Biology subjects

Wolfe, A. D.

Publications and source records attributed to Wolfe, A. D..

11 recordsLinked to original sources

bioRxiv2026

iG6PSnFR: A genetically encoded fluorescent sensor for observing glucose-6-phosphate dynamics in living preparations

Glucose-6-phosphate (G6P) is a key intermediate in multiple energetic and anabolic pathways, and quantifying its dynamics is essential for understanding cellular physiology. We have previously developed a syndicate of intensity-based, genetically encoded sensors based on the insertion of circularly permuted GFP into a Venus-flytrap-like analyte-binding protein. Here we use the same approach to develop an intensity-based G6P Sensing Fluorescent Reporter (iG6PSnFR). We present two variants: a G6P-activated sensor that increases fluorescence and a G6P-inactivated sensor that decreases fluorescence. We validate performance across progressively more complex preparations, including purified protein in vitro, immortalized and primary neuronal cultures, isolated pancreatic islets, in an intravital liver model, and finally in vivo in C. elegans neurons. In each context, iG6PSnFR reports G6P changes consistent with expected responses to physiological perturbations.

biochemistry↗

bioRxiv2026

Glycolytic Specialization Shapes Neuronal Physiology and Function in vivo

Neurons perform diverse functions that impose distinct energetic demands, but how energy-metabolic pathways are matched to these functions in vivo remains unknown. Here we show that two functionally divergent sister chemosensory neurons in C. elegans, ASEL and ASER, exhibit asymmetric glycolytic flux, with ASER exhibiting high and ASEL having low levels of glycolysis. Metabolic imaging, metabolic network modeling, and electrophysiology measurements reveal that ASERs elevated glycolysis supports a hyperpolarized resting potential, low input resistance, and rapid repolarization that enable a distinct functional role compared to ASEL. Impairing glycolysis collapses these electrophysiological specializations without abolishing neuronal excitability, and selectively disrupts ASERs calcium responses while leaving ASEL largely unaffected. These findings demonstrate that neuron-specific glycolytic programs shape core biophysical properties and are required for functional identity in vivo, establishing metabolism as an active determinant of neuronal physiology.

neuroscience↗

bioRxiv2025

Spatial Partitioning of Core Glycolysis Enables Tissue-Specific Metabolic Programs In Vivo

Tissues exhibit metabolic heterogeneity that tailors conserved pathways to distinct physiological demands, yet how this heterogeneity is achieved in vivo remains poorly understood. Here, we use Caenorhabditis elegans to investigate tissue-specific requirements for glucose-6-phosphate isomerase (GPI-1), a conserved reversible enzyme that links glycolysis and the pentose phosphate pathway (PPP). Tissue-specific metabolic-network modeling predicted differential glycolytic and PPP flux potential across adult tissues and identified tissue-specific biases in GPI-1 reaction directionality. Genetic disruption of gpi-1 produced germline defects consistent with impaired PPP-associated anabolic metabolism and somatic defects consistent with impaired glycolysis, indicating that GPI-1 supports distinct metabolic functions across tissues. We further discovered that two GPI-1 isoforms are differentially expressed and localized: GPI-1A is broadly expressed and cytosolic, whereas GPI-1B is enriched in the germline and localizes to endoplasmic reticulum-associated compartments. Isoform-specific perturbations revealed distinct requirements for GPI-1A and GPI-1B in somatic glycolysis and reproductive physiology. These findings implicate isoform-specific subcellular localization as a possible contributor to the partition of the functions of a conserved reversible enzyme, enabling tissue-specific anabolic and catabolic metabolism in vivo.

cell biology↗

bioRxiv2025

Glycogen metabolism acts in neurons to support glycolytic plasticity

Glycogen is the largest energy reserve in the brain, but the specific role of glycogen in supporting neuronal energy metabolism in vivo is not well understood. We established a system in C. elegans to dynamically probe glycolytic states in single cells of living animals via the use of the glycolytic sensor HYlight and determined that neurons can dynamically regulate glycolysis in response to activity or transient hypoxia. We performed an RNAi screen and identified that PYGL-1, an ortholog of the human glycogen phosphorylase, is required in neurons for glycolytic plasticity. We determined that neurons employ at least two mechanisms of glycolytic plasticity: glycogen-dependent glycolytic plasticity (GDGP) and glycogen-independent glycolytic plasticity (GIGP). We uncover that GDGP is employed under conditions of mitochondrial dysfunction, such as transient hypoxia or in mutants for mitochondrial function. We find that the ability of neurons to plastically regulate glycolysis through cell-autonomous GDGP is important for sustaining the synaptic vesicle cycle. Together, our study reveals that, in vivo, neurons can directly use glycogen as a fuel source to sustain glycolytic plasticity and synaptic function.

