A series of spontaneously blinking dyes for super-resolution microscopy.

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

Oligomerization state of the functional bacterial twin-arginine translocation (Tat) receptor complex.

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typically low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

Super-resolved 3D tracking of cargo transport through nuclear pore complexes.

Nuclear pore complexes (NPCs) embedded within the nuclear envelope mediate rapid, selective and bidirectional traffic between the cytoplasm and the nucleoplasm. Deciphering the mechanism and dynamics of this process is challenged by the need for high spatial and temporal resolution. We report here a multicolour imaging approach that enables direct three-dimensional visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring structure of the NPC scaffold. The success of this approach is enabled by the high positional stability of NPCs within permeabilized cells, as verified by a combined experimental and simulation analysis. Hourglass-shaped translocation conduits for two cargo complexes representing different nuclear transport receptor pathways indicate rapid migration through the permeability barrier on or near the NPC scaffold. Binding sites for cargo complexes extend more than 100 nm from the pore openings, which is consistent with a wide distribution of the phenylalanine-glycine polypeptides that bind nuclear transport receptors.

Tuning axial and lateral localization precision in 3D super-resolution microscopy with variable astigmatism.

Astigmatism imaging is a three-dimensional (3D) single molecule fluorescence microscopy approach that yields super-resolved spatial information on a rapid time scale from a single image. It is ideally suited for resolving structures on a sub-micrometer scale and temporal behavior in the millisecond regime. While traditional astigmatism imaging utilizes a cylindrical lens, adaptive optics enables the astigmatism to be tuned for the experiment. We demonstrate here how the precisions in x, y, and z are inter-linked and vary with the astigmatism, z-position, and photon level. This experimentally driven and verified approach provides a guide for astigmatism selection in biological imaging strategies.

Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation.

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 µM), and this monomer binds reversibly to spTorA (KD ≈ 1 µM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

The Tat protein transport system: intriguing questions and conundrums.

The Tat machinery catalyzes the transport of folded proteins across the cytoplasmic membrane in bacteria and the thylakoid membrane in plants. Transport occurs only in the presence of an electric field (Δψ) and/or a pH (ΔpH) gradient, and thus, Tat transport is considered to be dependent on the proton motive force (pmf). This presents a fundamental and major challenge, namely, that the Tat system catalyzes the movement of large folded protein cargos across a membrane without collapse of ion gradients. Current models argue that the active translocon assembles de novo for each cargo transported, thus providing an effective gating mechanism to minimize ion leakage. A limited structural understanding of the intermediates occurring during transport and the role of the pmf in stabilizing and/or driving this process have hindered the development of more detailed models. A fundamental question that remains unanswered is whether the pmf is actually 'consumed', providing an energetic driving force for transport, or alternatively, whether its presence is instead necessary to provide the appropriate environment for the translocon components to become active. Including addressing this issue in greater detail, we explore a series of additional questions that challenge current models, and, hopefully, motivate future work.

A Hinged Signal Peptide Hairpin Enables Tat-Dependent Protein Translocation.

The Tat machinery catalyzes the transport of folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane in plants. Using fluorescence quenching and cross-linking approaches, we demonstrate that the Escherichia coli TatBC complex catalyzes insertion of a pre-SufI signal peptide hairpin that penetrates about halfway across the membrane bilayer. Analysis of 512 bacterial Tat signal peptides using secondary structure prediction and docking algorithms suggest that this hairpin interaction mode is generally conserved. An internal cross-link in the signal peptide that blocks transport but does not affect binding indicates that a signal peptide conformational change is required during translocation. These results suggest, to our knowledge, a novel hairpin-hinge model in which the signal peptide hairpin unhinges during movement of the mature domain across the membrane. Thus, in addition to enabling the necessary recognition, the interaction of Tat signal peptides with the receptor complex plays a critical role in the transport process itself.

Investigating molecular crowding within nuclear pores using polarization-PALM.

The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC's internal organization consists of multiple dynamic environments with different local properties.

Deciphering the Structure and Function of Nuclear Pores Using Single-Molecule Fluorescence Approaches.

Due to its central role in macromolecular trafficking and nucleocytoplasmic information transfer, the nuclear pore complex (NPC) has been studied in great detail using a wide spectrum of methods. Consequently, many aspects of its architecture, general function, and role in the life cycle of a cell are well understood. Over the last decade, fluorescence microscopy methods have enabled the real-time visualization of single molecules interacting with and transiting through the NPC, allowing novel questions to be examined with nanometer precision. While initial single-molecule studies focused primarily on import pathways using permeabilized cells, it has recently proven feasible to investigate the export of mRNAs in living cells. Single-molecule assays can address questions that are difficult or impossible to answer by other means, yet the complexity of nucleocytoplasmic transport requires that interpretation be based on a firm genetic, biochemical, and structural foundation. Moreover, conceptually simple single-molecule experiments remain technically challenging, particularly with regard to signal intensity, signal-to-noise ratio, and the analysis of noise, stochasticity, and precision. We discuss nuclear transport issues recently addressed by single-molecule microscopy, evaluate the limits of existing assays and data, and identify open questions for future studies. We expect that single-molecule fluorescence approaches will continue to be applied to outstanding nucleocytoplasmic transport questions, and that the approaches developed for NPC studies are extendable to additional complex systems and pathways within cells.

