Biology subjects

Wallingford, J. B.

Publications and source records attributed to Wallingford, J. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Odad1 is Required for Deployment of Axonemal Outer Arm Dyneins and Embryonic Left-Right Patterning

Motile cilia generate fluid flow required for development and tissue homeostasis. Defects in ciliary motility cause Primary Ciliary Dyskinesia (PCD), presenting with situs inversus, infertility, chronic respiratory disease, and hydrocephalus. Ciliary beating requires outer dynein arms (ODAs), anchored to microtubules by the ODA docking complex (ODA-DC). ODAD1 is an ODA-DC component and a known PCD protein. ODAD1 alleles are often associated with human laterality defects, however Odad1 has not been reported to localize to 9+0 axonemes. Here, we show that GFP-Odad1 localizes to nodal cilia (9+0) in the gastrocoel roof plate (GRP), the frog's equivalent to the mammalian node where left-right asymmetry is first established. We found that Odad1 knockdown (KD) disrupts left-right patterning in Xenopus, with loss of pitx2 expression and abnormal gut looping reflecting defective 9+0 nodal cilia at the GRP. In 9+2 multiciliated cells (MCC) axonemes, Odad1 loss impaired deployment of ODA motors and ODA-DC subunits, disrupting ciliary motility. Two patient-derived truncating ODAD1 alleles were similarly unstable, failing to rescue ODA docking in MCCs or left-right patterning at the GRP. Together, these results show ODAD1 is required for ODA-DC assembly in both 9+0 and 9+2 cilia, linking its loss to ciliary motility and left-right patterning defects in ODAD1-associated ciliopathy.

developmental biology↗

bioRxiv2026

Predicted structure of the complete CPLANE complex reveals novel interactors and mechanisms of Jbts17 during ciliogenesis

Cilia are highly conserved organelles that use hundreds of unique proteins to drive extracellular motility and serve as signaling hubs for eukaryotic cells. The Ciliogenesis and Planar Polarity Effector (CPLANE) protein complex controls basal body docking during early ciliogenesis and recruits the intraflagellar transport (IFT) proteins to the base of growing cilia. Leveraging recent advantages in deep learning neural networks and high-performance computing, we predict the structure of JBTS17, the largest CPLANE subunit, and the tertiary structure of the complete CPLANE complex, revealing direct contacts between JBTS17 and three other CPLANE members. We then used this model for an in silico protein-protein interaction screen that identified several novel JBTS17 interactors. Experiments in Xenopus confirmed a functional link in vivo between Jbts17 and the Spinocerebellar ataxia-associated protein TTBK2. The data provide new insights into mechanisms of CPLANE function during ciliogenesis and demonstrate the power of modeling protein complexes for the discovery of physical and functional protein interactions in cell homeostasis and disease.

cell biology↗

bioRxiv2026

A pseudoenzyme controls the architecture of the extreme distal tip of motile cilia

Motile cilia are evolutionarily conserved organelles performing essential roles in development and tissue homeostasis. Unlike the core scaffold of motile cilia, the distal regions remain relatively less explored and display great diversity across species. Here, we describe a previously uncharacterized ciliary protein Jhc1, localizing at the extreme distal tip of multiciliated cell (MCC) cilia and encoded only in the genomes of non-mammalian vertebrates. Jhc1 is essential for cilia structure and length in Xenopus, an activity that is conserved in Jhc1 from reptiles and fish. Phylogenetic analysis and structure modeling suggests that Jhc1 arose by duplication and neofunctionalization of thiamine triphosphatase, and we show that residues crucial for that enzymes function have been lost and replaced by new residues essential for ciliary localization. These data provide new insights into the molecular mechanisms underlying the broad diversification of the structures at the tip of motile cilia during evolution.

cell biology↗

bioRxiv2025

Cilia.io: Computer vision and machine learning reveal spatial patterns of cilia beating dynamics in the spinal cord

