Yusuke T. Maeda

Development of an artificial cell, from self-organization to computation and self-reproduction

This article describes the state and the development of an artificial cell project. We discuss the experimental constraints to synthesize the most elementary cell-sized compartment that can self-reproduce using synthetic genetic information. The original idea was to program a phospholipid vesicle with DNA. Based on this idea, it was shown that in vitro gene expression could be carried out inside cell-sized synthetic vesicles. It was also shown that a couple of genes could be expressed for a few days inside the vesicles once the exchanges of nutrients with the outside environment were adequately introduced. The development of a cell-free transcription/translation toolbox allows the expression of a large number of genes with multiple transcription factors. As a result, the development of a synthetic DNA program is becoming one of the main hurdles. We discuss the various possibilities to enrich and to replicate this program. Defining a program for self-reproduction remains a difficult question as nongenetic processes, such as molecular self-organization, play an essential and complementary role. The synthesis of a stable compartment with an active interface, one of the critical bottlenecks in the synthesis of artificial cell, depends on the properties of phospholipid membranes. The problem of a self-replicating artificial cell is a long-lasting goal that might imply evolution experiments.

Ordered Patterns of Cell Shape and Orientational Correlation during Spontaneous Cell Migration

Background In the absence of stimuli, most motile eukaryotic cells move by spontaneously coordinating cell deformation with cell movement in the absence of stimuli. Yet little is known about how cells change their own shape and how cells coordinate the deformation and movement. Here, we investigated the mechanism of spontaneous cell migration by using computational analyses. Methodology We observed spontaneously migrating Dictyostelium cells in both a vegetative state (round cell shape and slow motion) and starved one (elongated cell shape and fast motion). We then extracted regular patterns of morphological dynamics and the pattern-dependent systematic coordination with filamentous actin (F-actin) and cell movement by statistical dynamic analyses. Conclusions/Significance We found that Dictyostelium cells in both vegetative and starved states commonly organize their own shape into three ordered patterns, elongation, rotation, and oscillation, in the absence of external stimuli. Further, cells inactivated for PI3-kinase (PI3K) and/or PTEN did not show ordered patterns due to the lack of spatial control in pseudopodial formation in both the vegetative and starved states. We also found that spontaneous polarization was achieved in starved cells by asymmetric localization of PTEN and F-actin. This breaking of the symmetry of protein localization maintained the leading edge and considerably enhanced the persistence of directed migration, and overall random exploration was ensured by switching among the different ordered patterns. Our findings suggest that Dictyostelium cells spontaneously create the ordered patterns of cell shape mediated by PI3K/PTEN/F-actin and control the direction of cell movement by coordination with these patterns even in the absence of external stimuli.

Assembly of MreB Filaments on Liposome Membranes: A Synthetic Biology Approach

The physical interaction between the cytoskeleton and the cell membrane is essential in defining the morphology of living organisms. In this study, we use a synthetic approach to polymerize bacterial MreB filaments inside phospholipid vesicles. When the proteins MreB and MreC are expressed inside the liposomes, the MreB cytoskeleton structure develops at the inner membrane. Furthermore, when purified MreB is used inside the liposomes, MreB filaments form a 4-10 μm rigid bundle structure and deform the lipid vesicles in physical contact with the vesicle inner membrane. These results indicate that the fibrillation of MreB filaments can take place either in close proximity of deformable lipid membrane or in the presence of associated protein. Our finding might be relevant for the self-assembly of cytoskeleton filaments toward the construction of artificial cell systems.

Insights into the micromechanical properties of the metaphase spindle

The microtubule-based metaphase spindle is subjected to forces that act in diverse orientations and over a wide range of timescales. Currently, we cannot explain how this dynamic structure generates and responds to forces while maintaining overall stability, as we have a poor understanding of its micromechanical properties. Here, we combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations to analyze the vertebrate metaphase spindles timescale- and orientation-dependent viscoelastic properties. We find that spindle viscosity depends on microtubule crosslinking and density. Spindle elasticity can be linked to kinetochore and nonkinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. These data suggest a quantitative model for the micromechanics of this cytoskeletal architecture and provide insight into how structural and functional stability is maintained in the face of forces, such as those that control spindle size and position, and can result from deformations associated with chromosome movement.

Dolichol-phosphate mannose synthase: Structure, function and regulation

Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and dolichol-phosphate-sugars, in which the sugar moiety is mannose or glucose. The sugar transfer from dolichol-phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of dolichol-phosphate-mannose, and particularly on the mammalian enzyme complex involved in the reaction.

Low-temperature molecular beam epitaxy of a ferromagnetic full-Heusler alloy Fe2MnSi on Ge(111)

We demonstrate the epitaxial growth of ferromagnetic full-Heusler alloy Fe2MnSi layers on group-IV semiconductor Ge(111) using molecular beam epitaxy at the growth temperatures of 130 and 200u2009°C. The Fe2MnSi/Ge(111) layers have an atomic-scale abrupt interface and include the ordered L21 phase. We also show ferromagnetic features with a saturation magnetization of ∼2.2u2002μB/f.u. and a Curie temperature of ∼210u2002K, which are nearly comparable to those of bulk Fe2MnSi.

