Live cell fluorescence microscopy offers a powerful way to examine structure and dynamics of proteins and protein complexes, and has been used to elucidate many of the spatial and temporal relationships between molecules within the cell. While most fluorescence microscopy relies solely on signal brightness, the photophysical properties of fluorescence provide many other parameters that can be utilized as well. These parameters include spectral shape, lifetime, and polarization. Fluorescence polarization, also referred to as anisotropy, has long been used in cuvette spectroscopy of proteins and lipids. While the basic infrastructure needed to make anisotropy measurements in the microscope has been developed, this approach has not yet found widespread use. This is somewhat surprising, considering that the polarizations of widely utilized fluorescence proteins are intrinsically high because of the fixed orientation of chromophore in the β-barrel structure (1). In this issue of the Biophysical Journal, a group at Rockefeller University describes the elegant utilization of fluorescence polarization to resolve structure of individual components of the nuclear pore complex (NPC) in live cells (2). This novel, to my knowledge, approach permits the researchers to distinguish between ordered and disordered domains within the NPC components. As the authors rightfully conclude, this information provides an important bridge between high-resolution structural information available from x-ray crystallography or cryo-electromagnetic studies and more-traditional cell biological studies that offer limited spatial resolution. This study also provides a comprehensive theoretical and experimental framework for anisotropy imaging experiments that should open this approach up for the study of many other intracellular protein complexes. Three experimental insights by Mattheyses et al. (2) made this study possible. First, the authors utilized GFP, which provides not only outstanding specificity of labeling, but also highly polarized fluorescence. Because the GFP chromophore is rigid within the overall protein structure, which is slowly rotating (10–20-ns rotational correlation time) with respect to the fluorescence lifetime of ∼3 ns, this results in highly polarized fluorescence that is easy to measure. Second, the authors used no extra amino acids between the GFP and the NPC component protein of interest. This led to a sufficiently rigid structure in which the GFP orientation reproduces the labeled protein orientation as well as possible. Third, they used the known symmetry of the NPC to obtain a full 360° orientation map at each NPC. Similar approaches have been used to map the plasma membrane as in, for instance, red blood cell ghosts (3), but this is the first such application to a single protein complex. Because many protein complexes have been shown to possess significant symmetry, this approach may prove quite general. As for any powerful technique, there are potential experimental pitfalls that must be carefully considered. For these detailed anisotropy imaging experiments, two important potential artifacts come from the possibility of resonance energy transfer between labels (homo-Forster resonance energy transfer, or homoFRET), and the use of a high numerical aperture objective. Both of these issues are addressed by the authors both theoretically and experimentally in exemplary fashion. First, because homoFRET can occur between two labels in different orientations, it can appear as a “rotation” and dramatically change the measured polarization (4). This is of particular concern at higher concentrations of labels, which might be expected within a single NPC. Fortunately, in this study, control experiments performed with different labeling concentrations showed that homoFRET was not an issue. Secondly, the sensitivity of any fluorescence microscopy experiment depends on the number of photons collected, and typically high numerical aperture objectives are used to facilitate maximal light collection. However, the use of polarized light with high NA lenses requires extra corrections, which were performed and validated as part of this study. One of the key difficulties in studying protein complexes inside the cell has been the lack of appropriate techniques for live cell imaging. Scanning near-field optical microscopy (SNOM) and total internal reflection fluorescence microscopy (TIRF) have been used to examine NPC in vitro, but because they are inherently surface measurement techniques, neither approach can be used to assay the structure or dynamics of the NPC inside the cell. Both single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) have been used to monitor molecules passing through the NPC pore, and these approaches can offer dynamic information at high spatial and temporal resolution, but they yield no information related directly to the structure of the NPC. Anisotropy microscopy with GFP labels, on the other hand, is somewhat analogous to the probe methods used to explore protein structure, such as luminescence resonance energy transfer (LRET) (5), tryptophan quenching of fluorescence (6), and site-directed spin-labeling electron paramagnetic resonance (EPR) (7). Most importantly, anisotropy imaging can be accomplished in live cells, yielding a new and complementary in vivo link among structure, dynamics, and the actual function of protein complexes in their native environment.
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Jul 22, 2024
Fabian Haake + 4
Open Access