Many innate immune receptors function collaboratively to detect and elicit immune responses to pathogens, but the physical mechanisms that govern the interaction and signaling crosstalk between the receptors are unclear. In this study, we report that the signaling crosstalk between Fc gamma receptor (FcγR) and Toll-like receptor (TLR)2/1 can be overall synergistic or inhibitory depending on the spatial proximity between the receptor pair on phagosome membranes. Using a geometric manipulation strategy, we physically altered the spatial distribution of FcγR and TLR2 on single phagosomes. We demonstrate that the signaling synergy between FcγR and TLR2/1 depends on the proximity of the receptors and decreases as spatial separation between them increases. However, the inhibitory effect from FcγRIIb on TLR2-dependent signaling is always present and independent of receptor proximity. The overall cell responses are an integration from these two mechanisms. This study presents quantitative evidence that the nanoscale proximity between FcγR and TLR2 functions as a key regulatory mechanism in their signaling crosstalk.
Industrial accidents, such as the Fukushima and Chernobyl disasters, release harmful chemicals into the environment, covering large geographical areas. Natural flora may serve as biological sensors for detecting metal contamination, such as cesium. Spectral detection of plant stresses typically employs a few select wavelengths and often cannot distinguish between different stress phenotypes. In this study, we apply hyperspectral reflectance imaging in the visible and near-infrared along with multivariate curve resolution (MCR) analysis to identify unique spectral signatures of three stresses in Arabidopsis thaliana: salt, copper, and cesium. While all stress conditions result in common stress physiology, hyperspectral reflectance imaging and MCR analysis produced unique spectral signatures that enabled classification of each stress. As the level of potassium was previously shown to affect cesium stress in plants, the response of A. thaliana to cesium stress under variable levels of potassium was also investigated. Increased levels of potassium reduced the spectral response of A. thaliana to cesium and prevented changes to chloroplast cellular organization. While metal stress mechanisms may vary under different environmental conditions, this study demonstrates that hyperspectral reflectance imaging with MCR analysis can distinguish metal stress phenotypes, providing the potential to detect metal contamination across large geographical areas.
Tsyrenova, Ayuna; Farooq, Muhammad Q.; Anthony, Stephen M.; Mollaeian, Keyvan; Li, Yifan; Liu, Fei; Miller, Kyle; Ren, Juan; Anderson, Jared L.; Jiang, Shan
This study reveals the unique role on Janus particles of the solid-solid interface at the boundary in determining particle interactions and assembly. In an aqueous ionic liquid (IL) solution, Janus spheres adopt intriguing orientations with their boundaries pinned on the glass substrate. It was further discovered that the orientation was affected by the particle amphiphilicity as well as the chemical structure and concentration of the IL. Further characterization suggests that the adsorption on the hydrophilic side is due to both an electrostatic interaction and hydrogen bonding, while adsorption on the hydrophobic side is due to hydrophobic attraction. Through the concerted interplay of all these interactions, the amphiphilic boundary may attract an excessive amount of IL cations, which guide the unique orientations of the Janus spheres. The results highlight the importance of the Janus boundary that has not been recognized previously. Adsorption at the solid-solid interfaces may inspire new applications in areas such as separation and catalysis.
When attempting to integrate single-molecule fluorescence microscopy with microfabricated devices such as microfluidic channels, fabrication constraints may prevent using traditional coverslips. Instead, the fabricated devices may require imaging through material with a different thickness or index of refraction. Altering either can easily reduce the quality of the image formation (measured by the Strehl ratio) by a factor of 2 or more, reducing the signal-to-noise ratio accordingly. In such cases, successful detection of single-molecule fluorescence may prove difficult or impossible. Here we provide software to calculate the effect of non-design materials upon the Strehl ratio or ensquared energy and explore the impact of common materials used in microfabrication.
Intracellular cargos are transported by molecular motors along actin and microtubules, but how their dynamics depends on the complex structure of the cytoskeletal network remains unclear. In this study, we investigated this longstanding question by measuring simultaneously the rotational and translational dynamics of cargos at microtubule intersections in living cells. We engineered two-faced particles that are fluorescent on one hemisphere and opaque on the other and used their optical anisotropy to report the rotation of cargos. We show that cargos undergo brief episodes of unidirectional and rapid rotation while pausing at microtubule intersections. Probability and amplitude of the cargo rotation depend on the geometry of the intersecting filaments. The cargo rotation is not random motion due to detachment from microtubules, as revealed by statistical analyses of the translational and rotational dynamics. Instead, it is an active rotation driven by motor proteins. Although cargos are known to pause at microtubule intersections, this study reveals a different dimension of dynamics at this seemingly static state and, more importantly, provides direct evidence showing the correlation between cargo rotation and the geometry of underlying microtubule intersections.
