The nuclear pore complex (NPC) forms a nanochannel for selective transport into and
out of the cells nucleus. Notably, the transport selectivity of the NPC critically depends on an assembly of intrinsically disordered proteins with multiple phenylalanine-glycine repeats (referred to as FG nups), grafted to the inner walls of the NPC. The focus of this work is to probe the biophysical nature of this indispensable nanochannel by using mimetic systems that emulate key properties of the NPC.
Using a DNA origami scaffold, it has been possible to study the configurations adopted by purifi ed FG-nups confi ned to a nanopore geometry that mimics the NPC central channel. In this thesis, atomic force microscopy (AFM) has been used extensively to compare different flavours of FG-nups and to monitor their stochastic behaviour as they form transient condensates that occlude the pore's central lumen. Rearrangements of these entanglements were observed on the timescale of seconds, suggesting that reordering occurs at little energetic cost. This supports the idea that the FG nups are sufficiently 'sticky' or 'cohesive' to form dense condensates that seal the NPC's transport barrier yet are sufficiently dynamic at the molecular scale to facilitate the transport of larger molecules. Furthermore, the dynamics of two different FG-nup (the more cohesive Nup100 and the less cohesive Nsp1) was quantifed using autocorrelation analysis and compared to analogous data acquired on native NPCs. As predicted Nup100 condensates were found to be longer lived than those formed by its less cohesive counterpart Nsp1. By contrast, no fluctuations above the background noise were observed in native NPCs. A possible explanation being due to the presence of soluble transport receptor proteins and molecules caught in transport that trap the FG-nups in given morphologies.
To further probe this hypothesis additional components of the transport system have
been added to the mimetic nuclear pore complexes and their binding has been visualised with single molecule resolution, both by AFM and by total internal reflection fluorescence microscopy (TIRFM). Importin was seen to bind to both Nup100 and Nsp1 whereby a systematic increase in the number of nucleoporins led to greater binding.Open Acces
