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It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAG⌬C) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAG⌬C compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAG⌬C was expressed exclusively in the cytosol. Furthermore, cells expressing BAG⌬C were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Fö rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new allsolid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of ␣-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant ␣-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation. Molecular & Cellular Proteomics 6:1446 -1454, 2007.Above and beyond the isolation and identification of proteins, the field of proteomics faces the challenges of detecting protein cellular localization and quantifying molecular states such as protein conformations, protein-protein interactions, and post-translational modifications. In the past decade, Fö rster resonance energy transfer (FRET) 1 and fluorescence lifetime imaging microscopy (FLIM) have proven to be instrumental for the quantitative imaging of these biochemical states in single cells (1). Similarly the analysis of different cellular populations (cellomes) will also benefit from these imaging methods. Quantitative multiparametric microscopy is a very young field in which advances in liquid/sample handling robotics and information technology are gradually being integrated into automated microscopes (2, 3). These automated imaging systems merge the high content image information with the high throughput volumes provided by their automation and unsupervised operation.Screening techniques have now reached (ultra)high throughput levels, i.e. they are capable of performing more than 10 5 assays/day in microliter volumes. Such a high throughput is necessary for applications where (bio)chemical libraries are tested, e.g. for drug discovery and interactomics research (4). Although the advance in throughput scale is neces...

Bax ihibitor-1 (BI-1) has been characterized as an inhibitor of Bax-induced cell death in plants and various mammalian cell systems. To explore the function of BI-1 in neurons, we overexpressed BI-1 tagged to HA or GFP in rat nigral CSM14.1 and human SH-SY5Y neuroblastoma cells. Stable BI-1 expression proved marked protection from cell death induced by thapsigargine, a stress agent blocking the Ca2+-ATPase of the endoplasmic reticulum (ER) but failed to inhibit cell death induced by staurosporine, a kinase inhibitor initiating mitochondria-dependent apoptosis. Moreover, BI-1 was neuroprotective in a paradigm mimicking ischemia, namely oxygen-glucose as well as serum deprivation. Examination of the subcellular distribution revealed that BI-1 predominantly locates to the ER and nuclear envelope but not mitochondria. Taken together, BI-1 overexpression in the ER is protective in neurons, making BI-1 an interesting target for future studies aiming at the inhibition of neuronal cell death during neurodegenerative diseases and stroke.