Protein and 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. Here 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. Strikingly, we found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.Gene expression is often stochastic because gene regulation takes place at a single DNA locus within a cell. Such stochasticity is manifested in fluctuations of mRNA and protein copy numbers within a cell lineage over time, and in variations of mRNA and protein copy numbers among a population of genetically identical cells at a particular time (1,2,3,4). Because both manifestations of stochasticity are connected, measurement of the latter allows the deduction of the gene expression dynamics in a cell (5). We aim to characterize such mRNA and protein distributions in single bacteria cells at a system-wide level.While single cell mRNA profiling has been carried out with cDNA microarray (6) and mRNA-seq (7), these studies did not have single molecule sensitivity and are not suitable for bacteria, which express mRNA at low copy numbers (8). A fluorescent protein reporter library of Saccharomyces cerevisiae (9) has proven to be extremely useful in protein profiling (10,11). However, the lack of sensitivity in existing flow cytometry or fluorescence microscopy techniques prevented the quantification of one third of the labeled proteins because of their low copy numbers. In recent years, single-molecule fluorescence ** Publisher's Disclaimer: This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS." † To whom correspondence should be addressed. xie@chemistry.harvard.edu. * These authors contributed equally to this work. NIH Public Access Author ManuscriptScience. Author manuscript; available in PMC 2010 August 17. Single-molecule imaging of a YFP reporter libraryWe created a chromosomal YFP fusion library (Fig. 1A), in which each strain has a particular gene tagged with the YFP coding sequence. YFP can be detected with single molecule sensitivity in live bacterial cells (8,18). We converted the C-terminus tags of an existing chromosomally affinity-tagged E. coli library (19,20...

Intratumoral heterogeneity is a major obstacle to cancer treatment and a significant confounding factor in bulk-tumor profiling. We performed an unbiased analysis of transcriptional heterogeneity in colorectal tumors and their microenvironments using single-cell RNA-seq from 11 primary colorectal tumors and matched normal mucosa. To robustly cluster single-cell transcriptomes, we developed reference component analysis (RCA), an algorithm that substantially improves clustering accuracy. Using RCA, we identified two distinct subtypes of cancer-associated fibroblasts (CAFs). Additionally, epithelial-mesenchymal transition (EMT)-related genes were found to be upregulated only in the CAF subpopulation of tumor samples. Notably, colorectal tumors previously assigned to a single subtype on the basis of bulk transcriptomics could be divided into subgroups with divergent survival probability by using single-cell signatures, thus underscoring the prognostic value of our approach. Overall, our results demonstrate that unbiased single-cell RNA-seq profiling of tumor and matched normal samples provides a unique opportunity to characterize aberrant cell states within a tumor.

We investigate the molecular mechanism of how an E. coli cell with the lac operon switches from one phenotype to another by monitoring fluorescently labeled lactose permease with single-molecule sensitivity. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We find that this basal level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic, single molecular event determines a cell's phenotype.Genetically identical cells in the same environment can exhibit different phenotypes, and a single cell can switch between distinct phenotypes in a stochastic manner (1-4). In the classic example of lactose metabolism in E. coli, the lac genes are fully expressed for every cell in a population under high extracellular concentrations of inducers, such as the lactose analog methyl-β-D-thiogalactoside (TMG). However, at moderate inducer concentrations, the lac genes are highly expressed in only a fraction of a population, which may confer a fitness advantage for the entire population (5). Here we study the molecular mechanism that controls the stochastic phenotype switching of a single cell.Lactose metabolism is controlled by the lac operon (6,7), which consists of the lacZ, lacY, and lacA genes encoding beta-galactosidase, lactose permease, and transacetylase, respectively. Expression of the operon is regulated by a transcription factor, the lac repressor (8), which dissociates from its specific binding sequences of DNA, the lac operators, in the presence of inducer to allow transcription (Fig. 1A). The production of the permease increases inducer influx (9), resulting in positive feedback on permease expression level. Above a certain threshold of permease numbers, a cell will be in a phenotype capable of lactose metabolism, and below this threshold, a cell will be in a phenotype incapable of lactose metabolism (10, 11). The former has high fluorescence from the cell membrane, whereas the latter has low fluorescence, when the permease is labeled with a yellow fluorescent protein (YFP).

Recent developments on fluorescent proteins and microscopy techniques have allowed the probing of single molecules in a living bacterial cell with high specificity, millisecond time resolution, and nanometer spatial precision. Recording movies and analyzing dynamics of individual macromolecules have brought new insights into the mechanisms of many processes in molecular biology, such as DNA-protein interactions, gene regulation, transcription, translation, and replication, among others. Here we review the key methods of single-molecule detection and highlight numerous examples to illustrate how these experiments are contributing to the quantitative understanding of the fundamental processes in a living cell.