High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors.

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors.

Large cargo transport by nuclear pores: implications for the spatial organization of FG-nucleoporins.

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine-glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non-uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG-NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.

Choreography of importin-α/CAS complex assembly and disassembly at nuclear pores.

Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the cytoplasm and the nucleoplasm. Soluble nuclear transport receptors bind signal-dependent cargos to form transport complexes that diffuse through the NPC and are then disassembled. Although transport receptors enable the NPC's permeability barrier to be overcome, directionality is established by complex assembly and disassembly. Here, we delineate the choreography of importin-α/CAS complex assembly and disassembly in permeabilized cells, using single-molecule fluorescence resonance energy transfer and particle tracking. Monitoring interaction sequences in intact NPCs ensures spatiotemporal preservation of structures and interactions critical for activity in vivo. We show that key interactions between components are reversible, multiple outcomes are often possible, and the assembly and disassembly of complexes are precisely controlled to occur at the appropriate place and time. Importin-α mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-α/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-α/CAS complexes after export occurs in the cytoplasmic filament region of the NPC. Mathematical models derived from our data emphasize the intimate connection between transport and the coordinated assembly and disassembly of importin-α/CAS complexes for generating productive transport cycles.

Effect of cargo size and shape on the transport efficiency of the bacterial Tat translocase.

The Tat machinery translocates fully-folded and oligomeric substrates. The passage of large, bulky cargos across an ion-tight membrane suggests the need to match pore and cargo size, and therefore that Tat transport efficiency may depend on both cargo size and shape. A series of cargos of different sizes and shapes were generated using the natural Tat substrate pre-SufI as a base. Four (of 17) cargos transported with significant (>20% of wild-type) efficiencies. These results indicate that cargo size and shape significantly influence Tat transportability.

Kinetics of precursor interactions with the bacterial Tat translocase detected by real-time FRET.

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.

Position-dependent effects of polylysine on Sec protein transport.

The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.

Single molecule studies of nucleocytoplasmic transport.

Molecular traffic between the cytoplasm and the nucleoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). Hundreds, if not thousands, of molecules interact with and transit through each NPC every second. The pore is blocked by a permeability barrier, which consists of a network of intrinsically unfolded polypeptides containing thousands of phenylalanine-glycine (FG) repeat motifs. This FG-network rejects larger molecules and admits smaller molecules or cargos bound to nuclear transport receptors (NTRs). For a cargo transport complex, minimally consisting of a cargo molecule plus an NTR, access to the permeability barrier is provided by interactions between the NTR and the FG repeat motifs. Numerous models have been postulated to explain the controlled accessibility and the transport characteristics of the FG-network, but the amorphous, flexible nature of this structure has hindered characterization. A relatively recent development is the ability to monitor the real-time movement of single molecules through individual NPCs via single molecule fluorescence (SMF) microscopy. A major advantage of this approach is that it can be used to continuously monitor a series of specific molecular interactions in an active pore with millisecond time resolution, which therefore allows one to distinguish between kinetic and thermodynamic control. Novel insights and prospects for the future are outlined in this review. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.

Signal peptides target protein cargos for secretion from the bacterial cytoplasm. These signal peptides contain a tri-partite structure consisting of a central hydrophobic domain (h-domain), and two flanking polar domains. Using a recently developed in vitro transport assay, we report here that a central h-domain position (C17) of the twin arginine translocation (Tat) substrate pre-SufI is especially sensitive to amino acid hydrophobicity. The C17I mutant is transported more efficiently than wild type, whereas charged substitutions completely block transport. Transport efficiency is well-correlated with Tat translocon binding efficiency. The precursor protein also binds to non-Tat components of the membrane, presumably to the lipids. This lipid-bound precursor can be chased through the Tat translocons under conditions of high proton motive force. Thus, the non-Tat bound form of the precursor is a functional intermediate in the transport cycle. This intermediate appears to directly equilibrate with the translocon-bound form of the precursor.

Bacterial Sec protein transport is rate-limited by precursor length: a single turnover study.

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.

Single-molecule measurements of importin alpha/cargo complex dissociation at the nuclear pore.

Macromolecules are transported between the cytoplasm and the nucleoplasm of eukaryotic cells through nuclear pore complexes (NPCs). Large (more than approximately 40 kDa) transport cargoes imported into the nucleus typically form a complex with at least one soluble transport cofactor of the importin (Imp) beta superfamily. Many cargoes require an accessory cofactor, Imp alpha, which binds to Imp beta and to the nuclear localization sequence on the cargo. We previously reported the use of narrow-field epifluorescence microscopy to directly monitor cargoes in transit through NPCs in permeabilized cells. We now report an expanded approach in which single-molecule fluorescence resonance energy transfer (FRET) is used to detect the disassembly of Imp alpha/cargo complexes as they transit through NPCs. We found that CAS, the recycling cofactor for Imp alpha, and RanGTP are essential for this dissociation process. After Imp alpha/cargo complex dissociation, most Imp alpha and cargo molecules entered the nucleoplasm. In contrast, the majority of Imp alpha/cargo complexes that did not dissociate at the NPC in the presence of CAS and RanGTP returned to the cytoplasm. These data are consistent with a model in which Imp alpha/cargo complexes are dissociated on the nucleoplasmic side of the NPC, and this dissociation requires both CAS and RanGTP.