MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly.

The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.

Treadmilling analysis reveals new insights into dynamic FtsZ ring architecture.

FtsZ, the primary protein of the bacterial Z ring guiding cell division, has been recently shown to engage in intriguing treadmilling dynamics along the circumference of the division plane. When coreconstituted in vitro with FtsA, one of its natural membrane anchors, on flat supported membranes, these proteins assemble into dynamic chiral vortices compatible with treadmilling of curved polar filaments. Replacing FtsA by a membrane-targeting sequence (mts) to FtsZ, we have discovered conditions for the formation of dynamic rings, showing that the phenomenon is intrinsic to FtsZ. Ring formation is only observed for a narrow range of protein concentrations at the bilayer, which is highly modulated by free Mg2+ and depends upon guanosine triphosphate (GTP) hydrolysis. Interestingly, the direction of rotation can be reversed by switching the mts from the C-terminus to the N-terminus of the protein, implying that the filament attachment must have a perpendicular component to both curvature and polarity. Remarkably, this chirality switch concurs with previously shown inward or outward membrane deformations by the respective FtsZ mutants. Our results lead us to suggest an intrinsic helicity of FtsZ filaments with more than one direction of curvature, supporting earlier hypotheses and experimental evidence.

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.

Toward Absolute Molecular Numbers in DNA-PAINT.

Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future.

Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy.

We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates.

FCS Analysis of Protein Mobility on Lipid Monolayers.

In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions.

Direct characterization of the evanescent field in objective-type total internal reflection fluorescence microscopy.

Total internal reflection fluorescence (TIRF) microscopy is a commonly used method for studying fluorescently labeled molecules in close proximity to a surface. Usually, the TIRF axial excitation profile is assumed to be single-exponential with a characteristic penetration depth, governed by the incident angle of the excitation laser beam towards the optical axis. However, in practice, the excitation profile does not only comprise the theoretically predicted single-exponential evanescent field, but also an additional non-evanescent contribution, supposedly caused by scattering within the optical path or optical aberrations. We developed a calibration slide to directly characterize the TIRF excitation field. Our slide features ten height steps ranging from 25 to 550 nanometers, fabricated from a polymer with a refractive index matching that of water. Fluorophores in aqueous solution above the polymer step layers sample the excitation profile at different heights. The obtained excitation profiles confirm the theoretically predicted exponential decay over increasing step heights as well as the presence of a non-evanescent contribution.

Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA Spacers.

DNA origami nanotechnology is being increasingly used to mimic membrane-associated biophysical phenomena. Although a variety of DNA origami nanostructures has already been produced to target lipid membranes, the requirements for membrane binding have so far not been systematically assessed. Here, we used a set of elongated DNA origami structures with varying placement and number of cholesteryl-based membrane anchors to compare different strategies for their incorporation. Single and multiple cholesteryl anchors were attached to DNA nanostructures using single- and double-stranded DNA spacers of varying length. The produced DNA nanostructures were studied in terms of their membrane binding and diffusion. Our results show that the location and number of anchoring moieties play a crucial role for membrane binding of DNA nanostructures mainly if the cholesteryl anchors are in close proximity to the bulky DNA nanostructures. Moreover, the use of DNA spacers largely overcomes local steric hindrances and thus enhances membrane binding. Fluorescence correlation spectroscopy measurements demonstrate that the distinct physical properties of single- and double-stranded DNA spacers control the interaction of the amphipathic DNA nanostructures with lipid membranes. Thus, we provide a rational basis for the design of amphipathic DNA origami nanostructures to efficiently bind lipid membranes in various environments.

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy.

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.

Optical Control of a Biological Reaction-Diffusion System.

