Biology subjects

Wideman, J. G.

Publications and source records attributed to Wideman, J. G..

13 recordsLinked to original sources

bioRxiv2026

Subcellular spatial proteomics of the heterotrophic nanoflagellate, Cafeteria roenbergensis

Cafeteria roenbergensis is the best-studied free-living heterotrophic nanoflagellate, an abundant marine bacterivore and the host of the giant virus CroV, yet almost nothing is known about how its cell is organized. To provide broad insight into the localization of its proteome, we used subcellular spatial proteomics (SSP), based on a label-free version of LOPIT-DC (Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation), in which organelles are separated by differential centrifugation and each protein is assigned to a compartment from its abundance profile across fractions. We detected 5,948 proteins (5,889 protein groups), corresponding to 69.7% of the predicted C. roenbergensis proteome, and assigned high-confidence localizations to 2,945 proteins across 14 subcellular compartments, including basal body/cilia, cytosol, ER, Golgi, lysosome, membrane-trafficking, mitochondria, nucleoplasm, nucleus, peroxisome, plasma membrane, proteasome, ribosome, and spliceosome. This study serves as a proteomic dataset to resolve protein compartmentalization in a free-living heterotrophic stramenopile, enabling evolutionary, biological and functional insight and analysis. Investigators interested in using these data prior to publication are encouraged to contact us to avoid overlap and to facilitate coordinated and collaborative use of this resource.

cell biology↗

bioRxiv2026

Subcellular spatial proteomics of the close animal relative, Capsaspora owczarzaki

Capsaspora owczarzaki is one of the closest relatives of animals and an increasingly tractable model for investigating the evolution of multicellularity. However, very little cell biological work has been done on the sister linages to animals. Subcellular spatial proteomics (SSP) is a technique that can localize thousands of proteins to subcellular compartments in a single experiment by combining cell fractionation with sensitive mass spectrometry. To provide broad insight into the localization of the Capsaspora proteome, we performed SSP based on a label-free version of LOPIT-DC (Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation). We detected 6,466 proteins, corresponding to 68.4% of the predicted Capsaspora proteome, and assigned high-confidence localizations to 3,106 of them across 13 subcellular compartments. This study serves as a proteomic dataset to resolve cellular compartmentalization in a close relative of animals, enabling downstream evolutionary, biological, and functional analyses. Investigators interested in using these data prior to publication are encouraged to contact us to avoid overlap and to facilitate coordinated and collaborative use of this resource.

cell biology↗

bioRxiv2026

Global mapping of protein localization in vegetative Dictyostelium discoideum by subcellular spatial proteomics

Dictyostelium discoideum is a genetically tractable amoebozoan model organism widely used to study conserved eukaryotic cellular processes, yet a comprehensive map of its subcellular proteome has been lacking. Here, we combined subcellular fractionation with label-free mass spectrometry to resolve a global protein localization map for D. discoideum. We detected 6,337 proteins (~50% of the predicted proteome) and generated protein abundance profiles across 10 subcellular fractions. Using a curated marker set and a support vector machine classifier with a median cutoff, we assigned high-confidence localizations to 3,169 proteins across 17 subcellular compartments. Independent validation using sequence-based targeting predictions strongly supported the compartment assignments. Together, this study provides the first global subcellular localization map of the Dictyostelium proteome, establishing a foundational resource for functional, cell biological, and evolutionary analyses. We are currently preparing manuscripts that further analyze (i) the membrane trafficking system of Dictyostelium, including detailed investigation of the contractile vacuole, Jotnarlogs, and patchy proteins, and (ii) the composition of the Dictyostelium peroxisome, with a focus on the subcellular localization of sterol biosynthesis enzymes. Investigators interested in using these data are encouraged to contact us prior to publication to avoid overlap and to facilitate coordinated and collaborative use of this resource.

