Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments.

Cell Culture and Transfections Mouse 10T1/2 cells, human intestinal smooth muscle cells, human lung cancer cells, human neuroblastoma cells, or Indian muntjac fibroblasts were plated onto glass coverslips and cultured 1–2 d in growth medium until 50% confluent. At this point, complete medium was removed and cells were transfected using lipofectamine (GIBCO BRL) and an ASF–GFP fusion protein construct described previously ( Sleeman et al. 1998 ). After transfection, cells were cultured in complete medium for ∼24 h before imaging. In some cases, staurosporine (Sigma-Aldrich) or 5,6-dichlorobenzimidazole riboside (DRB; Sigma-Aldrich) were added at 100 μg/ml and 75 μg/ml, respectively, between 2 and 4 h before imaging.

Live Cell Imaging

Coverslips were placed on glass slides containing several drops of media surrounded by vacuum grease. The vacuum grease allows an airtight seal to form. Cells are capable of growing in these conditions for least 24 h at 22°C. For 4-D imaging, a Zeiss AxioPlan II microscope, a 100× 1.4 NA lens, and a 12-bit cooled CCD (Cooke SensiCam) were used to collect images. Images were further processed by digital deconvolution using AutoQuant Autodeblur. For FRAP, the laser scanning microscope (Zeiss LSM 510) was set to laser scanning mode and the initial imaging conditions were determined. A 25× 0.8 NA lens was used for these experiments and pixel sampling was set between 90 and 120 nm/pixel. The argon laser spectral line at a wavelength of 488 nm was set to an intensity of no greater than 1.25% of its total power (15 mW) for image collection. A mask, which typically covered half of the cell nucleus, was then photobleached using 100 iterations at 3.75 mW laser power. 12-bit images were collected before, immediately after, and at defined intervals after bleaching. Our total experiment time was set to 500 s, which was sufficient for complete recovery from photobleaching. For quantitative imaging, a 3-μm wide strip of the cell was photobleached and then scanned during recovery. Photobleaching was completed in ∼2 s. Similar proportions of GFP (∼25%) and ASF–GFP (∼30%) total nuclear fluorescence were lost during photobleaching. Because the GFP protein recovered very rapidly, it was necessary to only scan the photobleached region in order to adequately sample GFP recovery. For simplicity in the graphical presentation of the data, the initial post-bleach value of the photobleached region was normalized to zero by subtraction and the maximal value obtained during recovery was normalized to 100. The t 1/2 of recovery can then be directly read off of the graph as the 50% value. Identical results were obtained when 2-μm spots, rather than strips, were bleached. To determine the immobile population of ASF–GFP, the maximal recovered value was divided by the starting value of the photobleached region after correction for total nuclear fluorescence lost during photobleaching. This correction factor was determined by measuring the total nuclear fluorescence immediately after photobleaching and dividing this value by the total nuclear fluorescence immediately before bleaching. The laser intensity during recovery was set so that no measurable loss of fluorescence was observed during the monitoring period. To track the mobility of HDAC4–GFP domains and small foci enriched in ASF–GFP we used the track points function in Universal Imaging's MetaMorph 4.0 imaging software. The image sequences were viewed on screen at 200% magnification and the center of each focus was identified with a mouse. For HDAC4 domains, the structures sometimes moved out of the plane of focus. In this instance, the foci could still be followed by identifying slightly elevated regions of signal intensity. Although difficult to identify in single frames, these could be easily identified by rapidly moving between the several frames where the domains move into and out of focus. This occurred for two of the five foci tracked. These foci, however, remained in focus for most of the time course.

Photobleaching and Cell Viability

Photobleaching experiments have an undeserved reputation of being photodestructive to living cells. Excessive photodamage, if it occurred, would complicate collection and interpretation of FRAP-based experimental data. However, bleaching GFP using the monochromatic laser at low intensities needed for bleaching and image acquisition, does not noticeably perturb live cells (reviewed White and Stelzer 1999 ). After repeated bleaching, cells expressing GFP-tagged trafficking proteins ( Nakata et al. 1998 ) and centrosome proteins ( Khodjakov and Rieder 1999 ) have remained viable for periods of hours, even to continue through mitosis, an event noted for its sensitivity to cell toxicity. As for cells remaining viable for periods long enough to complete photobleaching experiments, the conditions we have used are sufficient for cells to grow for >24 h and to undergo mitosis (our unpublished observations). To assess that cells remain viable during the initial photobleaching and recovery period, we monitored cells by differential interference contrast (DIC) optics for changes in cellular morphology as well as the maintenance of cytoplasmic organelle transport and the absence of detectable changes in mitochondrial structure (e.g., swelling). Therefore, we are confident that our experimental conditions and/or design does not erroneously influence the collection of FRAP or fluorescence loss in photobleaching (FLIP)–based data. Online Supplemental Material Video 1 further depicts Fig. 1 . A cell expressing ASF–GFP is shown in 3-D red/green stereo time-lapse. Individual frames were captured at 2-min intervals. Videos 2 and 3 further depict Fig. 2 . A cell expressing an HDAC4–GFP fusion protein (video 2) or ASF–GFP (video 3) was captured as a 2-D time-lapse experiment at 1-s intervals for 60 s. These videos are available at http://www.jcb.org/cgi/content/full/150/1/41/DC1.

Online Supplemental Material Video 1 further depicts Fig. 1 . A cell expressing ASF–GFP is shown in 3-D red/green stereo time-lapse. Individual frames were captured at 2-min intervals. Videos 2 and 3 further depict Fig. 2 . A cell expressing an HDAC4–GFP fusion protein (video 2) or ASF–GFP (video 3) was captured as a 2-D time-lapse experiment at 1-s intervals for 60 s. These videos are available at http://www.jcb.org/cgi/content/full/150/1/41/DC1.

Supplemental Material [Supplemental Material Index]