We examined the encoding and decoding of odor identity and intensity by neurons in the antennal lobe and the mushroom body, first and second relays, respectively, of the locust olfactory system. Increased odor concentration led to changes in the firing patterns of individual antennal lobe projection neurons (PNs), similar to those caused by changes in odor identity, thus potentially confounding representations for identity and concentration. However, when these time-varying responses were examined across many PNs, concentration-specific patterns clustered by identity, resolving the apparent confound. This is because PN ensemble representations changed relatively continuously over a range of concentrations of each odorant. The PNs’ targets in the mushroom body-Kenyon cells (KCs)-had sparse identity-specific responses with diverse degrees of concentration invariance. The tuning of KCs to identity and concentration and the patterning of their responses are consistent with piecewise decoding of their PN inputs over oscillation-cycle length epochs.

Eukaryotic cells are organized into membrane-bound organelles. These organelles communicate with one another through vesicular trafficking pathways and membrane contact sites (MCSs). MCSs are sites of close apposition between two or more organelles that play diverse roles in the exchange of metabolites, lipids and proteins. Organelle interactions at MCSs also are important for organelle division and biogenesis. For example, the division of several organelles, including mitochondria and endosomes, seem to be regulated by contacts with the endoplasmic reticulum (ER). Moreover, the biogenesis of autophagosomes and peroxisomes involves contributions from the ER and multiple other cellular compartments. Thus, organelle-organelle interactions allow cells to alter the shape and activities of their membrane-bound compartments, allowing them to cope with different developmental and environmental conditions.

R7 UV photoreceptors (PRs) are divided into yellow (y) and pale (p) subtypes. yR7 PRs express the Dpr11 cell surface protein and are presynaptic to Dm8 amacrine neurons (yDm8) that express Dpr11's binding partner DIP-g, while pR7 PRs synapse onto DIP-g-negative pDm8. Dpr11 and DIP-g expression patterns define 'yellow' and 'pale' color vision circuits. We examined Dm8 neurons in these circuits by electron microscopic reconstruction and expansion microscopy. and mutations affect the morphologies of yDm8 distal ('home column') dendrites. yDm8 neurons are generated in excess during development and compete for presynaptic yR7 PRs, and interactions between Dpr11 and DIP-g are required for yDm8 survival. These interactions also allow yDm8 neurons to select yR7 PRs as their appropriate home column partners. yDm8 and pDm8 neurons do not normally compete for survival signals or R7 partners, but can be forced to do so by manipulation of R7 subtype fate.

The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature.

The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.

In the field of biomedical imaging analysis on single-cell level, reliable and fast segmentation of the cell nuclei from the background on three-dimensional images is highly needed for the further analysis. In this work we propose an interactive cell segmentation toolkit that first establishes a set of exemplar regions from user input through an easy and intuitive interface in both 2D and 3D in real-time, then
extracts the shape and intensity features from those exemplars. Based on a local contrast-constrained region growing scheme, each connected component in the whole image would be filtered by the features from exemplars, forming an “exemplar-matching” group which passed the filtering and would be part of the final segmentation result, and a “non-exemplar-matching” group in which components
would be further segmented by the gradient vector field based algorithm. The results of the filtering process are visualized back to the user in near real-time, thus enhancing the experience in exemplar selecting and parameter tuning. The toolkit is distributed as a plugin within the open source Vaa3D system (http://vaa3d.org).

The point spread function (PSF) is fundamental to any type of microscopy, most importantly so for single-molecule localization techniques, where the exact PSF shape is crucial for precise molecule localization at the nanoscale. Optical aberrations and fixed fluorophore dipoles often result in non-isotropic and distorted PSFs, impairing and biasing conventional fitting approaches. Further, PSF shapes are deliberately modified in PSF engineering approaches for providing improved sensitivity, e.g., for 3D localization or determination of dipole orientation. As this can lead to highly complex PSF shapes, a tool for visualizing expected PSFs would facilitate the interpretation of obtained data and the design of experimental approaches. To this end, we introduce a comprehensive and accessible computer application that allows for the simulation of realistic PSFs based on the full vectorial PSF model. Our tool incorporates a wide range of microscope and fluorophore parameters, including orientationally constrained fluorophores, as well as custom aberrations, transmission and phase masks, thus enabling an accurate representation of various imaging conditions. An additional feature is the simulation of crowded molecular environments with overlapping PSFs. Further, our app directly provides the Cramér–Rao bound for assessing the best achievable localization precision under given conditions. Finally, our software allows for the fitting of custom aberrations directly from experimental data, as well as the generation of a large dataset with randomized simulation parameters, effectively bridging the gap between simulated and experimental scenarios, and enhancing experimental design and result validation.

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation and Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

RNAs have been shown to undergo transfer between mammalian cells, although the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g., exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions characteristic of membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple coculture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse β-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all coculture models tested (e.g., between primary cells, immortalized cells, and in cocultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin but is independent of de novo protein synthesis and is modulated by stress conditions and gene-expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues as well as for host-parasite or symbiotic interactions.

Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.