Use of New Scanning Technology to Elucidate Ion Channel Mobility

Fluorescent chromophores fused to specific proteins have greatly increased our ability to image dynamic events within the living cell. However, conventional fluorescence images alone do not provide much information about the turnover rate and mobility of individual molecules within a population. Fluorescence recovery after photobleaching (FRAP) is a technique that has been used since 1976 to study diffusion by measuring the rate of replacement of molecules within a photobleached region of the cell by fluorescent molecules from neighboring locations.1 FRAP measurements of diffusion rates and mobility have yielded insights into the organization of proteins in receptor–ligand binding, macromolecular complexes, and membrane structure.

The green fluorescent protein (GFP) has been mutated for optimal imaging and generally exhibits an excitation peak coincident with the commonly used 488-nm line of an argon laser and emissions that can be collected by standard fluorescein barrier filters. Further modification of this protein has produced a photoactivatable GFP (PA-GFP) in which exposure to an intense light pulse photoconverts the GFP chromophore, causing a 100-fold increase in the protein’s fluorescence. 2 Thus, only regions that have been photoactivated will emit above-background green fluorescence when excited with 488-nm laser light. Photoactivated GFP-tagged proteins can then be tracked as they move within the cell.

This recent derivation of PA-GFP enables distinct pools of molecules to be highlighted within live cells. Using light microscopy, photoactivation can be targeted to a defined region within the field of view. GFP-tagged proteins within the targeted region can be monitored by fluorescent imaging , while GFP-tagged proteins located outside the targeted region remain only marginally fluorescent.

Thus, through the use of targeted laser stimulation, both FRAP and PA-GFP can be utilized as complementary monitors of protein trafficking in live cell imaging studies.

Laser scanning mechanisms

Most studies of the type described here are performed using laser scanning confocal microscope systems. Photobleaching is typically performed using a high-intensity pulse of the same laser employed to image the fluorochrome: 488 nm in the case of GFP, and 514 nm for yellow fluorescent protein (YFP). Targeted photoactivation of GFP is achieved using brief exposure from a 405-nm diode laser or the 413-nm line of a krypton laser. In point scanning confocal microscopes, galvanometer-driven mirrors scan the laser beam over a rectangular field of view in a raster pattern. For stimulation (photoactivation or photobleaching), the angle traversed by the mirrors can be reduced in order to concentrate the photon flux of the bleaching or activating laser. After a period of stimulation, the mirrors are returned to their full field of view for conventional imaging. Due to the time lag between the stimulus scanning and image acquisition, rapid cellular events may be missed before imaging resumes.

Figure 1 - FluoView FV1000 confocal laser scanning microscope.

The FluoView FV1000 confocal laser scanning microscope (Olympus Corp., Tokyo, Japan) has addressed this issue by adding a second, independent scanner for laser stimulation while the main imaging scanner continues to capture image information before, during, and after the stimulus (Figure 1). This simultaneous (SIM) scanner is synchronized in time and space with the main scanner so that the SIM scanner precisely stimulates the defined regions of interest.


FRAP and PA-GFP used in ion channel studies

Ion channels are organized in membrane microdomains close to the signaling molecules that interact with them. Dynamic interactions such as protein binding, turnover, and sequestration are important mechanisms for modulating ion channel activity. Understanding these mechanisms is critical to the development of therapeutic agents for neurological disorders. The Tamkun Laboratory at Colorado State University (Fort Collins, CO) has used FRAP and PAGFP to elucidate the different organization and trafficking of functionally distinct voltage gated potassium channels.3,4 Voltage gated ion channels are important components of the electrical signaling pathway in excitable cells such as neurons. These channels open and close in response to changes in transmembrane voltage, allowing ions such as Na+, K+, and Ca++ to enter or leave the cell.

