Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Abstract

The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.

Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore

“…In earlier work, we used narrow-field epifluorescence microscopy to visualize the transport of individual cargo molecules thorough NPCs in permeabilized HeLa cells (21,28,29). Here, we have labeled the four solvent accessible cysteines of human Imp ␣1 and the model cargo NLS-2xGFP(4C) with Alexa Fluor 568 (donor dye) or Alexa Fluor 647 (acceptor dye).…”

“…39 and 40, using an expression plasmid obtained from Y.-M. Chook (University of Texas Southwestern, Dallas, TX). The solvent accessible cysteines on NLS-2ϫGFP(4C), Imp ␣1, and CAS were reacted with a 20-fold molar excess of maleimide dye (Invitrogen) for 2 h. Labeling ratios of Ϸ3.5, Ϸ4, and Ϸ3.4 dye molecules per protein molecule, respectively, were obtained from single-molecule photobleaching histograms (29). No additional labeling was observed with up to a 160-fold molar excess of dye.…”

“…The methodology to monitor the interaction of single molecules with NPCs in permeabilized HeLa cells was established (21,28,29). In brief, single-molecule narrow-field epifluorescence measurements (400 m excitation pinhole) were performed in transport buffer [20 mM Hepes (pH 7.3), 1.5% polyvinylpyrrolidone (360 kDa) 110 mM KOAc, 5 mM NaOAc, 2 mM MgOAc, and 1 mM EDTA], including 25% glycerol unless otherwise indicated by using a Cascade 128ϩ camera (Roper Scientific) and 2.5 W ArKr mixed-gas ion laser (Spectra Physics).…”

“…To distinguish between the above Imp ␣/cargo complex dissociation mechanisms, we expanded the previously developed single-molecule narrow-field epifluorescence approach (21,28,29) to measure FRET between the individual protein components of Imp ␣/cargo complexes. The major advantage of this approach is the ability to monitor the position and oligomerization state of Imp ␣ and the cargo during real time trafficking of these molecules through intact NPCs.…”

Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots

“…Wide-and narrow-field epifluorescence microscopy have been used to track transport receptors and a model cargo complex through the NPCs at the single-molecule level (13)(14)(15)(16)(17). The transport times of Imp β1 (≈7 ms) and its cargo complex (≈9 ms) through the NPCs were successfully visualized.…”