cell biology↗

bioRxiv2026

A subcellular spatial proteome of the non-photosynthetic euglenid Euglena longa

Euglena longa is a heterotrophic model euglenid, of significant research interest as a unicellular eukaryote. To provide broad insight into the localisation of its proteome we used subcellular spatial proteomics (SSP), based on a label-free version of LOPIT-DC (Localisation of Organelle Proteins by Isotope Tagging after Differential Centrifugation), in which organelles are separated by differential centrifugation and each protein is assigned to a compartment from its abundance profile across the fractions. We detected 5,909 proteins, using protein abundance profiles across fractions to resolve 16 different subcellular compartments, such as the plastid, pellicle, and peroxisomes, and assigned high-confidence localisations to 2,955 of them. This study serves as a proteomic dataset to resolve protein compartmentalisation for this euglenid, enabling evolutionary, biological and functional insight and analysis. We are drafting manuscripts concerning the metabolic nature of the plastid, the evolutionary origins of the pellicle, and the functions of the peroxisome. Investigators interested in using this data prior to publication are encouraged to contact us to avoid overlap and to facilitate coordinated and collaborative use of this resource.

cell biology↗

bioRxiv2025

Phylogenomic placement and morphological description of a novel phagotrophic euglenid from Hawaii: Hokulea waialensis n. gen. et sp.

Euglenids are a diverse group of flagellated protists that include phagotrophic, osmotrophic, and phototrophic lineages. Understanding the phylogenetic relationships of phagotrophic euglenids is crucial in understanding euglenid evolution as a whole. Yet many relationships within euglenids remain unclear, and further resolution requires extensive sampling, particularly from the deep-branching, paraphyletic group known as ploeotids. Improved resolution of evolutionary relationships among ploeotid taxa is necessary to elucidate the origin and diversification of complex ultrastructural traits (e.g., pellicle, feeding apparatus). Here, we isolated, cultivated and characterized a novel ploeotid species named Hokulea waialensis n. gen. et sp. using light and scanning electron microscopy, single-cell sequencing, and phylogenomic analyses. This new species is relatively small (10-12 {micro}m long) compared to related euglenids, and shares several morphological traits with related species of Alistosa. Both single and multigene phylogenetic reconstructions from single amplified genome data show that Hokulea waialensis n. gen. et sp. is closely related to several environmental small subunit ribosomal DNA (SSU rDNA) sequences, and more broadly to Lentomonas and Decastava.

evolutionary biology↗

bioRxiv2025

Subcellular proteomics of Paradiplonema papillatum reveals digestive capacity of the cell membrane and the plasticity of peroxisomes across euglenozoans

Diplonemids are among the most diverse and abundant protists in the deep ocean, have extremely complex and ancient cellular systems, and exhibit unique metabolic capacities. Despite this, we know very little about this major group of eukaryotes. To establish a model organism for comprehensive investigation, we performed subcellular proteomics on Paradiplonema papillatum and localized 4,870 proteins to 22 cellular compartments. We additionally confirmed the predicted location of several proteins by epitope tagging and fluorescence microscopy. To probe the metabolic capacities of P. papillatum, we explored the proteins predicted to the cell membrane compartment in our subcellular proteomics dataset. Our data revealed an accumulation of many carbohydrate active enzymes (CAZymes). Our predictions suggest that these CAZymes are exposed to extracellular space, supporting proposals that diplonemids may specialize in breaking down carbohydrates in plant and algal cell walls. Further exploration of carbohydrate metabolism revealed an evolutionary divergence in the function of glycosomes (modified peroxisomes) in diplonemids versus kinetoplastids. Our subcellular proteome provides a resource for future investigations into the unique cell biology of diplonemids.

cell biology↗

bioRxiv2025

Subcellular proteomics of Paramecium tetraurelia reveals mosaic localization of glycolysis and gluconeogenesis

Ciliates are unicellular heterotrophic eukaryotes, most of which consume other microbes as prey. They exhibit nuclear dimorphism which requires reconstruction of a transcriptionally active macronucleus from the germline micronucleus after sexual recombination. This complex genomic structure has prevented the development of highly tractable genetic models leaving much of ciliate cell biology unexplored. To complicate matters further, some ciliates tend to accumulate many gene duplicates either singly or via whole genome duplications. Thus, extensive insight into the cell biology of ciliates requires the use of high-throughput tools like subcellular proteomics. Here, we use a subcellular proteomics workflow to classify over 9,000 proteins to 16 subcellular compartments in Paramecium tetraurelia. From these data, we identify a small but robust subcellular cluster containing canonical mitochondrial outer membrane proteins as well as some ER proteins, putatively at membrane contact sites. Within this cluster, we identified the important glycolytic enzyme phosphofructokinase, which contained a transmembrane domain. Further investigation revealed that several latter-acting glycolytic enzymes were localized to the mitochondrial cluster. The location of phosphoenol pyruvate carboxykinase and pyruvate carboxylase in the mitochondria but pyruvate kinase in the cytosol suggests that ciliates prefer gluconeogenesis over glycolysis. The localization of these enzymes was confirmed in a preliminary subcellular proteome of Tetrahymena thermophila. In sum, our findings suggest that mitochondrial localization of glycolytic/gluconeogenic enzymes is widespread across ciliates and that several may preferentially undergo gluconeogenesis over glycolysis using amino acids as a primary carbon source in both catabolic and anabolic metabolism. HighlightsSubcellular proteomics of Paramecium tetraurelia revealed that glycolytic and gluconeogenic enzymes are mosaically distributed between the cytosol, mitochondrial matrix, and mitochondrial outer membrane. A distinct mitochondrial outer membrane compartment was identified with 105 classified proteins, including core mitochondrial biogenesis proteins and a putative Tom70-like protein. Phosphofructokinase, a key glycolytic enzyme, was found embedded in the mitochondrial outer membrane. Localization of biochemical pathways suggest ciliates favor gluconeogenesis over glycolysis. In total, over 9000 Paramecium proteins were identified using subcellular proteomics and classified into 16 different cellular compartments.

cell biology↗

bioRxiv2023

A DRP-like pseudoenzyme coordinates with MICOS to promote cristae architecture

Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial inner membrane-localized MICOS complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS complexes to promote normal mitochondrial morphology and respiratory function. Bioinformatic analyses reveal that Mmc1 is a distant relative of the Dynamin-Related Protein (DRP) family of GTPases, which are well established to shape and remodel membranes. We find that, like DRPs, Mmc1 self-associates and forms high molecular weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting it does not dynamically remodel membranes. These data are consistent with a model in which Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans.

cell biology↗

bioRxiv2023

Single-cell genomics reveals the divergent mitochondrial genomes of Retaria (Foraminifera and Radiolaria)

Mitochondria originated from an ancient bacterial endosymbiont that underwent reductive evolution by gene loss and endosymbiont gene transfer to the nuclear genome. The diversity of mitochondrial genomes published to date has revealed that gene loss and transfer processes are ongoing in many lineages. Most well-studied eukaryotic lineages are represented in mitochondrial genome databases, except for the superphylum Retaria--the lineage comprising Foraminifera and Radiolaria. Using single-cell approaches, we present two complete mitochondrial genomes of Foraminifera and two near-complete mitochondrial genomes of radiolarians. We report the complete coding content of an additional 14 foram species. We show that foraminiferan and radiolarian mitochondrial genomes encode a nearly fully overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. In contrast to animals and fungi, many protists encode a diverse set of proteins on their mitochondrial genomes, including several ribosomal genes; however, some aerobic eukaryotic lineages (euglenids, myzozoans, and chlamydomonas-like algae) have reduced mitochondrial gene content and lack all ribosomal genes. Similar to these reduced outliers, we show that retarian mitochondrial genomes lack ribosomal protein and tRNA genes, contain truncated and divergent small and large rRNA genes, and encode only 14-15 protein-coding genes, including nad1, 3, 4, 4L, 5, 7, cob, cox1, 2, 3, atp1, 6, and 9, with forams and radiolarians additionally encoding nad2 and nad6, respectively. In radiolarian mitogenomes, a non-canonical genetic code was identified in which all three stop codons encode amino acids. Collectively, these results add to our understanding of mitochondrial genome evolution and fill in one of the last major gaps in mitochondrial sequence databases.

