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.

Protein-Assisted Large-Scale Assembly and Differential Patterning of DNA Origami Lattices.

Nanofabrication has experienced a big boost with the invention of DNA origami, enabling the production and assembly of complex nanoscale structures that may be able to unlock fully new functionalities in biology and beyond. The remarkable precision with which these structures can be designed and produced is, however, not yet matched by their assembly dynamics, which can be extremely slow, particularly when attached to biological templates, such as membranes. Here, the rapid and controlled formation of DNA origami lattices on the scale of hundreds of micrometers in as little as 30 minutes is demonstrated, utilizing active patterning by the E.coli Min protein system, thereby yielding a remarkable improvement over conventional passive diffusion-based assembly methods. Various patterns, including spots, inverse spots, mazes, and meshes can be produced at different scales, tailored through the shape and density of the assembled structures. The differential positioning accomplished by Min-induced diffusiophoresis even allows the introduction of "pseudo-colors", i.e., complex core-shell patterns, by simultaneously patterning different DNA origami species. Beyond the targeted functionalization of biological surfaces, this approach may also be promising for applications in plasmonics, catalysis, and molecular sensing.

Mass-sensitive particle tracking to elucidate the membrane-associated MinDE reaction cycle.

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-ß-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

3D printed protein-based robotic structures actuated by molecular motor assemblies.

Upscaling motor protein activity to perform work in man-made devices has long been an ambitious goal in bionanotechnology. The use of hierarchical motor assemblies, as realized in sarcomeres, has so far been complicated by the challenges of arranging sufficiently high numbers of motor proteins with nanoscopic precision. Here, we describe an alternative approach based on actomyosin cortex-like force production, allowing low complexity motor arrangements in a contractile meshwork that can be coated onto soft objects and locally activated by ATP. The design is reminiscent of a motorized exoskeleton actuating protein-based robotic structures from the outside. It readily supports the connection and assembly of micro-three-dimensional printed modules into larger structures, thereby scaling up mechanical work. We provide an analytical model of force production in these systems and demonstrate the design flexibility by three-dimensional printed units performing complex mechanical tasks, such as microhands and microarms that can grasp and wave following light activation.

Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation.

Compartmentalization is key to many cellular processes and a critical bottleneck of any minimal life approach. In cells, a complex chemistry is responsible for bringing together or separating biomolecules at the right place at the right time. Lipids, nucleic acids and proteins self-organize, thereby creating boundaries, interfaces and specialized microenvironments. Exploiting reversible RNA-based liquid-liquid phase separation (LLPS) inside giant unilamellar vesicles (GUVs), we present an efficient system capable of propagating an RNA-based enzymatic reaction across a population of GUVs upon freezing-thawing (FT) temperature cycles. We report that compartmentalization in the condensed RNA-rich phase can accelerate such an enzymatic reaction. In the decondensed state, RNA substrates become homogeneously dispersed, enabling content exchange between vesicles during freeze-thawing. This work explores how a minimal reversible phase separation system in lipid vesicles could help to implement spatiotemporal control in cyclic processes, as required for minimal cells.

Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nanotechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in out-of-equilibrium DNA nanodevices with precise functions.

Prebiotic Foam Environments to Oligomerize and Accumulate RNA.

When water interacts with porous rocks, its wetting and surface tension properties create air bubbles in large number. To probe their relevance as a setting for the emergence of life, we microfluidically created foams that were stabilized with lipids. A persistent non-equilibrium setting was provided by a thermal gradient. The foam's large surface area triggers capillary flows and wet-dry reactions that accumulate, aggregate and oligomerize RNA, offering a compelling habitat for RNA-based early life as it offers both wet and dry conditions in direct neighborhood. Lipids were screened to stabilize the foams. The prebiotically more probable myristic acid stabilized foams over many hours. The capillary flow created by the evaporation at the water-air interface provided an attractive force for molecule localization and selection for molecule size. For example, self-binding oligonucleotide sequences accumulated and formed micrometer-sized aggregates which were shuttled between gas bubbles. The wet-dry cycles at the foam bubble interfaces triggered a non-enzymatic RNA oligomerization from 2',3'-cyclic CMP and GMP which despite the small dry reaction volume was superior to the corresponding dry reaction. The found characteristics make heated foams an interesting, localized setting for early molecular evolution.

