DNA constructs, cell transfections, and fluorescent probes
COS-7 cells were maintained in DME supplemented with 10% fetal calf serum at 5% CO2
and 37°C. Cells were transfected using FuGENE 6 (Roche Diagnostics) according manufacturer's protocol and imaged 1 and 2 d after transfection. cDNA for GFP-Ras chimeras (EGFP-CVLS, EGFP-CLLL, EGFP-HRas C181S, C184S, EGFP-NRas C181S, EGFP-HRas, and EGFP-NRas) and GFP-GPI were as previously described (Choy et al., 1999
; Nichols et al., 2001
). Note that no linkers were used in the construction of EGFP-CLLL and EGFP-CVSL. For simplicity, EGFP is referred to as GFP in the text. Control experiments confirmed that the CAAX motif of overexpressed GFP-Ras is quantitatively processed, and that in the absence of farnesylation the protein is not associated with any intracellular membranes (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200502063/DC1
). CTXB (Sigma-Aldrich) was fluorescently labeled with Cy3 according to the manufacturer's instructions (GE Healthcare). Cells were labeled with 1 μg/ml Cy3-CTXB for 15 min on ice and washed several times before imaging or drug treatments.
Nocodazole and cycloheximide treatments
To disrupt microtubules, cells were preincubated for 5 min on ice in DME containing 10% fetal calf serum and 50 mM Hepes. The cells were then treated with 5 μg/ml nocodazole (Sigma-Aldrich) for 15 min on ice, warmed for 5 min to 37°C, and imaged in the continued presence of nocodazole at 37°C. Control experiments were performed using vehicle alone (DMSO). To inhibit new protein synthesis, cells were treated with 200 μg/ml cycloheximide (Sigma-Aldrich) in DME, 10% fetal calf serum, and 50 mM Hepes for 4 h at 37°C. The cells were then imaged at 37°C in the cycloheximide solution.
Fluorescence microscopy and photobleaching measurements
Cells were imaged with an inverted laser scanning confocal microscope (model 510; Carl Zeiss MicroImaging, Inc.) equipped with the Confocor2 for FCS (Carl Zeiss MicroImaging, Inc.). Where indicated, an Air Stream Stage Incubator (Nevtek) was used for imaging at 37°C. GFP was excited with an argon laser with excitation at 488 nm and emission was detected with a GFP long-pass (LP) 505 or 530 or band-pass (BP) 505–530 filter. A Plan-Neofluar 40×/1.3 oil immersion lens was used for imaging all samples. Cells were maintained in phenol-red free DME containing 10% fetal calf serum and 50 mM Hepes for live-cell imaging experiments.
Confocal FRAP measurements were performed using a previously described protocol (Kenworthy et al., 2004
). In brief, a strip 4 μm wide was photobleached using high laser intensity and fluorescence recovery monitored at low intensity. Diffusion coefficients were calculated from whole-cell recoveries using a program that simulates diffusion (Siggia et al., 2000
). Mf was calculated as described previously (Ellenberg et al., 1997
). Statistical differences were evaluated using the t
In experiments measuring kinetics of Golgi refilling, an area containing the entire Golgi was bleached (Nichols et al., 2001
). Halftimes of recovery were calculated as described in Feder et al. (1996)
, and the final percentage of recovered fluorescence was calculated as for Mf after correcting for the loss of fluorescence due to the photobleaching event. Control experiments on fixed cells confirmed that the loss of fluorescence was confined to the bleached region.
All quantitative image analysis was performed using unprocessed images. For presentation purposes, image contrast was adjusted using Adobe Photoshop.
Quantitation of Ras localization after 2BP treatment
Cells were treated with 25 μM 2BP (Sigma-Aldrich) or vehicle (DMSO) at 37°C, either immediately after transfection or 18 h after transfection for the indicated times (30 min, 2 h, or 5 h). After treatment, cells were imaged live with the confocal pinhole fully open for quantitation or set at 1–2 Airy units for presentation purposes. The subcellular distribution of Ras in ~20 cells/treatment was quantitated in two ways. (1) Localization of Ras in the ER/nuclear envelope versus plasma membrane. Cells were scored for the relative amounts of ER/nuclear envelope labeling ranging from ER labeling but no apparent plasma membrane stain (++++) to no apparent ER or nuclear envelope label (−−−−). These scores were converted to a numeric value as follows: ++++ (1 pt), +++/− (0.75 pt), ++/−− (0.5 pt), +/−−− (0.25 pt), or −−−− (0 pt). The total numeric score for all cells at a given time point was calculated for each experiment. (2) Fraction of Ras localized to the Golgi complex. Images were converted to tiff format, and the average fluorescence in the Golgi region versus in the entire cell was calculated using NIH Image. After background subtraction, the ratio of fluorescent material in the Golgi region versus the entire cell was calculated.
Fluorescence correlation spectroscopy
FCS measures time-dependent fluorescence fluctuations in a diffraction-limited (0.1 femtoliter) volume defined using confocal microscope optics with a sensitive avalanche photodiode detector. Intensity fluctuations corresponding to the movements of individual molecules in and out of the confocal volume are recorded over time. Fluorescence fluctuations reflect the average residence time of the fluorescent species in the confocal volume, which in turn are a function of its characteristic diffusional mobility. The diffusion of fluorescently tagged proteins through the sampling volume occurs with a characteristic τD. This is related to the diffusion coefficient D by τD = (ω02)/(4D), where ω0 is the radius of the laser beam. Thus, a longer τD corresponds to a slower D. The intensity fluctuations are characterized by their average value <I> and their fluctuations δI(t) = I(t) − <I> and can be analyzed using an autocorrelation function G(τ). The normalized autocorrelation function is given by G(τ) = 1 + <δI(t) * δI(t + τ)> * <I>−2, where I(t) is the time-dependent fluorescence intensity, τ is a short time interval after any arbitrary time t, and <I> is the mean value of fluorescence intensity.
FCS experiments were performed on a microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) outfitted with ConfoCor2 (Carl Zeiss MicroImaging, Inc.), combining both FCS and confocal laser scanning capabilities. A C-Apochromat 40× 1.2 NA water objective was used in conjunction with a dichroic filter and 520-nm long-pass filter to focus and separate exciting and emitting radiation. GFP-tagged constructs were excited at 488 nm with a 40-mW argon laser. Aqueous rhodamine 6G solutions were used to calculate the confocal volume radius ω0 = 1.44 × 10−7 m, resulting in a confocal volume element of 0.1 fl.
Cells were imaged in LSM mode and, after selection of an appropriate cell, whole cells were repeatedly bleached to reduce the fluorescence to allow for FCS measurements. A line scan in the axial direction was performed to set the volume element at an appropriate position in the cell. FCS measurements were made for 10 s each and repeated 10 times per cell. The autocorrelation curves were fit using software provided by the manufacturer. The autocorrelation function G(τ) for a two-component model is described by the following equation: G(τ) = 1 + (1/N) [(1 − Y) (1 + τ/τD1)−1 (1 + τ/S2 τD1)−1/2 + Y (1 + τ/τD2)−1 (1 + τ/S2 τD2)−1/2].
Here, N is the number of fluorescent particles in the confocal volume; S is the structure parameter (defined by the dimensions of the confocal volume); τD1 and τD2 are the average residence times of the first and second component, respectively; and Y and 1 − Y are the fraction of particles in the confocal volume with diffusion time τD2 and τD1, respectively. Data were fit assuming a constant structure parameter of 5.0. Diffusion coefficients were calculated from the fitted values of τD and the known confocal radius ω0 as described above. Data were obtained from 10–20 cells from two independent experiments.
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