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. 2009 Sep 2;28(17):2554-67.
doi: 10.1038/emboj.2009.199. Epub 2009 Aug 13.

FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties

Steffen Frey  1 , Dirk Görlich
Affiliations

FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties

Steffen Frey et al. EMBO J. .

Abstract

The permeability barrier of nuclear pore complexes (NPCs) controls all nucleo-cytoplasmic exchange. It is freely permeable for small molecules. Objects larger than approximately 30 kDa can efficiently cross this barrier only when bound to nuclear transport receptors (NTRs) that confer translocation-promoting properties. We had shown earlier that the permeability barrier can be reconstituted in the form of a saturated FG/FxFG repeat hydrogel. We now show that GLFG repeats, the other major FG repeat type, can also form highly selective hydrogels. While supporting massive, reversible importin-mediated cargo influx, FG/FxFG, GLFG or mixed hydrogels remained firm barriers towards inert objects that lacked nuclear transport signals. This indicates that FG hydrogels immediately reseal behind a translocating species and thus possess 'self-healing' properties. NTRs not only left the barrier intact, they even tightened it against passive influx, pointing to a role for NTRs in establishing and maintaining the permeability barrier of NPCs.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Resealing modes of the permeability barrier. Cartoons sketch conceivable behaviours of the barrier towards nuclear transport receptors (NTRs) and inert material. (A) A scenario in which NTRs can penetrate into and through the barrier, but where no resealing behind the translocating species occurs. In this case, NTRs would cause a breakdown of the barrier. The problem should occur already at low NTR concentrations and would worsen with time. Eventually, the gel would disintegrate. (B) A scenario where resealing behind the translocating species is slow. In this case, NTRs would transiently collapse the barrier. The problem would increase with the load of facilitated gel entry. (C) A scenario where the barrier reseals immediately behind a translocating species. In this case, the barrier would stay tight against inert material, even at the highest transport load.
Figure 2
Figure 2
Experimental set-up for studying influx into an FG hydrogel. (A) Illustration of the experimental set-up. (B) A saturated FG/FxFG2–601Nsp1 hydrogel within the imaging chamber after completion of an influx experiment. Photographs were taken using a macro lens either under white light or UV illumination and overlaid. Note that the green NTR·cargo complex (IBB-MBP-mEGFP·scImpβ) entered the gel, whereas the red inert reference molecule (MBP-mCherry) stayed out. (C) Influx of MBP-mCherry alone into a saturated FG/FxFG2–601Nsp1 hydrogel followed by laser scanning confocal microscopy. Time elapsed after addition is indicated. Upper panels show the gel as detected by an incorporated Atto647N-labelled tracer molecule. Lower panels show 3 μM of MBP-mCherry added to the buffer side of the gel. The gel contained 200 mg/ml of FG/FxFG2–601Nsp1. For quantification see Figure 3 and Table I. For false-colour code, see panel E. (D) Experiment shows simultaneous influx of MBP-mCherry and IBB-MBP-mEGFP·scImpβ complex into the same batch of hydrogel as shown in panel C. Note that the rapid influx of the NTR·cargo complex did not detectably increase the entry of the non-receptor-bound inert reference molecule MBP-mCherry. For quantification, see Figure 3. (E) Look-up table used for translation of grey scale into false-colour images.
Figure 3
Figure 3
Pre-incubation of the FG/FxFG hydrogel with scImpβ tightens the barrier against passive influx. Panels A–C show concentration profiles of mobile species during their entry into a saturated FG/FxFG2–601Nsp1 hydrogel. The three time points (30 s, 10 min and 30 min) are colour-coded. For best comparison, the free concentration of the mobile species in buffer was scaled to 1. (A) Influx of MBP-mCherry alone. The comparison of the three time points indicates a slow, but still clearly detectable influx and a partition coefficient between gel and buffer of 0.18. (B) Simultaneous influx of 3 μM of MBP-mCherry and 1 μM of an IBB-MBP-mEGFP·scImpβ complex. The NTR·cargo complex entered the gel ≈100 times faster than the passive species. The presence of the NTR·cargo complex did not increase influx of the passive species as compared with panel A. (C) The influx experiment was performed as in panel B, the difference being that the FG hydrogel had been pre-incubated for 180 min with 10 μM of unlabelled, cargo-free scImpβ before the fluorescent mobile species was added. The pretreatment had only a minor effect on influx of the NTR·cargo complex (slight reduction in entry rate and intra-gel diffusion coefficient), indicating that this type of FG hydrogel is very robust against competition and can sustain a very high load of facilitated transport. However, the pretreatment had the striking effect of tightening the barrier against passive influx, and thus improving the performance of the barrier greatly. The labelled scImpβ·cargo complex now entered the gel at least 5000 times faster than the inert reference molecule MBP-mCherry. (D) Analytical gel filtration revealed a Stokes radius (RS) of 3.6 nm for the above used inert reference molecule (MBP-mCherry) and 5.1 nm for the IBB-MBP-mEGFP·scImpβ complex.