An order of magnitude faster DNA-PAINT imaging by optimized sequence design and buffer conditions.

DNA points accumulation in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement super-resolution technique. However, image acquisition is slow compared to most other approaches. Here, we overcome this limitation by designing optimized DNA sequences and buffer conditions. We demonstrate our approach in vitro with DNA origami and in situ using cell samples, and achieve an order of magnitude faster imaging speeds without compromising image quality or spatial resolution. This improvement now makes DNA-PAINT applicable to high-throughput studies.

Hydration Layer of Only a Few Molecules Controls Lipid Mobility in Biomimetic Membranes.

Self-assembly of biomembranes results from the intricate interactions between water and the lipids' hydrophilic head groups. Therefore, the lipid-water interplay strongly contributes to modulating membrane architecture, lipid diffusion, and chemical activity. Here, we introduce a new method of obtaining dehydrated, phase-separated, supported lipid bilayers (SLBs) solely by controlling the decrease of their environment's relative humidity. This facilitates the study of the structure and dynamics of SLBs over a wide range of hydration states. We show that the lipid domain structure of phase-separated SLBs is largely insensitive to the presence of the hydration layer. In stark contrast, lipid mobility is drastically affected by dehydration, showing a 6-fold decrease in lateral diffusion. At the same time, the diffusion activation energy increases approximately 2-fold for the dehydrated membrane. The obtained results, correlated with the hydration structure of a lipid molecule, revealed that about six to seven water molecules directly hydrating the phosphocholine moiety play a pivotal role in modulating lipid diffusion. These findings could provide deeper insights into the fundamental reactions where local dehydration occurs, for instance during cell-cell fusion, and help us better understand the survivability of anhydrobiotic organisms. Finally, the strong dependence of lipid mobility on the number of hydrating water molecules opens up an application potential for SLBs as very precise, nanoscale hydration sensors.

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.

Probing Biomolecular Interactions by a Pattern-Forming Peptide-Conjugate Sensor.

As a key mechanism underpinning many biological processes, protein self-organization has been extensively studied. However, the potential to apply the distinctive, nonlinear biochemical properties of such self-organizing systems to biotechnological problems such as the facile detection and characterization of biomolecular interactions has not yet been explored. Here, we describe an in vitro assay in a 96-well plate format that harnesses the emergent behavior of the Escherichia coli Min system to provide a readout of biomolecular interactions. Crucial for the development of our approach is a minimal MinE-derived peptide that stimulates MinD ATPase activity only when dimerized. We found that this behavior could be induced via any pair of foreign, mutually binding molecular entities fused to the minimal MinE peptide. The resulting MinD ATPase activity and the spatiotemporal nature of the produced protein patterns quantitatively correlate with the affinity of the fused binding partners, thereby enabling a highly sensitive assay for biomolecular interactions. Our assay thus provides a unique means of quantitatively visualizing biomolecular interactions and may prove useful for the assessment of domain interactions within protein libraries and for the facile investigation of potential inhibitors of protein-protein interactions.

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.

Reversible membrane deformations by straight DNA origami filaments.

Membrane-active cytoskeletal elements, such as FtsZ, septin or actin, form filamentous polymers able to induce and stabilize curvature on cellular membranes. In order to emulate the characteristic dynamic self-assembly properties of cytoskeletal subunits in vitro, biomimetic synthetic scaffolds were here developed using DNA origami. In contrast to our earlier work with pre-curved scaffolds, we specifically assessed the potential of origami mimicking straight filaments, such as actin and microtubules, by origami presenting cholesteryl anchors for membrane binding and additional blunt end stacking interactions for controllable polymerization into linear filaments. By assessing the interaction of our DNA nanostructures with model membranes using fluorescence microscopy, we show that filaments can be formed, upon increasing MgCl2 in solution, for structures displaying blunt ends; and can subsequently depolymerize, by decreasing the concentration of MgCl2. Distinctive spike-like membrane protrusions were generated on giant unilamellar vesicles at high membrane-bound filament densities, and the presence of such deformations was reversible and shown to correlate with the MgCl2-triggered polymerization of DNA origami subunits into filamentous aggregates. In the end, our approach reveals the formation of membrane-bound filaments as a minimal requirement for membrane shaping by straight cytoskeletal-like objects.

Active shape oscillations of giant vesicles with cyclic closure and opening of membrane necks.

