Biology subjects

Papoulas, O.

Publications and source records attributed to Papoulas, O..

At least 19 recordsLinked to original sources

bioRxiv2026

La1: an evolutionarily conserved player in the Arabidopsis telomerase complex

Analysis of yeast and mammalian telomerase ribonucleoprotein (RNP) complexes reveals striking divergence in their biogenesis and protein complements. However, little is known about telomerase in plants. In addition to the catalytic subunit TERT and the templating RNA TR, we previously reported Arabidopsis thaliana contains two telomerase accessory factors, AtNAP57, a dyskerin homolog that in mammals is essential for telomerase activity, and the telomeric DNA binding protein AtPOT1a. Both proteins stimulate Arabidopsis telomerase repeat addition processivity. Here we employ quantitative mass spectrometry (MS) to further examine telomerase in Arabidopsis. Unexpectedly, dyskerin and AtPOT1a were not detected in our purified complexes, but AtLa1, an RNA-binding factor that recognizes the UUU-3'OH of RNA Pol III transcripts, was highly enriched. RNA-IP assays confirmed AtLa1 association with AtTR in vivo. RNAi-mediated knockdown of AtLa1 strongly diminished telomerase activity, indicating AtLa1 is required for its function in vivo. In vitro binding studies revealed that AtLa1 contacts AtTR via the UUU-3'OH and a plant-specific P1a-P1b-P4 three-way junction (TWJ). Since the TWJ is also required for AtNAP57 binding, the data suggesting that AtNAP57 and AtLa1 compete for AtTR binding or sequentially associate during RNP during biogenesis. In contrast to AtNAP57, AtLa1 did not stimulate telomerase activity when TERT and TR were assembled in vitro, consistent with function during a different step in telomerase assembly. We conclude Arabidopsis telomerase employs multiple accessory factors utilized by both mammalian and single-celled relatives. Further exploration of Arabidopsis telomerase may offer novel insight into telomerase evolution and mechanisms of biogenesis. Significance of ResearchQuantitative mass spectrometry of Arabidopsis telomerase uncovered AtLa1, a homolog of ciliate and yeast proteins that promotes telomerase maturation. AtLa1 is essential for telomerase function in vivo, and in vitro it engages the same region of AtTR bound by AtNAP57, homologous to a telomerase accessory from mammals.

plant biology↗

bioRxiv2025

The CAGE complex: a hollow, megadalton, protein assembly in prokaryotic and eukaryotic microbes

We report the discovery and structure of a previously unknown [~]1 MDa hollow protein assembly, identified during a survey of ciliary complexes from the ciliate Tetrahymena thermophila. By combining mass spectrometry, structure prediction, and cryo-electron microscopy, we define a homotetrameric cage-like complex with a distinctive elliptical architecture and a large internal cavity. A sequence survey revealed several thousand homologs spanning diverse unicellular eukaryotes--including green algae, fungi, amoebozoans, choanoflagellates, and SAR lineages--as well as predominantly gram-negative bacteria, indicating an ancient evolutionary origin and arguing against a eukaryote-specific function. We determined a near-atomic resolution structure of the complex from the slime mold Dictyostelium discoideum, demonstrating conservation of overall architecture and cavity despite low sequence identity. Together, these results establish the CAGE complex (Conserved Assembly in Gram-negative bacteria and Eukaryotes) as a new class of large protein cage broadly distributed across the tree of life. While its biological function remains unknown, its size, architecture, and conservation suggest possible roles in transport or protein/RNA homeostasis.

molecular biology↗

bioRxiv2025

Comparative Proteomic Profiling of Receptor Kinase Signaling Reveals Key Trafficking Components Enforcing Plant Stomatal Development

Receptor kinases are pivotal for growth, development, and environmental response of plants. Yet, their regulatory mechanisms and spatial dynamics are still underexplored. The ERECTA-family receptor kinases coordinate diverse developmental processes, including stomatal development. To understand the proteomic landscape of the ERECTA-mediated signaling pathways, we here report comparative analyses of the ERECTA interactome and proximitome by epitope-tagged affinity-purification (ET-AP) and TurboID-based proximity labeling (TbID-PL) mass-spectrometry, respectively. While ET-AP successfully recovered receptor complex components (e.g., TOO MANY MOUTHS), TbID-PL effectively captured transient associations with the components of endosomal trafficking, i.e., clathrin-mediated endocytosis (CME) machinery. We further identify that specific subfamily members of phosphatidylinositol-binding clathrin assembly proteins (PICALMs) interact with and synergistically regulate ERECTA internalization. Mutations of these PICALMs impair ERECTA endocytosis and lead to excessive stomatal clustering by dampening the downstream signaling output. Taken together, our work provides a proteomic atlas of the ERECTA signaling network and suggests that timely removal of receptor kinase by the endocytosis machinery is essential for active signal transduction enforcing stomatal patterning.

