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Role of molecular charge in nucleocytoplasmic transport.

Goryaynov A, Yang W - PLoS ONE (2014)

Bottom Line: We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route.A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors.Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Temple University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Transport of genetic materials and proteins between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). A selective barrier formed by phenylalanine-glycine (FG) nucleoporins (Nups) with net positive charges in the NPC allows for passive diffusion of signal-independent small molecules and transport-receptor facilitated translocation of signal-dependent cargo molecules. Recently, negative surface charge was postulated to be another essential criterion for selective passage through the NPC. However, the charge-driven mechanism in determining the transport kinetics and spatial transport route for either passive diffusion or facilitated translocation remains obscure. Here we employed high-speed single-molecule fluorescence microscopy with an unprecedented spatiotemporal resolution of 9 nm and 400 µs to uncover these mechanistic fundamentals for nuclear transport of charged substrates through native NPCs. We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route. A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors. Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.

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3D pathways of Imp β1 alone and Imp α/Imp β1 in complex with differently charged GFP cargo (−30 NLS-GFP), −7 NLS-GFP and +36 NLS-GFP).(A) Imp β1 alone. Calculated electrostatic surface potentials of Imp β1 range from −15 kT/e (dark blue) to +15 kT/e (dark red), and neutral charge 0 is shown in green (I). Superimposed plots of localizations of single molecules located primarily within a rectangular area of 240×160 nm (II). N, the nucleoplasmic side of the NPC; C, the cytoplasmic side of the NPC. The locations in each 10×10 nm area were quantized and filtered with a Gaussian blur function to generate the 2D probability density map overlaid onto the NPC architecture (light blue). The highest density was 1.7×105 locations/µm2 and the lowest was 0 locations/µm2, shown in gray (III). A 3D probability density map (green cloud; brighter color indicates higher density) is shown in both side-view and a top-view orientations superimposed on the NPC architecture (blue). The length of pathway and the diameter at the central plane of NPC was measured and is labeled in nanometers. N, the nucleoplasmic side of the NPC. C, the cytoplasmic side of the NPC. (B–D) Nucleocytoplasmic transport pathways of −30NLS-GFP/Impα/Impβ1, −7NLS-GFP/Impα/Impβ1 and +36NLS-GFP/Impα/Impβ1.
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pone-0088792-g003: 3D pathways of Imp β1 alone and Imp α/Imp β1 in complex with differently charged GFP cargo (−30 NLS-GFP), −7 NLS-GFP and +36 NLS-GFP).(A) Imp β1 alone. Calculated electrostatic surface potentials of Imp β1 range from −15 kT/e (dark blue) to +15 kT/e (dark red), and neutral charge 0 is shown in green (I). Superimposed plots of localizations of single molecules located primarily within a rectangular area of 240×160 nm (II). N, the nucleoplasmic side of the NPC; C, the cytoplasmic side of the NPC. The locations in each 10×10 nm area were quantized and filtered with a Gaussian blur function to generate the 2D probability density map overlaid onto the NPC architecture (light blue). The highest density was 1.7×105 locations/µm2 and the lowest was 0 locations/µm2, shown in gray (III). A 3D probability density map (green cloud; brighter color indicates higher density) is shown in both side-view and a top-view orientations superimposed on the NPC architecture (blue). The length of pathway and the diameter at the central plane of NPC was measured and is labeled in nanometers. N, the nucleoplasmic side of the NPC. C, the cytoplasmic side of the NPC. (B–D) Nucleocytoplasmic transport pathways of −30NLS-GFP/Impα/Impβ1, −7NLS-GFP/Impα/Impβ1 and +36NLS-GFP/Impα/Impβ1.

