Biophysical coarse-grained modeling provides insights into transport through the nuclear pore complex

Abstract

The nuclear pore complex (NPC) is the gatekeeper of the nucleus, capable of actively discriminating between the active and inert cargo while accommodating a high rate of translocations. The biophysical mechanisms underlying transport, however, remain unclear due to the lack of information about biophysical factors playing role in transport. Based on published experimental data, we have established a coarse-grained model of an intact NPC structure to examine nucleocytoplasmic transport with refined spatial and temporal resolutions. Using our model, we estimate the transport time versus cargo sizes. Our findings suggest that the mean transport time of cargos smaller than 15 nm is independent of size, while beyond this size, there is a sharp increase in the mean transport time. The model confirms that kap-FG hydrophobicity is sufficient for active cargo transport. Moreover, our model predicts that during translocation, small and large cargo-complexes are hydrophobically attached to FG-repeat domains for 86 and 96% of their transport time, respectively. Inside the central channel FG-repeats form a thick layer on the wall leaving an open tube. The cargo-complex is almost always attached to this layer and diffuses back and forth, regardless of the cargo size. Finally, we propose a plausible model for transport in which the NPC can be viewed as a lubricated gate. This model incorporates basic assumptions underlying virtual-gate and reduction-of-dimensionality models with the addition of the FG-layer inside the central channel acting as a lubricant.

Figure 1

Putative view of the NPC along with different biochemical factors. For a recent comprehensive review of biochemical and structural aspects of the NPC, see Jamali et al. (5).

Figure 2

Different components of the NPC along with their sizes. Cytoplasmic filaments, cytoplasmic ring, central channel, nuclear ring, nuclear basket, distal ring, and intranuclear filaments are depicted. Dimensions are for Xenopus oocyte (23,41).

Figure 3

Initial configuration of our coarse-grained model of the NPC with FG-repeats (solid strings) attached to cytoplasmic filaments all the way to nuclear basket. (a) Magnification of a section of the cytoplasmic filament: the linear springs are depicted. A harmonic bending potential energy is also applied between each two consecutive bonds. (b) Magnification of a section of an FG-repeat: the FG-repeats are modeled as beads and series of discrete WLC springs. (Dashed line) Arbitrary configuration of the chain. The value r represents the end-to-end length of a spring.

Figure 4

Snapshot of a cargo-complex interacting with FG-repeat domains. (a) Cargo-complex interacting with FG-repeats during transport via hydrophobic affinity between kap-β and FG-domains. Interactions are limited to the half-circle of the right side of the cargo-complex. In visualization, when a kap-FG hydrophobic interaction is active, the color of the cargo-complex becomes green and the corresponding FG-repeats become red (see Movie S1). (b) Magnification of cargo-complex along with interacting FG-repeats in its vicinity. Short-range repulsive potential energy between cargo-complex surface and FG-repeats prevents them from penetrating each other. A half-circle on the right side of the cargo-complex represents the boatlike shape of kap-β. (c) Crystal structure of the kap-β (green) interacting with FG-repeats (red) on its convex surface (1F59 in the Protein DataBank). Our supposition that kap-β is a half-circle matches well with this structure.

Figure 5

Dependency of transport time on size of the cargo-complex (average time mean ± SE). When the cargo size exceeds 15 nm, a sharp increase in time is observed.

Figure 6

Attachment of the cargo-complex to FG-repeats during transport as a function of size. The red graph shows how much of the transport time, the cargo-complex is hydrophobically attached to FG-repeats. During translocation, small and large cargo-complexes are attached to FG-repeats for 86% and 96% of their transport time, respectively. The black graph shows similar information when the cargo-complex is inside the central channel. When the cargo-complex is inside the central channel, it is almost always attached to the FG-layer. Small and large cargo-complexes attach 97% and 99.8% of their time inside the channel to FG-layer, respectively. (Points) Simulation results. (Solid lines) Linear fit.

Figure 7

Typical presence contour of an (a) inert and (b) active cargo as well as (c) FG-repeat domains in the course of simulation. The brightness is proportional to presence time. (a) Gray contour shows the typical presence of an inert cargo during ∼8 ms. To obtain this pattern, 10,000 snapshots were superimposed. The brighter the color, the more time is spent in that region. As it can be seen, inert cargo is rejected by cytoplasmic and central FG-repeats. Although it can reach the entry of the central channel, it is finally rejected (see Movie S2). FG-repeat domains are removed for the sake of clarity. Cytoplasmic filaments and nuclear rods are chosen from 20 frames during transport. (b) (Green) Contour of the active cargo presence in different locations during transport. It is obtained by superimposing of more than 3100 frames during a ∼2.7-ms import. Note that, the brighter the color, the greater the portion of transport time that is spent in that region. It can be seen that the active cargo spends more time in the central channel, especially in its upper part (see Movie S1). Sporadic detachments of cargo from the FG-domains can be distinguished by observing the dimmer protrusions from the main path. (Yellow lines, top) Wavy motions of cytoplasmic filaments from 20 frames of the same simulation. The movement of the nuclear rods of the basket is also shown (bottom), which corresponds to the same 20 frames. The central channel is shown only by one frame and FG-repeat domains are removed for the sake of clarity. (c) The area covered by FG-repeat domains in the cytoplasmic periphery, central channel, and nuclear periphery is obtained by superimposing of the same 3100 frames (shown in red). Note that the brightness of the red color shows the average presence of FG-repeats over time. FG-repeats with the help of wavy motions of cytoplasmic filaments and nuclear rods can span a wide area in the cytoplasmic and nuclear peripheries during transport, resembling a nonuniform cloud which is more dense near the filaments (and rods). This can effectively reject inert cargos while attracting active cargos. (Transparent yellow) Pliable cytoplasmic filaments and nuclear basket.

Figure 8

Average trajectories of (a) centers-of-mass and (b) right edges of cargo-complexes with different sizes. Each trajectory is averaged over, at least, 50 independent simulations. As it can be seen, the cargo-complex, regardless of its size, is almost always attached to the channel wall. However, outside the channel, specifically in the nuclear basket, it has more fluctuations and detaches more repeatedly from the FG-repeats. Because the cargo-complex interacts with FG-repeats via its right-hand edge (see Fig. 4), in panel b) the right-hand edges' trajectories of different sizes overlap, specifically inside the central channel. The high degree of detachments inside the nuclear basket is because the cargo releases there and loses its hydrophobic affinity for FG-repeats. Cytoplasmic filaments and nuclear rods are chosen from 20 frames during a typical transport (shown in transparent gray). Central channel wall (red). FG-repeat domains are removed for the sake of clarity.