The majority of the DNA constructs used in the current protocol can be obtained from the addgene website (www.​addgene.​org, search for reagents from Dr. Klaus Hahn’s laboratory) or from our lab upon request (Table 1). PA-Rac contains the following protein sequences: LOV(404-546)-Rac(4-192, Q61L/E91H/N92H). Q61L is a constitutively active mutation in Rac that renders it GTP hydrolysis deficient. The mutations E91H and N92H were introduced empirically to disrupt Rac-GAP interactions. Overexpression of the LOV-Rac fusion that harbors only the Q61L mutation led to apparent membrane ruffles in cells in the dark, consistent with an elevated background of Rac activation. We speculated that this is due to a dominant-negative effect of this earlier construct on endogenous Rac because it may sequester GAP proteins. The GAP-binding mutations in PA-Rac resulted in improved binding of effector PAK in the light yet substantially reduced the background Rac activity in the dark.

We generated PA-Rac constructs with various fluorescent protein tags, including mCerulean, mVenus, and mCherry, to facilitate the identification of cells expressing PA-Rac in transient experiments. Based on the excitation wavelengths of these fluorescent proteins, mCherry (~590 nm) can be monitored without concern for excitation of the LOV domain. The standard commercial excitation filters for mVenus pass light below 500 nm which can activate the LOV domain. As a result, we recommend using different, somewhat suboptimal, excitation filters that blocks transmission below 510 nm, together with imaging routines that use attenuated excitation intensity and brief exposure time, to avoid unintended photoconversion of the LOV domain. Because the excitation wavelength of mCerulean (433 nm max) falls in the photoactivation spectrum, fluorescence imaging of mCerulean is best used for confirmation of expression at the end of experiments.

For transient transfections, plasmids in pTriEx-4 vector (Novagen) should be used. Alternatively plasmids in tetracycline-inducible expression (Tet-Off) retroviral vector pBabe-TetCMV-puro can be used to generate cell lines that have stable expression. With retroviral gene delivery, lower but more uniform expression of PA-Rac can be achieved, which is beneficial in obtaining consistent photoactivation responses among different cells. The inducible expression system is of favor because of the lack of expression of PA-Rac in cells in the presence of low concentration of doxycycline in the medium. The cells can be maintained and passaged in the light without worrying about unintended photoactivation. We recommend using the included fluorescent protein tag to gauge expression level, determining optimum expression for light-induced Rac activation with minimal background activity in the dark. The expression level, if needed, can be further controlled through titration with different concentrations of doxycycline. Alternatively, populations of stable cells with optimal expression level can be isolated using fluorescence activated cell sorting (FACS).

Other common reagents for cell culture can be obtained from Invitrogen. Additional reagents for specific imaging applications will be described in Subheading 3.

Localized illumination with precise computer control can be accomplished using most laser scanning confocal microscopes, and applications using PA-Rac on these systems should be relatively straightforward (see Note 1 ). Alternatively, localized illumination can be achieved using most conventional wide-field microscopes that have a field stop/diaphragm in place or can be modified to incorporate a pinhole in a conjugate image plane. The broad-spectrum light source used for epifluorescence excitation can be used to illuminate a small region of the cell. Commercial vendors have devised a variety of add-on solutions for laser irradiation of small portions of the field of view, usually for FRAP studies. A laser beam can be coupled into the light path and focused on the focal plane to a diffraction-limited spot or dilated to bigger areas through z offset. These spots can also be mobilized to scan across different shapes either manually or using galvanometer-driven mirrors. Complex patterns can be achieved using digital micromirror-based devices such as the commercially available Mosaic active illumination system (Andor) or more advanced methods based on liquid-crystal spatial light modulators, realized in applications involving laser tweezers [17] and adaptive optics [18]. We opted for the Mosaic active illumination system because of its ability to simultaneously illuminate multiple regions of interest and fast switching time. One of the concerns with this method is insufficient intensity output. Because the LOV domain has a reasonable extinction coefficient (ε ₄₄₉ = 12, 200 mol⁻¹ cm⁻¹) but high quantum yield (0.44) [19], we found that even collimated consumer LED light source (<1 W output) can induce membrane protrusion in live cells upon brief (seconds), localized illumination. We found that recent versions (TE2000 and Ti) of Nikon inverted epifluorescence microscopes are convenient for such applications because they have a “stage-up” design (see Note 2 ), proven advantageous over systems from other microscope manufacturers. The stage-up design allows an ideal incorporation of the Mosaic system so that epifluorescence excitation and Mosaic active illumination can be incorporated independently in the infinity space between objective and camera. Two motorized turrets for filter blocks also permit rapid and independent control of filter settings for imaging and photoactivation (see Fig. 2). Additional components needed for the customization of such an imaging system are:

We also installed a small CO₂ incubator near the microscope in the darkroom and yellow or red consumer LEDs (PAR38) for darkroom lightings for convenience in sample handling.