Cytotoxic lymphocytes eliminate virus-infected and cancerous cells by immune recognition and killing through the perforin-granzyme pathway. Traditional killing assays measure average target cell lysis at fixed times and high effector:target ratios. Such assays obscure kinetic details that might reveal novel physiology. We engineered target cells to report on granzyme activity, used very low effector:target ratios to observe potential serial killing, and performed low magnification time-lapse imaging to reveal time-dependent statistics of natural killer (NK) killing at the single-cell level. Most kills occurred during serial killing, and a single NK cell killed up to 10 targets over a 6-h assay. The first kill was slower than subsequent kills, especially on poor targets, or when NK signaling pathways were partially inhibited. Spatial analysis showed that sequential kills were usually adjacent. We propose that NK cells integrate signals from the previous and current target, possibly by simultaneous contact. The resulting burst kinetics and spatial coordination may control the activity of NK cells in tissues.

Evolutionary traces of thermophilic adaptation are manifest, on the whole-genome level, in compositional biases toward certain types of amino acids. However, it is sometimes difficult to discern their causes without a clear understanding of underlying physical mechanisms of thermal stabilization of proteins. For example, it is well-known that hyperthermophiles feature a greater proportion of charged residues, but, surprisingly, the excess of positively charged residues is almost entirely due to lysines but not arginines in the majority of hyperthermophilic genomes. All-atom simulations show that lysines have a much greater number of accessible rotamers than arginines of similar degree of burial in folded states of proteins. This finding suggests that lysines would preferentially entropically stabilize the native state. Indeed, we show in computational experiments that arginine-to-lysine amino acid substitutions result in noticeable stabilization of proteins. We then hypothesize that if evolution uses this physical mechanism as a complement to electrostatic stabilization in its strategies of thermophilic adaptation, then hyperthermostable organisms would have much greater content of lysines in their proteomes than comparably sized and similarly charged arginines. Consistent with that, high-throughput comparative analysis of complete proteomes shows extremely strong bias toward arginine-to-lysine replacement in hyperthermophilic organisms and overall much greater content of lysines than arginines in hyperthermophiles. This finding cannot be explained by genomic GC compositional biases or by the universal trend of amino acid gain and loss in protein evolution. We discovered here a novel entropic mechanism of protein thermostability due to residual dynamics of rotamer isomerization in native state and demonstrated its immediate proteomic implications. Our study provides an example of how analysis of a fundamental physical mechanism of thermostability helps to resolve a puzzle in comparative genomics as to why amino acid compositions of hyperthermophilic proteomes are significantly biased toward lysines but not similarly charged arginines.

Purpose. We have developed a method to study the molecular and cellular events underlying delayed skeletal repair in a model that utilizes distraction osteogenesis.Methods. The clinical states of delayed union and non-union were reproduced in this murine model by altering distraction parameters such as the inclusion and exclusion of a latency phase and variations in the rate and rhythm of distraction. Radiographic, cellular, and molecular analyses were performed on the distraction tissues.Results. Eliminating the latency period delayed bony union, but did not appreciably alter the extent of platelet endothelial cell adhesion marker (PECAM) immunostaining. Following elimination of a latency phase and a threefold increase in the rate of distraction, there was a further delay in bone regeneration and a higher rate of non-union (60%). Instead of bone, the distraction gap was comprised of adipose or fibrous tissue. Once again, despite the rigorous distraction protocol, we detected equivalent PECAM staining within the distraction gap. In a minority of cases, cartilage and osseous tissues occupied the distraction gap likely by a prolonged process of endochondral ossification.Conclusions. Here, we have altered the mechanical environment in such a way to reproducibly create delays in skeletal regeneration. These delays in skeletal tissue regeneration appear to develop even in the presence of endothelial cells, which suggests that mechanisms other than a disruption to the vascular network can account for some cases of non-union.

Previous studies have revealed that delayed internal fixation can stimulate fracture callus formation and decrease the rate of nonunion. However, the effect of delayed stabilization on stem cell differentiation is unknown. To address this, we created fractures in mouse tibiae and applied external fixation immediately, at 24, 48, 72, or 96 h after injury. Fracture healing was analyzed at 10 days by histological methods for callus, bone, and cartilage formation, and the mechanical properties of the calluses were assessed at 14 days postinjury by tension testing. The results demonstrate that delaying stabilization for 24-96 h does not significantly affect the volume of the callus tissue (TV) and the new bone (BV) that formed by 10 days, or the mechanical properties of the calluses at 14 days, compared to immediate stabilization. However, delaying stabilization for 24-96 h induces 10-40Â more cartilage in the fracture calluses compared with fractures stabilized immediately. These findings suggest that delaying stabilization during the early phase of fracture healing may not significantly stimulate bone repair, but may alter the mode of bone repair by directing formation of more cartilage. Fractures that are not rigidly stabilized form a significantly larger amount of callus tissue and cartilage by 10 days postinjury than fractures stabilized at 24-96 h, indicating that mechanical instability influences chondrocytes beyond the first 96 h of fracture healing. ß