Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity.

Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime. Adding to the sensitivity gains realized from rejecting non-modulatable background, the selective signal recovery of immobilized vs diffusing intracellular rsFastLime suggests that DM-SAFIRe can detect weak protein-protein interactions that are normally obscured by large fractions of unbound FPs.

Using bleach-chase to measure protein half-lives in living cells.

Protein removal has a central role in numerous cellular processes. Obtaining systematic measurements of multiple protein removal rates is necessary to understand the principles that govern these processes, but it is currently a major technical challenge. To address this, we developed 'bleach-chase', a noninvasive method for measuring the half-lives of multiple proteins at high temporal resolution in living cells. The method uses a library of annotated human reporter cell clones, each with a unique fluorescently tagged protein expressed from its native chromosomal location. In this protocol, we detail a simple procedure that bleaches the cells and uses time-lapse fluorescence microscopy and automated image analysis to systematically measure the half-life dynamics of multiple proteins. The duration of the protocol is 4-5 d. The method may be applicable to a wide range of fluorescently tagged proteins and cell lines.

Proteome half-life dynamics in living human cells.

Cells remove proteins by two processes: degradation and dilution due to cell growth. The balance between these basic processes is poorly understood. We addressed this by developing an accurate and noninvasive method for measuring protein half-lives, called "bleach-chase," that is applicable to fluorescently tagged proteins. Assaying 100 proteins in living human cancer cells showed half-lives that ranged between 45 minutes and 22.5 hours. A variety of stresses that stop cell division showed the same general effect: Long-lived proteins became longer-lived, whereas short-lived proteins remained largely unaffected. This effect is due to the relative strengths of degradation and dilution and suggests a mechanism for differential killing of rapidly growing cells by growth-arresting drugs. This approach opens a way to understand proteome half-life dynamics in living cells.

Generation of double-labeled reporter cell lines for studying co-dynamics of endogenous proteins in individual human cells.

Understanding the dynamic relationship between components of a system or pathway at the individual cell level is a current challenge. To address this, we developed an approach that allows simultaneous tracking of several endogenous proteins of choice within individual living human cells. The approach is based on fluorescent tagging of proteins at their native locus by directed gene targeting. A fluorescent tag-encoding DNA is introduced as a new exon into the intronic region of the gene of interest, resulting in expression of a full-length fluorescently tagged protein. We used this approach to establish human cell lines simultaneously expressing two components of a major antioxidant defense system, thioredoxin 1 (Trx) and thioredoxin reductase 1 (TrxR1), labeled with CFP and YFP, respectively. We find that the distributions of both proteins between nuclear and cytoplasmic compartments were highly variable between cells. However, the two proteins did not vary independently of each other: protein levels of Trx and TrxR1 in both the whole cell and the nucleus were substantially correlated. We further find that in response to a stress-inducing drug (CPT), both Trx and TrxR1 accumulated in the nuclei in a manner that was highly temporally correlated. This accumulation considerably reduced cell-to-cell variability in nuclear content of both proteins, suggesting a uniform response of the thioredoxin system to stress. These results indicate that Trx and TrxR1 act in concert in response to stress in regard to both time course and variability. Thus, our approach provides an efficient tool for studying dynamic relationship between components of systems of interest at a single-cell level.

Dynamic Proteomics: a database for dynamics and localizations of endogenous fluorescently-tagged proteins in living human cells.

Recent advances allow tracking the levels and locations of a thousand proteins in individual living human cells over time using a library of annotated reporter cell clones (LARC). This library was created by Cohen et al. to study the proteome dynamics of a human lung carcinoma cell-line treated with an anti-cancer drug. Here, we report the Dynamic Proteomics database for the proteins studied by Cohen et al. Each cell-line clone in LARC has a protein tagged with yellow fluorescent protein, expressed from its endogenous chromosomal location, under its natural regulation. The Dynamic Proteomics interface facilitates searches for genes of interest, downloads of protein fluorescent movies and alignments of dynamics following drug addition. Each protein in the database is displayed with its annotation, cDNA sequence, fluorescent images and movies obtained by the time-lapse microscopy. The protein dynamics in the database represents a quantitative trace of the protein fluorescence levels in nucleus and cytoplasm produced by image analysis of movies over time. Furthermore, a sequence analysis provides a search and comparison of up to 50 input DNA sequences with all cDNAs in the library. The raw movies may be useful as a benchmark for developing image analysis tools for individual-cell dynamic-proteomics. The database is available at http://www.dynamicproteomics.net/.

Protein dynamics in individual human cells: experiment and theory.

A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

A histone H3 lysine 27 demethylase regulates animal posterior development.

The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.

UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development.

The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, we show that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation.

Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth.

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by "peptide walking".

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b(559)), composed of gp91(phox) and p22(phox) subunits, and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91(phox). NADPH oxidase activation is the consequence of assembly of cytochrome b(559) with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22(phox) is believed to act as a linker between the cytosolic components and gp91(phox). We applied "peptide walking" to mapping of domains in p22(phox) participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22(phox) subunit of cytochrome b(559) serves as an anchor for both p47(phox) and p67(phox). 2) p47(phox) binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22(phox), but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67(phox) shares with p47(phox) the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67(phox) for p22(phox) peptides is lower than that of p47(phox). 5) Binding of both p47(phox) and p67(phox) to proline-rich p22(phox) peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22(phox) is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).