In vivo analysis of protein crowding within the nuclear pore complex in interphase and mitosis

Abstract

The central channel of the nuclear pore complex (NPC) is occupied by non-structured polypeptides with a high content of Phe-Gly (FG) motifs. This protein-rich environment functions as an entropic barrier that prevents the passage of molecules, as well as the binding sites for karyopherins, to regulate macromolecular traffic between the nucleoplasm and the cytoplasm. In this study, we expressed individual Nups fused with a crowding-sensitive probe (GimRET) to determine the spatial distribution of protein-rich domains within the central channel in vivo, and characterize the properties of the entropic barrier. Analyses of the probe signal revealed that the central channel contains two protein-rich domains at both the nucleoplasmic and cytoplasmic peripheries, and a less-crowded central cavity. Karyopherins and other soluble proteins are not the constituents of the protein-rich domains. The time-lapse observation of the post-mitotic reassembly process also revealed how individual protein-rich domains are constructed by a sequential assembly of nucleoporins.

Interactions between non‐structured domains of FG‐ and non‐FG‐nucleoporins coordinate the ordered assembly of the nuclear pore complex in mitosis

“…14 A recent study reported that FG-domains of Nup98 spontaneously self-assemble into particles in vitro at physiologic concentrations (≈10 µM). 6 This in vivo evidence suggests that even though FG-Nups self-assemble into protein-rich phases, several sets of Nups preferentially assemble into distinct protein-rich phases, possibly one at the cytoplasmic rim and another at the nucleoplasmic rim of the central channel. Another study using a crowding-sensitive fluorescent probe demonstrated that protein crowding is not homogeneous throughout the channel; "protein-rich" domains lie at both rims of the channel but not in the central cavity.…”

“…6 Nups containing a large number of phenylalanine-glycine (FG) motifs (FG-Nups) in a long intrinsically disordered region (IDR-Nups) 7,8 form a molecular sieve-like environment that functions as a selective barrier with respect to molecular transport. 6 Nups containing a large number of phenylalanine-glycine (FG) motifs (FG-Nups) in a long intrinsically disordered region (IDR-Nups) 7,8 form a molecular sieve-like environment that functions as a selective barrier with respect to molecular transport.…”

“…14 These findings suggest that the central channel of the NPC forms via self-assembly of non-structured FG-Nups. Furthermore, live cell imaging of individual Nups during postmitotic assembly of the NPC demonstrated that individual FG-Nups assemble orderly on the chromosome surface, 6,15,16 suggesting that the assembly of FG-Nups is driven by a specific molecular mechanism and does not proceed solely via a random self-assembly-driven mechanism. 6 This in vivo evidence suggests that even though FG-Nups self-assemble into protein-rich phases, several sets of Nups preferentially assemble into distinct protein-rich phases, possibly one at the cytoplasmic rim and another at the nucleoplasmic rim of the central channel.…”

“…In contrast to the scaffold, the central channel is formed primarily by a number of non-structured domains of Nups, | 1533 KONISHI aNd YOSHIMURa which assemble via promiscuous interactions to form a highly crowded environment within the pore. 6 Nups containing a large number of phenylalanine-glycine (FG) motifs (FG-Nups) in a long intrinsically disordered region (IDR-Nups) 7,8 form a molecular sieve-like environment that functions as a selective barrier with respect to molecular transport. Small molecules (<40 kDa) diffuse passively, but large molecules (>40 kDa) are repelled unless they are carried by an appropriate transport receptor, such as a karyopherin.…”

Roles of Nup133, Nup153 and membrane fenestrations in assembly of the nuclear pore complex at the end of mitosis

“…ELYS is crucial for NPC assembly because it recruits the Nup107-160 subcomplex to the chromatin where the NPC forms (Doucet & Hetzer, 2010;Franz et al, 2007). Kinetic analysis using live cell imaging of dividing cells showed that subcomplexes of the NPC are sequentially assembled on the chromatin to form a functional NPC (Anderson, Vargas, Hsiao, & Hetzer, 2009;Dultz et al, 2008;Konishi et al, 2016); the Nup107-160 subcomplex, and some fraction of Nup153, assembles in the earliest step of NPC assembly during telophase . In contrast, it has been suggested that the NPC is inserted into the sheetlike membrane generated from the ER cisternae during telophase (Lu, Ladinsky, & Kirchhausen, 2011).…”