Serine incorporator protein 5 (SERINC5) is the host anti-retroviral factor that reduces HIV-1 infectivity by incorporating into virions and inhibiting the envelope glycoprotein (Env) mediated virus fusion with target cells. We and others have shown that SERINC5 incorporation into virions alters the Env structure and sensitizes the virus to broadly neutralizing antibodies targeting cryptic Env epitopes. We have also found that SERINC5 accelerates the loss of Env function over time compared to control viruses. However, the exact mechanism by which SERINC5 inhibits HIV-1 fusion is not understood. Here, we utilized 2D and 3D super-resolution microscopy to examine the effect of SERINC5 on the distribution of Env glycoproteins on single HIV-1 particles. We find that, in agreement with a previous report, Env glycoproteins form clusters on the surface of mature virions. Importantly, incorporation of SERINC5, but not SERINC2, which lacks antiviral activity, disrupted Env clusters without affecting the overall Env content. We also show that SERINC5 and SERINC2 also form clusters on single virions. Unexpectedly, Env and SERINCs molecules exhibited poor co-distribution on virions, as evidenced by much greater Env-SERINC pairwise distances compare to Env-Env distances. This observation is inconsistent with the previously reported interaction between Env and SERINC5 and suggests an indirect effect of SERINC5 on Env cluster formation. Collectively, our results reveal a multifaceted mechanism of SERINC5-mediated restriction of HIV-1 fusion that, aside from the effects on individual Env trimers, involves disruption of Env clusters, which likely serve as sites of viral fusion with target cells.

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from mRNA and the transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

Information within the brain travels from neuron to neuron across synapses. At any given moment, only a few synapses within billions will be active and are thought to transmit key information about the environment, a behavior being executed or memory being recalled. Here we present SynTagMA, which marks active synapses within a ~2 s time window. Upon violet illumination, the genetically expressed tag converts from green to red fluorescence if bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we developed an analysis program that automatically identifies and tracks the fluorescence of thousands of individual synapses in tissue. Together, these tools provide a high throughput method for repeatedly mapping active synapses in vitro and in vivo.

In this work, we address the problem of precisely localizing key frames of an action, for example, the precise time that a pitcher releases a baseball, or the precise time that a crowd begins to applaud. Key frame localization is a largely overlooked and important action-recognition problem, for example in the field of neuroscience, in which we would like to understand the neural activity that produces the start of a bout of an action. To address this problem, we introduce a novel structured loss function that properly weights the types of errors that matter in such applications: it more heavily penalizes extra and missed action start detections over small misalignments. Our structured loss is based on the best matching between predicted and labeled action starts. We train recurrent neural networks (RNNs) to minimize differentiable approximations of this loss. To evaluate these methods, we introduce the Mouse Reach Dataset, a large, annotated video dataset of mice performing a sequence of actions. The dataset was collected and labeled by experts for the purpose of neuroscience research. On this dataset, we demonstrate that our method outperforms related approaches and baseline methods using an unstructured loss.

Tissue repair is a complex process that requires effective communication and coordination between cells across multiple tissues and organ systems. Two of the initial intracellular signals that encode injury signals and initiate tissue repair responses are calcium and extracellular signal-regulated kinase (ERK). However, calcium and ERK signaling control a variety of cellular behaviors important for injury repair including cellular motility, contractility, and proliferation, as well as the activity of several different transcription factors, making it challenging to relate specific injury signals to their respective repair programs. This knowledge gap ultimately hinders the development of new wound healing therapies that could take advantage of native cellular signaling programs to more effectively repair tissue damage. The objective of this review is to highlight the roles of calcium and ERK signaling dynamics as mechanisms that link specific injury signals to specific cellular repair programs during epithelial and stromal injury repair. We detail how the signaling networks controlling calcium and ERK can now also be dissected using classical signal processing techniques with the advent of new biosensors and optogenetic signal controllers. Finally, we advocate the importance of recognizing calcium and ERK dynamics as key links between injury detection and injury repair programs that both organize and execute a coordinated tissue repair response between cells across different tissues and organs. This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Biological Mechanisms > Cell Signaling Laboratory Methods and Technologies > Imaging Models of Systems Properties and Processes > Organ, Tissue, and Physiological Models.

