Acetylation of α-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here, we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35 Å TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule interprotofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes.

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.

Phenotypic plasticity allows organisms to quickly adapt in response to changing environments. Little is known of the genetic, environmental and epigenetic contribution to the expression of alternative adaptive developmental outcomes. We study aphid polyphenisms, which offer a unique, compelling opportunity to study multiple levels of biological organization, especially insect epigenetics. The pea aphid, Acyrthosiphon pisum, exhibits an adaptive reproductive polyphenism whereby genetically identical individuals reproduce either sexually (meiosis) or asexually (parthenogenesis) depending on environmental conditions during maternal development (short or long photoperiod, respectively). To understand how facultative asexuality evolved in aphids, we first determined meiosis gene activity in sexuals and asexuals. I determined that the pea aphid genome encodes single copies of homologs for the majority of the core meiotic machinery, suggesting that meiotic plasticity is not due simply to gene loss or expansion. Next, we determined if these core meiosis genes are expressed using PCR spanning across at least one intron from cDNA isolated from asexual and sexual ovaries. Surprisingly, meiosis specific genes (e.g., Spo11, Msh4, Msh5, Hop2 and Mnd1) are expressed in not only in asexual ovaries but also in somatic tissue and an obligately asexual aphid strain. Interestingly, the Spo11 PCR product contained intronic sequence, thus representing unspliced mRNA. Germline expression of Spo11, Mnd1 and Hop2 was confirmed by in situ analysis. Preliminary results identified candidate methylation sites in the Spo11 locus, indicating an epigenetic basis for this expression difference. Further characterization will help us better understand the molecular and epigenetic mechanisms underlying this adaptive facultative plasticity.

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.

Meiosis is a highly conserved process in which a diploid genome is recombined and assorted into haploid gametes. Remarkably, the pea aphid Acyrthosiphon pisum exhibits a reproductive polyphenism whereby environmental signals trigger a switch between apomixis (parthenogenetic reproduction) and meiosis (sexual reproduction). Aphid apomixis results in daughter embryo clones with 2n genome content without male contribution or recombination. This important adaptation allows aphid populations to not only rapidly expand upon abundant resources during summer but also survive winter. How aphids have evolved this ability to switch between parthenogenesis and sexual meiosis is unknown. To arrive at a mechanistic explanation for this developmental plasticity, I determined meiosis gene activity in sexuals and asexuals. I first identified homologs of a core set of meiosis genes from the pea aphid genome. Next, I tested the expression of these core meiosis genes by PCR spanning across at least one intron from cDNA isolated from asexual and sexual ovaries. Surprisingly, meiosis specific genes (e.g., Spo11, Msh4, Msh5, Hop2 and Mnd1) are expressed in asexual ovaries. Additionally, the Spo11 PCR product contained intronic sequence, thus representing unspliced mRNA. Future experiments looking at the quantities and localizations of mRNA and protein will help to distinguish among several possible explanations for these results. Further molecular characterization of this phenotypic plasticity will be helpful in understanding how multiple interacting pathways can evolve to create alternate developmental phenotypes.

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.

Recent studies suggest that the fly uses the inositol lipid signaling system for visual excitation and that the Drosophila transient receptor potential (trp) mutation disrupts this process subsequent to the production of IP3. In this paper, we show that trp encodes a novel 1275 amino acid protein with eight putative transmembrane segments. Immunolocalization indicates that the trp protein is expressed predominantly in the rhabdomeric membranes of the photoreceptor cells.

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.