Figure 4
Figure 4
Facilitated translocation tightens the FG/FxFG hydrogel even against passive influx of GFP-sized objects. The influx experiment was carried out as in Figure 3, but two differences were implemented: First, we used a smaller inert reference molecule (mCherry, Stokes radius (RS)=2.4 nm) in order to enhance the sensitivity for small changes in passive permeability. Second, for pre-incubation, the ‘empty' scImpβ was replaced by an IBB-MBP-mEGFP·scImpβ complex. The receptor used for the pre-incubation was therefore also detectable in the GFP channel. As expected from its smaller size, mCherry entered the untreated gel considerably faster than the MBP-mCherry fusion. The pre-incubation with the NTR·cargo complex diminished mCherry influx to very low levels. The pretreatment did, however, not abolish the NTR-mediated cargo entry (see Supplementary Figure 1).
Figure 5
Figure 5
GLFG repeat domains also form a highly selective permeability barrier. Panels show influx of mobile species into a hydrogel made of 200 mg/ml of a fusion between the GLFG repeat domains of Nup49p and Nup57p. Quantification was carried out analogous to Figure 3. (A) Influx of 3 μM of MBP-mCherry alone. (B) Simultaneous influx of 3 μM of MBP-mCherry and 1 μM of IBB-MBP-mEGFP·scImpβ complex. (C) As panel B, however, the gel was pre-incubated for 180 min with 10 μM of scImpβ. This pre-incubation tightened the barrier against passive influx, but also diminished receptor-mediated cargo entry as well as intra-gel diffusion of the NTR·cargo complex, suggesting that the GLFG gel is more sensitive to competition by scImpβ than the FG/FxFG gel (see Figure 3C). (D) As panel B, however, the gel was pre-incubated with the dominant-negative human Impβ45-462 fragment. This pre-incubation lead to a virtually complete block of passive influx and to a 100-fold reduction in receptor-mediated gel entry. Intra-gel movement of the scImpβ·cargo complex was essentially blocked (also see Supplementary Figure S4). The effect of the dominant-negative mutant on the GLFG gel was much stronger than on an FG/FxFG gel (see Supplementary Figure S4).
Figure 6
Figure 6
The GLFG hydrogel allows facilitated entry not only of scImpβ but also of other nuclear transport receptors (NTRs). The entry of the six indicated GFP-NTR fusions (1 μM concentration on buffer side) into a saturated GLFG hydrogel was studied. Left panels show microscopic images in the GFP channel at the 30-s and 30-min time points, all taken at identical settings. Right panels show quantifications. Note that all tested NTRs entered the gel rapidly and showed a similar intra-gel movement, corresponding to a passage time through an NPC of ≈10 to 20 ms.
Figure 7
Figure 7
Behaviour of a mixed FG/FxFG/GLFG hydrogel. A triple fusion comprising the FG repeat domains of the central Nsp1p·Nup49p·Nup57p complex was used to prepare a saturated FG hydrogel. Panels show quantification of influx into such gel, using the same probes and experimental conditions as in Figure 3A–C.
Figure 8
Figure 8
Comparison of scImpβ-mediated facilitated entry into different types of FG hydrogels. To allow a direct comparison between FG/FxFG, GLFG, and mixed hydrogels, influx of IBB-MBP-mEGFP·scImpβ complex into these gels was measured at identical settings and quantifications were plotted to identical scales. The FG/FxFG gel (left panel) showed the weakest enrichment of the NTR·cargo complex and fast intra-gel diffusion. The GLFG gel (middle panel) was characterised by a much stronger enrichment and slower intra-gel diffusion of the mobile species. The mixed FG/FxFG/GLFG gel (right panel) showed an intermediate enrichment of the transport receptor and yet fast intra-gel diffusion. It appeared to be the most efficient gel in terms of absorbing the transport receptor.
Figure 9
Figure 9
Entry of NTR·cargo complexes into an FG hydrogel is reversible. (A) A saturated FG/FxFG2–601Nsp1 hydrogel was preloaded overnight with an NTR·cargo complex (1 μM of GFP-scImpβ·IBB-MBP-mCherry in the buffer). The free complex was removed by several buffer changes and phenyl–sepharose beads were placed in front of the gel. Then, confocal scans detecting cargo and receptor were started. The beads served as a trap for scImpβ and strongly accumulated NTR and cargo over time. The accumulated signal was strongest on the side that faced the gel, identifying the hydrogel as the source of the NTR·cargo complex. Bar diagrams translate false colour look-up tables into grey scale. (B) An FG hydrogel was loaded with 1 μM of NTR·cargo complex as in panel A. The first scan (1) shows the loading of the gel. (2) A preloaded gel immediately after removing the free NTR·cargo complex by three buffer changes. (3) A preloaded gel after 3 h incubation with buffer and (4) after incubation with 4 μM of GTP-Gsp1p (S. cerevisiae Ran). Gsp1p increased the efflux of the cargo and of scImpβ from the gel. Two different scan settings of the experiment are shown, optimised to visualise either low or high protein concentrations.
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