Brain networks that mediate motivated behavior in the context of aversive and rewarding experiences involve the prefrontal cortex (PFC) and ventral tegmental area (VTA). Neurons in both regions are activated by stress and reward, and by learned cues that predict aversive or appetitive outcomes. Recent studies have proposed that separate neuronal populations and circuits in these regions encode learned aversive versus appetitive contexts. But how about the actual experience? Do the same or different PFC and VTA neurons encode unanticipated aversive and appetitive experiences? To address this, we recorded unit activity and local field potentials (LFP) in the dorsomedial PFC (dmPFC) and VTA of male rats as they were exposed, in the same recording session, to reward (sucrose) or stress (tail pinch) spaced one hour apart. As expected, experience-specific neuronal responses were observed. About 15-25% of single units in each region responded by excitation or inhibition to either stress or reward, and only stress increased LFP theta oscillation power in both regions and coherence between regions. But the largest number of responses (29% dmPFC and 30% VTA units) involved dual-valence neurons that responded to both stress and reward exposure. Moreover, the temporal profile of neuronal population activity in dmPFC and VTA as assessed by principal component analysis were similar during both types of experiences. These results reveal that aversive and rewarding experiences engage overlapping neuronal populations in the dmPFC and the VTA. These populations may provide a locus of vulnerability for stress related disorders, which are often associated with anhedonia. Animals must recognize unexpected harmful and rewarding events in order to survive. How the brain represents these competing experiences is not fully understood. Two interconnected brain regions implicated in encoding both rewarding and stressful events are the dmPFC and the VTA. In either region, separate neurons and associated circuitry are assumed to respond to events with positive or negative valence. We find, however, that a significant subpopulation of neurons in dmPFC and VTA encode both rewarding and aversive experiences. These dual-valence neurons may provide a computational advantage for flexible planning of behavior when organisms face unexpected rewarding and harmful experiences.

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, we investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and D. gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), we identified a ∼440 kb region on the X chromosome that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior. This article is protected by copyright. All rights reserved.

In Neuroscience, the structure of a circuit has often been used to intuit function - an inversion of Louis Kahn's famous dictum, `Form follows function' (Kristan and Katz 2006). However, different brain networks may utilize different network architectures to solve the same problem. The olfactory circuits of two insects, the Locust, and the fruit fly, , serve the same function - to identify and discriminate odors. The neural circuitry that achieves this shows marked structural differences. Projection neurons (PN) in the antennal lobe (AL) innervate Kenyon cells (KC) of the mushroom body (MB). In locust, each KC receives inputs from ∼50% PNs, a scheme that maximizes the difference between inputs to any two of ∼50,000 KCs. In contrast, in drosophila, this number is only 5% and appears sub-optimal. Using a computational model of the olfactory system, we show the activity of KCs is sufficiently high-dimensional that it can separate similar odors regardless of the divergence of PN-KC connections. However, when temporal patterning encodes odor attributes, dense connectivity outperforms sparse connections.Increased separability comes at the cost of reliability. The disadvantage of sparse connectivity can be mitigated by incorporating other aspects of circuit architecture seen in drosophila. Our simulations predict that drosophila and locust circuits lie at different ends of a continuum where the drosophila gives up on the ability to resolve similar odors to generalize across varying environments, while the locust separates odor representations but risks misclassifying noisy variants of the same odor. How does the structure of a network affect its function? We address this question in the context of two olfactory systems that serve the same function, to distinguish the attributes of different odorants, but do so using markedly distinct architectures. In the locust, the probability of connections between projection neurons and Kenyon cells - a layer downstream - is nearly 50%. In contrast, this number is merely 5% in drosophila. We developed computational models of these networks to understand the relative advantages of each connectivity. Our analysis reveals that the two systems exist along a continuum of possibilities that balance two conflicting goals - separating the representations of similar odors while grouping together noisy variants of the same odor.