Article

Reconstitution of Contractile FtsZ Rings in Liposomes

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710-3709, USA.
Science (Impact Factor: 33.61). 06/2008; 320(5877):792-4. DOI: 10.1126/science.1154520
Source: PubMed
ABSTRACT
FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which
contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted
FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated
into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions
along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the
liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly
and force generation.

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    • "As the natural load for actin-driven processes is biological membrane, researchers began to incorporate supported lipid bilayers and giant unilamellar vesicles in their reconstituted systems (Co, Wong, Gierke, Chang, & Taunton, 2007; Liu & Fletcher, 2006; Liu et al., 2008). Taking the first steps toward building true artificial cells, purified proteins and various cofactors were encapsulated into liposomes and used to investigate actin network formation and bacterial cell division (Jiménez, Martos, Vicente, & Rivas, 2011; Merkle, Kahya, & Schwille, 2008; Osawa, Anderson, & Erickson, 2008; Tsai, Stuhrmann, & Koenderink, 2011). With the advances made in cell-free protein expression over the years (Carlson, Gan, Hodgman, & Jewett, 2012), several groups have been able to encapsulate a bacterial cell-free expression system inside liposomes and demonstrated expression of proteins within the liposome (Tawfik & Griffiths, 1998; Yu et al., 2001). "
    [Show abstract] [Hide abstract] ABSTRACT: Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell-free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Dec 2015 · Methods in cell biology
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    • "The Z ring spans the circumference of the cell and serves as a scaffold upon which the division apparatus or divisome is assembled. The Z ring also provides a contractile force during cytokinesis that helps to 'pull in' the cell envelope12131415. This ultimately splits the parental cell compartment into two distinct newborn cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Fluctuations in nutrient availability are a fact of life for bacterial cells in the 'wild'. To survive and compete, bacteria must rapidly modulate cell-cycle processes to accommodate changing nutritional conditions and concomitant changes in cell growth. Our understanding of how this is achieved has been transformed in recent years, with cellular metabolism emerging as a central player. Several metabolic enzymes, in addition to their normal catalytic functions, have been shown to directly modulate cell-cycle processes in response to changing nutrient levels. Here we focus on cell division, the final event in the bacterial cell cycle, and discuss recent compelling evidence connecting division regulation to nutritional status and metabolic activity. Cellular metabolism has emerged as a key player in the nutritional regulation of bacterial division and other cell-cycle processes.A number of metabolic enzymes have been shown to directly influence division in response to nutrient status by modulating the activity of FtsZ, the master orchestrator of bacterial cytokinesis.The ability of metabolic enzymes to affect division is controlled at least in part by substrate levels, which serve as an intracellular reporter of nutrient availability.A diversity of metabolic regulatory mechanisms has been observed for division in different bacteria, hinting at evolution on a species-specific level.
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    • "The ESCRT-III Snf7 filament-mediated membrane remodeling is conceptually reminiscent of other membrane remodeling machinery, including bacterial FtsZ (Osawa et al., 2008). Interestingly, both Snf7 and FtsZ/FtsA can drive cytokinetic abscission, and they share at least three distinct structural characteristics: electrostatic protein-membrane interactions, membrane insertion of an amphipathic helix, and oligomeric protein scaffolding. "
    [Show abstract] [Hide abstract] ABSTRACT: The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling.
    Full-text · Article · Dec 2015 · eLife Sciences
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