Gene Expression within Cell‐Sized Lipid Vesicles

Nomura, Shin Ichiro M.; Tsumoto, K.; Hamada, Tsutomu; Akiyoshi, Kazunari; Nakatani, Yôichi; Yoshikawa, Kenichi

doi:10.1002/cbic.200300630

Cited by 304 publications

(251 citation statements)

References 19 publications

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“…In recent years, an increasing variety of materials have been encapsulated within lipid bilayer vesicles. For example, enzymatic reactions including replication and protein synthesis have been demonstrated within the aqueous interior of lipid vesicles (9)(10)(11)(12)(13)(14)(15). Cytoplasmic extracts captured within vesicles maintained enzymatic activity over several days when pore-forming proteins were incorporated in the bilayer (16).…”

mentioning

confidence: 99%

Dynamic microcompartmentation in synthetic cells

Long

Jones

Helfrich

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

230

271

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An experimental model for cytoplasmic organization is presented. We demonstrate dynamic control over protein distribution within synthetic cells comprising a lipid bilayer membrane surrounding an aqueous polymer solution. This polymer solution generally exists as two immiscible aqueous phases. Protein partitioning between these phases leads to microcompartmentation, or heterogeneous protein distribution within the ''cell'' interior. This model cytoplasm can be reversibly converted to a single phase by slight changes in temperature or osmolarity, such that local protein concentrations can be manipulated within the vesicle interior.aqueous phase separation ͉ intracellular organization ͉ vesicle T he interior of living cells is a crowded milieu of macromolecules, cytoskeletal filaments, and organelles. Even in cytoplasmic regions not separated by obvious barriers such as lipid membranes, differences in local composition are common. This phenomenon, referred to as microcompartmentation, is thought to have profound implications for cell function (1, 2). Understanding its role in living cells has been complicated by the lack of an experimental model system in which hypotheses could be tested. Even the mechanism(s) by which microcompartmentation is maintained remain unclear. Several possibilities have been proposed, including specific targeting and processes driven by macromolecular crowding, such as multiprotein complex formation, binding to intracellular surfaces, or phase separation (3). Aqueous phase separation occurs readily in bulk solutions of macromolecules even at much lower weight percents than are present in living cells (2). Thus, the question has been posed as to whether cytoplasm can exist without undergoing phase separation (4). Phase separation, and the accompanying partition of solutes between phases, could account for microcompartmentation of macromolecules, metabolites, and ions. Thus far, the complexity of living cells has precluded direct testing of the phase separation hypothesis. † We have encapsulated a poly(ethylene glycol) (PEG)͞dextran aqueous two-phase system (ATPS) within lipid vesicles to construct synthetic cells capable of dynamic protein and nucleic acid microcompartmentation. Substantial local variations in protein concentration can be maintained in the absence of intervening membranous barriers within these ATPS-containing vesicles. Our synthetic cytoplasm is promising as an experimental model for intracellular organization in general and demonstrates that aqueous phase separation is a viable mechanism for microcompartmentation.This work represents a bottom-up approach to understanding cell biology, in contrast to the top-down approach often adopted in biochemistry and perhaps best exemplified by efforts to generate the ''minimal cell'' through gene disruption in already simple organisms (6). Experimental model systems such as this one enable us to begin to test hypotheses in cell biology such as that of cytoplasmic phase separation. An analogy is lipid bilayer models of cell membrane...

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mentioning

confidence: 99%

Dynamic microcompartmentation in synthetic cells

Long

Jones

Helfrich

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

230

271

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“…By regulating the timely synthesis of the multiple proteins, the gene expression machinery is arguably at the heart of a programmable, genetically controlled, artificial cell. Cell-free protein synthesis inside liposomes has first been performed using cellular extracts derived from cytoplasmic parts of Escherichia coli bacteria (Nomura et al 2003;Noireaux and Libchaber 2004). Despite many advantages, including reduced costs (Jewett and Forster 2010), high yield of protein expression (Caschera and Noireaux 2014) and the possibility to use the endogenous bacterial RNA polymerases (Shin and Noireaux 2010), crude extracts are poorly characterized mixtures containing plenty of undesired components that can interfere with the intended reactions.…”

Section: Mimicking Cell Division In Its Most Simple Formmentioning

confidence: 99%

Toward the assembly of a minimal divisome

2014

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The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication.

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“…spatial separation, is essential to life for simple processes, such as the formation of chemical gradients or the protection of biomolecules from degradation. It also is a prerequisite for the appearance for well-documented emergent properties such as the link between a phenotype out of a genotype [4,5], or the arrangements of metabolic networks in a vectorial fashion [6]. Today, these boundary structures are formed primarily of complex lipids, which are an evolutionary product of living systems.…”

Section: Open Accessmentioning

confidence: 99%

Primitive Membrane Formation, Characteristics and Roles in the Emergent Properties of a Protocell

Maurer

Monnard

2011

Entropy

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All contemporary living cells are composed of a collection of self-assembled molecular elements that by themselves are non-living but through the creation of a network exhibit the emergent properties of self-maintenance, self-reproduction, and evolution. This short review deals with the on-going research that aims at either understanding how life emerged on the early Earth or creating artificial cells assembled from a collection of small chemicals. In particular, this article focuses on the work carried out to investigate how self-assembled compartments, such as amphiphile and lipid vesicles, contribute to the emergent properties as part of a greater system.

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Gene Expression within Cell‐Sized Lipid Vesicles

Cited by 304 publications

References 19 publications

Dynamic microcompartmentation in synthetic cells

Dynamic microcompartmentation in synthetic cells

Toward the assembly of a minimal divisome

Primitive Membrane Formation, Characteristics and Roles in the Emergent Properties of a Protocell

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