Single molecule microscopy approch provided novel insights into nuclear transport.
1. Preparation of the cell system for single molecule experiment
2. Preparation of essential proteins for nuclear transport experiment
3. Single molecule microscopy
4. Representative results
Performed correctly, our protocol allowed us to collect a series of videos demonstrating the translocation of single cargo molecules interacting with the nuclear pore complex in permeabilized HeLa POM 121 cells. (Fig 1). We set out to image and track the transient transport steps of dextran molecules through the NPC in digitonin-permeabilized HeLa cells stably expressing GFP-POM121. Cells were incubated with low concentrations of the labeled dextran molecules. The equatorial plane of the nucleus was brought into focus with a clear image of green ring of GFP fluorescence excited by a 488-nm mercury lamp light. A long-time exposure of GFP-NE enables us to obtain an excellent signal-to-noise (SNR) ratio to localize the middle plane of the NE. Previously 10 kDa dextran molecules were found to diffuse through the NE within approximately 2 ms with a spatial resolution of about 20-40 nm by a detection frame rate of 500 or 1000 Hz. Such a spatiotemporal resolution allows capture of only one to two diffusion steps of dextran molecules through the NPC which is not enough to elucidate more spatial information for passive diffusion. To capture more fine steps of dextran molecules within the NPC with a better spatiotemporal resolution, we have conducted two major improvements: (i) to adapt a faster detection frame rate of 2500 Hz to capture more fine diffusion steps; and (ii) to employ a higher illumination optical density to excite more photons from single molecules to obtain a higher spatial resolution. These improvements enable us to capture multiple steps of transient diffusion of alexa647-labeled dextran molecules across the NE by a 633-nm laser light at irradiance of 100 kW/cm2 with a spatiotemporal resolution of 12 nm and 400 ms.
As shown in Figure 1, a series of still images of typical transport events of dextran molecules were recorded. By tracking the spatial trajectories of individual transiting dextran molecules, the fine steps of diffusion through the NE were distinguished. We found that there are approximately 30% of interaction events with the NE have recognized originating and destination compartments. Among them, dextran molecules moving from the cytoplasm to the NE have two destinations: end in the nucleus after diffusing through the NE or move back to the cytoplasm after interacting with the NE The similar situations occurred for export events: dextran molecules starting from the nucleus enter the cytoplasm or diffuse back to the nucleus after interacting with the NE. Such image series contain information on the spatial position of the single molecules with regard to the NPC, as well as temporal information on the dwell time of the molecules through the NPC. By analyzing the trajectories of dextran molecules around the area of NE, the transport time, efficiency and entrance frequency can be determined. The transport time in our work can be identified as the import or the export time. They were defined as the average dwell time of dextran molecules within the NPCs that undergo the import or export. The import or export efficiency was defined as the percentage of the complete import or the export events over the sum of the complete and abortive events. The import or export entrance frequency refers the number of molecules interacting with the NE during a second.
Figure 1. Selected video frames of typical transport events of dextran molecules through the NE. (A) A cytoplasm-to-nucleus event. A single dextran molecule (red spot) started from the cytoplasm, interacted with the NE (green pixel line), and ended in the nucleus. The trajectories of the event (blue lines and open dots) were determined and superimposed with the NE (black line). The green and red lines represent 100 nm-distance from the NE on the cytoplasm and the nuclear side. C, the cytoplasm. N, the nucleus. Scale bar: 2 mm. (B) A cytoplasm-to-cytoplasm event. (C) A nucleus-to-nucleus event. (D) A nucleus-to-cytoplasm event.
Care must be taken to insure a proper colocalization of NE and single molecules during the experiment. It is also important to provide a high signal noise ratio, especially when the signal is low due to poor labeling or photobleaching. Localization precision is limited by background noise (arising from out-of-focus fluorescence, CCD readout noise, dark current and other factors).
The authors have nothing to disclose.
This project was supported by the Research Capacity Enhancement Grant (Bowling Green State University).