Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SynDiv (A nanophysics approach to synthetic cell division)

Teaser

Imagine building a living cell from basic components, a vesicle filled with biomolecules that can sustain itself and reproduceinto similar offspring. Can this be done?This proposal addresses the most tantalizing aspect: synthetic cell division. We aim to build liposomes (lipid...

Summary

Imagine building a living cell from basic components, a vesicle filled with biomolecules that can sustain itself and reproduce
into similar offspring. Can this be done?
This proposal addresses the most tantalizing aspect: synthetic cell division. We aim to build liposomes (lipid vesicles
enclosing an aqueous solution with proteins and DNA) that can spontaneously divide through a contractile protein ring at
the vesicle perimeter. To realize this, we employ an experimental biophysics approach that addresses both the actual
division and the prerequisite spatial control, with:
1. Cells in nanofabricated shapes. We will study cell-division proteins and DNA in live E.coli bacteria that are molded into
user-defined arbitrary shapes and sizes. Clarifying the effects of cell shape will elucidate the guiding principles for the
spatiotemporal organization of the cell-division machinery.
2. Proteins and DNA in nanofabricated chambers. We will use a bottom up approach to study the basic divisome
components in vitro exploiting the full control provided by nanochambers. This will resolve the spatial organization of the
fascinating patterns of Min proteins and chromatin that dictate the localization of the division ring.
3. Liposomes on chip. We will develop a chip-based technology to generate liposomes for exploring synthetic cell division.
We will use both microfluidic constrictions and a biomimetic approach (encapsulation of divisome proteins such as FtsZ) to
induce autonomous liposome splitting, thus enabling a simplified but tightly controlled form of synthetic cell division.
To our knowledge, this nanofabrication-based approach to synthetic division is unique. We expect to be able to make
important contributions to understanding cell division, and anticipate that on a 5-year scale we indeed can master synthetic
division. We believe that our mix of nanophysics and synthetic biology is bound to yield deep insight into the biophysical
underpinnings of cellular reproduction.

We 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.

Work performed

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 the behavior of the Min system in fully confined three-dimensional chambers that are lithography-defined lipid-bilayer coated and 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 that are also observed on 2D surfaces. This work was published in Elife and other journals.

Furthermore, we made significant progress in understanding of 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 th

Final results

As planned, we have developed an innovative chip-based technology to generate liposomes for exploring synthetic cell division, which is superior to earlier techniques.

Furthermore, we have expanded the shape manipulation of bacterial cells using microfabricated structures and antobiotics.

The project is well underway and no change of course is foreseen for the second phase of the project.