Co-Opting COPAS

Though COPAS™ is a significantly less familiar acronym than FACS™, the technology is proving to be just as useful to researchers. COPAS, which stands for Complex Object Parametric Analyzer Sorter, is essentially a scaled-up flow cytometer. Instead of sorting single cells, COPAS-based devices can analyze and dispense embryos, seeds, and bead-based combichem libraries. For instance, COPAS is widely used for sorting C. elegans, Drosophila, and even zebrafish; like a traditional flow cytometer, the instrument allows researchers to hone in on subsets of the population that are tagged with a fluorescent marker. The capabilities of COPAS technology attracted the interest of Miller et al., who wished to investigate the molecular heterogeneity of various substructures of the kidney collecting duct. Their original workflow was based on methods published in the 1960s and that involved tedious, highly skilled, and time-consuming manual microdissection. Not surprisingly, they were eager to find an alternative strategy to allow them to move more quickly to answering questions about differences in gene expression and proteome representation between the various tubular fragments, and in a recent paper, they show that a COPAS-based strategy is an effective solution. Kidneys were isolated from transgenic mice expressing GFP in a desired subset of collecting duct cells. All the procedure required was disassociation of the kidney tissue and COPAS sorting to isolate pure populations of GFP positive or negative tubules. Most cells in the isolated tubules remained viable, and the samples were able to be used for real-time RT-PCR and immunoblot analyses. The strategy should be applicable to other defined tissue segments of a size on the order of 250 m, would allow sorting on the basis of YFP and RFP in addition to (or instead of) GFP, and is sufficiently high-throughput to allow pooling of samples from replicate animals. –ND

Miller et al 2006 Automated method for the isolation of collecting ducts. American Journal of Physiology-Renal Physiology [Epub ahead of print, February 7, 2006.

Suicidal Tendencies

Identifying cells in the process of committing programmed cell death is of ongoing interest to researchers in a variety of fields. A popular method for in vitro studies is the use of fluorescently labeled Annexin V, which binds phosphatidylserine that has become redistributed to the outer layer of the cell membrane during apoptosis. Although annexins are most commonly used for in vitro analyses that rely upon flow cytometry and fluorescence microscopy, there are some studies that have employed radiolabeled Annexin V for in vivo imaging in human subjects. A synthetic functional mimetic of annexin would offer greater flexibility for in vivo imaging of apoptosis. To that end, Quinti et al., describe the synthesis and in vitro validation of a fluorescent nanosensor for apoptosis. The sensing molecule is the previously described Zn(II) di-2-picolylamine (DPA) complex, which, like Annexin V, binds phosphatidylserine. For enhanced binding affinity, multiple DPA units were bound to the unnatural amino acid diaminopropionic acid (Dpr), which itself formed part of a peptide that could be conjugated to dextran-coated iron-oxide nanoparticles. Because the magnetic nanoparticles had previously been reacted under conditions that resulted in the attachment of a TAMRA label, the resulting particles could be tracked by fluorescence. The authors establish that these functionalized nanoparticles compare favorably with traditional fluorescent annexin labeling for detection of apoptotic cells. Significantly, the magnetic properties of the nanoparticles would also be compatible with in vivo MRI procedures. Furthermore, the synthetic strategy, described in detail in the paper's freely available online supplementary methods, should be extendible to the creation of other nanosensors, –ND

Quinti et al. 2006. A fluorescent nanosensor for apoptotic cells. Nano Letters 6:488-490.

Phosphate Detection Goes (Di)Nuclear

Phosphorylation is a ubiquitous and critical on off toggle switch used by most living organisms to control all manner of cellular functions, from regulating embryogenesis and cell cycle checkpoints, to apoptosis and necrosis Mutations that affect phosphorylation of the protein members of these essential pathways have been implicated in a broad range of diseases, including numerous cancers Increasingly sophisticated and sensitive tools and techniques have been developed to allow researchers to unlock the information held in the phosphorylation states of the important protein protagonists, but as usual there is a constant push to improve speed, accuracy, and sensitivity of detection In a recent paper a group from Hiroshima University has demonstrated the application of two new phosphate detection assays, based on dinuclear metal containing tags. The first was able to specifically detect phosphorylated, but not unphosphorylated, proteins through a biotinylated Zn^2 +-Phos-tag that could recognize the phosphomonester dianion and was subsequently bound by a streptavidin-HRP molecule and visualized using chemiluminescence. This was shown on both one- and two-dimensional SDS-PAGE gels following a change in phosphorylation status induced by EG F stimulation of a carcinoma cell line. The second detection assay utilized a Mn²⁺-Phos-tag conjugated to acrylamide; this configuration allowed the addition of the tag to polyacrylamide gels as a co polymer in such a way that it would bind and retard the progress of electrophoresed proteins, which could then be visualized by staining with Coomassie® Brilliant Blue. The retention of phosphorylated proteins by the tag resulted in a marked mobility shift when compared to their unphosphorylated counterparts. Some clear advantages of these systems include the ability to detect all phosphorylation sites without the requirement for sometimes temperamental antibodies specific to each phosphorylated amino acid and the avoidance of dangerous and expensive radioactivity. –SS

Kinoshita et al. 2006. Phosphate binding tag, a new tool to visualize phosphorylated proteins Molecular & Cellular Proteomics 5(4): 749-757.

Hi-Res Arrays

It is an important goal in the genomics field to determine the role that small variations in gene copy number may play in phenotypic differences between individuals, as well as in the etiology of disease This is realistically only achievable through the continued development of more accurate and higher resolution gene mapping technologies Large deletions, insertions, or translocations of chromosome fragments can be routinely detected by comparative genome hybridization (CGH) and other array based systems; however, the sensitivity to identify smaller aberrations, usually of <100 kb, is currently beyond the ability of most of these technologies. The recent discovery that copy number polymorphisms may be more prevalent in the average population—not just in the small number of individuals with phenotypic manifestations—has driven the development of methodologies capable of drilling down deeper in to the genome to an increasingly fine resolution. Now, Michael Snyder and his group at Yale, in collaboration with others in both industry and academia, have described their high resolution CGH (HR-CGH), which they claim can resolve chromosomal defects down to the sub-200 bp range. They achieved this by creating dense isothermal arrays that tile either through specific regions of a chromosome (in this case, the authors used part of the short arm of chromosome 11) with probes designed to be 9 bp apart, or through almost an entire chromosome (here, the long arm of chromosome 22) at 85-bp intervals. Using the former, an approximately 600-bp deletion in the β-globin gene was confirmed from the blood of an affected patient, while the latter allowed identification of both known and novel copy number polymorphisms. This type of hi-res array will prove its worth not only in the detection of new chromosomal defects linked with serious physical defects, but also in the identification of more subtle copy number differences with less obvious phenotypic signs that will enhance our understanding of human diversity and disease. –SS

Urban et al. 2006. High-resolution mapping of DNA copy alterations in human chromosome 22 using high density tiling oligonucleotide arrays. Proceedings of the National Academy of Sciences of the USA 103(12): 4534-4539.