Articles in 2011

Adenosine-to-inosine RNA editing modifies expressed sequences and enhances functional protein diversity. The authors report an in vivo fluorescent reporter that provides a readout of adenosine deaminase RNA-editing activity in Drosophila melanogaster neurons, showing evidence of inter-individual variability in editing activity.

Bayesian analysis of fluorophore blinking and bleaching in image data collected from simple xenon arc lamp illumination and high-speed wide-field imaging of standard fluorescent proteins allows localization microscopy in living cells with a 50 nm spatial and a 4 s temporal resolution.

In this paper, the authors report GFP reconstitution across synaptic partners (GRASP) adapted for synapse visualization in the mammalian brain.

Site-directed seamless modification of bacterial artificial chromosomes is enhanced more than tenfold in efficiency by improving the counterselection step. A set of plasmids and oligonucleotide design software also make this E. coli recombineering approach markedly faster and easier.

The microbial rhodopsin protein, Archaerhodopsin 3, can function as a rapid and highly sensitive genetically encoded voltage indicator in mammalian cells that is capable of detecting single action potentials with a signal-to-noise ratio greater than 10. A mutant lacking proton pumping displays greater sensitivity but a slowed response.

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.

A quantitative proteomics approach to characterize protein palmitoylation dynamics on a global scale in cells, as well as to identify enzymes responsible for the regulation of palmitoylation, is described.

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.

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

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.

Owing to a lack of tools and a lack of a consensus sequence for O-glycosylation, studies of the O-glycoproteome have been few and far between, despite the biological importance of O-glycosylation. This method to analyze O-glycan attachment sites to proteins using glycoengineered cell lines with simplified, homogenous O-glycoproteomes should facilitate future O-glycoproteomics studies.

Soft hydrogel microwell arrays with controllably variable stiffness are spotted with proteins of interest, individually or in combination. The system permits in vitro study of the biophysical and biochemical aspects of the stem-cell niche.

The question of whether transcription factories containing RNA polymerases exist has been controversial, owing to the fact that they have not been isolated previously. Now, a method to carefully isolate these complexes and analyze their proteomes by mass spectrometry is described.

Reported is the robust directed differentiation of mouse epiblast stem cells to oligodendrocyte precursor cells, which then differentiate into myelinating oligodendrocytes in vitro and in vivo. The system should prove useful for basic research and for drug screens.

Implementation of pair correlation analysis for photoactivated localization microscopy allows quantitative analysis of protein clustering in the plasma membrane, revealing the degree to which different perturbations alter protein arrangements.

An integrated, miniature (1.9 g) fluorescence microscope containing light source, optics and sensor allows high-speed, wide field of view imaging of calcium spiking in hundreds of neurons in freely moving mice. The mass-producible portable microscope is also useful for a variety of fluorescence assays for which size, cost and portability can be concerns.

This paper reports transgenesis by genetic modification of gametes in the domestic cat. The approach is used to generate transgenic cats expressing a virus restriction factor from rhesus macaque.

Destabilized mutants of firefly luciferase are characterized as sensors for protein homeostasis (proteostasis). Their use as tools for comparisons of proteostasis capacity is demonstrated in cells and in Caenorhabditis elegans.

Presented is an experimental analysis of the stability of transgene expression, the perturbation of endogenous expression and the perturbation of epigenetic organization upon site-directed delivery of transgenes to the CCR5 and AAVS1 loci in human cells. It provides guidelines for optimal cassette design for stable and nonperturbative gene transfer.