Deciphering the conformations and dynamics of FG-nucleoporins in situ

Abstract

The ~120 MDa nuclear pore complex (NPC) acts as a gatekeeper for the molecular traffic between the nucleus and the cytosol. Small cargo readily passes through the transport channel, yet large cargo requires specialized nuclear transport receptors. While the scaffold structure that anchors the NPC in the double-layered nuclear envelope has been resolved to remarkable details, the spatial organization of intrinsically disordered nucleoporins (NUPs) within the central channel remains enigmatic. These so-called FG-NUPs account for about one-third of the total mass of the NPC and form the actual transport barrier. Here we combined site-specific fluorescent labeling in non-fixed cells and fluorescent lifetime imaging microscopy (FLIM) to directly decipher the conformations of an essential constituent of the permeability barrier, NUP98, inside the functioning NPCs using Fluorescence resonance energy transfer (FRET). With detailed measurements of the distance distribution of eighteen NUP98 segments combined with coarse-grained modeling, we mapped the uncharted biochemical environment inside the nanosized transport channel. We found that "good-solvent" conditions for a polymer dominate the inside of the nanosized NPC, expand the FG-domain in situ and facilitate nuclear transport, in sharp contrast to the collapsed NUP98 FG-chain in aqueous solution. The combination of fluorescence microscopy, high-resolution electron tomography, and molecular simulation opens a window into the so-far unresolved organization of the FG-NUPs at the center of NPC function, allowing us to reconcile scientific models of nuclear transport.

Dynamic molecular mechanism of the nuclear pore complex permeability barrier

“…Importantly, these results collectively agree with fluorescence-based measurements of dynamic FG Nup behaviour in intact NPCs in vivo (Atkinson et al, 2013;Yu et al, 2022) and in silico (Moussavi-Baygi and Mofrad, 2016;Winogradoff et al, 2022), and the restricted motion of FG domains near their anchor sites as inferred from cryo-EM data (Akey et al, 2022;.…”

“…Hence, two basic characteristics distinguish biomolecular condensates and polymer brushes: (i) the ability to self-assemble in the absence or presence of an anchoring scaffold, and (ii) the dynamic motion of the FG domains. An appreciation of these contextual differences has motivated varied efforts to examine the authentic behavior of the FG domains within the NPC (Atkinson et al, 2013;Cardarelli et al, 2012;Ma et al, 2016;Mattheyses et al, 2010;Mohamed et al, 2017;Stanley et al, 2018;Yu et al, 2022).…”

Improving the hole picture: towards a consensus on the mechanism of nuclear transport

“…Taking all this together, it seems reasonable to suggest that the polymer brush FG repeats of the CT form a "restrained concentrate", where a concentrating effect of FG repeats is achieved by the anchoring of FG Nups in the CT walls, and the density and selective transport behavior of the CT contents results mainly from this constraint plus the recruitment of NTRs/cargoes (above), without necessarily having a specific requirement for internal cohesion necessary to form a gel. This view is most consistent with solution studies of different isolated FG repeat flavors which show a picture of highly mobile, minimally cohesive IDPs (61,65,67,112) with fast, low affinity interactions with NTRs and with very high mobility of tagged FG repeat regions in vivo (34,53,80,113). Notable in this regard, the Lim lab has also contributed key observations that the central portion of the in situ CT is dynamic using high speed atomic force microscopy, definitively establishing the movement of the center of the NPC in the 100 ms and faster range and showing that intermingling FG Nups do not appear to cohere into a highly crosslinked meshwork (35,66).…”

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

“…The feasibility of this idea is supported by the successful delivery of dye-labelled DNA and proteins into cells via physical methods (e.g., electroporation, microinjection), e.g., for single-molecule studies [156][157][158][159]. Furthermore, in-cell labelling has become possible using bio-orthogonal labelling approaches [160] and various self-labelling protein tags [161][162][163][164]. However, the feasibility of in-cell PIFE assays to derive meaningful information is impacted by two major factors: (i) cells feature distinct and quite heterogeneous viscosities in different compartments and strongly differ from dilute buffer conditions, (ii) nonspecific and unwanted interactions of macromolecules with the PIFE-probe are possible within a cell.…”