Bench Marks

While it is possible to analyze the global lipid composition of a cell, a deeper understanding of what lipids are doing within that cell is more difficult to come by. Though the lipid components may be known, finding their exact position, how dynamically they change location, and how rapidly they are metabolized presents an experimental challenge. The obvious approach would be the addition a fluorescent tag, which would allow for imaging of lipids in cells. Unfortunately, most commonly used fluorescent tags are as large as the lipid itself and are likely to have a strong effect on lipid location and metabolism.

In the July issue of Cold Spring Harbor Protocols, Joachim Goedhart and colleagues present a suite of protocols to get around these problems and allow for live imaging of lipids in cells. Their introduction to the topic explains the approach:

To circumvent this problem, two solutions have been developed–namely, the use of fluorescently labeled proteins that specifically recognize lipids and a chemical method to introduce the fluorescent tag inside the cell.

Protocols are provided for Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain, Labeling Lipids for Imaging in Fixed Cells, and Labeling Lipids for Imaging in Live Cells.

The zebrafish (Danio rerio) has rapidly become a favored model organism for studying developmental biology. One of the most commonly used methods for genetic manipulation in the zebrafish is the delivery of plasmids or oligonucleotides to cells within the living embryo via electroporation. When cells are exposed to brief electrical fields, transient membrane destabilization occurs and nucleic acids can cross the plasma membrane. When the electrical field is removed, the membrane seals and the nucleic acids are trapped inside the cell. In vivo electroporation has proven particularly effective for delivering fluorescent protein expression vectors for imaging and loss-of-function reagents such as morpholinos or RNA interference (RNAi) constructs for the knockdown of gene function. In the July issue of Cold Spring Harbor Protocols, Jack Horne and colleagues present Targeting the Zebrafish Optic Tectum Using In Vivo Electroporation, a modification of the technique that can be used to specifically target the developing optic tectum, the midbrain’s visual processing center. Instructions are given for the construction of electroporation electrodes, preparation and injection of DNA, and electroporation of the DNA into the embryonic brain.

Cold Spring Harbor Laboratory Press’ new Drosophila Neurobiology laboratory manual covers the three main approaches taught in the CSHL course: studying neural development, recording and imaging the nervous system, and studying behavior. The featured electrophysiology paper is part of the recording/imaging section, while the second featured article in the July issue of Cold Spring Harbor Protocols comes from a neural development chapter.

The larval Drosophila brain has been a valuable model for investigating the role of stem cells in development. These neural stem cells, called “neuroblasts,” have provided insight into the role of cell polarity in influencing cell fate. Identifying neuroblasts and their progeny requires a method capable of recognizing cell polarity and cell fate markers. Immunofluorescent Staining of Drosophila Larval Brain Tissue, provided by Cheng-Yu Lee and colleagues, describes procedures for the collection and processing of Drosophila larval brains for analysis of these markers. Neuroblasts are identified via immunolocalization, the use of labeled antibodies that specifically bind the marker proteins of interest. As one of our featured articles, it is freely available to subscribers and non-subscribers alike.

Since the early days of the 20th century and Thomas Hunt Morgan’s famous “Fly Room” at Columbia University, the fruit fly Drosophila melanogaster has been at the forefront of biological research. The powerful arsenal of experimental methods developed for this model organism is now being used to tackle one of the great scientific challenges of a new century: understanding the nervous system. Cold Spring Harbor Laboratory’s Neurobiology of Drosophila course has served as the training ground for a generation of scientists tackling these complex problems. A new laboratory manual based on the protocols and background information taught in the course promises to spread these techniques to a wider audience. Methods from the manual are featured in the July issue of Cold Spring Harbor Protocols.

When a fly is confronted with danger, it jumps into the air and flies away. The giant fiber system (GFS) of Drosophila is a neuronal circuit that mediates this escape response. The neurons in the GFS are readily identified and easily accessible for experimental assay. Electrophysiological Recordings from the Drosophila Giant Fiber System, from Marcus Allen and Tanja Godenschwege, describes a simple procedure for stimulating neurons directly in the brain of the adult fly and obtaining recordings from the output muscles of the GFS. As one of our featured articles, the protocol is freely available to subscribers and nonsubscribers alike.