Articles in 2008

In vitro studies of neuronal function have mainly been limited to two-dimensional networks of cultured neurons. Use of transparent colloids as a moveable support for neuronal growth allows user-guided construction of optically accessible three-dimensional networks whose function can be manipulated and measured.

Single-particle tracking methods allow detailed analysis of protein movement in cells, but existing tracking algorithms have substantial limitations, particularly at high particle densities. A new software tool overcomes some of these limitations and is used to track CD36 receptors and assay the lifetime of clathrin-coated pits. Also in this issue, Sergé et al. describe an alternative software tool for high-density single-particle tracking.

A library of universal Saccharomyces cerevisiae Barcoder strains for efficient tagging is presented. It is used to tag a collection of hypomorphic alleles of essential yeast genes and applied to chemical genetic screens. Also in this issue, Breslow et al. present a similar collection of hypomorphic alleles, coupled with a sensitive growth assay for improved genetic interaction studies.

To increase the range and precision of genetic interaction studies in Saccharomyces cerevisiae, a collection of hypomorphic alleles of essential yeast genes and a highly sensitive flow cytometry–based growth competition assay are presented. Also in this issue, Yan et al. present a similar strain collection, tagged with unique bar-code identifiers, and use this collection in pooled chemical genetic screens.

Single-particle tracking methods allow detailed analysis of protein movement in cells, but existing tracking algorithms have substantial limitations, particularly at high particle densities. A new software tool overcomes some of these limitations and can be used to track high-density particles in cell membranes. Also in this issue, Jaqaman et al. describe an alternative software tool for high-density single-particle tracking.

Automated imaging of the Caenorhabditis elegans embryo now allows monitoring of the timing and relative expression of individual reporter genes at single-cell resolution over almost all of embryonic development. Future systematic analysis could be used to reveal gene expression patterns of every cell during development.

Many proteins, including G protein–coupled receptors (GPCRs), interact to form oligomers at the cell surface. A combination of bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential resonance energy transfer (SRET) extends these methods to study higher-order oligomers of GPCRs or other proteins.

A major bottleneck for genetic approaches in model organisms is the application of state-of-the-art technologies to phenotyping. Now, using a microfluidic chip, high-resolution imaging of fluorescent reporters and accurate sorting is demonstrated in an automated manner in Caenorhabditis elegans.

Cells in vivo are exposed not only to soluble factors but also to immobilized ligands. Controlled immobilization of functional growth factors yields dose-dependent responses in mouse embryonic stem cells in vitro and allows the effects of immobilized versus soluble ligands to be studied.

The mouse transcriptome in three tissue types has been analyzed using Illumina next-generation sequencing technology. This quantitative RNA-Seq methodology has been used for expression analysis and splice isoform discovery and to confirm or extend reference gene models. Also in this issue, another paper reports application of the ABI SOLiD technology to sequence the transcriptome in mouse embryonic stem cells.

Application of next-generation sequencing using the ABI SOLiD technology to mammalian transcriptome analysis enabled a survey of the content, the complexity and the developmental dynamics of the embryonic stem cell transcriptome in the mouse. Also in this issue, Mortazavi et al. report Illumina technology–based RNA-Seq analysis of the mouse transcriptome in three different tissues.

A fluorescence microscope relying entirely on focused light allows the generation of spherical focal fluorescence spots much smaller than the wavelength of light. This development, termed isoSTED, overcomes the resolution limitation imposed by the diffraction of light and permits three-dimensional nanoscale imaging inside cells with common fluorophores.

Many extracellular receptors are organized into complexes that may have functional implications. A combination of snap-tag protein labeling technology with time-resolved fluorescence resonance energy transfer (FRET) provides a method for the systematic analysis of higher-order protein-protein interactions on the surface of living cells.

Analysis of intracellular redox-based processes is constrained by the limited choice of appropriate biosensors. Fusion of human glutaredoxin-1 to an existing redox-sensitive GFP results in a ratiometric biosensor that allows rapid and sensitive dynamic imaging of glutathione redox potential in living cells.

Improved photostability of fluorescent proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved fluorescent proteins yielded highly photostable variants of existing orange and red fluorescent proteins without compromising other beneficial characteristics.