What does Lady Gaga have in common with researchers from MIT's Department of Mechanical Engineering? They both use sound to get things moving. In an article appearing in Lab on a Chip, Agarwal and Livermore present the idea of acoustic excitation for sorting cells by size. Over the years, a panoply of nonmechanical approaches for moving cells has emerged, including optical tweezers, trapping in microfluidic flows, and dielectrophoretic positioning. More recently, acoustic tweezers have been investigated as a simpler, safer, and more flexible tool for cell manipulation. In such approaches, standing surface waves in the ultrasonic range (1–3 MHz) have been shown to trap cells. Agarwal and Livermore hypothesized that high-frequency acoustic waves could also be used for size-selective sorting in a high-density, parallel format. The technique they developed, called TASR (templated assembly by selective removal), uses ultrasonic fluid removal forces to place single cells in an array of cell-sized cavities on a patterned silicon substrate. At low voltages, the ultrasonic transducer is unable to circulate the cell culture medium enough to lift the cells from the silicon substrate. As the voltage increases, the cells detach from the surface and only come to rest again when retention effects (from a high contact area between the cell and a well-matched well) exceed the ultrasonic fluidic removal forces. The authors, who had previously shown TASR accurately sorts nonliving objects, applied this strategy to SF9 insect cells, which are commonly used for expressing recombinant proteins from baculovirus vectors. The diameter of SF9 cells varies from around 12 to 22 µm, depending on whether the cells have been infected with baculovirus, so holes of 12, 15, or 22 µm were tested. Stained cells were added to the arrays at a 100-fold excess of cells to wells. After 3 minutes of acoustic excitation, the array was imaged to grade sorting. Regardless of the interwell spacing of the array, sorting was highly efficient, matching cells to wells that differed by no more than 0.5 µm, and cell densities of 900 cells per square millimeter could be obtained using the most closely spaced arrays. At the voltages corresponding to the best sorting yields, only 1% loss in viability was observed, and temperature increases were contained within 0.5°C. The results suggest that TASR could be adapted for use with mammalian cells, and the authors propose that TASR be combined with existing chip-based analytic platforms for efficient single-cell– based study of biological phenomena.
G. Agarwal and C. Livermore. Chip-based size-selective sorting of biological cells using high frequency acoustic excitation. Lab Chip. [Epub ahead of print, May 26, 2011; doi:10.1039/c1lc20050j].A Cut Above
Protein splicing sounds like an elegant means of posttranslational regulation, but the reality falls short of the ideal in mammalian cells. While a RecA-based intein that is induced to splice in the presence of 4-hydroxytamoxifen has been shown to work efficiently in yeast, the higher temperatures needed for mammalian cell culture lead to less-efficient, higher-background splicing. As a result, few researchers have been able to take advantage of the desirable features of inducible inteins, which include independence from cellular cofactors, the flexibility to work well within a large variety of “host” proteins, and quick production of excised, active protein following addition of a cell-permeable inducer. In an article published in Chemistry and Biology, Peck et al. describe a directed evolution effort to adapt the yeast-proven 4-hydroxytamoxifen–dependent intein to mammalian cells. As each excision event leaves behind a cysteine residue, the intein was inserted in place of cysteine 108 in GFP to provide the raw material for selection. In the unspliced protein, the intein disrupts fluorescence, but adding 4-hydroxytamoxifen triggers excision and restoration of GFP signal. By combining rounds of mutagenic PCR, cloning into yeast for fluorescence-based cell sorting, and selecting for cells without or with fluorescence in the absence or presence of inducer, respectively, the authors hoped to isolate efficient, selective splicers. Since the point of the selection was to identify inteins suitable for mammalian cells, screening was carried out at 37°C (with a parallel 30°C selection for comparison). After several selective steps, candidate inteins were expressed in mammalian cells to see how they performed compared with their first-generation counterparts. Splicing efficiency reached 73% after 24 hours, more than double the efficiency of the original inteins. Importantly, the efficiency gains did not come at the cost of selectivity, as background excision remained 3% or less. Up to eight-fold performance enhancements were observed in three other proteins, suggesting the new inteins work well in a variety of sequence and structure contexts. The authors conclude that the improved inteins should extend small molecule– dependent protein splicing to mammalian cells, enabling unprecedented precision in disrupting a single protein in order to infer its function.
S.H. Peck et al. 2011. Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells. Chem Biol. 18:619–30.