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1.  A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening 
PLoS ONE  2011;6(12):e28338.
Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.
PMCID: PMC3230607  PMID: 22162763
2.  The Compartmentalized Bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins 
PLoS Biology  2010;8(1):e1000281.
Compartmentalized bacteria have proteins that are structurally related to eukaryotic membrane coats, and one of these proteins localizes at the membrane of vesicles formed inside bacterial cells.
The development of the endomembrane system was a major step in eukaryotic evolution. Membrane coats, which exhibit a unique arrangement of β-propeller and α-helical repeat domains, play key roles in shaping eukaryotic membranes. Such proteins are likely to have been present in the ancestral eukaryote but cannot be detected in prokaryotes using sequence-only searches. We have used a structure-based detection protocol to search all proteomes for proteins with this domain architecture. Apart from the eukaryotes, we identified this protein architecture only in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum, many members of which share a compartmentalized cell plan. We determined that one such protein is partly localized at the membranes of vesicles formed inside the cells in the planctomycete Gemmata obscuriglobus. Our results demonstrate similarities between bacterial and eukaryotic compartmentalization machinery, suggesting that the bacterial PVC superphylum contributed significantly to eukaryogenesis.
Author Summary
Despite decades of research, the origin of eukaryotic cells remains an unsolved issue. The endomembrane system defines the eukaryotic cell, and its origin is linked to that of eukaryotes. A search was conducted within all known sequences for proteins that are characteristic of the eukaryotic endomembrane system, using a combination of fold types that is uniquely found in the membrane coat proteins. Outside eukaryotes, such proteins were solely found in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum. By immuno-electron microscopy, one of these bacterial proteins was found to localize adjacent to the membranes of vesicles found within the cells of one member of the PVC superphylum. Thus, there appear to be similarities between bacterial and eukaryotic compartmentalization systems, suggesting that the bacterial PVC superphylum may have contributed significantly to eukaryogenesis.
PMCID: PMC2799638  PMID: 20087413
3.  Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p 
The Journal of Cell Biology  2002;159(6):971-982.
Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex.
PMCID: PMC2173977  PMID: 12499354
myosin-V; RNA localization; FLIP; myosin regulation; ASH1

Results 1-3 (3)