Endosomes in cells are known to move directionally along microtubules, but their rotational dynamics have rarely been investigated. Even less is known, specifically, about the rotation of nonspherical endosomes. Here we report a single-Janus rod rotational tracking study to reveal the rich rotational dynamics of rod-shaped endosomes in living cells. The rotational reporters were Janus rods that display patches of different fluorescent colors on opposite sides along their long axes. When the Janus rods are wrapped tightly inside endosomes, their shape and optical anisotropy allow the simultaneous measurements of all three rotational angles (in-plane, out-of-plane, and longitudinal) and the translational motion of single endosomes with high spatiotemporal resolutions. We demonstrate that endosomes undergo in-plane rotation and rolling during intracellular transport and that such rotational dynamics are driven by rapid microtubule fluctuations. We reveal for the first time the "rock-and-roll" of endosomes in living cells and how the intracellular environment modifies such rotational dynamics. This study demonstrates a unique application of Janus particles as imaging probes in the elucidation of fundamental biological questions.
Miller, Kyle; Tsyrenova, Ayuna; Anthony, Stephen M.; Qin, Shiyi; Yong, Xin; Jiang, Shan
Amphiphilic Janus particles demonstrate unique assembly structures when dried on a hydrophilic substrate. Particle orientations are influenced by amphiphilicity and Janus balance. A three-stage model is developed to describe the process. Simulation further indicates the dominant force is capillary attraction due to the interface pinning at rough Janus boundaries.
Cosmic ray spikes are especially problematic for hyperspectral imaging because of the large number of spikes often present and their negative effects upon subsequent chemometric analysis. Fortunately, while the large number of spectra acquired in a hyperspectral imaging data set increases the probability and number of cosmic spikes observed, the multitude of spectra can also aid in the effective recognition and removal of the cosmic spikes. Zhang and Ben-Amotz were perhaps the first to leverage the additional spatial dimension of hyperspectral data matrices (DM). They integrated principal component analysis (PCA) into the upper bound spectrum method (UBS), resulting in a hybrid method (UBS-DM) for hyperspectral images. Here, we expand upon their use of PCA, recognizing that principal components primarily present in only a few pixels most likely correspond to cosmic spikes. Eliminating the contribution of those principal components in those pixels improves the cosmic spike removal. Both simulated and experimental hyperspectral Raman image data sets are used to test the newly developed UBS-DM-hyperspectral (UBS-DM-HS) method which extends the UBS-DM method by leveraging characteristics of hyperspectral data sets. A comparison is provided between the performance of the UBS-DM-HS method and other methods suitable for despiking hyperspectral images, evaluating both their ability to remove cosmic ray spikes and the extent to which they introduce spectral bias.
Chambers, Mariah; Mallory, Stewart A.; Malone, Heather; Gao, Yuan; Anthony, Stephen M.; Yi, Yi; Cacciuto, Angelo; Yu, Yan
Amphiphilic Janus particles self-assemble into complex metastructures, but little is known about how their assembly might be modified by weak interactions with a nearby biological membrane surface. Here, we report an integrated experimental and molecular dynamics simulation study to investigate the self-assembly of amphiphilic Janus particles on a lipid membrane. We created an experimental system in which Janus particles are allowed to self-assemble in the same medium where zwitterionic lipids form giant unilamellar vesicles (GUVs). Janus particles spontaneously concentrated on the inner leaflet of the GUVs. They exhibited biased orientation and heterogeneous rotational dynamics as revealed by single particle rotational tracking. The combined experimental and simulation results show that Janus particles concentrate on the lipid membranes due to weak particle-lipid attraction, whereas the biased orientation of particles is driven predominantly by inter-particle interactions. This study demonstrates the potential of using lipid membranes to influence the self-assembly of Janus particles.
We investigated the rotational dynamics of single microparticles during their internalization by macrophage cells. The microparticles used were triblock patchy particles that display two fluorescent patches on their two poles. The optical anisotropy made it possible to directly visualize and quantify the orientation and rotation of the particles. We show that particles exhibit a mixture of fast and slow rotation as they are uptaken by macrophages and transiently undergo directional rotation during their entry into the cell. The size of the particles and the surface presentation of ligands exerted a negligible influence on this heterogeneity of particle rotation.
Photosynthetic organisms use complex and regulated multichromophore assemblies, called lightharvesting (LH) antennas, to capture, concentrate and direct solar radiation to reaction centers that then carry out concomitant chemistry. Nature’s LH antennas are remarkable, operating with high efficiency in fluctuating environmental and photic conditions as well as being assembled with nanoscale precision thus, they often serve as inspiration in material design. The presented work was inspired by a natural LH antenna. We show that a diblock copolymer amphiphile enables the generation and integration of optically dense chromophore arrays, within a biomimetic polymer membrane. The entire construct is solution-processable, scalable and exhibits intra and inter-supramolecular energy transfer in a completely noncovalent design. This work demonstrates the potential of polymer membrane materials in generating spatial-energetic landscapes for photonic applications.