Single Nuclear Pore Complexes Research Articles

Optical recording of signal-mediated protein transport through single nuclear pore complexes.

Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of Xenopus oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate in vivo conditions as closely as possible, a Xenopus egg extract was applied to the cytosolic side and a Xenopus oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, "patched" NPCs depended on signal sequences, extracts, and metabolic energy, as in vivo. All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were approximately 2 dimers/s for the import protein and approximately 4 dimers/s for the export protein ( approximately 15 microM substrate concentration, 22-24 degrees C), in good agreement with in vivo rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms.

Permeability of Single Nuclear Pores

In this first application of optical single transporter recording (OSTR), a recently established technique for optically monitoring the activity of single transporters in membrane patches (Tschödrich-Rotter and Peters. 1998. J. Microsc. 192:114–125), the passive permeability of the nuclear pore complex (NPC) was measured for a homologous series of hydrophilic probe molecules. Nuclei were isolated from Xenopus oocytes and firmly attached to filters containing small cylindrical pores. Transport through membrane patches spanning filter pores was measured by scanning microphotolysis. Thus the permeability coefficients of single NPCs were determined for fluorescently labeled dextrans of ∼4, 10, and 20kDa. Dextrans of ≥40kDa could not permeate the NPC. The data were consistent with a model in which the NPC contains a single diffusion channel. By application of established theories for the restricted diffusion through small pores, the diffusion channel was approximated as a cylinder with a radius of 4.4–6.1nm (mean 5.35nm). Because the transport rate constant of the single NPC was known, the equivalent length of the channel could be also determined and was found to be 40–50nm (mean 44.5nm). The symmetry of the NPC implies that a singular component such as the diffusion channel is located at the center of the NPC. Therefore a common transport pathway apparently mediates both passive and signal-dependent transport. To test this hypothesis, measurements of signal-dependent transport and of the mutual effects signal-dependent and passive transport may exert on each other are in progress.

Aldosterone and nuclear signaling in kidney

Initiation of transcription is an early step in steroid hormone action. We investigated by means of atomic force microscopy (AFM) and fluorescence imaging the role of nuclear pore complexes (NPCs) in mediating signal transduction of the mineralocorticoid hormone aldosterone from the extracellular space into the cell nucleus. With AFM, we imaged single NPCs of isolated nuclear envelopes under native conditions. We observed that individual NPCs contract in response to a Ca 2+ signal, which is known to occur in seconds after aldosterone exposure. In living kidney cells in culture (MDCK cells), aldosterone led within seconds to the contraction of the whole nucleus measured by DNA-fluorescence imaging. Nuclear contraction was elicited at similar time scale and to similar extent by bradykinin, a peptide hormone known to mobilize Ca 2+ from internal stores, and by ionomycin, a Ca 2+ ionophore known to directly increase intracellular Ca 2+. Nuclear contraction is explained by the individual contraction of calcium-sensitive NPCs that occur in high density in the nuclear envelope. We present a model in which nuclear pore complexes play a key role as barrier molecules of high plasticity in the control of aldosterone-induced gene expression.

Chapter 5 Localization of Single Nuclear Pore Complexes by Confocal Laser Scanning Microscopy and Analysis of Their Distribution

The nuclear pore complex (NPC) undoubtedly assumes a central role in the communication between nucleus and cytoplasm. It controls both the export of mature nuclei acids from the nucleus and the import of large proteins, such as transcription factors, into the nucleus. Light microscopy enables the visualization of single NPCs as bright diffraction-limited spots. Combination of image analysis methods and distribution analysis allows the assessment and characterization of three-dimensional arrangement on the nuclear envelope. This is made possible by two features of the NPC system: (1) the large diameter of the pore complexes, which nearly approach the lateral optical resolution and (2) a relatively low pore density on the nuclear envelope surface. This was determined to be 4–5/μm 2 for 3T3 cells. Because many cell types have higher NPC densities (up to more than 50/μm 2 ), a detailed analysis of the pore distribution can be applied only in a limited number of systems.

Single nuclear pores visualized by confocal microscopy and image processing

How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arranged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image processing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was tested by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localized with a lateral accuracy of <20 nm and an axial accuracy of approximately 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti-nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution. Herein the nuclear pore complexes appeared as bright diffraction-limited spots whose centers were localized by fitting them by three-dimensional gaussians. The nearest-neighbor distribution function and the pair correlation function were calculated and found to agree well with those of randomly distributed hard cylinders of 138 +/- 17 nm diameter, but not with those of randomly distributed points or nonrandomly distributed cylinders. This was supported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed.

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex.

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10(-15)-10(-10)M) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, gamma, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pM. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.

Imaging nuclear pores of aldosterone-sensitive kidney cells by atomic force microscopy.

In nuclei of renal target cells, aldosterone enhances transcriptional activity followed by the translocation of specific RNA molecules across the nuclear envelope. Trafficking between cell nucleus and cytoplasm occurs via nuclear pore complexes (NPCs) located in the double-layered nuclear envelope. We investigated the nucleocytoplasmic transport route by structure-function analysis at subcellular level in quiescent and aldosterone-stimulated cells. With atomic-force microscopy (AFM) we imaged individual pores of the nuclear surface of cultured kidney cells and related the number of pores per micron2 to nuclear envelope conductance (Gn, per micron2) evaluated electrically by current injection into the isolated nucleus. NPCs were equally distributed resembling "donut-like" structures with outer diameters of 134 +/- 12 nm (n = 50), each equipped with a central channel. Six hours of aldosterone exposure (0.1 microM) increased the number of NPCs per micron 2 of nuclear surface from 7.4 +/- 0.4 to 9.8 +/- 0.4 (n = 12; P < 0.01). At the same time Gn rose from 6900 +/- 520 to 9600 +/- 610 pS/micron2 paralleled by an increase of the intranuclear electrical potential from -2.8 +/- 0.2 to -6.2 +/- 0.4 mV (n = 18; P < 0.01). Assuming that NPCs represent the sole conductive pathway in the nuclear envelope, we calculate a mean single NPC conductance of 932 and 980 pS, in the absence and presence of aldosterone, respectively. We conclude that aldosterone facilitates nucleocytoplasmic transport by increasing the number of NPCs but not by modifying their biophysical properties. Possibly, aldosterone controls similar transport mechanisms in both plasma membrane and nuclear envelope.