How to operate a nuclear pore complex by Kap-centric control - PubMed (original) (raw)

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How to operate a nuclear pore complex by Kap-centric control

Roderick Y H Lim et al. Nucleus. 2015.

Abstract

Nuclear pore complexes (NPCs) mediate molecular transport between the nucleus and cytoplasm in eukaryotic cells. Tethered within each NPC lie numerous intrinsically disordered proteins known as FG nucleoporins (FG Nups) that are central to this process. Over two decades of investigation has converged on a view that a barrier mechanism consisting of FG Nups rejects non-specific macromolecules while promoting the speed and selectivity of karyopherin (Kaps) receptors (and their cargoes). Yet, the number of NPCs in the cell is exceedingly small compared to the number of Kaps, so that in fact there is a high likelihood the pores are always populated by Kaps. Here, we contemplate a view where Kaps actively participate in regulating the selectivity and speed of transport through NPCs. This so-called "Kap-centric" control of the NPC accounts for Kaps as essential barrier reinforcements that play a prerequisite role in facilitating fast transport kinetics. Importantly, Kap-centric control reconciles both mechanistic and kinetic requirements of the NPC, and in so doing potentially resolves incoherent aspects of FG-centric models. On this basis, we surmise that Kaps prime the NPC for nucleocytoplasmic transport by fine-tuning the NPC microenvironment according to the functional needs of the cell.

Keywords: FG Nucleoporin; Karyopherin; Nuclear pore complex; Nucleocytoplasmic transport.

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Figures

Figure 1.

Figure 1.

The NPC-bound fraction of endogenous Kapβ1 is stable and long-lived. (A) Row 1: HeLa cells were fixed and digitonin-permeabilized as described in ref. 56. As a control, anti-Kapβ1 (Abcam) staining reveals the steady state distribution of endogenous Kapβ1, a fraction of which is localized at the nuclear envelope. This further co-localizes with anti-Nup153 (Sigma) staining, thereby signifying the presence of Kapβ1 at the NPCs. Row 2 to 4: To test for the retention of the NPC-bound pool of endogenous Kapβ1, cells were first digitonin-permeabilized then incubated in PBS buffer for 15 mins, 1 hr and 5 hr respectively, followed by fixation. The persistence of anti-Kapβ1 staining at the nuclear envelope up to 5h incubation indicates that the NPC-bound fraction of Kapβ1 is very stable and long-lived. Scale bar, 5 μm. (B) The immunofluorescent staining of Kapβ1 localized at the nuclear envelope was calibrated against InSpeck™ Red calibration beads (Life Technologies) to exclude intensity variations between measurements. The data shown corresponds to Rows 1 to 4 in (A). Final intensity ratios are normalized to the control sample (Row 1) to facilitate comparisons between experiments. Student's t-test analysis (p>0.05) shows a neglectable difference among these experimental outcomes. All images were obtained using a point scanning laser confocal microscope (LSM700, Zeiss AG).

Figure 2.

Figure 2.

Hypothetical translocation scenarios in the NPC. Left to right: Small passive molecules that diffuse into the NPC exhibit long 3D search trajectories (and therefore long dwell times) due to a lack of FG-Nup binding. The dirty velcro effect reduces the travel distance of Kaps by promoting 2D diffusion along the luminal surface of the translocation corridor. This reduction of dimensionality leads to shorter dwell times and rapid translocation rates. Large non-specific molecules experience hindered diffusion and have a lower probability to enter the pore. Increased pore confinement and the dirty velcro effect expedite the translocation of large cargoes with multiple Kaps due to reduced search trajectories that stem from 1D diffusion. Note: For clarity of illustration, other diffusing entities have been omitted from each drawing. However, overlaying these scenarios does reveal how different spatiotemporal routes might co-exist in the pore. C = cytoplasm, N = nucleus.

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