Isolating live cells after high-throughput, long-term, time-lapse microscopy.

Single-cell genetic screens can be incredibly powerful, but current high-throughput platforms do not track dynamic processes, and even for non-dynamic properties they struggle to separate mutants of interest from phenotypic outliers of the wild-type population. Here we introduce SIFT, single-cell isolation following time-lapse imaging, to address these limitations. After imaging and tracking individual bacteria for tens of consecutive generations under tightly controlled growth conditions, cells of interest are isolated and propagated for downstream analysis, free of contamination and without genetic or physiological perturbations.

Microfluidics and single-cell microscopy to study stochastic processes in bacteria.

Bacteria have molecules present in low and fluctuating numbers that randomize cell behaviors. Understanding these stochastic processes and their impact on cells has, until recently, been limited by the lack of single-cell measurement methods. Here, we review recent developments in microfluidics that enable following individual cells over long periods of time under precisely controlled conditions, and counting individual fluorescent molecules in many cells.

Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform.

Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as Escherichia coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. We developed a platform for microfluidics-assisted cell screening (MACS) that overcomes this trade-off by temporarily immobilizing suspension cells within a microfluidics chip. This enables high-throughput and automated single-cell microscopy for a wide range of cell types and sizes.

RNase J participates in a pentatricopeptide repeat protein-mediated 5' end maturation of chloroplast mRNAs.

Nucleus-encoded ribonucleases and RNA-binding proteins influence chloroplast gene expression through their roles in RNA maturation and stability. One mechanism for mRNA 5' end maturation posits that sequence-specific pentatricopeptide repeat (PPR) proteins define termini by blocking the 5'→3' exonucleolytic activity of ribonuclease J (RNase J). To test this hypothesis in vivo, virus-induced gene silencing was used to reduce the expression of three PPR proteins and RNase J, both individually and jointly, in Nicotiana benthamiana.

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA.

Ribonuclease J is an essential enzyme, and the Bacillus subtilis ortholog possesses both endoribonuclease and 5' → 3' exoribonuclease activities. Chloroplasts also contain RNase J, which has been postulated to participate, as both an exo- and endonuclease, in the maturation of polycistronic mRNAs. Here we have examined recombinant Arabidopsis RNase J and found both 5' → 3' exoribonuclease and endonucleolytic activities.

HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B.

Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii.