De novo vesicle formation and growth: an integrative approach to artificial cells.

The assembly of artificial cells provides a novel strategy to reconstruct life's functions and shed light on how life emerged on Earth and possibly elsewhere. A major challenge to the development of artificial cells is the establishment of simple methodologies to mimic native membrane generation. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of highly ordered membrane architectures with defined functionality. This perspective will cover recent advances and the current state-of-the-art of minimal lipid architectures that can faithfully reconstruct the structure and function of living cells. Specifically, we will overview work related to the de novo formation and growth of biomimetic membranes. These studies give us a deeper understanding of the nature of living systems and bring new insights into the origin of cellular life

A minimal biochemical route towards de novo formation of synthetic phospholipid membranes.

All living cells consist of membrane compartments, which are mainly composed of phospholipids. Phospholipid synthesis is catalyzed by membrane-bound enzymes, which themselves require pre-existing membranes for function. Thus, the principle of membrane continuity creates a paradox when considering how the first biochemical membrane-synthesis machinery arose and has hampered efforts to develop simplified pathways for membrane generation in synthetic cells. Here, we develop a high-yielding strategy for de novo formation and growth of phospholipid membranes by repurposing a soluble enzyme FadD10 to form fatty acyl adenylates that react with amine-functionalized lysolipids to form phospholipids. Continuous supply of fresh precursors needed for lipid synthesis enables the growth of vesicles encapsulating FadD10. Using a minimal transcription/translation system, phospholipid vesicles are generated de novo in the presence of DNA encoding FadD10. Our findings suggest that alternate chemistries can produce and maintain synthetic phospholipid membranes and provides a strategy for generating membrane-based materials

Applications of Lipid Assemblies in Artificial Cell Development

It is of great fundamental interest to develop experimental model systems to reconstruct design principles underlying formation of living cells and shed light on how life emerged on Earth and possibly elsewhere. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of basic building blocks with defined functionality to construct a life-like entity. This dissertation will explore the roles of miscellaneous lipid assemblies as building blocks of artificial cellular compartments. The first part (Chapters 2 and 3) of this dissertation shows how lipid environments facilitate chemoselective reactions between amphiphilic partners through physical partitioning. Through examples of histidine ligation, and acyl phosphate chemistry we show functionalized micelle-forming amphiphiles react to form amidophospholipids which self-assemble into micron-sized vesicles.The second part (Chapter 3) of the dissertation seeks to understand the origins of cellular phospholipid synthesis pathways by repurposing soluble fatty acid activating enzymes (fatty acyl adenylate ligase and fatty acyl CoA ligase) to synthesize phospholipids. Such simplified biochemical pathways may provide a hint at how lipids were likely synthesized in a minimal protocell without the necessity of transmembrane protein enzymes. The third part (Chapters 4 and 5) of the dissertation discusses the vesicle formation from a novel class of single-chain amphiphiles derived from galactopyranosyl head groups and unsaturated fatty acid tails. The geometric parameters and thermodynamic properties of these amphiphilic assemblies are characterized by a host of physical techniques. The vesicles are further shown to sustain model biochemical reactions.The final part (Chapters 6) of the dissertation describes the formation of sponge phase droplets from fatty acyl galactopyranosylamides and non-ionic detergents. The droplets contain a dense bicontinuous network of bilayers and nanometric aqueous channels, which facilitates molecules to partition into them based on their size, polarity, and specific binding motifs. The sequestration of biomolecules can be programmed by doping the droplets with suitably functionalized amphiphiles. The droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled

