Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy

Abstract

Nuclear pore complexes (NPCs) are biological nanomachines that mediate the bidirectional traffic of macromolecules between the cytoplasm and nucleus in eukaryotic cells. This process involves numerous intrinsically disordered, barrier-forming proteins known as phenylalanine-glycine nucleoporins (FG Nups) that are tethered inside each pore. The selective barrier mechanism has so far remained unresolved because the FG Nups have eluded direct structural analysis within NPCs. Here, high-speed atomic force microscopy is used to visualize the nanoscopic spatiotemporal dynamics of FG Nups inside Xenopus laevis oocyte NPCs at timescales of ∼100 ms. Our results show that the cytoplasmic orifice is circumscribed by highly flexible, dynamically fluctuating FG Nups that rapidly elongate and retract, consistent with the diffusive motion of tethered polypeptide chains. On this basis, intermingling FG Nups exhibit transient entanglements in the central channel, but do not cohere into a tightly crosslinked meshwork. Therefore, the basic functional form of the NPC barrier is comprised of highly dynamic FG Nups that manifest as a central plug or transporter when averaged in space and time.

Integrative structure and functional anatomy of a nuclear pore complex

“…Each of the FG repeat domains was represented as a flexible string of beads; a bead had a radius of 6 Å and encompassed 20 residues to achieve a compromise between computational efficiency and accuracy 118,146–149 . Consecutive beads were restrained by a bond with an equilibrium length of 18 Å and a constant force of 1.0 kcal/mol/Å, approximating the spring-like nature of flexible polymers 150 in general and FG repeat domains 118,147–149,151–153 in particular. The freely diffusing molecules included 1,600 NTRs and 1,600 inert macromolecules (0.33 mM each), each consisting of eight subgroups of 200 macromolecules, ranging in radius from 4 to 28 Å in 2 Å increments (10 to 75 kDa, assuming constant protein density of 1.38 g/cm 2 ).…”

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review

“…High-speed AFM [26] has been commercially available, and the time of capturing an AFM image has reduced to about sub-100 ms [156]. However, high-speed AFM is commonly adequate for small size imaging with rigid and flat surface, such as isolated molecules on substrate [157] and microbial cells possessing stiff cell wall [158]. Wide-area high-speed AFM has been developed for imaging mammalian cells [159], but in this case the time of capturing an AFM image is about 5 s which is still much larger than the response time of cell to external stimulation.…”

High-speed atomic force microscopy and its future prospects

“…Therefore, the NE is considered to be rigid compared to the plasma membranes. Recently, the surfaces of nuclei isolated from Xenopus laevis oocytes were observed by HS-AFM (Sakiyama et al 2016). Nuclear pore complexes (NPCs) act as a central regulator of transport between the nucleus and cytoplasm.…”