Nanoparticles for Interrogation of Cell Signaling.

Cell functions rely on signal transduction-the cascades of molecular interactions and biochemical reactions that relay extracellular signals to the cell interior. Dissecting principles governing the signal transduction process is critical for the fundamental understanding of cell physiology and the development of biomedical interventions. The complexity of cell signaling is, however, beyond what is accessible by conventional biochemistry assays. Thanks to their unique physical and chemical properties, nanoparticles (NPs) have been increasingly used for the quantitative measurement and manipulation of cell signaling. Even though research in this area is still in its infancy, it has the potential to yield new, paradigm-shifting knowledge of cell biology and lead to biomedical innovations. To highlight this importance, we summarize in this review studies that pioneered the development and application of NPs for cell signaling, from quantitative measurements of signaling molecules to spatiotemporal manipulation of cell signal transduction.

Immobile ligands enhance FcγR-TLR2/1 crosstalk by promoting interface overlap of receptor clusters.

Innate immune cells detect pathogens through simultaneous stimulation of multiple receptors, but how cells use the receptor crosstalk to elicit context-appropriate responses is unclear. Here, we reveal that the inflammatory response of macrophages from FcγR-TLR2/1 crosstalk inversely depends on the ligand mobility within a model pathogen membrane. The mechanism is that FcγR and TLR2/1 form separate nanoclusters that interact at their interfaces during crosstalk. Less mobile ligands induce stronger interactions and more overlap between the receptor nanoclusters, leading to enhanced signaling. Different from the prevailing view that immune receptors colocalize to synergize their signaling, our results show that FcγR-TLR2/1 crosstalk occurs through interface interactions between non-colocalizing receptor nanoclusters, which are modulated by ligand mobility. This suggests a mechanism by which innate immune cells could use physical properties of ligands to fine-tune host responses.

Real-Time Simultaneous Imaging of Acidification and Proteolysis in Single Phagosomes Using Bifunctional Janus-Particle Probes.

The digestion of pathogens inside phagosomes by immune cells occurs through a sequence of reactions including acidification and proteolysis, but how the reactions are orchestrated in the right order is unclear due to a lack of methods to simultaneously measure more than one reaction in phagosomes. Here we report a bifunctional Janus-particle probe to simultaneously monitor acidification and proteolysis in single phagosomes in live cells. Each probe consists of a pH reporter and a proteolysis reporter that are spatially separated but function concurrently. Using the Janus probes, we found the acidic pH needed to initiate and maintain proteolysis, revealing the mechanism for the sequential occurrence of both reactions during pathogen digestion. We showed how bacterium-derived lipopolysaccharides alter the acidification and proteolysis in phagosomes. This study showcases Janus-particle probes as a generally applicable tool for monitoring multiple reactions in intracellular vesicles.

Mitochondria localization induced self-assembly of peptide amphiphiles for cellular dysfunction.

Achieving spatiotemporal control of molecular self-assembly associated with actuation of biological functions inside living cells remains a challenge owing to the complexity of the cellular environments and the lack of characterization tools. We present, for the first time, the organelle-localized self-assembly of a peptide amphiphile as a powerful strategy for controlling cellular fate. A phenylalanine dipeptide (FF) with a mitochondria-targeting moiety, triphenyl phosphonium (Mito-FF), preferentially accumulates inside mitochondria and reaches the critical aggregation concentration to form a fibrous nanostructure, which is monitored by confocal laser scanning microscopy and transmission electron microscopy. The Mito-FF fibrils induce mitochondrial dysfunction via membrane disruption to cause apoptosis. The organelle-specific supramolecular system provides a new opportunity for therapeutics and in-depth investigations of cellular functions.Spatiotemporal control of intracellular molecular self-assembly holds promise for therapeutic applications. Here the authors develop a peptide consisting of a phenylalanine dipeptide with a mitochondrial targeting moiety to form self-assembling fibrous nanostructures within mitochondria, leading to apoptosis.

Position-Dependent Three-Dimensional Diffusion in Nematic Liquid Crystal Monitored by Single-Particle Fluorescence Localization and Tracking.

Anisotropic mass diffusion in liquid crystals (LCs) is important from the point of both basic LC physics and their applications in optoelectronic devices. We use super-resolution fluorescence microscopy with astigmatic imaging to track 3D diffusion of quantum dots (QDs) in an ordered nematic LC. The method allowed us to evaluate the diffusion coefficients independently along the three spatial axes as well as to determine the absolute position of the QD with respect to the cell wall. We found variations of the diffusion coefficient along the different directions across the cell thickness and explained these as being due to changes of a tilt angle of the LC director. Close to the surface, the diffusion is slowed down due to the confinement effect of the cell wall. Overall, the QD diffusion is much slower than expected for a corresponding particle size. This phenomenon is suggested to originate from reorientation of the LC director in the vicinity of the particle.