iRFP is a real time marker for transformation based assays in high content screening

Anchorage independent growth is one of the hallmarks of oncogenic transformation. Here we show that infrared fluorescent protein (iRFP) based assays allow accurate and unbiased determination of colony formation and anchorage independent growth over time. This protocol is particularly compatible with high throughput systems, in contrast to traditional methods which are often labor-intensive, subjective to bias and do not allow further analysis using the same cells. Transformation in a single layer soft agar assay could be documented as early as 2 to 3 days in a 96 well format, which can be easily combined with standard transfection, infection and compound screening setups to allow for high throughput screening to identify therapeutic targets

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail

Modular construction of mammalian gene circuits using TALE transcriptional repressors

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator–like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches using feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50GM098792)National Institutes of Health (U.S.) (Grant 1R01CA173712-01

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.Peer reviewe

A practical guide to photoacoustic tomography in the life sciences

The life sciences can benefit greatly from imaging technologies that connect microscopic discoveries with macroscopic observations. One technology uniquely positioned to provide such benefits is photoacoustic tomography (PAT), a sensitive modality for imaging optical absorption contrast over a range of spatial scales at high speed. In PAT, endogenous contrast reveals a tissue's anatomical, functional, metabolic, and histologic properties, and exogenous contrast provides molecular and cellular specificity. The spatial scale of PAT covers organelles, cells, tissues, organs, and small animals. Consequently, PAT is complementary to other imaging modalities in contrast mechanism, penetration, spatial resolution, and temporal resolution. We review the fundamentals of PAT and provide practical guidelines for matching PAT systems with research needs. We also summarize the most promising biomedical applications of PAT, discuss related challenges, and envision PAT's potential to lead to further breakthroughs

Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More.

The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-fluorescent molecules called "fluorogens" that become fluorescent upon binding to genetically encodable RNAs called "light-up aptamers." Yet, the development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is presented and discussed.journal article2020importe

A reference map of murine cardiac transcription factor chromatin occupancy identifies dynamic and conserved enhancers

Mapping the chromatin occupancy of transcription factors (TFs) is a key step in deciphering developmental transcriptional programs. Here we use biotinylated knockin alleles of seven key cardiac TFs (GATA4, NKX2-5, MEF2A, MEF2C, SRF, TBX5, TEAD1) to sensitively and reproducibly map their genome-wide occupancy in the fetal and adult mouse heart. These maps show that TF occupancy is dynamic between developmental stages and that multiple TFs often collaboratively occupy the same chromatin region through indirect cooperativity. Multi-TF regions exhibit features of functional regulatory elements, including evolutionary conservation, chromatin accessibility, and activity in transcriptional enhancer assays. H3K27ac, a feature of many enhancers, incompletely overlaps multi-TF regions, and multi-TF regions lacking H3K27ac retain conservation and enhancer activity. TEAD1 is a core component of the cardiac transcriptional network, co-occupying cardiac regulatory regions and controlling cardiomyocyte-specific gene functions. Our study provides a resource for deciphering the cardiac transcriptional regulatory network and gaining insights into the molecular mechanisms governing heart development