Abstract: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 alig… Show more
“…Having a detector that does not rely on vesicle shape is valuable, as shape control and GUV deformation are essential aspects of synthetic cell engineering. 9, 40, 73, 74 We demonstrate the use of FF on GUVs deformed by encapsulated stiff actin bundles (fig. 3C).…”
Section: Resultsmentioning
confidence: 89%
“…In this way, membrane deformation by fluores- cent structures can be quantified in an automated way for vesicle populations, enabling an accessible and quantitative approach in GUV deformation studies. Besides its use in actinmediated GUV deformation studies, 40,44,[77][78][79] this analysis is also valuable in other studies on global vesicle shape deformation, for example by other proteins involved in cytokinesis such as the bacterial division proteins FtsZ 80 and Min system, 73 by other membrane-binding proteins, 9 DNA origami, 81 by microfluidic traps, 74,82 or by spontaneous membrane fluctuations. 83 Furthermore, RMD could be applied to characterize local membrane deformations, such as protrusions 84 or nanotubes.…”
Giant Unilamellar Vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis 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 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.
“…Having a detector that does not rely on vesicle shape is valuable, as shape control and GUV deformation are essential aspects of synthetic cell engineering. 9, 40, 73, 74 We demonstrate the use of FF on GUVs deformed by encapsulated stiff actin bundles (fig. 3C).…”
Section: Resultsmentioning
confidence: 89%
“…In this way, membrane deformation by fluores- cent structures can be quantified in an automated way for vesicle populations, enabling an accessible and quantitative approach in GUV deformation studies. Besides its use in actinmediated GUV deformation studies, 40,44,[77][78][79] this analysis is also valuable in other studies on global vesicle shape deformation, for example by other proteins involved in cytokinesis such as the bacterial division proteins FtsZ 80 and Min system, 73 by other membrane-binding proteins, 9 DNA origami, 81 by microfluidic traps, 74,82 or by spontaneous membrane fluctuations. 83 Furthermore, RMD could be applied to characterize local membrane deformations, such as protrusions 84 or nanotubes.…”
Giant Unilamellar Vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis 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 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.
“…Deformation of the membrane was also achieved by de novo synthesis of FtsZ and ZipA in GUVs, but not with the FtsZ-mts chimera [ 115 ]. Finally, lipid vesicles produced by manual emulsion were deformed into rod-shaped containers by trapping them in microfluidic devices, allowing visualization studies of FtsZ-mts filament arrangement in bacterial cell-like geometries [ 142 ].…”
Section: Reconstruction Of Cellular Ftsz Subsystemsmentioning
FtsZ is an essential and central protein for cell division in most bacteria. Because of its ability to organize into dynamic polymers at the cell membrane and recruit other protein partners to form a “divisome”, FtsZ is a leading target in the quest for new antibacterial compounds. Strategies to potentially arrest the essential and tightly regulated cell division process include perturbing FtsZ’s ability to interact with itself and other divisome proteins. Here, we discuss the available methodologies to screen for and characterize those interactions. In addition to assays that measure protein-ligand interactions in solution, we also discuss the use of minimal membrane systems and cell-like compartments to better approximate the native bacterial cell environment and hence provide a more accurate assessment of a candidate compound’s potential in vivo effect. We particularly focus on ways to measure and inhibit under-explored interactions between FtsZ and partner proteins. Finally, we discuss recent evidence that FtsZ forms biomolecular condensates in vitro, and the potential implications of these assemblies in bacterial resistance to antibiotic treatment.
“…For instance, due to the exceptionally low surface-tovolume ratio within microdroplets, spontaneous localization of MinE has to be modulated for a stable wave emergence 21,22 . In and on vesicles, FtsZ was able to deform membrane surfaces [23][24][25][26] and dynamically reorganize dependent on membrane shape and anchoring features 26,27 .…”
Section: Introductionmentioning
confidence: 99%
“…Despite the importance of the interactions between the Min proteins and FtsZ for proper FtsZ-ring placement, the co-reconstitution of these proteins, especially in con ned spaces, has rarely been accomplished so far [28][29][30][31] . This di culty is mainly due to a large number of components and environmental factors to be controlled and their intrinsic complex dynamics that are still not fully understood 27,32 .…”
Constructing a minimal machinery for autonomous self-division of synthetic cells is a major goal of bottom-up synthetic biology. One paradigm has been the E. coli divisome, with the MinCDE protein system guiding assembly and positioning of a presumably contractile ring based on FtsZ and its membrane adaptor FtsA. Here, we demonstrate the full in vitro reconstitution of this machinery consisting of five proteins within lipid vesicles, allowing to observe the following sequence of events in real time: 1) Assembly of an isotropic filamentous FtsZ network, 2) its condensation into a ring-like structure, along with pole-to-pole mode selection of Min oscillations resulting in equatorial positioning, and 3) onset of ring constriction, deforming the vesicles from spherical shape. Besides demonstrating these essential features, we highlight the importance of decisive experimental factors, such as macromolecular crowding. Our results provide an exceptional showcase of the emergence of cell division in a minimal system, and may represent a major breakthrough towards developing a synthetic cell.
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