Figure 2 - Rapid recovery of fluorescence in FRAP experiments using GFP-tagged Kv1.4. Similar FRAP experiments using GFP-tagged Kv2.1 showed no visible recovery. Images from a FRAP series in a GFP-Kv1.4 expressing HEK cell. ROI#2 (red circle) was bleached using the 405-nm line of a FluoView FV1000 laser scanning confocal microscope in Tornado Scanning mode and recovery monitored every 5 sec for 250 sec. ROI#3 (cyan line) was used for quantification (data not shown). All research images courtesy of Dr. Kristen M.S. O’Connell and Prof. Michael M. Tamkun (Colorado State University).

Figure 3 - Comparison of Kv1.4 and Kv2.1 mobility using PA-GFP. Scale bars: 10 μm. a) Rapid diffusion of activated PA-GFP-labeled channels from the site of photoactivation throughout the membrane. Approx. 13 min after photoactivation, PA-GFP-Kv1.4 is evenly distributed over the entire cell membrane. b) Slow organization of activated PA-GFP-labeled channels into discrete puncta and restricted movement to membrane sites outside of the activated region.

FRAP studies indicate that the channel known as Kv1.4 is localized to the plasma membrane and diffuses rapidly (Figure 2). In contrast, Kv2.1 appears to be less mobile within the membrane. Using PA-GFP, Kv1.4 again appears to diffuse rapidly to surrounding areas of the membrane (Figure 3a), whereas Kv2.1 organizes into puncta that move slowly to adjacent membrane (Figure 3b). Corroborative studies indicate that Kv1.4 sediments as a small tetramer, whereas Kv2.1 is part of a large macromolecular complex.

Additional advantages

Along with the FluoView FV1000 SIM scanner, a technical development known as Tornado Scanning (Olympus) has been added to address the bleaching limitations of conventional raster scanning. With ordinary raster scanners, the mirrors must slow down and reverse direction at the end of each line. This results in an increased dwell time for the laser at the edges of the scanned rectangle. To counter this uneven exposure, the laser beam is blocked outside of the field of view, during the mirror turnaround.

Figure 4 - Comparison of bleaching rates between Tornado Scanning and conventional raster scanning using the same laser intensity on test targets.

Such beam-blanking at the edge of the image is important during fluorescence imaging. However, when beam-blanking occurs during photobleaching studies, fluorescence recovery can begin at multiple time points, potentially confounding the true kinetics of the cellular process. Tornado Scanning, an option provided by the Fluo View FV1000, is a continuous spiraling scan, described by a circle drawn on the image. The relentless laser exposure results in a faster bleach rate (Figure 4). The same loss of fluorescence occurs in a shorter time, arguably reducing the overall laser exposure for the cell.

Conclusion

Important to both FRAP and PA-GFP experiments, rapid dynamic molecular movements within living cells can now be simultaneously observed during an efficient stimulation process. Molecular biology and optoelectronics technology are coming together to elucidate the dynamic world of the living cell.

References

  1. Jacobson K, Derzko Z, Wu ES, Hou Y, Poste G. Measurement of the lateral mobility of cell surface components in single, living cells by fluorescence recovery after photobleaching. J Supramol Struct 1976; 5(4):565(417)–76(428).
  2. Patterson GH, Lippincott-Schwartz J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 2002; 297:1873–7.
  3. O’Connell KMS, Tamkun MM. Role of the N- and C-terminal domains of Kv2.1 in channel trafficking and cell surface localization. 49th Annual Meeting of the Biophysical Society, Long Beach, CA, Feb 12–16, 2005.
  4. O’Connell KMS, Tamkun MM. Distinct subcellular localization and trafficking of Kv2.1, Kv1.4 and Kv1.3 channel isoforms. 49th Annual Meeting of the Biophysical Society, Long Beach, CA, Feb 12–16, 2005.

Ms. Goodacre is Applications Specialist, Olympus America Inc., 2 Corporate Park Dr., Melville, NY 11747, U.S.A.; tel.: 800-455-8236; fax: 631-844-5111; e-mail: angela.goodacre@olympus.com. Dr. O’Connell is a Fellowship Grant Trainee in the laboratory of Prof. Michael M. Tamkun, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, U.S.A.

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