Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions.

DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) has recently significantly enhanced observation times in in vitro SPT experiments by overcoming the constraints of fluorophore photobleaching. However, with the reported implementation, only a single target can be imaged and the technique cannot be applied straight to live cell imaging. Here we report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-color DNA-PAINT-SPT for quantifying protein dimerization and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

Exploring Giant Unilamellar Vesicle Production for Artificial Cells - Current Challenges and Future Directions.

Creating an artificial cell from the bottom up is a long-standing challenge and, while significant progress has been made, the full realization of this goal remains elusive. Arguably, one of the biggest hurdles that researchers are facing now is the assembly of different modules of cell function inside a single container. Giant unilamellar vesicles (GUVs) have emerged as a suitable container with many methods available for their production. Well-studied swelling-based methods offer a wide range of lipid compositions but at the expense of limited encapsulation efficiency. Emulsion-based methods, on the other hand, excel at encapsulation but are only effective with a limited set of membrane compositions and may entrap residual additives in the lipid bilayer. Since the ultimate artificial cell will need to comply with both specific membrane and encapsulation requirements, there is still no one-method-fits-all solution for GUV formation available today. This review discusses the state of the art in different GUV production methods and their compatibility with GUV requirements and operational requirements such as reproducibility and ease of use. It concludes by identifying the most pressing issues and proposes potential avenues for future research to bring us one step closer to turning artificial cells into a reality.

The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging.

Imaging the dynamics and interactions of biomolecules at the single-molecule level in live cells and reconstituted systems has generated unprecedented knowledge about the biomolecular processes underlying many cellular functions. To achieve the speed and sensitivity needed to detect and follow individual molecules, these experiments typically require custom-built microscopes or custom modifications of commercial systems. The costs of such single-molecule microscopes, their technical complexity and the lack of open-source documentation on how to build custom setups therefore limit the accessibility of single-molecule imaging techniques. To advance the adaptation of dynamic single-molecule imaging by a wider community, we present the "K2": an open-source, simultaneous triple-color total internal reflection fluorescence (TIRF) microscope specifically designed for live-cell and single-molecule imaging. We explain our design considerations and provide step-by-step building instructions, parts list and full CAD models. The modular design of this TIRF microscope allows users to customize it to their scientific and financial needs, or to re-use parts of our design to improve the capabilities of their existing setups without necessarily having to build a full copy of the K2 microscope.

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles.

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

FtsZ Reorganization Facilitates Deformation of Giant Vesicles in Microfluidic Traps*.

The geometry of reaction compartments can affect the local outcome of interface-restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell-sized, membrane-bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar-like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane-bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially-sensitive reactions inside them, such as that of protein self-organization.

Imaging rRNA Methylation in Bacteria by MR-FISH.

Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~104 to 107 cells to be pooled to generate sufficient material for analysis. Here we describe a method-methylation-sensitive RNA in situ hybridization (MR-FISH)-that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy. We outline step-by-step protocols for designing probes, in situ hybridization, and analysis of data using freely available code.

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.

Synthetic cell division via membrane-transforming molecular assemblies.

Reproduction, i.e. the ability to produce new individuals from a parent organism, is a hallmark of living matter. Even the simplest forms of reproduction require cell division: attempts to create a designer cell therefore should include a synthetic cell division machinery. In this review, we will illustrate how nature solves this task, describing membrane remodelling processes in general and focusing on bacterial cell division in particular. We discuss recent progress made in their in vitro reconstitution, identify open challenges, and suggest how purely synthetic building blocks could provide an additional and attractive route to creating artificial cell division machineries.

More from less - bottom-up reconstitution of cell biology.

The ultimate goal of bottom-up synthetic biology is recreating life in its simplest form. However, in its quest to find the minimal functional units of life, this field contributes more than its main aim by also offering a range of tools for asking, and experimentally approaching, biological questions. This Review focusses on how bottom-up reconstitution has furthered our understanding of cell biology. Studying cell biological processes in vitro has a long tradition, but only recent technological advances have enabled researchers to reconstitute increasingly complex biomolecular systems by controlling their multi-component composition and their spatiotemporal arrangements. We illustrate this progress using the example of cytoskeletal processes. Our understanding of these has been greatly enhanced by reconstitution experiments, from the first in vitro experiments 70 years ago to recent work on minimal cytoskeleton systems (including this Special Issue of Journal of Cell Science). Importantly, reconstitution approaches are not limited to the cytoskeleton field. Thus, we also discuss progress in other areas, such as the shaping of biomembranes and cellular signalling, and prompt the reader to add their subfield of cell biology to this list in the future.

Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex.

