Biomarker Analysis: Quantitating Phosphoproteins in Fixed Tissue Samples

Phosphoproteins offer a window into the physiological status of tissues and hold promise as a means for diagnosing pathologies that involve aberrant phosphoprotein-mediated signaling. However, the conditions required for preserving phosphoprotein profiles in tissue samples are not well defined. In this report, the authors characterize tissue handling conditions that are conducive to the preservation of phosphoprotein biomarkers in tissue samples, and describe a quantum dot–based immunofluorescence assay for their reliable quantitation. They demonstrate that phosphoprotein levels in fixed tissues mirror the in vivo state; phosphoprotein levels as determined by quantum dot immunolabeling and fluorescence analysis showed good agreement with values determined independently.

Bodo J, Durkin L, Hsi ED (2009) Quantitative in situ detection of phosphoproteins in fixed tissues using quantum dot technology. J Histochem Cytochem 57:701–708.

Cell Communication: Following the Path of Alexa Fluor Dye–labeled Prions Between Cells

The mechanisms by which the prions that cause Creutzfeldt-Jakob disease move to the nervous system are poorly understood; this study examines the role of tunnelling nanotubes (TnTs) in the transduction of infectious prions. After loading mouse CAD neuronal cells with LysoTracker Red reagent, the authors observed the movement of lysosomal vesicles between cells connected by TnTs. After transfecting CAD cells with GFP-labeled prions (wild type and infectious), they demonstrated that TnTs did affect the transport of prions from infected to uninfected cells. Coculture experiments revealed that Alexa Fluor dye–labeled prions were transferred from uninfected CAD cells to bone marrow–derived dendritic cells as well as to cerebellar granule neurons.

Gousset K, Schiff E, Langevin C et al. (2009) Prions hijack tunnelling nanotubes for intercellular spread. Nat Cell Biol 11:328–336.

Ion Indicators: Probing Redox Capacity with Sodium Green and Rhod-5N Dyes

Although mutations in the mitochondrial kinase PINK1 have been associated with Parkinson’s disease, the role of this kinase in mitochondrial function has not been identified. Gandhi and colleagues use fluorescence-based analysis to demonstrate changes in respiration and redox capacity in PINK1-deficient neuronal cells. Reduced calcium capacity in the mitochondria of these cells was demonstrated using neurons co-loaded with Sodium Green and rhod-5N indicators. PINK1-deficient neurons showed a decreased ability to maintain mitochondrial membrane polarization in response to a rapid rise in cytosolic calcium. The authors describe the pathophysiology of PINK1-deficient cells as part of a mechanism leading to opening of the mitochondrial permeability transition pore (PTP) and increased susceptibility to neuronal apoptosis.

Gandhi S, Wood-Kaczmar A, Yao Z et al. (2009) PINK1-associated Parkinson’s disease is caused by neuronal vulnerability to calcium-induced cell death. Molecular Cell 33:627–638.

Stem Cells: Click-iT EdU Analysis of Germ Cell Regulation

The ultimate fate of germ line stem cells—mitotic self-renewal or meiotic gamete formation—is balanced through strict but incompletely characterized genetic control mechanisms. Dorsett and coworkers examine the role of the putative methyltransferase METT-10 in the regulation of germ line stem cell development in C. elegans. The authors used the Click-iT EdU assay with Alexa Fluor 488 labeling to characterize germ cell fate in METT-10 wild type and mutant nematodes. They observed that the mett-10 gene product both inhibits germ cell proliferation and promotes the cell cycle progression of those cells that do enter mitosis. This dual role for METT-10 suggests that the mechanisms that specify germ line stem cell fate are distinct from those that control the execution of a particular chosen path.

Dorsett M, Westlund B, Schedl T (2009) METT-10, a putative methyltransferase, inhibits germ cell proliferative fate in Caenorhabditis elegans. Genetics 183:233–247.

For Research Use Only. Not for use in diagnostic procedures.