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Using photoactivatable fluorescent protein Dendra2 to track protein movement
 
Dmitriy M. Chudakov, Sergey Lukyanov, and Konstantin A. Lukyanov
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The time interval between the images should be selected depending on the speed of the target protein movement. Based on the proposed rate of protein redistribution, or on the preliminary data concerning this rate, the frequency of image acquisition within a time series should be reduced as much possible. Indeed, for long time series, too often image acquisition will require many images to be captured, leading to undesirable photobleaching of the photoactivated protein. Generally, 10–30 consecutive images are enough to measure protein mobility and at the same time to avoid undesirable photobleaching/photoconversion effects during visualization.

Examples of Dendra2 Tracking

Here we provide examples of photoactivation and tracking of Dendra2 using different laser scanning confocal microscopes.

Example 1: tracking of Dendra2 fast redistribution within HeLa nucleus; photoactivation by 405-nm laser. A Zeiss LSM 5 LIVE Duoscan confocal microscope with Plan Neofluar 40×/1.3 oil objective (Carl Zeiss, Jena, Germany) was used. HeLa cells were transiently transfected with pDendra2-C vector (Evrogen). Photoactivation was performed at the edge of the HeLa cell nucleus by a short pulse of 405-nm laser light. Further time series was obtained, 1 frame/0.6 s, in two channels: channel 1, excitation 532 nm 8%, emission collected BP 560–675; channel 2, excitation 488 nm 0.1%, emission collected BP 495–555. Diffusion of the photoactivated red fluorescent protein within the nucleus could be accurately monitored, along with its replacement with the nonactivated green fluorescent form (Figure 2).



Example 2: tracking of Dendra2 redistribution from nucleus to cytosol of HeLa cell; photoactivation by 405-nm laser.An Olympus FluoView™ FV1000 with objective UPLSAPO 60 × O NA:1.35 (Olympus, Tokyo, Japan) was used. HeLa cells were transiently transfected with pDendra2-C vector. The protein was photoactivated in the center of nucleus by a 15% 405-nm laser line, SIM Tornado (Olympus) for 200 ms. Further redistribution of both protein forms was tracked. We used the following settings to obtain a time series (1 frame/20 s): zoom 2.4 (88.064 × 88.064 µm image size); 1024 × 1024 pixels; sampling speed 2 µs per pixel; line sequential mode: channel 1, excitation with 488-nm laser line, emission 500–541 nm; channel 2, excitation with 561-nm laser line, emission 576–676 nm; DM405/488/561/633; integration count 3. Redistribution of both the photoactivated red fluorescent protein and the nonactivated green fluorescent form between the nucleus and cytosol was observed (Figure 3).



Example 3: activation and tracking Dendra2 fused with nucleolus protein fibrillarin; photoactivation by 488-nm laser. This experiment was performed using a Leica confocal inverted microscope DMIRE2 TCS SP2 equipped with HCX PL APO Ibd.BL 63× 1.4 NA oil objective (Leica, Wetzlar, Germany) and 125 mW Ar and 1 mW HeNe lasers. Figure 4 shows Dendra2-fibrillarin tracking in nucleus of a HeLa cell transiently transfected with pDendra2-fibrillarin vector (Evrogen). We used the following settings: mode, xyt; format, 512 × 512 pixels; zoom, 13 (18 × 18 µm field of view); scan speed, 400 Hz; beam expander, 3; pinhole, 140 µm; laser, 488 nm 1% power, PMT1, 500–535 nm, gain 725 V (for green fluorescence detection); or laser, 543 nm 20% power, PMT2, 560–680 nm; gain 700 V (for red fluorescence detection). Activation was done at a point within smaller nucleolus using point bleach mode by 25% 488-nm laser for 200 ms. After that, 20 images in red channel were taken with 3-s time interval. As a result, we were able to observe a drastic increase of red signal in the activated nucleolus [region of interest (ROI)1] and further migration of red signal in the nucleoplasm and adjacent nucleolus. A transient wave of Dendra2-fibrillarin migration was clearly detected in a nucleoplasm region near the activation point (ROI 2); only in about 45 s red signal became equalized across the nucleoplasm (compare ROI 2 and 3). Analysis of red signal within the nonactivated nucleolus showed that Dendra2-fibrillarin easily migrates through the nucleolus with a rate comparable to that in the nucleoplasm. So, Dendra2-fibrillarin accumulation occurred first at the side closest to the activated nucleolus (ROI 4), then in the central part (ROI 5), then at the opposite side (ROI 6), but not first at the periphery, then in the central part (as it could be expected). Apparently, such detailed information about migration of fibrillarin in nucleus cannot be obtained in a single experiment using classical approaches based on photobleaching.



We are grateful to T. Zimmermann (Centre de Regulació Genòmica, Barcelona, Spain), D. Ossipov and W. Hempell (Olympus), R. Wolleschensky and M. Kempe (Zeiss), and J. Schroeder (Leica-Microsystems). This work was supported by Russian Academy of Sciences for the program Molecular and Cell Biology, EC FP-6 Integrated Project LSHG-CT-2003-503259, National Institutes of Health (GM070358). D.M.C. and K.A.L. are supported by Grants of the President of Russian Federation MK-8236.2006.4 and Russian Science Support Foundation.

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