Reaction-diffusion systems encapsulated within giant unilamellar vesicles (GUVs) can lead to shape oscillations of these vesicles as recently observed for the bacterial Min protein system. This system contains two Min proteins, MinD and MinE, which periodically attach to and detach from the GUV membranes, with the detachment being driven by ATP hydrolysis. Here, we address these shape oscillations within the theoretical framework of curvature elasticity and show that they can be understood in terms of a spontaneous curvature that changes periodically with time. We focus on the simplest case provided by a attachment-detachment kinetics that is laterally uniform along the membrane. During each oscillation cycle, the vesicle shape is transformed from a symmetric dumbbell with two subcompartments of equal size to an asymmetric dumbbell with two subcompartments of different size, followed by the reverse, symmetry-restoring transformation. This sequence of shapes is first analyzed within the spontaneous curvature model which is then extended to the area-difference-elasticity model by decomposing the spontaneous curvature into a local and nonlocal component. For both symmetric and asymmetric dumbbells, the two subcompartments are connected by a narrow membrane neck with a circular waistline. The radius of this waistline undergoes periodic oscillations, the time dependence of which can be reasonably well fitted by a single Fourier mode with an average time period of 56 s.

Membrane-coated 3D architectures for bottom-up synthetic biology.

One of the great challenges of bottom-up synthetic biology is to recreate the cellular geometry and surface functionality required for biological reactions. Of particular interest are lipid membrane interfaces where many protein functions take place. However, cellular 3D geometries are often complex, and custom-shaping stable lipid membranes on relevant spatial scales in the micrometer range has been hard to accomplish reproducibly. Here, we use two-photon direct laser writing to 3D print microenvironments with length scales relevant to cellular processes and reactions. We formed lipid bilayers on the surfaces of these printed structures, and we evaluated multiple combinatorial scenarios, where physiologically relevant membrane compositions were generated on several different polymer surfaces. Functional dynamic protein systems were reconstituted in vitro and their self-organization was observed in response to the 3D geometry. This method proves very useful to template biological membranes with an additional spatial dimension, and thus allows a better understanding of protein function in relation to the complex morphology of cells and organelles.

MinE conformational switching confers robustness on self-organized Min protein patterns.

Protein patterning is vital for many fundamental cellular processes. This raises two intriguing questions: Can such intrinsically complex processes be reduced to certain core principles and, if so, what roles do the molecular details play in individual systems? A prototypical example for protein patterning is the bacterial Min system, in which self-organized pole-to-pole oscillations of MinCDE proteins guide the cell division machinery to midcell. These oscillations are based on cycling of the ATPase MinD and its activating protein MinE between the membrane and the cytoplasm. Recent biochemical evidence suggests that MinE undergoes a reversible, MinD-dependent conformational switch from a latent to a reactive state. However, the functional relevance of this switch for the Min network and pattern formation remains unclear. By combining mathematical modeling and in vitro reconstitution of mutant proteins, we dissect the two aspects of MinE's switch, persistent membrane binding and a change in MinE's affinity for MinD. Our study shows that the MinD-dependent change in MinE's binding affinity for MinD is essential for patterns to emerge over a broad and physiological range of protein concentrations. Mechanistically, our results suggest that conformational switching of an ATPase-activating protein can lead to the spatial separation of its distinct functional states and thereby confer robustness on an intracellular protein network with vital roles in bacterial cell division.

Fine-Tuning Protein Self-Organization by Orthogonal Chemo-Optogenetic Tools.

A universal gain-of-function approach for the spatiotemporal control of protein activity is highly desirable when reconstituting biological modules in vitro. Here we used orthogonal translation with a photocaged amino acid to map and elucidate molecular mechanisms in the self-organization of the prokaryotic filamentous cell-division protein (FtsZ) that is highly relevant for the assembly of the division ring in bacteria. We masked a tyrosine residue of FtsZ by site-specific incorporation of a photocaged tyrosine analogue. While the mutant still shows self-assembly into filaments, dynamic self-organization into ring patterns can no longer be observed. UV-mediated uncaging revealed that tyrosine 222 is essential for the regulation of the protein's GTPase activity, self-organization, and treadmilling dynamics. Thus, the light-mediated assembly of functional protein modules appears to be a promising minimal-regulation strategy for building up molecular complexity towards a minimal cell.