Chromophore-assisted laser inactivation (CALI) inactivates target proteins by generating reactive photoproducts such as reactive oxygen species (ROS) by intense irradiation of photosensitizer chromophores, such as EGFP. Capping protein (CapZ, abbreviated CP throughout) is an obligate heterodimer of alpha and beta subunits and is the predominant actin filament capping protein in most cells. Rat2 cells deficient in endogenous CP, but rescued with EGFP-CP (KDR cells), were subjected to CALI. After administration of a 100-ms dose of 1.5 mW/[μm]2 laser light, KDR cells exhibited dynamic changes on the dorsal surface of the cell, forming numerous protrusions as seen in the DIC time-lapse movie (CIL# 25821). This is a movie of a zoomed area of the irradiated region from CIL# 25821 and control movies are CIL#s 25823, 25824. This movie is Movie2 in Supporting Information and still images are in Fig 2B, top panels, in Proc Natl Acad Sci (2007) 104: 6702-6707.
EGFP-CP CALI experiments were performed by using a Spectra Physics Stabilite 2017 (Spectra Physics Laser Incorporated) argon ion laser (488 nm line, 2 W of beam power at the laser head) focused onto a 23.4 μm diameter spot (1/[e]2 diameter) through a 60x 1.45 N.A. PlanApo TIRF objective (Olympus). Irradiation was controlled with a fast Uniblitz (Vincent Associates) shutter. Laser power at the specimen plane dropped to 625 mW because of optical losses and was measured by placing the sensor of a laser power meter (Model FM with LS 10 head, Coherent Inc.) directly above the objective. Irradiation time was 100 ms, resulting in a 62.5-mJ dose of energy for the CALI experiment. Laser light was focused onto the specimen plane through a 488-nm laser filter set (Z488/10, HQ525/50, Z488RDC, Chroma Technology Corporation). Rat2 cells were plated onto 35-mm glass-bottomed dishes coated with 10 mg/ml fibronectin. Twenty minutes after plating, media was changed to DMEM without phenol red supplemented with 10% FBS, l-glutamine, and penicillin/streptomycin. DIC time-lapse imaging was performed on an Olympus IX81 inverted microscope equipped with a cooled 12-bit digital CCD camera (Sensicam QE, Cooke Corporation), and a heated chamber (Werner Instruments) with CO2 flow. All images were acquired with MetaMorph software (Version 6.0, Universal Imaging Corporation).
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