neuroscience↗

bioRxiv2023

Local and dynamic regulation of neuronal glycolysis in vivo

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here we adapted a biosensor for glycolysis, HYlight, for use in C. elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons perform glycolysis cell-autonomously, and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function, and uncovers new relationships between neuronal identities and metabolic landscapes in vivo. Significance statementWhile it is generally accepted that energy metabolism underpins neuronal function, how it is distributed and dynamically regulated in different tissues of the brain to meet varying energy demands is not well understood. Here we utilized a fluorescent biosensor, HYlight, to observe glycolytic metabolism at cellular and subcellular scales in vivo. By leveraging both the stereotyped identities of individual neurons in C. elegans, and genetic tools for manipulating glycolytic metabolism, we determined that neurons perform and dynamically regulate glycolysis to match changing cellular demands for energy. Our findings support a model whereby glycolytic states should be considered distinct and related to individual neuron identities in vivo, and introduce new questions about the interconnected nature of metabolism and neuronal function.

neuroscience↗

bioRxiv2021

Asynchronous rates of lineage, phenotype, and niche diversification in a continental-scale adaptive radiation

Rapidly diversifying clades are central to the study of diversification dynamics. This central importance is perhaps most apparent when rapid evolution occurs across several axes of diversification (e.g., lineage, phenotype, and niche); such clades facilitate investigations into the interplay between adaptive and non-adaptive diversification mechanisms. Yet, empirical evidence from rapidly evolving clades remains unclear about the relationships, if any, across diversification axes. This is especially apparent regarding the timing of diversification rate shifts. We address this knowledge gap through comparisons of the rate and timing of lineage, phenotypic, and niche diversification in Penstemon, a rapidly-evolving angiosperm genus. We find that diversification rate shifts in Penstemon are asynchronous; while we identify a burst and subsequent slowdown in lineage diversification rate [~]2.0-2.5 MYA, shifts in phenotypic and niche diversification rates either lagged behind temporally or did not occur at all. We posit that this asynchronicity in diversification rate shifts is the result of initial niche-neutral diversification followed by adaptive, density-dependent processes. Our findings contribute to a growing body of evidence that asynchronous shifts in diversification rates may be common and question the applicability of expectations for diversification dynamics across disparate empirical systems.

evolutionary biology↗

bioRxiv2021

Phylogenetics of a Rapid, Continental Radiation: Diversification, Biogeography, and Circumscription of the Beardtongues (Penstemon; Plantaginaceae)

Penstemon (Plantaginaceae), the largest genus of plants native to North America, represents a recent continental evolutionary radiation. We investigated patterns of diversification, phylogenetic relationships, and biogeography, and determined the age of the lineage using 43 nuclear gene loci. We also assessed the current taxonomic circumscription of the ca. 285 species by developing a phylogenetic taxonomic bootstrap method. Penstemon originated during the Pliocene/Pleistocene transition. Patterns of diversification and biogeography are associated with glaciation cycles during the Pleistocene, with the bulk of diversification occurring from 1.0-0.5 mya. The radiation across the North American continent tracks the advance and retreat of major and minor glaciation cycles during the past 2.5 million years with founder-event speciation contributing the most to diversification of Penstemon. Our taxonomic bootstrap analyses suggest the current circumscription of the genus is in need of revision. We propose rearrangement of subgenera, sections, and subsections based on our phylogenetic results. Given the young age and broad distribution of Penstemon across North America, it offers an excellent system for studying a rapid evolutionary radiation in a continental setting.

evolutionary biology↗

bioRxiv2020

Phylogeographic analysis of shrubby beardtongues reveals range expansions during the Last Glacial Maximum and implicates the Klamath Mountains as a hotspot for hybridization

Quaternary glacial cycles often altered species geographic distributions, which in turn altered the geographic structure of a species genetic diversity. In many cases, glacial expansion forced species in temperate climates to contract their ranges and reside in small pockets of suitable habitat (refugia), where they were likely to interact closely with other species, setting the stage for potential gene exchange. These introgression events, in turn, would have degraded species boundaries, making the inference of phylogenetic relationships challenging. Using high-throughput sequence data, we employ a combination of species distribution models, models of demographic history, and hybridization tests to assess the effect of glaciation on the geographic distributions, phylogenetic relationships, and patterns of gene flow of five species of Penstemon subgenus Dasanthera, long-lived shrubby angiosperms distributed throughout the Pacific Northwest of North America. Surprisingly, we find that rather than reducing their ranges to small refugia, most Penstemon subgenus Dasanthera species experienced increases in suitable habitat during the Last Glacial Maximum. We also find substantial evidence for gene exchange between species, with the bulk of introgression events occurring in or near the Klamath Mountains of southwestern Oregon and northwestern California. Subsequently, our phylogenetic inference reveals blurred taxonomic boundaries in the Klamath Mountains, where introgression is most prevalent. Our results question the classical paradigm of temperate species responses to glaciation, and highlight the importance of contextualizing phylogenetic inference with the demographic histories of the species of interest.

