Volume 8 Issue 12, December 2011

Fusing light-activated proteins for precise optogenetic control

Retaining the recipient oocyte genome after human somatic cell nuclear transfer permits development to the blastocyst stage and derivation of triploid human embryonic stem cell lines.

Precise amounts of DNA and quantum dots can be moved into cells through tiny channels.

The hypothesis that many glycans may have a regular sequence gains support, with new evidence of a sequence for the simplest proteoglycan, bikunin.

A self-imaging Petri dish monitors cell cultures as they grow in the incubator.

Long noncoding RNA interactions with chromatin can be mapped genome-wide using biotinylated tiling oligos.

Researchers pave the way to high-throughput studies of intact protein isoforms.

Better-performing imaging sensors have arrived, but putting them to use is not easy.

Daisy-chaining light-sensitive ion channels, pumps and fluorescent proteins extends the possibilities for control of neuronal activity.

A cardiac-specific reporter genetically engineered into human embryonic stem cells allows the optimization of differentiation protocols and the identification of cell-surface markers—a welcome new tool to help isolate and define cardiac cell lineages.

Chromatin immunoprecipitation and yeast one-hybrid systems are complementary approaches to identify protein-DNA interactions. Improvements to these methods now make them more versatile and high-throughput, and should lead to the generation of rich datasets for the study of gene regulation.

In this Perspective the authors discuss strategies for the development of improved fluorescent proteins, with a focus on probes at the red end of the spectrum. They synthesize the literature on chromophore photochemistry and protein structure to identify residues for targeted mutagenesis, and consider improvements in molecular evolution methodologies to enable improved screening for desired probes.

A quantitative characterization of the switching properties of 26 organic dyes relates these properties to the quality of localization-based super-resolution images they generate. The data are a useful resource for selecting dyes and point to avenues for future analysis.

A human embryonic stem cell line expressing a fluorescent reporter of cardiac differentiation is described. The authors use this tool to optimize differentiation methods and to identify cell-surface markers in the cardiac lineage.

An empirical approach for identifying optimal proteotypic peptides and fragmentation patterns from in vitro–synthesized proteins, for targeted proteomics applications, is described.

Live-cell volumetric super-resolution imaging with 120-nm lateral and 360-nm axial resolution using structured-illumination microscopy at speeds of up to 5 s per cell volume over >50 time points captures fine cellular dynamics using only low illumination intensities.

The combination of light-sheet microscopy and localization-based super-resolution imaging allows deep subdiffraction resolution imaging in thick scattering specimens as demonstrated by three-dimensional super-resolution imaging of proteins in live 150-μm-diameter cell spheroids.

A sequence-verified collection of human transcription factors is reported. The authors used it in the enhanced yeast-one hybrid (eY1H) assay to map human gene regulatory networks. Also in this issue, Reece-Hoyes et al. describe the eY1H pipeline.

An almost-complete, sequence-verified collection of Arabidopsis thaliana root stele transcription factors is reported. The authors use it in the enhanced yeast-one hybrid (eY1H) assay to map gene regulatory interactions in the plant. Also in this issue, Reece-Hoyes et al. describe the eY1H pipeline.

Presented is a study of gene regulation during development using a combination of chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) and directed differentiation of mouse embryonic stem cells inducibly expressing epitope-tagged transcription factors.

The authors describe the enhanced yeast one-hybrid platform for large-scale screening of protein-DNA interactions and test its performance by mapping Caenorhabditis elegans gene regulatory networks. Also in this issue, Hens et al. describe an alternative platform for this purpose and apply it to screen for transcription factor–DNA interactions in Drosophila melanogaster.

Described is a high-throughput yeast one-hybrid platform for mapping protein-DNA interactions and a sequence-verified clone collection of Drosophila transcription factors. Also in this issue, Reece-Hoyes et al. report enhanced yeast one-hybrid assays, an alternative system for large-scale protein-DNA interaction screens.

This paper describes a method to enrich for homozygous mutant mouse embryonic stem cells without an inherently selectable phenotype. It is used to construct a bank of homozygous and heterozygous mutant cells and should prove useful for cellular phenotypic screens.

Reported is a recombineering pipeline for efficient and large-scale gene modification in the mouse malarial parasite Plasmodium berghei.

Molecular engineering allows stoichiometric and co-localized expression of two optogenetic actuators, spaced by a fluorescent protein and an additional transmembrane helix in a single protein fusion. The method provides modular optogenetic tools for bidirectional membrane potential control or synergistic effects on neuronal activity.