Applications of Lipid Assemblies in Artificial Cell Development

It is of great fundamental interest to develop experimental model systems to reconstruct design principles underlying formation of living cells and shed light on how life emerged on Earth and possibly elsewhere. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of basic building blocks with defined functionality to construct a life-like entity. This dissertation will explore the roles of miscellaneous lipid assemblies as building blocks of artificial cellular compartments. The first part (Chapters 2 and 3) of this dissertation shows how lipid environments facilitate chemoselective reactions between amphiphilic partners through physical partitioning. Through examples of histidine ligation, and acyl phosphate chemistry we show functionalized micelle-forming amphiphiles react to form amidophospholipids which self-assemble into micron-sized vesicles.The second part (Chapter 3) of the dissertation seeks to understand the origins of cellular phospholipid synthesis pathways by repurposing soluble fatty acid activating enzymes (fatty acyl adenylate ligase and fatty acyl CoA ligase) to synthesize phospholipids. Such simplified biochemical pathways may provide a hint at how lipids were likely synthesized in a minimal protocell without the necessity of transmembrane protein enzymes. The third part (Chapters 4 and 5) of the dissertation discusses the vesicle formation from a novel class of single-chain amphiphiles derived from galactopyranosyl head groups and unsaturated fatty acid tails. The geometric parameters and thermodynamic properties of these amphiphilic assemblies are characterized by a host of physical techniques. The vesicles are further shown to sustain model biochemical reactions.The final part (Chapters 6) of the dissertation describes the formation of sponge phase droplets from fatty acyl galactopyranosylamides and non-ionic detergents. The droplets contain a dense bicontinuous network of bilayers and nanometric aqueous channels, which facilitates molecules to partition into them based on their size, polarity, and specific binding motifs. The sequestration of biomolecules can be programmed by doping the droplets with suitably functionalized amphiphiles. The droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled

Applications of Lipid Assemblies in Artificial Cell Development

It is of great fundamental interest to develop experimental model systems to reconstruct design principles underlying formation of living cells and shed light on how life emerged on Earth and possibly elsewhere. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of basic building blocks with defined functionality to construct a life-like entity. This dissertation will explore the roles of miscellaneous lipid assemblies as building blocks of artificial cellular compartments. The first part (Chapters 2 and 3) of this dissertation shows how lipid environments facilitate chemoselective reactions between amphiphilic partners through physical partitioning. Through examples of histidine ligation, and acyl phosphate chemistry we show functionalized micelle-forming amphiphiles react to form amidophospholipids which self-assemble into micron-sized vesicles.The second part (Chapter 3) of the dissertation seeks to understand the origins of cellular phospholipid synthesis pathways by repurposing soluble fatty acid activating enzymes (fatty acyl adenylate ligase and fatty acyl CoA ligase) to synthesize phospholipids. Such simplified biochemical pathways may provide a hint at how lipids were likely synthesized in a minimal protocell without the necessity of transmembrane protein enzymes. The third part (Chapters 4 and 5) of the dissertation discusses the vesicle formation from a novel class of single-chain amphiphiles derived from galactopyranosyl head groups and unsaturated fatty acid tails. The geometric parameters and thermodynamic properties of these amphiphilic assemblies are characterized by a host of physical techniques. The vesicles are further shown to sustain model biochemical reactions.The final part (Chapters 6) of the dissertation describes the formation of sponge phase droplets from fatty acyl galactopyranosylamides and non-ionic detergents. The droplets contain a dense bicontinuous network of bilayers and nanometric aqueous channels, which facilitates molecules to partition into them based on their size, polarity, and specific binding motifs. The sequestration of biomolecules can be programmed by doping the droplets with suitably functionalized amphiphiles. The droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition, however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display compositional variability on the order of 5-15 mol percent

De novo vesicle formation and growth: an integrative approach to artificial cells.

The assembly of artificial cells provides a novel strategy to reconstruct life's functions and shed light on how life emerged on Earth and possibly elsewhere. A major challenge to the development of artificial cells is the establishment of simple methodologies to mimic native membrane generation. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of highly ordered membrane architectures with defined functionality. This perspective will cover recent advances and the current state-of-the-art of minimal lipid architectures that can faithfully reconstruct the structure and function of living cells. Specifically, we will overview work related to the de novo formation and growth of biomimetic membranes. These studies give us a deeper understanding of the nature of living systems and bring new insights into the origin of cellular life