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A nanophysics approach to synthetic cell division

Periodic Reporting for period 4 - SynDiv (A nanophysics approach to synthetic cell division)

Reporting period: 2020-01-01 to 2020-06-30

This project was aimed at building liposomes (lipid vesicles enclosing an aqueous solution with proteins and DNA) that can divide through a contractile protein ring at the vesicle perimeter. To realize this, we employed an experimental biophysics approach that addresses both the actual division and the prerequisite spatial control. This project made important contributions to understanding cell division, and helped to estimate the feasibility of synthetic division.
We indeed have made significant progress towards the goals stated in the project proposal. We have succeeded in studying cell-division proteins and DNA in live E.coli bacteria that were molded into user-defined arbitrary shapes and sizes. Significant results were obtained, as we resolved the treadmilling dynamics of the FtsZ filaments in the divisome of cells, as well as realized the first-ever imaging of the circular genome of bacteria. We used a bottom up approach for Min proteins in vitro in nanochambers, yielding the first study in the spatial organization of the fascinating Turing patterns of Min proteins in full spatial confinement. We have developed an innovative chip-based technology to generate liposomes for exploring synthetic cell division and managed to obtain a range of results on biophysical manipulation including mechanical division of liposomes.
A significant number of scientific results were obtained, in line with the research proposal. We studied, both in vivo and in vitro, how the Min protein spatial regulators of the divisome function. In vivo, we studied the emergence, stability, and state transitions of multistable Min protein oscillation patterns in live Escherichia coli bacteria during growth up to defined large dimensions in microfabricated molds. Transitions between multistable Min patterns are found to be rare events induced by strong intracellular perturbations. In vitro, we realized the first study of behavior of the Min system in fully confined three-dimensional lithography-defined lipid-bilayer coated chambers, isolated through soft-lithography pressure valves. We identified three typical dynamical behaviors that occur dependent on the geometrical parameters of the chambers: pole-to-pole oscillations, spiral rotations, and running waves. We established the geometrical selection rules and showed that the larger part of the geometrical phase diagram is governed by Min-protein rotations, a manifestation of the Min spirals also observed on 2D surfaces. This work was published in Elife and other journals. Furthermore, we made significant progress in understanding the spatial organization of DNA in confinement. We made important discoveries regarding SMC proteins that play a key role in chromosome organization and compaction. We used single-molecule imaging to demonstrate that yeast condensin is a molecular motor capable of ATP hydrolysis-dependent translocation along double-stranded DNA. Our results suggested that condensin takes steps comparable in length to its ~50-nanometer coiled-coil subunits, suggestive of a translocation mechanism that is distinct from any reported DNA motor protein. The finding that condensin is a mechanochemical motor provided important evidence for a DNA loop extrusion model. This work was published in Science. Recently, we resolved, for the first time, the spatial organization of the circular chromosome of bacteria by directly imaging the chromosome in live E. coli cells with a broadened cell shape. The chromosome was observed to open up into a ring-like torus topology, where, strikingly, we observed an intriguing heterogeneous DNA domain structure that was unanticipated from any earlier studies. We even managed to obtain movies that show that these Mbp-size domains undergo major dynamic rearrangements, splitting and merging at a minute timescale. We discovered that bacterial cytokinesis is controlled by the circumferential treadmilling of FtsAZ filaments that drives the insertion of new cell wall. The mechanism by which bacteria divide is still incompletely understood. Cell division is mediated by filaments of FtsZ that recruit septal peptidoglycan synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsZ filaments treadmilled circumferentially around the division ring, driving the motions of the peptidoglycan synthesizing enzymes. This work was published in Science. We developed synthetic cells through an innovative new technique to create liposomes on chip. We managed to realize mechanical division of liposomes. We started studies of the FtsZ- and ESCRT-based divisome in these synthetic cells. We developed a novel microfluidics-based method, octanol-assisted liposome assembly, to form monodisperse, cell-sized (5–20 µm), unilamellar liposomes with excellent encapsulation efficiency. Akin to bubble blowing, an inner aqueous phase and a surrounding lipid-carrying 1-octanol phase was pinched off by outer fluid streams. Such hydrodynamic flow focusing resulted in double-emulsion droplets that spontaneously develop a side-connected 1-octanol pocket that splitted off to yield fully assembled solvent-free liposomes within minutes. This solved the long-standing fundamental problem of prolonged presence of residual oil in the liposome bilayer. The technique offers a versatile platform for future analytical tools, delivery systems, nanoreactors, and synthetic cells. This work was published in Nature Communications. Indeed, we are currently underway with studying various divisome components in these synthetic cells systems.