UC Berkeley

The nuclear pore complexes (NPCs) are the sole channels known on the nuclear envelope in eukaryotes through which nucleocytoplasmic traffic is selectively and efficiently conducted. While the NPC has been the subject of extensive research for the past six decades and many of its structural, biochemical, and biophysical details are revealed, mechanistics of selective transport yet to be known. The main game-player in conducting the selective transport is a class of the NPC proteins rich in Phe-Gly (FG)-repeat domains that are localized mainly to the channel interior. FG-repeat domains are natively unfolded and thus belong to the family of intrinsically disordered proteins (IDPs). The conformational behavior of FG-repeats remains unknown because of lack of detailed information about their microdynamics, leaving plenty of room for speculation on how the selectivity barrier forms inside the NPC.

To tackle the microdynamics of FG-repeats, here I used polymer physics’ principles to develop a coarse-grained computational biophysical model, incorporating the full sequence of all amino-acids. The simulations were run under Brownian dynamics approach to generate long time-evolution of FG-repeat domains.

Under known physiological conditions and geometrical constraints, FG-repeats form a spatially nonuniform cohesive meshwork, percolating in radial and axial directions, with a dense hydrophobic zone in the middle and a low-density zone near the wall. The FG-meshwork is extremely dynamic, resembling a jerking plug with a fluctuating concentration in radial direction.

Being porous with the dominant pore sizes of 4 and 6 nm, this dynamic meshwork is permeable to the active cargos in a hydrophobic, and to a lesser extent, charge, stimuli-responsive manner, but strongly impermeable to inert cargos having the same size. An active cargo creates a big deformation inside the FG-meshwork, but because of rapid Brownian motions of the FG-repeats, it reconstructs itself in in a cargo size and shape dependent manner, suggesting the individual FG-repeats undergo reversible collapse. Significantly, the reconstruction process follows a saturating exponential pattern with rapid and slow phases. The characteristic time of reconstruction is a function of cargo size and shape, and is generally smaller for the elongated cargos compared to globular cargos having the same surface chemistry.

Next, I used computational microrheology via many-particle tracking without external probe to investigate the full mechanical spectrum of FG-repeats under different physical and geometrical conditions, including FG-repeats’ composition, FG-repeats’ length, geometrical confinement, shuttling cargo, and end-tethering. The results reveal that FG-repeats show a non-Newtonian behavior as manifested in their shear-thinning viscosity. The viscoelastic response of FG-repeats is strongly frequency-dependent, and is consistent with the function of the permeability barrier at different frequencies, or equivalently, at different timescales. At low frequencies, equivalent to timescale of nucleocytoplasmic transport, FG-repeats form a pseudo solid-like meshwork. At high frequencies, equivalent to the timescale of thermal diffusion of small molecules, FG-repeats behave like a predominantly viscous liquid.

The end-tethering is determined to be the most influential factor in shaping the mechanical spectrum of the FG-repeats. When the end-tethering is lifted and FG-repeats get free in the space, they invariably behave as a non-Newtonian viscous liquid over all frequencies. The other factors investigated are geometrical confinement, FG-repeats composition, i.e. hydrophobicity and charge content, length of FG-repeats, and shuttling cargo. Although these factors might shift the value of viscoelastic response in the frequency domain, they do not change the physics of the response, as end-tethering does. Comparing the viscoelastic response of FG-repeats with a general polymer melt show a remarkable consistency between FG-repeats and polymer melt over the range of frequencies accessible to the current model. Ultimately, the answer to the question of “do FG-repeats form gel?” is discussed in detail.