Intelligent Image-Activated Cell Sorting and Beyond

We present a groundbreaking machine intelligence technology called “intelligent image-activated cell sorting” that achieves high-throughput image-triggered sorting of single cells by integrating high-speed fluorescence microscopy, cell focusing, cell sorting, and deep learning

Intelligent Image-Activated Cell Sorting and Beyond

We present a groundbreaking machine intelligence technology called “intelligent image-activated cell sorting” that achieves high-throughput image-triggered sorting of single cells by integrating high-speed fluorescence microscopy, cell focusing, cell sorting, and deep learning

SINC-seq: correlation of transient gene expressions between nucleus and cytoplasm reflects single-cell physiology

We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level

Single-molecule imaging of full protein synthesis by immobilized ribosomes

How folding of proteins is coupled to their synthesis remains poorly understood. Here, we apply single-molecule fluorescence imaging to full protein synthesis in vitro. Ribosomes were specifically immobilized onto glass surfaces and synthesis of green fluorescent protein (GFP) was achieved using modified commercial Protein Synthesis using Recombinant Elements that lacked ribosomes but contained purified factors and enzyme that are required for translation in Escherichia coli. Translation was monitored using a GFP mutant (F64L/S65T/F99S/M153T/V163A) that has a high fluorophore maturation rate and that contained the Secretion Monitor arrest sequence to prevent dissociation from the ribosome. Immobilized ribosomal subunits were labeled with Cy3 and GFP synthesis was measured by colocalization of GFP fluorescence with the ribosome position. The rate of appearance of colocalized ribosome GFP was equivalent to the rates of fluorescence appearance coupled with translation measured in bulk, and the ribosome–polypeptide complexes were stable for hours. The methods presented here are applicable to single-molecule investigation of translational initiation, elongation and cotranslational folding

Equilibrium and Transition between Single- and Double-Headed Binding of Kinesin as Revealed by Single-Molecule Mechanics

Kinesin is a processive motor protein that “walks” on a microtubule toward its plus end. We reported previously that the distribution of unbinding force and elastic modulus for a single kinesin-microtubule complex was either unimodal or bimodal depending on the nucleotide states of the kinesin heads, hence showing that the kinesin may bind the microtubule either with one head or with both heads at once. Here, we found that the shape of the unbinding-force distribution depends both on the loading rate and on the manner of loading not only in the presence of AMP-PNP but also in the absence of nucleotides. Irrespective of the nucleotide state and the loading conditions examined here, the unbinding force obtained by loading directed toward the minus end of microtubule was 45% greater than that for plus end-directed loading. These results could be explained by a model in which equilibrium exists between single- and double-headed binding and the load (F) dependence of lifetime, τ(F), of each binding is expressed by τ(F) = τ(0)exp(−Fd/k(B)T), where τ(0) is the lifetime without external load and d a characteristic distance, both of which depend on single- or double-headed binding, k(B), the Boltzmann constant and T, the absolute temperature. The model analysis showed that the forward and backward rates of transition from single- to double-headed binding are 2 and 0.2/s for the AMP-PNP state, and 70 and 7/s for the nucleotide-free state. Moreover, in the presence of AMP-PNP, we detected the moment of transition from single- to double-headed binding through an abrupt increase in the elastic modulus and estimated the transition rate to be ∼1/s, which is consistent with the model analysis

Accumulation of TERT in mitochondria exerts two opposing effects on apoptosis

Telomerase reverse transcriptase (TERT) is a protein that catalyzes the reverse transcription of telomere elongation. TERT is also expected to play a non‐canonical role beyond telomere lengthening since it localizes not only in the nucleus but also in mitochondria, where telomeres do not exist. Several studies have reported that mitochondrial TERT regulates apoptosis induced by oxidative stress. However, there is still some controversy as to whether mitochondrial TERT promotes or inhibits apoptosis, mainly due to the lack of information on changes in TERT distribution in individual cells over time. Here, we simultaneously detected apoptosis and TERT localization after oxidative stress in individual HeLa cells by live‐cell tracking. Single‐cell tracking revealed that the stress‐induced accumulation of TERT in mitochondria caused apoptosis, but that accumulation increased over time until cell death. The results suggest a new model in which mitochondrial TERT has two opposing effects at different stages of apoptosis: it predetermines apoptosis at the first stage of cell‐fate determination, but also delays apoptosis at the second stage. As such, our data support a model that integrates the two opposing hypotheses on mitochondrial TERT's effect on apoptosis. Furthermore, detailed statistical analysis of TERT mutations, which have been predicted to inhibit TERT transport to mitochondria, revealed that these mutations suppress apoptosis independent of mitochondrial localization of TERT. Together, these results imply that the non‐canonical functions of TERT affect a wide range of mitochondria‐dependent and mitochondria‐independent apoptosis pathways

SINC-seq: correlation of transient gene expressions between nucleus and cytoplasm reflects single-cell physiology

1細胞RNA分画解読法の開発に成功 --細胞生物学の研究を加速--. 京都大学プレスリリース. 2018-06-07.We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level