plant biology↗

bioRxiv2025

Physical and functional interaction of Lrrc56 and Odad3 controls deployment of axonemal dyneins in vertebrate multiciliated cells

Primary ciliary dyskinesia is a genetically heterogeneous motile ciliopathy characterized chronic respiratory disease, laterality defects, hydrocephalus, and infertility, caused by impaired function of motile cilia. LRRC56 has recently emerged as a novel PCD candidate gene, but its role in vertebrate cilia remains poorly understood. Here, we used Xenopus laevis multiciliated cells, targeted knockdown, and in vivo imaging to investigate Lrrc56 function. We show that loss of lrrc56 causes specific depletion of outer dynein arms (ODAs) from the distal axoneme. In vivo affinity purification mass spectrometry revealed that Lrrc56 binds the ODA docking complex components, including Odad3. Consistently, Lrrc56 knockdown also led to distal loss of Odad3. Moreover, we show that disease-associated variants in LRRC56 and ODAD3 disrupted their localization and interaction, pointing to a shared functional pathway. Our work demonstrates that Lrrc56 is a critical regulator of distal ODAs and ODA docking complex deployment and provides new mechanistic insight into how LRRC56 mutations contribute to PCD.

cell biology↗

bioRxiv2025

VerteBrain reveals novel neural and non-neural protein assemblies conserved across vertebrate evolution

Protein-protein interactions underlie core brain functions, including neurotransmitter release, receptor activation, and intracellular signaling essential for learning, memory, and cognition. Here, we systematically map conserved brain protein interactions across five vertebrate species-rabbit, chicken, dolphin, pig, and mouse-using co-fractionation and immunoprecipitation mass spectrometry. From 2,197 biochemical fractions, we identify over 81,000 high-confidence interactions among 6,108 conserved proteins. This interaction map (VerteBrain) reveals both regulatory and structural complexes, including extensive synaptonemal protein associations likely involved in inter-neuronal coordination. Conservation across species underscores essential roles in neuronal and glial function, as well as in additional tissues for more widely expressed complexes. The VerteBrain dataset uncovers candidate disease mechanisms, including roles for ARHGEF1 in short stature syndromes, synaptic vesicle trafficking complexes in epilepsy, and RELCH in congenital deafness. VerteBrain provides a publicly accessible framework for investigating brain protein interactions and their relevance to human neurological disorders.

systems biology↗

bioRxiv2025

Molecular organization of the distal tip of vertebrate motile cilia

The beating of cilia on multi-ciliated cells (MCCs) is essential for normal development and homeostasis in animals. But while the structure and function of basal bodies and axonemes have received significant attention recently, the distal tips of MCC cilia remain relatively poorly defined. Here, we characterize the molecular organization of the distal tip of vertebrate MCC cilia, characterizing two distinct domains occupied by distinct protein constituents. Using frog, mouse, and human MCCs, we find that two largely uncharacterized proteins, Ccdc78 and Ccdc33 occupy a previously undefined region at the extreme distal tip, and these are required for the normal organization of all other known tip proteins. Ccdc78 and Ccdc33 each display robust microtubule-bundling activity both in vivo and in vitro, yet each is independently required for normal length regulation of MCC cilia. Moreover, loss of each protein elicits a distinct pattern of defective cilia beating and resultant fluid flow. Thus, two previously undefined proteins form a key module essential for organizing and stabilizing the distal tip of motile cilia in vertebrate MCCs. We propose that these ill-defined proteins represent potential disease loci for motile ciliopathies.

cell biology↗

bioRxiv2024

Structure and organization of full-length Epidermal Growth Factor Receptor in extracellular vesicles by cryo-electron tomography

