Recent methodological advances towards single-cell proteomics.

Studying the central dogma at the single-cell level has gained increasing attention to reveal hidden cell lineages and functions that cannot be studied using traditional bulk analyses. Nonetheless, most single-cell studies exploiting genomic and transcriptomic levels fail to address information on proteins that are central to many important biological processes. Single-cell proteomics enables understanding of the functional status of individual cells and is particularly crucial when the specimen is composed of heterogeneous entities of cells. With the growing importance of this field, significant methodological advancements have emerged recently. These include miniaturized and automated sample preparation, multi-omics analyses, and combined analyses of multiple techniques such as mass spectrometry and microscopy. Moreover, artificial intelligence and single-molecule detection technologies have advanced throughput and improved sensitivity limitations, respectively, over conventional methods. In this review, we summarize cutting-edge methodologies for single-cell proteomics and relevant emerging technologies that have been reported in the last 5 years, and provide an outlook on this research field.

iNucs: inter-nucleosome interactions.

MOTIVATION: Deciphering nucleosome-nucleosome interactions is an important step toward mesoscale description of chromatin organization but computational tools to perform such analyses are not publicly available. RESULTS: We developed iNucs, a user-friendly and efficient Python-based bioinformatics tool to compute and visualize nucleosome-resolved interactions using standard pairs format input generated from pairtools. AVAILABILITYAND IMPLEMENTATION: https://github.com/Karimi-Lab/inucs/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hi-CO: 3D genome structure analysis with nucleosome resolution.

The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with their orientations at every genomic locus in the nucleus. This technology consists of an experimental procedure for nucleosome proximity analysis and a computational procedure for 3D modeling. The experimental procedure is based on an improved method of high-throughput chromosome conformation capture (Hi-C) analysis. Whereas conventional Hi-C allows spatial proximity analysis among genomic loci with 1-10 kbp resolution, our Hi-CO allows proximity analysis among DNA entry or exit points at every nucleosome locus. This analysis is realized by carrying out ligations among the entry/exit points in every nucleosome in a micrococcal-nuclease-fragmented genome, and by quantifying frequencies of ligation products with next-generation sequencing. Our protocol has enabled this analysis by cleanly excluding unwanted non-ligation products that are abundant owing to the frequent genome fragmentation by micrococcal nuclease. The computational procedure is based on simulated annealing-molecular dynamics, which allows determination of optimized 3D positions and orientations of every nucleosome that satisfies the proximity ligation data sufficiently well. Typically, examination of the Saccharomyces cerevisiae genome with 130 million sequencing reads facilitates analysis of a total of 66,360 nucleosome loci with 6.8 nm resolution. The technique requires 2-3 weeks for sequencing library preparation and 2 weeks for simulation.

BHi-Cect: a top-down algorithm for identifying the multi-scale hierarchical structure of chromosomes.

High-throughput chromosome conformation capture (Hi-C) technology enables the investigation of genome-wide interactions among chromosome loci. Current algorithms focus on topologically associating domains (TADs), that are contiguous clusters along the genome coordinate, to describe the hierarchical structure of chromosomes. However, high resolution Hi-C displays a variety of interaction patterns beyond what current TAD detection methods can capture. Here, we present BHi-Cect, a novel top-down algorithm that finds clusters by considering every locus with no assumption of genomic contiguity using spectral clustering. Our results reveal that the hierarchical structure of chromosome is organized as 'enclaves', which are complex interwoven clusters at both local and global scales. We show that the nesting of local clusters within global clusters characterizing enclaves, is associated with the epigenomic activity found on the underlying DNA. Furthermore, we show that the hierarchical nesting that links different enclaves integrates their respective function. BHi-Cect provides means to uncover the general principles guiding chromatin architecture.

PHi-C: deciphering Hi-C data into polymer dynamics.

