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...

The survival and growth of tumor cells in a foreign environment is considered a rate-limiting step during metastasis. To identify genes that may be essential for this process, we isolated highly metastatic variants from a poorly metastatic human melanoma cell line and performed expression analyses of metastases and primary tumors from these cells. GPR56 is among the genes markedly downregulated in the metastatic variants. We show that overexpression of GPR56 suppresses tumor growth and metastasis, whereas reduced expression of GPR56 enhances tumor progression. Levels of GPR56 do not correlate with growth rate in vitro, suggesting that GPR56 may mediate growth suppression by interaction with a component in the tumor microenvironment in vivo. We show that GPR56 binds specifically to tissue transglutaminase, TG2, a widespread component of tissue and tumor stroma previously implicated as an inhibitor of tumor progression. We discuss the mechanisms whereby GPR56-TG2 interactions may suppress tumor growth and metastasis. (ii) some of the detached cells enter the circulation via blood vessels or lymphatics (intravasation); (iii) a fraction of the cells in the circulation arrest and transmigrate through blood vessels or lymphatics and invade into a distant tissue or organ (extravasation); (iv) some of the invading cells survive and proliferate in the new environment as metastases. To produce any clinically relevant metastases, a tumor cell must complete all these steps.Abundant clinical and experimental data suggest that the survival and growth step (step iv) is a rate-limiting step during metastasis (1, 2). Frequently, tumor cells are able to enter the circulation and settle in many organs but are not able to proliferate or are only able to proliferate in certain organs. Experimental metastasis assays have been developed to study these steps of metastasis. In these assays, a pool of poorly metastatic tumor cells is injected into the circulation of immunodeficient mice and gives rise to metastases at low frequency. Cells in these rare metastases can be selected from the original pool as variants which, through genetic or epigenetic changes, have gained the ability to invade, survive, and grow in a foreign environment. When these cells are isolated and amplified in vitro, they largely maintain their enhanced metastatic potential. Genes involved in metastasis can then be identified by comparing the gene expression profiles between the highly metastatic variants and the poorly metastatic pool through microarray analyses. With this method, RhoC has previously been discovered to play important roles during melanoma metastasis to lung (3), and a five-gene signature has been discovered to be essential for breast cancer metastasis to bone (4).In this article, we report that a member of a newly described family of G protein-coupled receptors (GPCRs), GPR56, contributes to suppression of melanoma metastasis and tumor growth. This suppression is not cell-autonomous, because cells with altered levels of GPR56 grow at similar rates ...

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The survival and growth of tumor cells in a foreign environment is considered a rate‐limiting step during metastasis. To identify genes that may be essential for this process, we isolated highly metastatic variants from a poorly metastatic human melanoma cell line and performed expression analyses of metastases and primary tumors from these cells. GPR56 is among the genes markedly down‐regulated in the metastatic variants. We show that overexpression of GPR56 suppresses tumor growth and metastasis, whereas reduced expression of GPR56 enhances tumor progression. Levels of GPR56 do not correlate with growth rate in vitro, suggesting that GPR56 may mediate growth suppression by interaction with a component in the tumor microenvironment in vivo. We show that GPR56 binds specifically to tissue transglutaminase, TG2, a widespread component of tissue and tumor stroma previously implicated as an inhibitor of tumor progression. We discuss the mechanisms whereby GPR56‐TG2 interactions may suppress tumor growth and metastasis. This work was supported by National Institutes of Health Grant CA17007 (to R.O.H.), the Virginia and Daniel K. Ludwig Fund for Cancer Research (R.O.H.), the Howard Hughes Medical Institute (R.O.H.), and an Anna Fuller Postdoctoral Fellowship (to L.X.).