Protein-free division of giant unilamellar vesicles controlled by enzymatic activity

Abstract

Cell division is one of the hallmarks of life. Success in the bottom-up assembly of synthetic cells will, no doubt, depend on strategies for the controlled autonomous division of protocellular compartments. Here, we describe the protein-free division of giant unilamellar lipid vesicles (GUVs) based on the combination of two physical principles -phase separation and osmosis. We visualize the division process with confocal fluorescence microscopy and derive a conceptual model based on the vesicle geometry. The model successfully predicts the shape transformations over time as well as the time point of the final pinching of the daughter vesicles. Remarkably, we show that two fundamentally distinct yet highly abundant processes -water evaporation and metabolic activity -can both regulate the autonomous division of GUVs. Our work may hint towards mechanisms that governed the division of protocells and adds to the strategic toolbox of bottom-up synthetic biology with its vision of bringing matter to life."Omni cellulae es cellulae." From the point of view of modern science, Raspail's realization from 1825 [1], popularized by Virchow [2], may state the obvious: Every living cell found on Earth today originates from a preexisting living cell. Bottom-up synthetic biology, however, is challenging this paradigm with the vision to create a synthetic cell from scratch [3,4]. Success unquestionably entails that the synthetic cells must have the capacity to produce offspring, making the implementation of synthetic cell division an exciting goal [5,6,7,8]. Over the course of evolution, living cells have developed a sophisticated machinery to divide their compartments in a highly regulated manner. The reconstitution of a minimal set of components from the procaryotic divisome seems to be a promising route towards synthetic cell division. FtsZ [9,6], ESCRT [10,11] and Min proteins [12] led to shape transformations in lipid vesicles, including budding [11] and the formation of tight constrictions [6]. However, reproducible minimal cell division based on common biological mechanisms has not yet been achieved. These challenges leave room for creative approaches, seeking solutions beyond the mimicry of today's biological cells. One exciting strategy is to assemble a division machinery de novo, by designing active, not necessarily protein-based nanomachines. DNA origami structures have been used to shape and remodel lipid vesicles [13,14,15], although active force-generating contractile motors remain a distant goal. A shortcut towards synthetic cell division is the mechanical division of liposomes, which was achieved with microfluidic splitters [16]. While this is not autonomous, it may jump start exciting new directions by circumventing a longstanding challenge. The exploitation of physico-chemical mechanisms, on the other hand, could lead to autonomous division. Noteworthy theoretical and experimental work describes the shape transformations of single-component [17,18,19] as well as phase-separated liposomes [20,21], determined by th...

Modelling the dynamics of vesicle reshaping and scission under osmotic shocks

“…Possible further applications of the approach taken here could thus be to study multiple endocytic events in fluctuating and heterogeneous environments, which could give rise to complex internal vesicle structures of different compositions. The model is also well suited for studying vesicle bursting cycles observed in hypotonic environments 24 or the different morphologies observed for multiphasic 8 and protein charged vesicles 11,19 , where the dynamical nature of MD simulations should prove useful.…”

“…In more evolved cells this process is usually very specifically driven by a set of proteins (such as actomyosin 2 , ESCRT-III 3,4 or FtsZ 5 ) composing the divisome machinery, responsible for exerting the required forces on the membrane. However, division of vesicles can also occur in spontaneous ways usually caused by environmental changes (shear stress 6 , osmotic shocks [7][8][9], compositional changes to the membrane (protein addition 10,11 ) or even motility of nascent cells 12 . † Electronic Supplementary Information (ESI) available:…”

“…It is thus known that hypertonic shocks (when the solute concentration is larger outside than inside the vesicle) induce strong deformations on GUVs with varying results depending on the specific composition and chemical properties of the membrane 9,20,21 . Division can thus occur when osmotic stress is coupled with phase separation in the lipid bilayer 7,8 . More important for cell compartmentalization and endocytosis, simple osmotic shocks can also drive inward budding of the membrane coupled with absorption of external medium into the newly created inner compartment 9,14,15,22,23 .…”

Modelling the dynamics of vesicle reshaping and scission under osmotic shocks

“…Possible further applications of the combination of the coarsegrained membrane model with explicit solute particles could be to study multiple encodytic events in fluctuating and heterogeneous environments, which could give rise to complex internal vesicle structures of different compositions. The model is also well suited for studying vesicle bursting cycles observed in hypotonic environments 22 or the different morphologies observed for multiphasic 8 and protein charged vesicles 11,19 , where the dynamical nature of MD simulations should prove useful.…”

“…In more evolved cells this process is usually very specifically driven by a set of proteins (such as actomyosin 2 , ESCRT-III 3,4 or FtsZ 5 ) composing the divisome machinery, responsible for exerting the required forces on the membrane. However, division of vesicles can also occur in spontaneous ways usually caused by environmental changes (shear stress 6 , osmotic shocks [7][8][9] ), compositional changes to the membrane (protein addition 10,11 ) or even motility of nascent cells 12 . a typical model system to study membrane reshaping events.…”