evolutionary biology↗

bioRxiv2020

Inferring Patterns of Hybridization and Polyploidy in the Plant Genus Penstemon (Plantaginaceae)

Reticulate evolutionary events are hallmarks of plant phylogeny, and are increasingly recognized as common occurrences in other branches of the Tree of Life. However, inferring the evolutionary history of admixed lineages presents a difficult challenge for systematists due to genealogical discordance caused by both incomplete lineage sorting (ILS) and hybridization. Methods that accommodate both of these processes are continuing to be developed, but they often do not scale well to larger numbers of species. An additional complicating factor for many plant species is the occurrence of whole genome duplication (WGD), which can have various outcomes on the genealogical history of haplotypes sampled from the genome. In this study, we sought to investigate patterns of hybridization and WGD in two subsections from the genus Penstemon (Plantaginaceae; subsect. Humiles and Proceri), a speciose group of angiosperms that has rapidly radiated across North America. Species in subsect. Humiles and Proceri occur primarily in the Pacific Northwest of the United States, occupying habitats such as mesic, subalpine meadows, as well as more well-drained substrates at varying elevations. Ploidy levels in the subsections range from diploid to hexaploid, and it is hypothesized that most of the polyploids are hybrids (i.e., allopolyploids). To estimate phylogeny in these groups, we first developed a method for estimating quartet concordance factors (QCFs) from multiple sequences sampled per lineage, allowing us to model all haplotypes from a polyploid. QCFs represent the proportion of gene trees that support a particular species quartet relationship, and are used for species network estimation in the program SNaQ (Solis-Lemus & Ane. 2016. PLoS Genet. 12:e1005896). Using phased haplotypes for nuclear amplicons, we inferred species trees and networks for 38 taxa from P. subsect. Humiles and Proceri. Our phylogenetic analyses recovered two clades comprising a mix of taxa from both subsections, indicating that the current taxonomy for these groups is inconsistent with our estimates of phylogeny. In addition, there was little support for hypotheses regarding the formation of putative allopolyploid lineages. Overall, we found evidence for the effects of both ILS and admixture on the evolutionary history of these species, but were able to evaluate our taxonomic hypotheses despite high levels of gene tree discordance. Our method for estimating QCFs from multiple haplotypes also allowed us to include species of varying ploidy levels in our analyses, which we anticipate will help to facilitate estimation of species networks in other plant groups as well.

evolutionary biology↗

bioRxiv2018

Fluidigm2PURC: automated processing and haplotype inference for double-barcoded PCR amplicons

Premise of the studyTargeted enrichment strategies for phylogenomic inference are a time- and cost-efficient way to collect DNA sequence data for large numbers of individuals at multiple, independent loci. Automated and reproducible processing of these data is a crucial step for researchers conducting phylogenetic studies.\n\nMethods and ResultsWe present Fluidigm2PURC, an open source Python utility for processing paired-end Illumina data from double-barcoded PCR amplicons. In combination with the program PURC (Pipeline for Untangling Reticulate Complexes), our scripts process raw FASTQ files for analysis with PURC and use its output to infer haplotypes for diploids, polyploids, and samples with unknown ploidy. We demonstrate the use of the pipeline with an example data set from the genus Thalictrum L. (Ranunculaceae).\n\nConclusionsFluidigm2PURC is freely available for Unix-like operating systems on GitHub [https://github.com/pblischak/fluidigm2purc] and for all operating systems through Docker [https://hub.docker.com/r/pblischak/fluidigm2purc].

bioinformatics↗

bioRxiv2017

HyDe: a Python package for genome-scale hybridization detection

The analysis of hybridization and gene flow among closely related taxa is a common goal for researchers studying speciation and phylogeography. Many methods for hybridization detection use simple site pattern frequencies from observed genomic data and compare them to null models that predict an absence of gene flow. The theory underlying the detection of hybridization using these site pattern probabilities exploits the relationship between the coalescent process for gene trees within population trees and the process of mutation along the branches of the gene trees. For certain models, site patterns are predicted to occur in equal frequency (i.e., their difference is 0), producing a set of functions called phylogenetic invariants. In this paper we introduce HyDe, a software package for detecting hybridization using phylogenetic invariants arising under the coalescent model with hybridization. HyDe is written in Python, and can be used interactively or through the command line using pre-packaged scripts. We demonstrate the use of HyDe on simulated data, as well as on two empirical data sets from the literature. We focus in particular on identifying individual hybrids within population samples and on distinguishing between hybrid speciation and gene flow. HyDe is freely available as an open source Python package under the GNU GPL v3 on both GitHub (https://github.com/pblischak/HyDe) and the Python Package Index (PyPI: https://pypi.python.org/pypi/phyde).

evolutionary biology↗