Dynamic reorganization of the actomyosin cytoskeleton allows fast modulation of the cell surface, which is vital for many cellular functions. Myosin-II motors generate the forces required for this remodeling by imparting contractility to actin networks. However, myosin-II activity might also have a more indirect contribution to cytoskeletal dynamics; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by enhancing disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but the reassembly of actin in these assays was limited by factors such as diffusional constraints and the use of stabilized actin filaments. Here, we present the reconstitution of a minimal dynamic actin cortex, where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which the actin network undergoes constant turnover. Our findings suggest a multifaceted role of myosin-II in the dynamics of the eukaryotic actin cortex. This article has an associated First Person interview with the first author of the paper.

Membrane association and remodeling by intraflagellar transport protein IFT172.

The cilium is an organelle used for motility and cellular signaling. Intraflagellar transport (IFT) is a process to move ciliary building blocks and signaling components into the cilium. How IFT controls the movement of ciliary components is currently poorly understood. IFT172 is the largest IFT subunit essential for ciliogenesis. Due to its large size, the characterization of IFT172 has been challenging. Using giant unilamellar vesicles (GUVs), we show that IFT172 is a membrane-interacting protein with the ability to remodel large membranes into small vesicles. Purified IFT172 has an architecture of two globular domains with a long rod-like protrusion, resembling the domain organization of coatomer proteins such as COPI-II or clathrin. IFT172 adopts two different conformations that can be manipulated by lipids or detergents: 1) an extended elongated conformation and 2) a globular closed architecture. Interestingly, the association of IFT172 with membranes is mutually exclusive with IFT57, implicating multiple functions for IFT172 within IFT.

Detecting RNA base methylations in single cells by in situ hybridization.

Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~104-107 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m62A, m1G and m3U) that destabilize Watson-Crick base pairs. Our method-methylation-sensitive RNA fluorescence in situ hybridization-detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging.

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading.

The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5-140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10(4), are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.

Single-molecule imaging reveals that small amyloid-ß1-42 oligomers interact with the cellular prion protein (PrP(C)).

Oligomers of the amyloid-ß peptide (Aß) play a central role in the pathogenesis of Alzheimer's disease and have been suggested to induce neurotoxicity by binding to a plethora of cell-surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aß42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single-molecule imaging to characterize Aß42 oligomers (oAß42) and to confirm the controversial interaction of oAß42 with the cellular prion protein (PrP(C)) on live neuronal cells. Our results show that, at nanomolar concentrations, oAß42 interacts with PrP(C) and that the species bound to PrP(C) are predominantly small oligomers (dimers and trimers). Single-molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor-mediated oAß-induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.

A quantitative comparison of single-dye tracking analysis tools using Monte Carlo simulations.

Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles' displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-ß-cyclodextrin and latrunculin B.

Single molecule characterization of the interactions between amyloid-ß peptides and the membranes of hippocampal cells.

Oligomers of the 40 and 42 residue amyloid-ß peptides (Aß40 and Aß42) have been implicated in the neuronal damage and impaired cognitive function associated with Alzheimer's disease. However, little is known about the specific mechanisms by which these misfolded species induce such detrimental effects on cells. In this work, we use single-molecule imaging techniques to examine the initial interactions between Aß monomers and oligomers and the membranes of live cells. This highly sensitive method enables the visualization of individual Aß species on the cell surface and characterization of their oligomerization state, all at biologically relevant, nanomolar concentrations. The results indicate that oligomers preferentially interact with cell membranes, relative to monomers and that the oligomers become immobilized on the cell surface. Additionally, we observe that the interaction of Aß species with the cell membrane is inhibited by the presence of ATP-independent molecular chaperones. This study demonstrates the power of this methodology for characterizing the interactions between protein aggregates and the membranes of live neuronal cells at physiologically relevant concentrations and opens the door to quantitative studies of the cellular responses to potentially pathogenic oligomers.

Identification of a hypochlorite-specific transcription factor from Escherichia coli.

Hypochlorite is a powerful oxidant produced by neutrophils to kill invading microorganisms. Despite this important physiological role of HOCl in fighting bacterial infections, no hypochlorite-specific stress response has been identified yet. Here, we identified a hypochlorite-responsive transcription factor, YjiE, which is conserved in proteobacteria and eukaryotes. YjiE forms unusual dodecameric ring-like structures in vitro that undergo large DNA-induced conformational changes to form dimers and tetramers as shown by transmission electron microscopy and analytical ultracentrifugation. Such smaller oligomers are predominant in hypochlorite-stressed cells and are the active species as shown by fluorescence anisotropy and analytical ultracentrifugation. YjiE regulates a large number of genes upon hypochlorite stress. Among them are genes involved in cysteine, methionine biosynthesis, and sulfur metabolism (up-regulated) and genes involved in iron acquisition and homeostasis (down-regulated), thus supposedly replenishing oxidized metabolites and decreasing the hypochlorite-mediated amplification of intracellular reactive oxygen species. As a result, YjiE specifically confers hypochlorite resistance to E. coli cells. Thus, to our knowledge, YjiE is the first described hypochlorite-specific transcription factor.