Genomes are spatiotemporally organized within the cell nucleus. Genome-wide chromosome conformation capture (Hi-C) technologies have uncovered the 3D genome organization. Furthermore, live-cell imaging experiments have revealed that genomes are functional in 4D. Although computational modeling methods can convert 2D Hi-C data into population-averaged static 3D genome models, exploring 4D genome nature based on 2D Hi-C data remains lacking. Here, we describe a 4D simulation method, PHi-C (polymer dynamics deciphered from Hi-C data), that depicts 4D genome features from 2D Hi-C data by polymer modeling. PHi-C allows users to interpret 2D Hi-C data as physical interaction parameters within single chromosomes. The physical interaction parameters can then be used in the simulations and analyses to demonstrate dynamic characteristics of genomic loci and chromosomes as observed in live-cell imaging experiments. PHi-C is available at https://github.com/soyashinkai/PHi-C.

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed 'labeling occupancy' (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Sub-nucleosomal Genome Structure Reveals Distinct Nucleosome Folding Motifs.

Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and ß-rhombus analogous to α helix and ß sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.

Extending Single Molecule Imaging to Proteome Analysis by Quantitation of Fluorescent Labeling Homogeneity in Complex Protein Samples.

Fluorescence-based electrophoresis has been widely used for proteome analysis in which every protein species in cells is labeled with a fluorescent dye, separated by electric migration, and quantified using fluorescence detection. The ultimate limit of sensitivity for this approach could be reached by single-molecule fluorescence imaging and counting individual proteins, requiring exhaustive fluorescent labeling of proteins across molecular populations and species. However, it remains unclear how homogeneous the fluorescence labeling of individual protein molecules of each species is across the proteome. To address this question, we developed a method to measure the labeling homogeneity based on a single-molecule fluorescence counting assay. Our results reveal that the proportion of proteins labeled with at least one dye, called labeling occupancy (LO), was 35% for fluorescently labeled BSA using existing protocols. We then found that the LO could be improved to 82% under high pH and surfactant-rich conditions. Furthermore, when a proteome sample from a human cell lysate was analyzed, the total LO was 71%, whereby the values varied between 50 and 90% for low and high molecular weight proteome fractions, respectively. The results support the possibility of sensitive detection of proteins using single-molecule counting with fluorescent labeling at the proteome scale.

Nucleosome-level 3D organization of the genome.

Nucleosomes are the unitary structures of chromosome folding, and their arrangements are intimately coupled to the regulation of genome activities. Conventionally, structural analyses using electron microscopy and X-ray crystallography have been used to study such spatial nucleosome arrangements. In contrast, recent improvements in the resolution of sequencing-based methods allowed investigation of nucleosome arrangements separately at each genomic locus, enabling exploration of gene-dependent regulation mechanisms. Here, we review recent studies on nucleosome folding in chromosomes from these two methodological perspectives: conventional structural analyses and DNA sequencing, and discuss their implications for future research.

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

Genome-Wide Analysis of Protein and mRNA Copy Numbers in Single Escherichia coli Cells with Single-Molecule Sensitivity.

Single-cell proteomic and transcriptomic analysis is an emerging approach for providing quantitative and comprehensive characterization of gene functions in individual cells. This analysis, however, is often hampered by insufficient sensitivity for detecting low copy gene expression products such as transcription factors and regulators. Here I describe a method for the quantitative genome-wide analysis of single-cell protein and mRNA copy numbers with single molecule sensitivity for the model organism Escherichia coli.

Protein expression analyses at the single cell level.

The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

Fabrication of metal half-shells using colloidal particle monolayer and their application in surface-enhanced Raman scattering.

Three types of Au shells, an isolated half-shell, one-dimensional strings of shells, and two-dimensional films, were fabricated by using a monolayer of polystyrene (PS) particles with diameters of 213, 560, and 1360 nm. The three types of Au shells that were removed from the PS particle monolayer and the as-deposited Au shells that adhered to PS particles were modified with 4-mercaptopyridine for use as platforms for surface-enhanced Raman scattering (SERS). We examined the effects of the shapes and sizes of Au shells on their SERS efficiency and found that the Au shells exhibited strong SERS signals and that Au shells prepared by using 560-nm PS particles were the most suitable platform for SERS at both 632.8- and 785-nm excitations. Further, we found that SERS enhancements depended on the shape of Au shells and on whether Au shells adhered to PS particles or not.

Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells.

Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules often exist in low copy numbers and are difficult to detect in single cells. We carried out quantitative system-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At high expression levels, the distributions are dominated by extrinsic noise. We found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.

Thermal fluctuations biased for directional motion in molecular motors.

Recently developed single molecule measurements have demonstrated that the mechanisms for numerous protein functions involve thermal fluctuation, or Brownian motion. Protein interactions bias the random thermal noise in a manner such that the protein can perform its given functions. This phenomenon has been observed in molecular motor unidirectional movement where Brownian motion is used to preferentially bind the motor heads in one direction causing directional motility. This is analogous to that used by proteins in which spontaneous structural fluctuations are used to switch function. Seeing that two very different systems implement similar mechanisms suggests there exists a general scheme applied by diverse proteins that exploits thermal fluctuations in order to achieve their respective functions.

The forward and backward stepping processes of kinesin are gated by ATP binding.

The kinesin motor converts the chemical energy from ATP turnover into mechanical work, which produces successive 8-nm steps in the forward and backward direction along a microtubule. A key problem for kinesin mechanochemistry is explaining how ATP turnover is coordinated with mechanical work. We investigated this by measuring the ATP dependent properties of kinesin forward and backward steps using optical trapping nanometry. The results showed that the rate for both forward and backward steps are ATP-dependent, indicating that ATP binding to kinesin triggers both forward and backward steps. This suggests that ATP turnover in kinesin is not rigidly coupled to total mechanical work at high load.

Single molecule thermodynamics in biological motors.

Biological molecular machines use thermal activation energy to carry out various functions. The process of thermal activation has the stochastic nature of output events that can be described according to the laws of thermodynamics. Recently developed single molecule detection techniques have allowed each distinct enzymatic event of single biological machines to be characterized providing clues to the underlying thermodynamics. In this study, the thermodynamic properties in the stepping movement of a biological molecular motor have been examined. A single molecule detection technique was used to measure the stepping movements at various loads and temperatures and a range of thermodynamic parameters associated with the production of each forward and backward step including free energy, enthalpy, entropy and characteristic distance were obtained. The results show that an asymmetry in entropy is a primary factor that controls the direction in which the motor will step. The investigation on single molecule thermodynamics has the potential to reveal dynamic properties underlying the mechanisms of how biological molecular machines work.

Entropy rectifies the Brownian steps of kinesin.

Kinesin is a stepping motor that successively produces forward and backward 8-nm steps along microtubules. Under physiological conditions, the steps powering kinesin's motility are biased in one direction and drive various biological motile processes. The physical mechanism underlying the unidirectional bias of the kinesin steps is not fully understood. Here we explored the mechanical kinetics and thermodynamics of forward and backward kinesin steps by analyzing their temperature and load dependence. Results show that the frequency asymmetry between forward and backward steps is produced by entropy. Furthermore, the magnitude of the entropic asymmetry is 6 k(B)T, more than three times greater than expected from a current model, in which a mechanical conformational change within the kinesin molecular structure directly biases the kinesin steps forward. We propose that the stepping direction of kinesin is preferably caused by an entropy asymmetry resulting from the compatibility between the kinesin and microtubule interaction based on their polar structures.

Single molecule processes on the stepwise movement of ATP-driven molecular motors.

Movement is a fundamental characteristic of all living things. This biogenic function that is attributed to the molecular motors such as kinesin, dynein and myosin. Molecular motors generate forces by using chemical energy derived from the hydrolysis reaction of ATP molecules. Despite a large number of studies on this topic, the chemomechanical energy transduction mechanism is still unsolved. In this study, we have investigated the chemomechanical coupling of the ATPase cycle to the mechanical events of the molecular motor kinesin using single molecule detection (SMD) techniques. The SMD techniques allowed to detection of the movement of single kinesin molecules along a microtubule and showed that kinesin steps mainly in the forward direction, but occasionally in the backward. The stepping direction is determined by a certain load-dependent process, on which the stochastic behavior is well characterized by Feynman's thermal ratchet model. The driving force of the stepwise movement is essentially Brownian motion, but it is biased in the forward direction by using the free energy released from the hydrolysis of ATP.