Amy C. Rowat

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼107. After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼108 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Biocompatible surfactants for water-in-fluorocarbon emulsions

Drops of water-in-fluorocarbon emulsions have great potential for compartmentalizing both in vitro and in vivo biological systems; however, surfactants to stabilize such emulsions are scarce. Here we present a novel class of fluorosurfactants that we synthesize by coupling oligomeric perfluorinated polyethers (PFPE) with polyethyleneglycol (PEG). We demonstrate that these block copolymer surfactants stabilize water-in-fluorocarbon oil emulsions during all necessary steps of a drop-based experiment including drop formation, incubation, and reinjection into a second microfluidic device. Furthermore, we show that aqueous drops stabilized with these surfactants can be used for in vitro translation (IVT), as well as encapsulation and incubation of single cells. The compatability of this emulsion system with both biological systems and polydimethylsiloxane (PDMS) microfluidic devices makes these surfactants ideal for a broad range of high-throughput, drop-based applications.

Designer emulsions using microfluidics

We describe new developments for the controlled fabrication of monodisperse emulsions using microfluidics. We use glass capillary devices to generate single, double, and higher order emulsions with exceptional precision. These emulsions can serve as ideal templates for generating well-defined particles and functional vesicles. Polydimethylsiloxane microfluidic devices are also used to generate picoliter-scale water-in-oil emulsions at rates as high as 10 000 drops per second. These emulsions have great potential as individual microvessels in high-throughput screening applications, where each drop serves to encapsulate single cells, genes, or reactants.

Drop-based microfluidic devices for encapsulation of single cells

We use microfluidic devices to encapsulate, incubate, and manipulate individual cells in picoliter aqueous drops in a carrier fluid at rates of up to several hundred Hz. We use a modular approach with individual devices for each function, thereby significantly increasing the robustness of our system and making it highly flexible and adaptable to a variety of cell-based assays. The small volumes of the drops enables the concentrations of secreted molecules to rapidly attain detectable levels. We show that single hybridoma cells in 33 pL drops secrete detectable concentrations of antibodies in only 6 h and remain fully viable. These devices hold the promise of developing microfluidic cell cytometers and cell sorters with much greater functionality, allowing assays to be performed on individual cells in their own microenvironment prior to analysis and sorting.

Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions

Background: The unusual nuclear shape of neutrophils has been speculated to facilitate their passage through confined spaces. Results: Levels of nuclear protein lamin A modulate cell passage through micron-scale pores. Conclusion: The unique protein composition of neutrophil nuclei facilitates their deformation; lobulated nuclear shape is not essential. Significance: Altered nuclear envelope composition, as reported in cancer cells, could impact cell passage through physiological gaps. Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.

Tracking lineages of single cells in lines using a microfluidic device

Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.

Towards an integrated understanding of the structure and mechanics of the cell nucleus

Changes in the shape and structural organization of the cell nucleus occur during many fundamental processes including development, differentiation and aging. In many of these processes, the cell responds to physical forces by altering gene expression within the nucleus. How the nucleus itself senses and responds to such mechanical cues is not well understood. In addition to these external forces, epigenetic modifications of chromatin structure inside the nucleus could also alter its physical properties. To achieve a better understanding, we need to elucidate the relationship between nuclear structure and material properties. Recently, new approaches have been developed to systematically investigate nuclear mechanical properties. These experiments provide important new insights into the disease mechanism of a growing class of tissue‐specific disorders termed ‘nuclear envelopathies’. Here we review our current understanding of what determines the shape and mechanical properties of the cell nucleus. BioEssays 30:226–236, 2008.

Divalent Cations Crosslink Vimentin Intermediate Filament Tail Domains to Regulate Network Mechanics

Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca(2+) and Mg(2+), showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l(P) approximately 0.5 mum, and Youngs modulus, Y approximately 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.

Electrodes on a budget: Micropatterned electrode fabrication by wet chemical deposition

Precise patterning of metals is required for diverse microfluidic and microelectromechanical system (MEMS) applications ranging from the separation of proteins to the manipulation of single cells and drops of water-in-oil emulsions. Here we present a very simple, inexpensive method for fabricating micropatterned electrodes. We deposit a thin metal layer of controlled thickness using wet chemistry, thus eliminating the need for expensive equipment typically required for metal deposition. We demonstrate that the resulting deposited metal can be used to fabricate functional electrodes: The wet-deposited metal film can sustain patterning by photolithography down to micron-sized features required for MEMS and microfluidic applications, and its properties are suitable for operative electrodes used in a wide range of microfluidic applications for biological studies.

Commentary : Farnesylated peptides in model membranes: a biophysical investigation (vol 33, pg 300, 2003)

Protein prenylation plays an important role in signal transduction, protein-protein interactions, and the localization and association of proteins with membranes. Using three different techniques, this study physically characterizes the interactions between model dimyristoylphosphatidylcholine membranes and a series of farnesylated peptides. Magic angle spinning nuclear Overhauser enhancement spectroscopy and differential scanning calorimetry reveal that both charged [Ac-Asn-Lys-Asn-Cys-(farnesyl)-OMe and Ac-Asn-Lys-Asn-Cys-(farnesyl)-NH(2)] and uncharged [Ac-Cys-(farnesyl)-OMe and farnesol] species partition into dimyristoylphosphatidylcholine bilayers. Calorimetry and vesicle fluctuation analysis of giant unilamellar vesicles show that the charged peptides modestly decrease the main gel-fluid phase transition and markedly increase the bending rigidity of large unilamellar vesicles. Uncharged species, on the other hand, dramatically decrease the main phase transition and modestly decrease the bending rigidity. No difference with carboxyl methylation is detected.