Patterns formed by reaction and diffusion are the foundation for many phenomena in biology. However, the experimental study of reaction-diffusion (R-D) systems has so far been dominated by chemical oscillators, for which many tools are available. In this work, we developed a photoswitch for the Min system of Escherichia coli, a versatile biological in vitro R-D system consisting of the antagonistic proteins MinD and MinE. A MinE-derived peptide of 19 amino acids was covalently modified with a photoisomerizable crosslinker based on azobenzene to externally control peptide-mediated depletion of MinD from the membrane. In addition to providing an on-off switch for pattern formation, we achieve frequency-locked resonance with a precise 2D spatial memory, thus allowing new insights into Min protein action on the membrane. Taken together, we provide a tool to study phenomena in pattern formation using biological agents.

Fluorescence Correlation Spectroscopy to Examine Protein-Lipid Interactions in Membranes.

Fluorescence correlation spectroscopy (FCS) is a versatile technique to study membrane dynamics and protein-lipid interactions. It can provide information about diffusion coefficients, concentrations, and molecular interactions of proteins and lipids in the membrane. These parameters allow for the determination of protein partitioning into different lipid environments, the identification of lipid domains, and the detection of lipid-protein complexes on the membrane. During the last decades, FCS studies were successfully performed on model membrane systems as also on living cells, to characterize protein-lipid interactions. Recent developments of the method described here improved quantitative measurements on membranes and decreased the number of potential artifacts. The aim of this chapter is to provide the reader with the necessary information and some practical guidelines to perform FCS studies on artificial and cellular membranes.

Stationary Patterns in a Two-Protein Reaction-Diffusion System.

Patterns formed by reaction-diffusion mechanisms are crucial for the development or sustenance of most organisms in nature. Patterns include dynamic waves, but are more often found as static distributions, such as animal skin patterns. Yet, a simplistic biological model system to reproduce and quantitatively investigate static reaction-diffusion patterns has been missing so far. Here, we demonstrate that the Escherichia coli Min system, known for its oscillatory behavior between the cell poles, is under certain conditions capable of transitioning to quasi-stationary protein distributions on membranes closely resembling Turing patterns. We systematically titrated both proteins, MinD and MinE, and found that removing all purification tags and linkers from the N-terminus of MinE was critical for static patterns to occur. At small bulk heights, dynamic patterns dominate, such as in rod-shaped microcompartments. We see implications of this work for studying pattern formation in general, but also for creating artificial gradients as downstream cues in synthetic biology applications.

The MinDE system is a generic spatial cue for membrane protein distribution in vitro.

The E. coli MinCDE system has become a paradigmatic reaction-diffusion system in biology. The membrane-bound ATPase MinD and ATPase-activating protein MinE oscillate between the cell poles followed by MinC, thus positioning the main division protein FtsZ at midcell. Here we report that these energy-consuming MinDE oscillations may play a role beyond constraining MinC/FtsZ localization. Using an in vitro reconstitution assay, we show that MinDE self-organization can spatially regulate a variety of functionally completely unrelated membrane proteins into patterns and gradients. By concentration waves sweeping over the membrane, they induce a direct net transport of tightly membrane-attached molecules. That the MinDE system can spatiotemporally control a much larger set of proteins than previously known, may constitute a MinC-independent pathway to division site selection and chromosome segregation. Moreover, the here described phenomenon of active transport through a traveling diffusion barrier may point to a general mechanism of spatiotemporal regulation in cells.

Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains.

We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel-wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging.

Fluorescence fluctuation microscopy: a diversified arsenal of methods to investigate molecular dynamics inside cells.

Fluorescence microscopy provides insight into the subcellular organization of biological functions. However, images are snap shots averaging over a highly dynamic molecular system. Fluorescence fluctuation microscopy, employing similar detection technology, encompasses a powerful arsenal of analysis tools that investigate the molecular heterogeneity in space and time. Analyzing signal fluctuations from small ensembles (several hundred particles) reveals their concentration, the stoichiometry, the stochastic motion, as well as superimposed signatures of the environment such as spatial confinement and binding events. Thus, fluctuation analysis provides access to dynamic molecular properties that can be used to build physical models of cellular processes. In the last decade these methods experienced a remarkable diversification, which we revisit here with a particular focus on live cell applications.