Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics

Abstract

Recent developments in reagent design can address problems in single cells that were not previously approachable. We have attempted to foresee what will become possible, and the sorts of biological problems that become tractable with these novel reagents. We have focused on the novel fluorescent proteins that allow convenient multiplexing, and provide for a time-dependent analysis of events in single cells. Methods for fluorescently labeling specific molecules, including endogenously expressed proteins and mRNA have progressed and are now commonly used in a variety of organisms. Finally, sensitive microscopic methods have become more routine practice. This article emphasizes that the time is right to coordinate these approaches for a new initiative on single cell imaging of biological molecules.

Figure 1

Major groups of RFPs, their photophysical properties, and potential applications are shown. (a) Conventional and split RFPs. Two non-fluorescent fragments of split FP when brought together form a complete FP barrel. (b) Large Stokes shift RFPs. Excited state proton transfer was shown to be responsible for large Stokes shift. (c) Fluorescent timers. (d) Three types of photoactivatable RFPs. Dark-to-red PAFPs irreversably convert from non-fluorescent state to the fluorescent state under violet light (photoactivation). Green-to-red PAFPs irreversably convert from green fluorescent state to red fluorescent state under violet light (photoconvertion). Red-to-dark photoswitchable FPs reversably convert from non-fluorescent state to the fluorescent state under different lights (photoswitching).

Figure 2

Advanced microscopy and spectroscopy techniques for imaging gene expression, nuclear localization, and dynamics. (a) Super-resolution microscopy: The first class of super-resolution microscopy exploits the nonlinear optics to reduce the illumination spot size in technique such as stimulated emission depletion (STED) microscopy, reversible saturable optical fluorescence transition (RESOLFT) microscopy [47], and saturated structured illumination microscopy (SSIM) [48]. The second class involves repeated activation and bleaching of sparsely selected fluorescent molecule and subsequently accurate localization to build up the high resolution images, such as photoactivation localization microscopy (PALM) and its close variants STORM and fPALM [25^•]. Single particle tracking PALM (sptPALM) allows tracking of high density molecules in live cell [29]. (b) MPM: Multiphoton microscopy [49] offers attractive feature over traditional confocal and widefield microscopy for live cell and thick tissue imaging for its increased penetration depth owing to less light scattering, reduced autofluorescence and photobleaching, minimal absorbance of hemoglobin and skin melanin at the longer wavelengths, and its optical sectioning effect. Development of RFPs with large Stokes shift and far-red spectrum enables multicolor in vivo MPM with subcellular resolution [12]. (c) FFS: Fluorescence fluctuation spectroscopy includes a variety of techniques that utilize the fluctuating fluorescence signal when molecules randomly diffuse through a subfemtoliter observation volume created by confocal or two-photon microscope. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) [31,50] exploit the temporal decay of correlation/crosscorrelation of the signal to extract the concentration, mobility, and the interaction information. Brightness analysis studies the amplitude of the fluctuation and provides stoichiometry and affinity information of interactions [33]. Image correlation spectroscopy (ICS) and cross correlation spectroscopy (ICCS) [32] measures spatially fluctuating signal from raster-scan laser confocal/two-photon microscopy. They are powerful tools to measure the clustering and dynamics of membrane proteins and receptors. (d) FRAP, FLIP, Photoactivation, Photoconversion: Molecules in a region of interest are optically highlighted by photobleaching or photoactivation [28]. As the highlighted molecule exchanges with the surrounding unhighlighted ones owing to diffusion and binding, the fluorescence in the ROI is monitored to obtain the kinetic information about mobility and interaction. (e) FRET: Fluorescence resonance energy transfer measures the effect of excited-state energy transfer from donor to an adjacent acceptor protein. FRET provides evidence for direct interaction since the energy transfer occurs only when donor and acceptor are within 10 nm of each other. Compared with FFS, FRET is independent of the mobility of the molecule under investigation. FRET can be measured simply by acceptor bleaching or ratiometric imaging. However ratiometric imaging is not appropriate for general purpose protein interaction assays since it depends on relative concentration of donor and acceptor. Fluorescence lifetime imaging microscopy (FLIM) based FRET assay is not limited by this and is commonly applied to detect protein interactions. (f) BiFC [38]: In bimolecular fluorescence complementation experiment, an FP is split into two segments and fused to two interacting molecules. The two segments remain dark until the interacting partners bring them together and form a complete FP. However, owing to the maturation of fluorophore, there is delay between the interaction and the appearance of fluorescence. In certain scenario, the formation of bimolecular complex is irreversible, which complicates the physiological process understudy. BiFC has been successfully applied to study protein–protein interaction. (g) Super-registration microscopy: Imaging two interacting molecules in different color with high spatial and temporal resolution is challenging. The super-registration microscopy [26] exploits a natural cellular marker to register positions in different detection channels beyond the diffraction limit. It has been applied to detect a single mRNA particle passing through a single nuclear pore. Currently, the technique is limited to the case that the cellular marker is relative immobile during the time of imaging.

Figure 3

The gene expression in eukaryotic cells involves many steps and numerous components. First transcription requires close cooperation between transcription factor, corregulator, mediator, chromatin remodeler, histone covalent modifier, and basal transcription machinery. After transcription, the mRNA is again subjected to post-transcription modification, export, localization, translation, and degradation. Each individual step can be visualized by tagging corresponding factors with different FPs. Quantitative microscopy techniques allow one to extract dynamic information as reviewed in the text.