Abstract
The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo.
Highlights
The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases
By combining single-point illumination and single-molecule fluorescence recovery after photobleaching, here we show that the spatial localizations of NETs on the inner nuclear membrane (INM) and outer nuclear membrane (ONM) are distinguished with a spatial resolution of o10-nm in real-time
One can determine if a NET transports into the nucleus, and its distribution along the INM and ONM within 30 min with a precision of o10 nm
Summary
The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. We demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo. Several super-resolution microscopy techniques (STORM, PALM and RESOLFT/STED) have been employed to obtain sub-diffraction images in live cells[17] Most of these techniques were shown to provide approximately a 50-nm imaging resolution in vivo[17], which render them unlikely to distinguish the real-time localizations of NETs on the INM and ONM, since the two membrane bilayers are separated by a 40-nm perimembrane space[17]. We have further developed the FRAP technique by adapting a diffraction-limit photobleaching area and recording the recovery of single NETs on the INM and ONM with super-high spatiotemporal resolutions in live cells. Several different NETs with unique NE localizations are used to verify and highlight the capabilities of this technique
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