence microscope was used because this tool is widely available and can be used by other investigators with minimal difficulty. Laser-mediated irradiation is very common in FALI, and light sources including the 488-nm laser line typically used in confocal microscopy might further reduce the time required for FALI irradiation (9).…”

“…For example, intracellular targeting of a photosensitizing compound to a protein of interest can be used to rapidly inactivate that protein upon irradiation (8). GFP is not an efficient photosensitizer, presumably because the protein shell of GFP prevents the generation or the efficacy of reactive oxygen species (ROS) that mediate the inactivation (9).…”

A minimal system allowing tubulation with molecular motors pulling on giant liposomes

“…Streptavidin beads (100 nm) were purchased from Bangs Laboratories (Carmel, IN). Biotinylated hemagglutinin-kinesin (a gift of F. Nédélec, European Molecular Biology Laboratory, Heidelberg) was purified as described (26). By dividing the number of kinesins by the number of beads, the number of kinesin molecules per bead was estimated at approximately 1,500.…”

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

“…Also MV D7 cells are Ϸ50% smaller than Rat2 fibroblasts. Lamellipodial protrusions were analyzed by using kymography, which characterizes the kinetic signature of lamellipodia undergoing cycles or protrusion and retraction (17,27). One of the hallmarks of using CALI to subcellularly inactivate target proteins is that the effect should be reversible providing that there is sufficient exchange of nonirradiated and irradiated protein: fluorescence recovery is indicative of nondamaged protein entering the effected region.…”