A magnetically powered nanomachine with a DNA clutch.

Machines found in nature and human-made machines share common components, such as an engine, and an output element, such as a rotor, linked by a clutch. This clutch, as seen in biological structures such as dynein, myosin or bacterial flagellar motors, allows for temporary disengagement of the moving parts from the running engine. However, such sophistication is still challenging to achieve in artificial nanomachines. Here we present a spherical rotary nanomotor with a reversible clutch system based on precise molecular recognition of built-in DNA strands. The clutch couples and decouples the engine from the machine's rotor in response to encoded inputs such as DNA or RNA. The nanomotor comprises a porous nanocage as a spherical rotor to confine the magnetic engine particle within the nanospace (∼0.004 µm3) of the cage. Thus, the entropically driven irreversible disintegration of the magnetic engine and the spherical rotor during the disengagement process is eliminated, and an exchange of microenvironmental inputs is possible through the nanopores. Our motor is only 200 nm in size and the clutch-mediated force transmission powered by an embedded ferromagnetic nanocrystal is high enough (∼15.5 pN at 50 mT) for the in vitro mechanical activation of Notch and integrin receptors, demonstrating its potential as nano-bio machinery.

Pre-organized landscape of T cell surface.

T cell activation is initiated by the recognition of specific antigenic peptides and subsequently accomplished by complex signaling cascades. These aspects have been extensively studied for decades as pivotal factors in the establishment of adaptive immunity. However, how receptors or signaling molecules are organized in the resting state prior to encountering antigens has received less attention. Recent advancements in super-resolution microscopy techniques have revealed topographically controlled pre-formed organization of key molecules involved in antigen recognition and signal transduction on microvillar projections of T cells before activation and substantial effort has been dedicated to characterizing the topological structure of resting T cells over the past decade. This review will summarize our current understanding of how key surface receptors are pre-organized on the T-cell plasma membrane and discuss the potential role of these receptors, which are preassembled prior to ligand binding in the early activation events of T cells.

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling.

Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production.

Atherosclerosis is an inflammatory disease of the artery walls and involves immune cells such as macrophages. Olfactory receptors (OLFRs) are G protein­coupled chemoreceptors that have a central role in detecting odorants and the sense of smell. We found that mouse vascular macrophages express the olfactory receptor Olfr2 and all associated trafficking and signaling molecules. Olfr2 detects the compound octanal, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and induces interleukin-1ß secretion in human and mouse macrophages. We found that human and mouse blood plasma contains octanal, a product of lipid peroxidation, at concentrations sufficient to activate Olfr2 and the human ortholog olfactory receptor 6A2 (OR6A2). Boosting octanal levels exacerbated atherosclerosis, whereas genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques. Our findings suggest that inhibiting OR6A2 may provide a promising strategy to prevent and treat atherosclerosis.

CD45 pre-exclusion from the tips of T cell microvilli prior to antigen recognition.

The tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for T cell receptor (TCR) signal transduction. Here, we use expansion super-resolution microscopy to reveal that CD45 is mostly pre-excluded from the tips of microvilli (MV) on primary T cells prior to antigen encounter. This pre-exclusion is diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration length, but is independent of the CD45 extracellular domain. We further show that brief MV-mediated contacts can induce Ca2+ influx in mouse antigen-specific T cells engaged by antigen-pulsed antigen presenting cells (APC). We propose that the scarcity of CD45 phosphatase activity at the tips of MV enables or facilitates TCR triggering from brief T cell-APC contacts before formation of a stable IS, and that these MV-mediated contacts represent the earliest step in the initiation of a T cell adaptive immune response.

Control over size, shape, and photonics of self-assembled organic nanocrystals.

The facile fabrication of free-floating organic nanocrystals (ONCs) was achieved via the kinetically controlled self-assembly of simple perylene diimide building blocks in aqueous medium. The ONCs have a thin rectangular shape, with an aspect ratio that is controlled by the content of the organic cosolvent (THF). The nanocrystals were characterized in solution by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering. The ONCs retain their structure upon drying, as was evidenced by TEM and atom force microscopy. Photophysical studies, including femtosecond transient absorption spectroscopy, revealed a distinct influence of the ONC morphology on their photonic properties (excitation energy transfer was observed only in the high-aspect ONCs). Convenient control over the structure and function of organic nanocrystals can enhance their utility in new and developed technologies.

Circulating T cell-monocyte complexes are markers of immune perturbations.

Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artifacts and thus ignored in data acquisition and analysis, are the result of biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell-monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.

A synthetic planar cell polarity system reveals localized feedback on Fat4-Ds1 complexes.

The atypical cadherins Fat and Dachsous (Ds) have been found to underlie planar cell polarity (PCP) in many tissues. Theoretical models suggest that polarity can arise from localized feedbacks on Fat-Ds complexes at the cell boundary. However, there is currently no direct evidence for the existence or mechanism of such feedbacks. To directly test the localized feedback model, we developed a synthetic biology platform based on mammalian cells expressing the human Fat4 and Ds1. We show that Fat4-Ds1 complexes accumulate on cell boundaries in a threshold-like manner and exhibit dramatically slower dynamics than unbound Fat4 and Ds1. This suggests a localized feedback mechanism based on enhanced stability of Fat4-Ds1 complexes. We also show that co-expression of Fat4 and Ds1 in the same cells is sufficient to induce polarization of Fat4-Ds1 complexes. Together, these results provide direct evidence that localized feedbacks on Fat4-Ds1 complexes can give rise to PCP.

Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies.

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.