We report here transport of the Epidermal Growth Factor Receptor (EGFR), Insulin Receptor, 7-pass transmembrane receptor Smoothened, and 13-pass Sodium-iodide symporter to extracellular vesicles (EVs) for structural and functional studies. Mass spectrometry confirmed the transported proteins as the most abundant in EV membranes, and the presence of many receptor-interacting proteins demonstrates the utility of EVs for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in the presence of EGF with a [~]3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular regions do not form regular arrays, suggesting that clustering is mediated by the intracellular region. Subtomogram averaging of the EGFR extracellular region (ECR) yielded a 15 [A] map into which the crystal structure of the ligand-bound EGFR ECR dimer fits well. These findings refine our understanding of EGFR activation, clustering, and signaling, and they establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in a native-like environment. Significance StatementAtomic or near-atomic resolution structural studies of proteins embedded in cell membranes have proven challenging. We show that transporting integral membrane proteins to cell-derived extracellular vesicles enables structural and functional studies of human membrane proteins in a native membrane environment. We have used this approach to visualize an active form of full-length Epidermal Growth Factor Receptor (EGFR) and show that it forms clusters in the membrane and projects its cytoplasmic signaling domains [~]3 nm away from the membrane surface. EGFR is essential for normal development, but abnormal EGFR activity is associated with several human cancers and is the target of many anticancer therapies. Our studies refine current models of how ligand binding to EGFR transmits signals across cell membranes.

biophysics↗

bioRxiv2024

Ancient eukaryotic protein interactions illuminate modern genetic traits and disorders

All eukaryotes share a single-celled ancestor from [~]1.5-1.8 billion years ago, the Last Eukaryotic Common Ancestor (LECA). Roughly half of gene families found in modern eukaryotes were already present in LECA, forming molecular systems that continue to influence genetic diseases and traits today. To investigate these systems, we compared genes across 156 organisms to define a core set of protein-coding gene families likely present in LECA, with a quarter remaining uncharacterized. Integrating >26,000 mass spectrometry proteomics analyses from 31 species, we inferred higher-order complexes among these ancient proteins. This reconstructed interactome reveals both established and novel assemblies, offering a biochemical snapshot of LECAs organization. Finally, by exploring these ancient protein interactions, we found new human gene-disease associations for bone density and congenital birth defects, illustrating the value of ancestral protein networks for modern functional genetics.

evolutionary biology↗

bioRxiv2023

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 19,000 XLs, we identified 4,757 unique amino acid interactions among 1,143 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease. HighlightsO_LIOver 4,700 distinct amino-acid cross-links reveal the composition, structural organization, and conformational dynamics of proteins in motile ciliary axonemes. C_LIO_LIDense interaction networks are defined for tubulins, Intraflagellar Transport complexes, axonemal dyneins, radial spokes, the CCDC39/40 96nm molecular ruler, and other complexes. C_LIO_LIThese data reveal the placement of multiple adenylate kinases in the central apparatus and radial spokes of motile axonemes, a new microtubule-associated protein complex of CFAP58 and CCDC146, and insights into the activity of ciliopathy protein MAATS1/CFAP91. C_LIO_LIThe data also provide the first known molecular defect resulting from loss of the human ciliopathy protein ENKUR. C_LI

cell biology↗

bioRxiv2023

Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism

Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the conserved nexin-dynein regulatory complex (N-DRC), which links adjacent doublet microtubules and regulates and coordinates the activity of outer doublet complexes. Despite its critical role in cilia motility, the assembly and molecular basis of the regulatory mechanism are poorly understood. Here, utilizing cryo-electron microscopy in conjunction with biochemical cross-linking and integrative modeling, we localized 12 DRC subunits in the N-DRC structure of Tetrahymena thermophila. We also found that the CCDC96/113 complex is in close contact with the N-DRC. In addition, we revealed that the N-DRC is associated with a network of coiled-coil proteins that most likely mediates N-DRC regulatory activity.

cell biology↗

bioRxiv2023

Label-free proteomic comparison reveals ciliary and non- ciliary phenotypes of IFT-A mutants

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila. A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

cell biology↗

bioRxiv2023

Protein nonadditive expression and solubility in Arabidopsis hybrids and allotetraploids

