Fluorescence anisotropy reveals order and disorder of protein domains in the nuclear pore complex

Abstract

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport.

Figure 1

NPC geometry and coordinate systems. (A) Distribution of nups in the NPC. Nups are arranged in rings containing eight copies, creating an eightfold symmetry axis. Sixteen copies arranged in two rings of eight are related by a pseudo-twofold symmetry axis. (B) A cross section of the yeast NE in the xyz microscope-fixed coordinate system. The position of an NPC in the NE is defined by the angle γ between N and the y axis. (C) A single fluorophore dipole, μ, in the NPQ coordinate system. The dipole μ is characterized by two angles, α and β. NPQ can be transformed into xyz by a rotation of γ around P.

Figure 2

Fluorophores ordered in the NPC lead to modulation of NE anisotropy. Four different possible orientations of GFP within the NPC are depicted: (A) α = 0°, (B) α = 90°, (C) α = 90°, and (D) α = 45°.

Figure 3

Measurement of anisotropy in individual yeast nuclei. Cells expressing (A) Nup57-GFPfolded or (B) Nup57-GFPtip. Images of individual nuclei are shown in I_∥ and I_⊥. I_⊥ is always lower than I_∥ (grayscale). The calculated anisotropy (r), with the NE mask applied (pseudocolor scale). (C) Schematic of population analysis.

Figure 4

Ordered and disordered protein domains in the NPC. Anisotropy mean ± SE plotted as a function of NE position and a pseudocolored representation of the NE, where anisotropy is represented by color in each of the 32 segments for (A) Nup57-GFPfolded, (B) Nup57-GFPtip, (C) Nup116-GFPboundary, (D) Nup116-GFPtip, (E) Nic96-GFPfolded, and (F) Nup159-GFPtip. (G) Diagrams of nup-GFP constructs. (H) Order scores.

Figure 5

Sources of error. (A) Varying percentage of labeled Nup57-GRPtip. 100% labeled (black), 50% labeled (gray). (B) Nup57-GFPfolded: individualized background (bg) subtraction (dark gray) and no bg subtraction (light gray). (C) Computationally generated data with no bg subtraction (light gray), correct bg subtraction (dark gray), and 2× correct bg subtraction (black). (D) Nup57-GFPtip: individualized bg subtraction (dark gray) and no bg subtraction (light gray). (E) Computationally generated data with no bg subtraction (light gray), correct bg subtraction (dark gray), and 2× correct bg subtraction (black).