Recently, there is growing evidence in the field of cell biology that membrane-less organelles or condensates are formed by liquid-liquid phase separation (LLPS), as well as some types of membrane-bound organelles like lipid rafts and lipid droplets. Biophysical analyses are powerful and indispensable to elucidate those structures, dynamics and (dys)functions. Here, we combine in vivo intracellular imaging with in vitro synthetic biology and soft-matter physics, to show a new avenue for understanding the biophysics of LLPS-driven organelles. In this review, we particularly discuss macro-to-micro phase separation in lipid rafts and liquid-liquid crystal phase transition in lipid droplets.
Snakes have lost their limbs and acquired the ability to move in various environments by using a simple elongated body structure through long-term evolutionary process. Specifically, snakes have various locomotion patterns (scaffold-based locomotion, concertina locomotion etc.) and change them in response to the environment. This ability is likely achieved by a decentralized control mechanism, yet it is still largely unknown. We have attempted to elucidate the decentralized control mechanism through a synthetic approach, an approach to unveil the mechanism through behavioral experiments, mathematical modeling, and robot experiments, for ten years. Here we review our works including some future scopes.
Cytochrome c oxidase in the mitochondrial respiratory chain mostly exists as an independent monomer, or as a monomer associated with the complexes I and III in the supercomplex, although its molecular mechanism has been elucidated based on the crystal structure of its dimeric form. Here, we report detailed comparison of the activity and crystal structure between the monomer and the dimer, and the possibility that the monomer is an activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane.
The TOM complex is the main entry gate for mitochondrial proteins. Recently we determined the Cryo-EM structure of the yeast TOM complex at 3.8 Å resolution. The structure shows the dimeric form consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits. The structure-based biochemical analysis revealed that presequence-containing preproteins pass through the Tom40 channel in the middle of the dimer and are transferred to the TIM23 complex. On the other hand, presequence-less preproteins leave the channel at the periphery of the TOM complex and are relayed to the chaperone proteins. Our results demonstrate the efficient transfer mechanism of preproteins.
In vitro reconstitution of cellular events is a promising strategy as a bottom-up approach to build an artificial cell in the field of synthetic biology. Also, the reconstituted system sometimes provides a useful biotechnology tool for various fields. We recently reconstituted a whole replication cycle of the Escherichia coli chromosome in vitro as a part of the bottom-up synthetic biology. This replication cycle reaction (RCR) propagates circular DNA exponentially and precisely by autonomous repetition of the cycle in an isothermal reaction. Even genome-sized DNA (1 Mbp) can be propagated as circular DNA in RCR. We also developed a DNA assembly tool. The combination of the DNA assembly and RCR amplification enables cell-free synthesis of artificial large circular DNA, providing a powerful tool for a top-down study of the synthetic biology, in which living cells are modified at a whole genome level.