Effects of long DNA folding and small RNA stem-loop in thermophoresis

In thermophoresis, with the fluid at rest, suspensions move along a gradient of temperature. In an aqueous solution, a PEG polymer suspension is depleted from the hot region and builds a concentration gradient. In this gradient, DNA polymers of different sizes can be separated. In this work the effect of the polymer structure for genomic DNA and small RNA is studied. For genome-size DNA, individual single T4 DNA is visualized and tracked in a PEG solution under a temperature gradient built by infrared laser focusing. We find that T4 DNA follows steps of depletion, ring-like localization, and accumulation patterns as the PEG volume fraction is increased. Furthermore, a coil–globule transition for DNA is observed for a large enough PEG volume fraction. This drastically affects the localization position of T4 DNA. In a similar experiment, with small RNA such as ribozymes we find that the stem–loop folding of such polymers has important consequences. The RNA polymers having a long and rigid stem accumulate, whereas a polymer with stem length less than 4 base pairs shows depletion. Such measurements emphasize the crucial contribution of the double-stranded parts of RNA for thermal separation and selection under a temperature gradient. Because huge temperature gradients are present around hydrothermal vents in the deep ocean seafloor, this process might be relevant, at the origin of life, in an RNA world hypothesis. Ribozymes could be selected from a pool of random sequences depending on the length of their stems.

Changes in the magnitude and distribution of forces at different Dictyostelium developmental stages.

The distribution of forces exerted by migrating Dictyostelium amebae at different developmental stages was measured using traction force microscopy. By using very soft polyacrylamide substrates with a high fluorescent bead density, we could measure stresses as small as 30 Pa. Remarkable differences exist both in term of the magnitude and distribution of forces in the course of development. In the vegetative state, cells present cyclic changes in term of speed and shape between an elongated form and a more rounded one. The forces are larger in this first state, especially when they are symmetrically distributed at the front and rear edge of the cell. Elongated vegetative cells can also present a front-rear asymmetric force distribution with the largest forces in the crescent-shaped rear of the cell (uropod). Pre-aggregating cells, once polarized, only present this last kind of asymmetric distribution with the largest forces in the uropod. Except for speed, no cycle is observed. Neither the force distribution of pre-aggregating cells nor their overall magnitude are modified during chemotaxis, the later being similar to the one of vegetative cells (F(0) approximately 6 nN). On the contrary, both the force distribution and overall magnitude is modified for the fast moving aggregating cells. In particular, these highly elongated cells exert lower forces (F(0) approximately 3 nN). The location of the largest forces in the various stages of the development is consistent with the myosin II localization described in the literature for Dictyostelium (Yumura et al.,1984. J Cell Biol 99:894-899) and is confirmed by preliminary experiments using a GFP-myosin Dictyostelium strain.

The Acidic Environment of the Golgi Is Critical for Glycosylation and Transport

Proteins and glycolipids are modified by various modes of glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. It is well known that the lumen of the Golgi is acidic and compromising acidification by chemical compounds causes impaired glycosylation and transport of proteins (Axelsson et al., 2001; Chapman and Munro, 1994; Palokangas et al., 1994; Presley et al., 1997; Puri et al., 2002; Reaves and Banting, 1994; Rivinoja et al., 2006; Tartakoff et al., 1978). The mechanisms by which glycosylation and transport are regulated by an acidic pH remain largely unknown. Recent findings that the impaired regulation of an acidic environment may be implicated in the pathology of several diseases emphasize the importance of pH regulation (Jentsch, 2007; Kasper et al., 2005; Kornak et al., 2001; Kornak et al., 2008; Piwon et al., 2000; Stobrawa et al., 2001; Teichgraber et al., 2008). We recently established a mutant cell line in which Golgi acidification was selectively impaired and the raised luminal Golgi pH caused impaired transport and glycosylation of proteins and altered Golgi morphology (Maeda et al., 2008). As alkalinizing compounds nonselectively affect all acidic organelles including lysosomes, endosomes, and the Golgi, the mutant cell is thought to be useful in analyzing how the acidic environment of the Golgi regulates glycosylation. In this chapter, we have introduced how we established mutant cells with impaired Golgi acidification and methods for measuring Golgi pH.

Directing and Boosting of Cell Migration by the Entropic Force Gradient in Polymer Solution

Noncontact manipulation of nano/micromaterials presents a great challenge in fields ranging from biotechnology to nanotechnology. In this study we developed a new strategy for the manipulation of molecules and cells based on diffusiophoresis driven by a concentration gradient of a polymer solute. By using laser focusing in a microfluidic device, we created a sharp concentration gradient of poly(ethylene glycol) (PEG) in a solution of this polymer. Because diffusiophoresis essentially depends on solute gradients alone, PEG solute contrast resulted in trapping of DNA and eukaryotic cells with little material dependence. Furthermore, quantitative analysis revealed that the motility of migrating cells was enhanced with the PEG concentration, consistent with a theoretical model of boosted cell migration. Our results support that a solute contrast of polymer can exert an interfacial force gradient that physically propels objects and may have application for the manipulation of soft materials.