NG108-15 cells (gifts of Drs. David Julius and Neil Smalheiser, University of California San Francisco) were cultured in GIBCO BRL DME H21 cell culture media supplemented with 10% FCS, penicillin/streptomycin, and 1× H.A.T. at 37°C and 5% CO2
. 4–5 d before microinjection, the media was supplemented with 1 mM dibutyryl cyclic AMP, an agent that induces formation of axons and growth cones in these cells (Furuya and Furuya 1983
). In preparation for microinjection, cells were briefly trypsinized (0.05% in 1 mM EDTA) and replated on a 25-mm-circular glass coverslip attached with silicone grease to the bottom of a 35-mm polystyrene tissue culture dish in which we had drilled a hole. These coverslips were precoated with poly-d
-lysine and coated with matrigel (a mixture of extracellular matrix proteins, primarily collagen and laminin) just before plating cells, essentially as described (Tanaka and Kirschner 1991
). Cells were transferred to coverslips 24–48 h before microinjection and incubated in 1 ml of media without phenol red. 30 min before microinjection, the media was supplemented with 25 mM sodium-Hepes, pH 7.4. On the microscope, the culture dishes were placed in a water-heated machine chamber maintained at 38°C. The temperature near the cells was close to 30°C because of loss of heat through contact with the microscope objective.
For photoactivation, we labeled rabbit muscle actin on its reactive thiol with the caged rhodamine derivative α-carboxy-dimethoxy-C2CQRd-IA (caged Q-rhodamine)1
as described (Mitchison et al. 1998
). This actin derivative has been shown to form filaments in vitro and to localize to actin-containing structures (Mitchison et al. 1998
). Caged Q-rhodamine is the rhodamine derived from 7-hydroxy-quinoline, caged as a bis-carbamate with two α-carboxy-dimethoxy-nitrobenzyl groups. None of the other caged fluorochromes we have worked with were satisfactory for actin labeling because of either poor photostability of the final fluorochrome (fluorescein and resorufin derivatives) or slow uncaging (caged rhodamines without α-carboxy substitution on the nitrobenzyl caging group). Caged Q-rhodamine actin was stored at 4–5 mg/ml in G buffer (5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2
, 1 mM ATP, 5 mM glutathione) in 5-μl aliquots at −80°C. For microinjection, it was diluted twofold with a solution of Cy5-conjugated dextran in G buffer. Injection was verified, and cells found after stage movement, using the Cy5 signal. Gerrard Marriot (Max Planck Institute, Martinsried, Germany) provided us with a plasmid encoding Dictyostelium discoideum
actin fused to GFP (Choidas et al. 1998
). Cells were transfected using a standard calcium phosphate method (Maniatis et al. 1989
A Zeiss Axiovert inverted microscope with a bottom port was modified for photoactivation and photobleaching experiments. A mercury arc lamp illuminating a slit in a conjugate focal plane was used to generate a bar of 360-nm light for photoactivation experiments. An argon ion laser focused through a cylindrical lens was used to generate a bar of 488-nm light for GFP photobleaching experiments. Descriptions of our photoactivation microscopes have been published elsewhere (Mitchison et al. 1998
Phase and fluorescence images were acquired through a 100× objective to cooled CCD cameras from Princeton Instruments (http://www.prinst. com). For photoactivation experiments, we used a Kodak KAF1400 chip with 6.9-μm pixels cooled to −40°C. For phase images, we used 2 × 2 binning and for fluorescence images, 4 × 4 binning. For photobleaching experiments, we used a Sony 768x512 interline chip with 8.3-μm pixels cooled to –10°C. Phase images were collected without binning, and fluorescence images were collected with 2 × 2 binning. In both cases, we typically used 200–400-ms exposures with 100 W Hg illumination and standard fluorescence filters.
Images were acquired with Princeton Instrument's WinView software with additional home-written software to control image acquisition. Delay between phase and fluorescence images was typically <2 s. Negligible changes occur in NG108 filopodia over this time-scale, so these image pairs effectively represent a single time point for our purposes.
Locations of filopodium tips and photo-marks were determined by measuring the position of each with respect to the substrate along the axis of the filopodium. In practice, a single reference line was overlaid on the phase image of each filopodium, and tip distances were calculated with respect to the end of the line for each time point in the sequence. Filopodia were sometimes observed to pivot about an axis located in the growth cone body, as previously reported (Bray and Chapman 1985
). Only filopodia that rotated <15° were included in our study. Generally, filopodia that exhibited minor pivoting behavior rotated smoothly from one position to another. Reference lines were drawn to bisect the angle of filopodium rotation and intersect with the axis of rotation. The position of the tip was projected via a perpendicular onto the reference line. Subsequently, an identical reference line was generated in the fluorescence channel, and similar measurements of the mark were taken. When we generated distance versus time plots of this data, all measurements were normalized with respect to the position or length recorded at t = 0, the time the mark was made.