Cell recognition requires interactions through molecules located on cell surface. The insect homolog of Down syndrome cell adhesion molecule (Dscam) manifests huge molecular diversity in its extracellular domain. High-affinity Dscam-Dscam interactions only occur between isoforms that carry identical extracellular domains. Homophilic Dscam signaling can, thus, vary in strength depending on the compositions of Dscams present on the opposing cell surfaces. Dscam abundantly exists in the developing nervous system and governs arborization and proper elaboration of neurites. Notably, individual neurons may stochastically and dynamically express a small subset of Dscam isoforms such that any given neurite can be endowed with a unique repertoire of Dscams. This allows individual neurites to recognize their sister branches. Self-recognition leads to self-repulsion, ensuring divergent migration of sister processes. By contrast, weak homophilic Dscam interactions may promote fasciculation of neurites that express analogous, but not identical, Dscams. Differential Dscam binding may provide graded cell recognition that in turn governs complex neuronal morphogenesis.

Glucose transporters (GLUTs) provide a pathway for glucose transport across membranes. Human GLUTs are implicated in devastating diseases such as heart disease, hyper- and hypo-glycemia, type 2 diabetes and caner. The human GLUT1 has been recently crystalized in the inward-facing open conformation. However, there is no other structural information for other conformations. The X-ray structures of E. coli Xylose permease (XylE), a glucose transporter homolog, are available in multiple conformations with and without the substrates D-xylose and D-glucose. XylE has high sequence homology to human GLUT1 and key residues in the sugar-binding pocket are conserved. Here we construct a homology model for human GLUT1 based on the available XylE crystal structure in the partially occluded outward-facing conformation. A long unbiased all atom molecular dynamics simulation starting from the model can capture a new fully opened outward-facing conformation. Our investigation of molecular interactions at the interface between the transmembrane (TM) domains and the intracellular helices (ICH) domain in the outward- and inward-facing conformation supports that the ICH domain likely stabilizes the outward-facing conformation in GLUT1. Furthermore, inducing a conformational transition, our simulations manifest a global asymmetric rocker switch motion and detailed molecular interactions between the substrate and residues through the water-filled selective pore along a pathway from the extracellular to the intracellular side. The results presented here are consistent with previously published biochemical, mutagenesis and functional studies. Together, this study shed light on the structure and functional relationships of GLUT1 in multiple conformational states.

Coordinated motor behaviors depend on feedback communication between peripheral sensory systems and central circuits in the brain and spinal cord. Relay of muscle and tendon-derived sensory information to the CNS is facilitated by functionally and anatomically diverse groups of spinocerebellar tract neurons (SCTNs), but the molecular logic by which SCTN diversity and connectivity is achieved is poorly understood. We used single cell RNA sequencing and genetic manipulations to define the mechanisms governing the molecular profile and organization of SCTN subtypes. We found that SCTNs relaying proprioceptive sensory information from limb and axial muscles are generated through segmentally-restricted actions of specific Hox genes. Loss of Hox function disrupts SCTN subtype-specific transcriptional programs, leading to defects in the connections between proprioceptive sensory neurons, SCTNs, and the cerebellum. These results indicate that Hox-dependent genetic programs play essential roles in the assembly of the neural circuits required for proprioception.

Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.

Synapses have undergone significant diversification and adaptation, contributing to the complexity of the central nervous system. Understanding their molecular architecture is essential for deciphering the brain's functional evolution. While nicotinic acetylcholine receptors (nAchRs) are widely distributed across metazoan brains, their associated protein networks remain poorly characterized. Using in vivo proximity labeling, we generated proteomic maps of subunit-specific nAchR interactomes in developing and mature brains. Our findings reveal a developmental expansion and reconfiguration of the nAchR interactome. Proteome profiling with genetic perturbations showed that removing individual nAchR subunits consistently triggers compensatory shifts in receptor subtypes, highlighting mechanisms of synaptic plasticity. We also identified the Rho-GTPase regulator Still life (Sif) as a key organizer of cholinergic synapses, with loss of Sif disrupting their molecular composition and structural integrity. These results provide molecular insights into the development and plasticity of central cholinergic synapses, advancing our understanding of synaptic identity conservation and divergence.

