Amorphous nanoparticles (a-NPs) have physicochemical properties distinctly different from those of the corresponding bulk crystals; for example, their solubility is much higher. However, many materials have a high propensity to crystallize and are difficult to formulate in an amorphous structure without stabilizers. We fabricated a microfluidic nebulator that can produce amorphous NPs from a wide range of materials, even including pure table salt (NaCl). By using supersonic air flow, the nebulator produces drops that are so small that they dry before crystal nuclei can form. The small size of the resulting spray-dried a-NPs limits the probability of crystal nucleation in any given particle during storage. The kinetic stability of the a-NPs—on the order of months—is advantageous for hydrophobic drug molecules.
The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.
We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.
The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease.