Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

Understanding live-cell behavior in part requires high precision mapping of molecular species in 3-D dynamic environments. Approaches like single-molecule localization microscopy (SMLM) offer high promise for challenges posed by molecular cartography. Effectively, the precision of these approaches is dependent on the how many photons / second a fluorescent marker is capable of emitting. For this reason, many SRLM experiments are typically done using fluorescent organic dyes (such as Alexa Fluors) in reducing chemical environments which cause some organic dyes to stochastically cycle through dark states, allowing single-molecule localization (e.g. (d)STORM). The need to couple these dyes to antibodies and the harsh reducing conditions makes their application to live cell work problematic. To overcome these limitations, we made use of modifications to Janelia Fluor-based dyes which make them spontaneously cycle through dark states (blink) under physiological imaging conditions. The dyes are spectrally compatible with photo-activatable fluorescent proteins such as mEos and allow for simultaneous 2-color superresolution microscopy. When conjugated to a HaloTag, these artificial dyes can bind genetically encodable targets in live samples, allowing subsequent measurement in a live-cell environment. To correct for nanoscale chromatic aberrations we developed a new machine-learning based approach with reconstruction errors below achievable localization precisions. We show that these methods allow the reconstruction of live synapse surfaces and a variety of the associated molecular machineries with up to 50 nm accuracy in 3 dimensions.

Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data, and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

Most traditional optical biosensors operate through molecular recognition, where ligand binding causes conformational changes that lead to optical perturbations in the emitting motif. Optical sensors developed from single-stranded DNA-functionalized single-walled carbon nanotubes (ssDNA–SWCNTs) have started to make useful contributions to biological research. However, the mechanisms underlying their function have remained poorly understood. In this study, we combine experimental and computational approaches to show that ligand binding alone is not sufficient for optical modulation in this class of synthetic biosensors. Instead, the optical response that occurs after ligand binding is highly dependent on the chemical properties of the ligands, resembling mechanisms seen in activity-based biosensors. Specifically, we show that in ssDNA–SWCNT catecholamine sensors, the optical response correlates positively with the electron density on the aryl motif, even among ligands with similar ligand binding affinities. Importantly, despite the strong correlations with electrochemical properties, we find that catechol oxidation itself is not necessary to drive the sensor optical response. We discuss how these findings could serve as a framework for tuning the performance of existing sensors and guiding the development of new biosensors of this class.

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.