In mammals, subplate neurons (SPNs) are among the first generated cortical neurons. While most SPNs exist only transiently during development, a number of SPNs persist among adult Layer 6b (L6b). During development, SPNs receive thalamic and intra-cortical input, and primarily project to Layer 4 (L4). SPNs are critical for the anatomical and functional development of thalamocortical connections and also pioneer corticothalamic projections. Since SPNs are heterogeneous, SPN subpopulations might serve different roles. Here, we investigate the connectivity of one subpopulation, complexin-3 (Cplx3)-positive SPNs (Cplx3-SPNs), in mouse whisker somatosensory (barrel) cortex (S1). We find that many Cplx3-SPNs survive into adulthood and become a subpopulation of L6b. Cplx3-SPNs axons project to thalamorecipient layers, that is, L4, 5a, and 1. The L4 projections are biased towards the septal regions between barrels in the second postnatal week. Thus, S1 Cplx3-SPN targets co-localize with the eventual projections of the medial posterior thalamic nucleus (POm). In addition to their cortical targets, Cplx3-SPNs also extend long-range axons to several thalamic nuclei, including POm. Thus, Cplx3-SPN/L6b neurons are associated with paralemniscal pathways and can potentially directly link thalamocortical and corticothalamic circuits. This suggests an additional key role for SPNs in the establishment and maintenance of thalamocortical processing.

This protocol describes how to apply appropriate pharmacological controls to induce mitochondrial fusion or fission in studies of mitochondria morphology for four different mammalian cell types, HepG2 human liver hepatocellular carcinoma cells, MCF7 human breast adenocarcinoma cells, HEK293 human embryonic kidney cells, and collagen sandwich culture of primary rat hepatocytes. The protocol provides methods of treating cells with these pharmacological controls, staining mitochondria with commercially available MitoTracker Green and TMRE dyes, and imaging the mitochondrial morphology in live cells using a confocal fluorescent microscope. It also describes the cell culture methods needed for this protocol. © 2018 by John Wiley & Sons, Inc.

Insects, like most animals, tend to steer away from imminent threats [1-7]. Drosophila melanogaster, for example, generally initiate an escape take-off in response to a looming visual stimulus, mimicking a potential predator [8]. The escape response to a visual threat is, however, flexible [9-12] and can alternatively consist of walking backward away from the perceived threat [11], which may be a more effective response to ambush predators such as nymphal praying mantids [7]. Flexibility in escape behavior may also add an element of unpredictability that makes it difficult for predators to anticipate or learn the prey's likely response [3-6]. Whereas the fly's escape jump has been well studied [8, 9, 13-18], the neuronal underpinnings of evasive walking remain largely unexplored. We previously reported the identification of a cluster of descending neurons-the moonwalker descending neurons (MDNs)-the activity of which is necessary and sufficient to trigger backward walking [19], as well as a population of visual projection neurons-the lobula columnar 16 (LC16) cells-that respond to looming visual stimuli and elicit backward walking and turning [11]. Given the similarity of their activation phenotypes, we hypothesized that LC16 neurons induce backward walking via MDNs and that turning while walking backward might reflect asymmetric activation of the left and right MDNs. Here, we present data from functional imaging, behavioral epistasis, and unilateral activation experiments that support these hypotheses. We conclude that LC16 and MDNs are critical components of the neural circuit that transduces threatening visual stimuli into directional locomotor output.

The accelerating pace of technological advancements necessitates specialised expertise and cutting-edge instruments to maintain competitive research in life sciences. Core facilities - collaborative laboratories equipped with state-of-the-art tools and staffed by expert personnel - are vital resources that support diverse scientific endeavours. However, their adoption in lower-income communities has been comparatively stagnant due to both financial and cultural challenges. This paper explores the perils of not supporting core facilities on national research enterprises, underscoring the need for balanced investments in discovery science and crucial infrastructure support. We explore the implications from the perspectives of funders, university leaders and lab heads. We advocate for a paradigm shift to recognise these facilities as essential components of national research efforts. Core facilities are positioned not as optional but as strategic investments that can catalyse breakthroughs, particularly in environments with limited resources.

The accelerating pace of technological advancements necessitates specialised expertise and cutting-edge instruments to maintain competitive research in life sciences. Core facilities - collaborative laboratories equipped with state-of-the-art tools and staffed by expert personnel - are vital resources that support diverse scientific endeavours. However, their adoption in lower-income communities has been comparatively stagnant due to both financial and cultural challenges. This paper explores the perils of not supporting core facilities on national research enterprises, underscoring the need for balanced investments in discovery science and crucial infrastructure support. We explore the implications from the perspectives of funders, university leaders and lab heads. We advocate for a paradigm shift to recognise these facilities as essential components of national research efforts. Core facilities are positioned not as optional but as strategic investments that can catalyse breakthroughs, particularly in environments with limited resources.