1: Delft University of Technology, the Netherlands; 2: University of Potsdam, Germany
Giant Unilamellar Vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs are an excellent chassis for endeavours to build a synthetic cell from the bottom up. While GUV experimentation has progressed substantially in the past decades, quantitative analysis of their images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, thereby making characterization time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVery’s three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple pre-defined analysis modules allow the user to extract general properties such as membrane fluorescence, vesicle shape and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of non-spherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in GUV research.
Max Planck Institute for Medical Research Heidelberg, Germany
Many fundamental cellular processes, including cell division, are highly dependent on the cell shape. Success in the bottom-up assembly of synthetic cells will, no doubt, depend on strategies to manipulate vesicle shapes in a controlled manner.
Here, we demonstrate mechanisms to engineer fundamental membrane properties to achieve controlled division of giant unilamellar lipid vesicles (GUVs) and induce morphological changes.
For GUV division, we rely on the line tension of phase-separated GUVs and a surface-to-volume ratio increase via osmosis to achieve controlled division of GUVs. We derive a conceptual model based on the vesicle geometry which makes four quantitative predictions that we verify experimentally.
Alternatively, we achieve GUV division based on osmotic deflation and an increase of the membrane spontaneous curvature. To controllably increase the spontaneous curvature, we use the photosensitizer chlorine e6 which self-assembles into lipid bilayers and leads to lipid peroxidation upon illumination.
Last but not least, we use DNA origami to restructure liquid-phased lipid membranes and locally induce the formation of gel-like lipid rafts. The latter lead to characteristic vesicle shapes observed for gel-phased lipid vesicles.
Overall, our work provides broadly applicable mechanisms to manipulate vesicle shapes and achieve controlled division of synthetic cell compartments and adds to the strategic toolbox of bottom-up synthetic biology.
1: TU Munich, Germany; 2: Northwestern University, United States of America; 3: Brandeis University, United States of America; 4: UC Santa Barbara, United States of America
The first thing cells need to do is to separate themselves from their environment through their membrane. At the same time however, in order to move, adapt or divide, cells need to be able to deform. This is accomplished by coupling the cell membrane to the cytoskeleton, a set of active polymers that not only act as a scaffold for the cells but, being able to self-organize and exert forces, also allow for dynamical shape changes. To study this interplay, we confine a microtubule-based active fluid, propelled by kinesins and fueled by ATP, inside giant unilamellar vesicles. This results in large, active shape deformations of the GUV’s membrane induced by microtubules pushing on the membrane. At the same time, the presence of a flexible boundary, affects the self-organization of the microtubules themselves. While the effect of hard confinement in active cytoskeletal system is starting to be understood, how they react to the presence of a soft, deformable boundary is still unclear. By studying not only the membrane’s fluctuation spectra, both in its spatial and temporal behavior, but also the 3D self-organization of the active fluid we establish direct links between the two. This allows to understand the intricate interplay between an active, cytoskeleton-like confined system and the plastic, cell-like confinement itself. Moreover, by using simulations of a deformable membrane containing active filaments, we establish a connection between the membrane’s deformability and the spatial self-organization of the active fluid. These results can pave the way both to the understanding of the extreme mechanical deformability of living cells and to the construction of self-propelled, cell-like, soft microrobots.
1: CEA/CNRS/Université Grenoble Alpes, France; 2: University of Helsinki, Institute of Biotechnology, Finland; 3: Courant Institute, New York University, United States of America
Cells constantly experience environmental changes requiring a fast adaptation of their structures in order to modify shape or type of movement for example. In particular, fast turnover of actin is of a primary importance in order to assemble and disassemble networks with various architectures. However, the basic principles of actin turnover are still largely unknown. In this study, we used a combination of purified proteins and cell-sized confinements to reconstitute actin turnover in vitro.
To reconstitute actin assembly, we coated micron-sized beads with an activator of Arp2/3 complex. In the presence of actin, profilin, Arp2/3 and capping protein, the beads move by assembling actin comet tails. In order to mimic the cellular environment, we set-up microwells with a volume of 160 pL and observable with fluorescence microscopy. We introduced the beads in those confined environments, allowing us to study the influence of a limited pool of proteins on actin turnover.
We first demonstrated that the comet assembly is quickly limited over time in those confined environments compared to bulk assembly. In the presence of disassembly (ADF/cofilin) and recycling factors (cyclase associated protein), we obtained structures in a dynamic steady state. The actin comets in this dynamic steady state were characterized by length and velocity maintained constant for several hours. This indicates that in those conditions, actin filaments are simultaneously polymerized and depolymerized at the same rate, leading to a continuous turnover of actin filaments and then to a dynamic steady state. To our knowledge, this is the first reconstitution of steady state dynamics on these time scales.
Delft University of Technology, The Netherlands
The design of a synthetic cell division mechanism inspired from animal cell division requires precise tuning of the crosstalk between the cell membrane, the actin cytoskeleton, and microtubules. An interesting candidate to mediate this crosstalk is the septin family of proteins. Septins, the recently acknowledged fourth component of the cytoskeleton, are a conserved family of filament–forming proteins that participate in many cellular functions such as cell polarization, cell migration, and cell division. They do so by interacting with many partners, such as the cell membrane, actin filaments, and microtubules. Despite their importance in cells, there has been not much research into the biochemistry and biophysics of septin’s cellular interactions. I will explain how we study these basic properties via cell-free in vitro reconstitution with the aim to design a method of synthetic cell division. We learned that human septins are recruited to lipid membranes by negatively charged lipids and that they need phosphatidylinositol 4,5-bisphosphate in order to properly bind and form a thin meshwork that stabilizes the membrane. Septins also bind and bundle actin filaments and protect these bundles from myosin activity. Additionally, they can also act as a membrane anchor to bind actin to a membrane, forming a cortex mimic. Finally, septin filaments interact with microtubules and modulate microtubule dynamics. We hence propose septin as a versatile protein that can link the cytoskeleton with the membrane to regulate synthetic cytokinesis.
References: 10.1242/jcs.258850 & 10.7554/ELIFE.63349 & 10.1242/JCS.258484 & 10.1101/2022.02.23.481653
1: Max Planck Institute for Medical Research, Germany; 2: Stuttgart University, Germany
The development and bottom-up assembly of synthetic cells with a functional cytoskeleton sets a major milestone to understand cell mechanics and to develop man-made cellular machines.
However, the combination of multiple elements and functions remained elusive, which stimulates endeavors to explore entirely synthetic bio-inspired and rationally designed solutions towards engineering life. To this end, DNA nanotechnology represents one of the most promising routes, given the inherent sequence speciﬁcity, addressability, and programmability of DNA. Here, we demonstrate functional DNA-based cytoskeletons operating in microfluidic cell-sized compartments and lipid vesicles. The synthetic cytoskeletons consist of either stimuli-responsive DNA origami (Jahnke et al., Nat. Commun. 2021) or DNA tiles self-assembled into ﬁlament networks (Zhan*, Jahnke* et al., accepted in Nat. Chem.; Jahnke et al., under review in ACS Nano). These synthetic cytoskeletons can be rationally designed and controlled to imitate features of natural cytoskeletons, including dynamic instability, ATP-triggered polymerization, morphology control and vesicle transport in cell-sized conﬁnement. Also, they possess engineerable characteristics, including assembly and disassembly powered by DNA hybridization, light or aptamer-target interactions and autonomous transport of gold nanoparticles. This work underpins DNA nanotechnology as a key player in building synthetic cells from the bottom up.