Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution

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.

Messages from the voices within: regulation of signaling by proteins of the nuclear lamina

“…Conventional subcellular fractionation and immunolocalization approaches have shown that some of these, including Myorg/Net37 [25] and Plpp7/Net39 [26], are enriched at the NE. New superresolution microscopy approaches are emerging [27] to evaluate whether such transmembrane proteins are concentrated at the NE relative to the peripheral ER ( de facto implying a distinctive nuclear function), or simply are ER residents that are not NE-excluded [28 • ]. …”

Determination of Membrane Protein Distribution on the Nuclear Envelope by Single‐Point Single‐Molecule FRAP

“…By using the full width at half maximum (FWHM) as determined by data analysis software (a built-in function in Origin 6.1 is used here), the range between where points can lie along either side of the NE can be determined for further analysis such as diffusion coefficient and protein concentration along the NE (see Mudumbi et al 2016 for the relevant formulae).…”

“…], including the lateral diffusion of NETs on the NE (Ellenberg et al, 1997) (25). Here 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 (Mudumbi et al, 2016). …”

“…Additionally, fluorescence recovery after photobleaching (FRAP) is a widely used technique where a cellular membrane, which contains membrane proteins tagged with a fluorophore, is photobleached with a high-powered laser and the recovery of fluorescence, through the diffusion of the protein of interest, is recorded (Axelrod, Koppel, Schlessinger, Elson, & Webb, 1976). Here, we combine single-molecule tracking, super-resolution imaging, and FRAP techniques to determine the spatial distribution of NETs in live cells (Mudumbi, Schirmer, & Yang, 2016). In general, we first pre-photobleach fluorescently tagged proteins in a small detection area (~0.5 µm), after which we track fluorescently intact single protein molecules moving into this photobleached area.…”

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

“…For their localization, SRM methods with an axial resolution of more than 10 nm have been developed [130, 131]. …”