Mentions: In this paper, our major goal is to examine the role played by molecular surface charge, compared to the influence of molecular size and specific signal, in determining the transport kinetics and spatial transport routes for both passive and facilitated transport. To this end, we first employed a new imaging approach recently developed in our lab, single-point edge-excitation sub-diffraction (SPEED) microscopy (Fig. 1A), that enables the following capabilities for mapping nucleocytoplasmic transport in HeLa cells [27]–[28], [30]: 1) capture real-time fast movements of transiting molecules (typically a few milliseconds) through the native NPCs in cells with high temporal resolution (0.4 ms in SPEED microscopy, as shown in Fig. 1B and Movie S1–S6); 2) spatially distinguish and localize the individual NPCs on the NE (1–3 nm localization precision for the centroid of NPC) and simultaneously track the transiting molecules through the NPCs with high spatial resolution (particle tracking precision <10 nm for substrates moving through the NPC, as observed in Fig. 1C); and 3) obtain a 3D view of real-time spatial transport routes for the inherently 3D moving substrates in the NPC via a deconvolution algorithm (demonstrated in Fig. 1D and Movie S7–S10). Second, to specifically examine charge-dependent nucleocytoplasmic transport, we selected proper substrates of the same sizes, the same specific signals, and similar surface hydrophobicity (another factor that is suggested to affect nuclear transport), but differ only in surface charge. Here, the chosen substrates include green fluorescence protein (−7GFP, its inherent net negative surface charges is −7) and its super-charged mutations with either a positive charge of +36 (+36GFP) or a negative charge of −30 (−30GFP) in our tests (Fig. 2). Previously, these super-charged GFPs were successfully applied to study the transfection process in live cells, showing their suitability in cellular fluorescence measurements [33]–[34]. With the same molecular weight of ∼27 kDa, the same hydrodynamic size of ∼ 6 nm in diameter (determined by dynamic light scattering as shown in Fig. S1) and differing only in surface charge, the above GFP candidates are well below the cut-off size of ∼ 40 kDa (∼ 10 nm in diameter) for passive diffusion and are thus suitable for testing the effect of molecular charge on passive diffusion. Additionally, to prepare charged signal-dependent cargos for facilitated translocation, we further fused an NLS (PKKKRKV) to each of the above charged GFPs and thus obtained differently charged import cargo complexes of the NLS-GFP/transport receptor (Fig. 3 and Table 1). Finally, we tested the effect of surface charge on the nucleocytoplasmic transport of a construct of the endogenous ribosomal protein rpL23 (Fig. 4).


Role of molecular charge in nucleocytoplasmic transport.

Goryaynov A, Yang W - PLoS ONE (2014)

3D pathways of Imp β1 alone and Imp α/Imp β1 in complex with differently charged GFP cargo (−30 NLS-GFP), −7 NLS-GFP and +36 NLS-GFP).(A) Imp β1 alone. Calculated electrostatic surface potentials of Imp β1 range from −15 kT/e (dark blue) to +15 kT/e (dark red), and neutral charge 0 is shown in green (I). Superimposed plots of localizations of single molecules located primarily within a rectangular area of 240×160 nm (II). N, the nucleoplasmic side of the NPC; C, the cytoplasmic side of the NPC. The locations in each 10×10 nm area were quantized and filtered with a Gaussian blur function to generate the 2D probability density map overlaid onto the NPC architecture (light blue). The highest density was 1.7×105 locations/µm2 and the lowest was 0 locations/µm2, shown in gray (III). A 3D probability density map (green cloud; brighter color indicates higher density) is shown in both side-view and a top-view orientations superimposed on the NPC architecture (blue). The length of pathway and the diameter at the central plane of NPC was measured and is labeled in nanometers. N, the nucleoplasmic side of the NPC. C, the cytoplasmic side of the NPC. (B–D) Nucleocytoplasmic transport pathways of −30NLS-GFP/Impα/Impβ1, −7NLS-GFP/Impα/Impβ1 and +36NLS-GFP/Impα/Impβ1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928296&req=5