genomics↗

bioRxiv2022

The persistent homology of mitochondrial ATP synthases

While mitochondrial ATP synthase has been thoroughly studied in animals and fungi, relatively little is known about the structures of protists. Among those that have been studied, protist ATP synthases possess divergent structures distinct from those of yeast or animals. Therefore, we aimed to clarify the subunit composition and evolution of ATP synthase across all major eukaryotic lineages. We used sensitive homology detection methods and molecular modelling tools to demonstrate the persistence of a near-complete ancestral set of 17 subunits in most major eukaryotic taxa even despite major divergence. These data demonstrate that most eukaryotes possess an ancestral-like ATP synthase structure similar to those of animals, fungi, and plants, but a number have diverged drastically (e.g., ciliates, myzozoans, euglenozoans, and likely retarians and heteroloboseans). In addition, we identified the first synapomorphy of the SAR (stramenopile, alveolate, rhizaria) supergroup - a ~1 billion-year-old gene fusion between ATP synthase stator subunits. Our comparative approach highlights the persistence of ancestral subunits even amidst major structural changes. We conclude by urging that more ATP synthase structures (e.g., from jakobids, heteroloboseans, stramenopiles, rhizarians) are needed to provide a complete picture of the evolution of structural diversity of this ancient and essential complex.

evolutionary biology↗

bioRxiv2021

Picozoa are archaeplastids without plastid

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we used single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids.

evolutionary biology↗

bioRxiv2020

Unanticipated diversity, undiscovered ancient paralogs, and Asgard origins of the ARF GTPase protein family

The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited. Sampling an extensive set of available genome and transcriptome sequences, we have assembled a dataset of over 2,000 manually curated ARF family genes from 114 eukaryotic species, including many deeply diverged protist lineages, and carried out comprehensive molecular phylogenetic analyses. These reconstructed as many as 16 ARF family members present in the last eukaryotic common ancestor (LECA), nearly doubling the previously inferred ancient system complexity. Evidence for the wide occurrence and ancestral origin of Arf6, Arl13 and Arl16 is presented for the first time. Moreover, Arl17, Arl18 and SarB, newly described here, are absent from well-studied model organisms and as a result their function(s) remain unknown. Analyses of our dataset revealed a previously unsuspected diversity of membrane association modes and domain architectures within the ARF family. We detail the step-wise expansion of the ARF family in the metazoan lineage, including discovery of several new animal-specific family members. Delving back to its earliest evolution in eukaryotes, the resolved relationship observed between the ARF family paralogs sets boundaries for scenarios of vesicle coat origins during eukaryogenesis. Altogether, our work fundamentally broadens the understanding of the diversity and evolution of a protein family underpinning the structural and functional complexity of the eukaryote cells. SignificanceARF Family GTPases are crucial regulations of a diversity of cellular compartments and processes and as such the extent of this system in eukaryotes reflects both cellular complexity in modern eukaryotes and its evolution. Strikingly, a comprehensive comparative genomic analysis of the protein family is lacking, leaving its recent and ancient evolution poorly resolved. We performed a comprehensive molecular evolutionary analysis, reconstructing a highly complex ARF family complement in the Last Eukaryotic Common Ancestor, including a number of paralogs never before identified as such, and we find resolved relationships between the paralogs. This work has implications for cellular evolution from eukaryogenesis to cellular complexity in metazoans.

evolutionary biology↗

bioRxiv2020

Depletion of voltage-dependent anion channel (VDAC) of Toxoplasma gondii affects multiple mitochondrial functions, but not calcium signalling.

The Voltage Dependent Anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contact between the mitochondria and ER. We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to the mitochondria, but does not appear to modulate calcium (Ca2+) signalling. Further, depletion of VDAC resulted in significant morphological changes of the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii.

cell biology↗