Interspecific hybridization in plants often leads to allopolyploids including most important crops such as wheat, cotton, and canola, while many other crops such as corn and sorghum are grown as hybrid. Both allopolyploids and hybrids show hybrid vigor or heterosis. The phenomenon has been widely applied in agriculture and extensively studied at the genetic and gene expression levels. However, proteomic changes in hybrids and polyploids remain poorly understood. Here, we report comparative analysis of soluble and insoluble proteomes in Arabidopsis intraspecific and interspecific hybrids or allotetraploids formed between A. thaliana and A. arenosa. Both allotetraploids and intraspecific hybrids displayed nonadditive expression (unequal to the sum of the two parents) of the proteins, most of which were involved in biotic and abiotic stress responses. In the allotetraploids, homoeolog-expression bias was not observed among all proteins examined but could occur among 17-20% of the nonadditively expressed proteins, consistent with the transcriptome results. Among expression-biased homoeologs, there were more A. thaliana-biased than A. arenosa-biased homoeologs. Analysis of the insoluble and soluble proteomes revealed more soluble proteins in the hybrids than their parents but not in the allotetraploids. Most proteins in ribosomal biosynthesis and in the thylakoid lumen, membrane, and stroma were in the soluble fractions, indicating a role of protein stability in photosynthetic activities for promoting growth. These results collectively support roles of nonadditive expression of stress-responsive proteins and increased solubility of photosynthetic proteins in Arabidopsis hybrids and allotetraploids, which may promote hybrid vigor. Plain Language SummaryWe analyzed fractionated proteomes to test protein abundance and solubility in Arabidopsis hybrids and polyploids. Many proteins in stress-responses are nonadditively expressed in intraspecific hybrids or allotetraploids. There are more soluble proteins of ribosomal biosynthesis and photosynthetic activities in the hybrids than in their parents but not in allotetraploids. Expression levels are equal among most protein homoeologs in the allotetraploids but are biased for ~20% of nonadditively expressed proteins. Nonadditive protein expression and solubility may play a role in heterosis.

genetics↗

bioRxiv2022

Does AlphaFold2 model proteins' intracellular conformations? An experimental test using cross-linking mass spectrometry of endogenous ciliary proteins

A major goal in structural biology is to understand protein assemblies in their biologically relevant states. Here, we investigate whether AlphaFold2 structure predictions match native protein conformations. We chemically cross-linked proteins in situ within intact Tetrahymena thermophila cilia and native ciliary extracts and identified 1,225 intramolecular cross-links within the 100 best-sampled proteins to provide a benchmark of distance restraints obeyed by proteins in their native assemblies. The corresponding AlphaFold2 structure predictions were highly concordant, positioning 86.2% of cross-linked residues within C-to-C distances of 30 [A], consistent with the known cross-linker length. 43% of the proteins showed no violations. Most inconsistencies occurred in low-confidence regions or between domains of the structure prediction. For basal body protein BBC118, cross-links combined with the predicted structure revealed domain packing satisfying both data. Overall, AlphaFold2 predicted biological structures with low predicted aligned error corresponded to more correct native structures. However, we observe cases where rigid body domains are oriented incorrectly, suggesting that combining structure prediction with experimental information will better reveal biologically relevant conformations.

bioinformatics↗

bioRxiv2022

Integrative modeling reveals the molecular architecture of the Intraflagellar Transport A (IFT-A) complex

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease. SummaryThe 3D structure of the six-subunit complex and its polymeric assembly gives insights into cargo transport in cilia and how specific mutations in these genes lead to ciliopathy birth defects.

biochemistry↗

bioRxiv2021

The protein organization of a red blood cell

Red blood cells (RBCs, erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles, and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we defined a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validated protein complexes through electron microscopy and chemical crosslinking, and with these data, built 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-link compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.

systems biology↗

bioRxiv2021

Cell adhesions link subcellular actomyosin dynamics to tissue scale force production during vertebrate convergent extension.

Axis extension is a fundamental biological process that shapes multicellular organisms. The design of an animals body plan is encoded in the genome and execution of this program is a multiscale mechanical progression involving the coordinated movement of proteins, cells, and whole tissues. Thus, a key challenge to understanding axis extension is connecting events that occur across these various length scales. Here, we use approaches from proteomics, cell biology, and tissue biomechanics to describe how a poorly characterized cell adhesion effector, the Armadillo Repeat protein deleted in Velo-Cardio-Facial syndrome (Arvcf) catenin, controls vertebrate head-to-tail axis extension. We find that Arvcf catenin is required for axis extension within the intact organism but is not required for extension of isolated tissues. We then show that the organism scale phenotype is caused by a modest defect in force production at the tissue scale that becomes apparent when the tissue is challenged by external resistance. Finally, we show that the tissue scale force defect results from dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to cell membranes. These results not only provide a comprehensive understanding of Arvcf function during an essential biological process, but also provide insight into how a modest cellular scale defect in cell adhesion results in an organism scale failure of development.

developmental biology↗

bioRxiv2020

Twinfilin1 controls lamellipodial protrusive activity and actin turnover during vertebrate gastrulation

The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1. We confirm previous results on the role of Twf1 in lamellipodia and extend those findings by linking Twf1, actin turnover, and cell polarization required for convergent extension during vertebrate gastrulation.

cell biology↗

bioRxiv2020

Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

cell biology↗