pone-0088792-g003: 3D pathways of Imp β1 alone and Imp α/Imp β1 in complex with differently charged GFP cargo (−30 NLS-GFP), −7 NLS-GFP and +36 NLS-GFP).(A) Imp β1 alone. Calculated electrostatic surface potentials of Imp β1 range from −15 kT/e (dark blue) to +15 kT/e (dark red), and neutral charge 0 is shown in green (I). Superimposed plots of localizations of single molecules located primarily within a rectangular area of 240×160 nm (II). N, the nucleoplasmic side of the NPC; C, the cytoplasmic side of the NPC. The locations in each 10×10 nm area were quantized and filtered with a Gaussian blur function to generate the 2D probability density map overlaid onto the NPC architecture (light blue). The highest density was 1.7×105 locations/µm2 and the lowest was 0 locations/µm2, shown in gray (III). A 3D probability density map (green cloud; brighter color indicates higher density) is shown in both side-view and a top-view orientations superimposed on the NPC architecture (blue). The length of pathway and the diameter at the central plane of NPC was measured and is labeled in nanometers. N, the nucleoplasmic side of the NPC. C, the cytoplasmic side of the NPC. (B–D) Nucleocytoplasmic transport pathways of −30NLS-GFP/Impα/Impβ1, −7NLS-GFP/Impα/Impβ1 and +36NLS-GFP/Impα/Impβ1.
Mentions: In this paper, our major goal is to examine the role played by molecular surface charge, compared to the influence of molecular size and specific signal, in determining the transport kinetics and spatial transport routes for both passive and facilitated transport. To this end, we first employed a new imaging approach recently developed in our lab, single-point edge-excitation sub-diffraction (SPEED) microscopy (Fig. 1A), that enables the following capabilities for mapping nucleocytoplasmic transport in HeLa cells [27]–[28], [30]: 1) capture real-time fast movements of transiting molecules (typically a few milliseconds) through the native NPCs in cells with high temporal resolution (0.4 ms in SPEED microscopy, as shown in Fig. 1B and Movie S1–S6); 2) spatially distinguish and localize the individual NPCs on the NE (1–3 nm localization precision for the centroid of NPC) and simultaneously track the transiting molecules through the NPCs with high spatial resolution (particle tracking precision <10 nm for substrates moving through the NPC, as observed in Fig. 1C); and 3) obtain a 3D view of real-time spatial transport routes for the inherently 3D moving substrates in the NPC via a deconvolution algorithm (demonstrated in Fig. 1D and Movie S7–S10). Second, to specifically examine charge-dependent nucleocytoplasmic transport, we selected proper substrates of the same sizes, the same specific signals, and similar surface hydrophobicity (another factor that is suggested to affect nuclear transport), but differ only in surface charge. Here, the chosen substrates include green fluorescence protein (−7GFP, its inherent net negative surface charges is −7) and its super-charged mutations with either a positive charge of +36 (+36GFP) or a negative charge of −30 (−30GFP) in our tests (Fig. 2). Previously, these super-charged GFPs were successfully applied to study the transfection process in live cells, showing their suitability in cellular fluorescence measurements [33]–[34]. With the same molecular weight of ∼27 kDa, the same hydrodynamic size of ∼ 6 nm in diameter (determined by dynamic light scattering as shown in Fig. S1) and differing only in surface charge, the above GFP candidates are well below the cut-off size of ∼ 40 kDa (∼ 10 nm in diameter) for passive diffusion and are thus suitable for testing the effect of molecular charge on passive diffusion. Additionally, to prepare charged signal-dependent cargos for facilitated translocation, we further fused an NLS (PKKKRKV) to each of the above charged GFPs and thus obtained differently charged import cargo complexes of the NLS-GFP/transport receptor (Fig. 3 and Table 1). Finally, we tested the effect of surface charge on the nucleocytoplasmic transport of a construct of the endogenous ribosomal protein rpL23 (Fig. 4).

Bottom Line: We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route.A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors.Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Temple University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Transport of genetic materials and proteins between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). A selective barrier formed by phenylalanine-glycine (FG) nucleoporins (Nups) with net positive charges in the NPC allows for passive diffusion of signal-independent small molecules and transport-receptor facilitated translocation of signal-dependent cargo molecules. Recently, negative surface charge was postulated to be another essential criterion for selective passage through the NPC. However, the charge-driven mechanism in determining the transport kinetics and spatial transport route for either passive diffusion or facilitated translocation remains obscure. Here we employed high-speed single-molecule fluorescence microscopy with an unprecedented spatiotemporal resolution of 9 nm and 400 µs to uncover these mechanistic fundamentals for nuclear transport of charged substrates through native NPCs. We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route. A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors. Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.

Show MeSH
Related in: MedlinePlus