Motile cilia coordinate fluid flows that are essential for normal tissue physiology and function. Cilia display diverse beating waveforms, and while pronounced defects are strongly associated with motile ciliopathies, more subtle alterations may also influence disease manifestations. Finer quantification of ciliary dynamics is critical for a full understanding of cilia-associated disorders, but the heterogeneity of cilia beating dynamics makes accurate and robust characterization challenging. Because existing tools have proven to be limiting in noisy in vivo environments, we developed Cilia.io, a machine learning (ML)-based quantification tool that uses state-of-the-art vision transformers to segment cilia out of the background based on their biological features. Cilia.io enables fast, accurate, and reproducible quantification of motile cilia morphodynamics and outperforms existing tools. Indeed, using Cilia.io, we discovered distinct regional differences in ciliary waveforms in the zebrafish spinal cord. The ability of Cilia.io to capture subtle ciliary defects was further demonstrated by analyzing a novel allele in the ciliopathy gene bbs2 that causes a highly heterogeneous scoliosis phenotype. In these mutants, only dorsal cilia displayed altered beating dynamics, while ventral cilia remained largely unaffected. Our new tool therefore represents a substantial advance on existing methods and suggests that additional fine-scale analyses of ciliary beating will be important for understanding organismal phenotypes and cilia-driven disease.

developmental 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

Stressed Actin Binding by the Prickle2 LIM Domains and itsRegulation in the Full-Length Protein

Cells are capable of sensing mechanical changes in their cytoskeletal network via stress-sensitive actin-binding proteins. Recently, a novel stress-sensing mechanism was described whereby LIM domains from diverse protein families bind directly to stressed actin filaments. It remains unclear, however, how the interaction of these domains with actin is regulated in the context of full-length proteins. Here, we show that the LIM domain containing region (LCR) of the planar cell polarity protein Prickle2 (Pk2) associated with stressed actin filaments in Xenopus mesoderm alongside known stress-sensitive LIM domains. By contrast, the full-length Pk2 did not exhibit similar recruitment along actin filaments. Structure function analysis revealed that both the structured PET domain and unstructured C-terminal region of Pk2 suppress recruitment of Pk2s LCR to stressed actin and promote recruitment to Pk2-rich nodes. Finally, we show that two human patient-derived variants associated with epilepsy result in a loss of Pk2-LCR recruitment to actin filaments. These data provide new insights into the regulation of stress-sensitive LIM domains and may inform our understanding of planar cell polarity.

cell 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

The human ciliopathy protein RSG1 links the CPLANE complex to transition zone architecture

Cilia are essential organelles and variants in genes governing ciliary function result in ciliopathic diseases. The Ciliogenesis and PLANar polarity Effectors (CPLANE) protein complex is essential for ciliogenesis in animals models but remains poorly defined. Notably, all but one subunit of the CPLANE complex have been implicated in human ciliopathy. Here, we identify three families in which variants in the remaining CPLANE subunit CPLANE2/RSG1 also cause ciliopathy. These patients display cleft palate, tongue lobulations and polydactyly, phenotypes characteristic of Oral-Facial-Digital Syndrome. We further show that these alleles disrupt two vital steps of ciliogenesis, basal body docking and recruitment of intraflagellar transport proteins. Moreover, APMS reveals that Rsg1 binds the CPLANE and also the transition zone protein Fam92 in a GTP-dependent manner. Finally, we show that CPLANE is generally required for normal transition zone architecture. Our work demonstrates that CPLANE2/RSG1 is a causative gene for human ciliopathy and also sheds new light on the mechanisms of ciliary transition zone assembly.

cell biology↗

bioRxiv2024

Emergence of cellular nematic order is a conserved feature of gastrulation in animal embryos

Cells undergo dramatic morphological changes during embryogenesis, yet how these changes affect the formation of ordered tissues remains elusive. Here, we show that a phase transition leading to the formation of a nematic liquid crystal state during gastrulation in the development of embryos of fish, frogs, and fruit flies occurs by a common mechanism despite substantial differences between these evolutionarily distant animals. Importantly, nematic order forms early before any discernible changes in the shapes of cells. All three species exhibit similar propagation of the nematic phase, reminiscent of nucleation and growth mechanisms. The spatial correlations in the nematic phase in the notochord region are long-ranged and follow a similar power-law decay (y~x-) with less than unity, indicating a common underlying physical mechanism. To explain the common physical mechanism, we created a theoretical model that not only explains the experimental observations but also predicts that the nematic phase should be disrupted upon loss of planar cell polarity (frog), cell adhesion (frog), and notochord boundary formation (zebrafish). Gene knockdown or mutational studies confirm the theoretical predictions. The combination of experiments and theory provides a unified framework for understanding the potentially universal features of metazoan embryogenesis, in the process shedding light on the advent of ordered structures during animal development.

developmental biology↗

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

New tools reveal PCP-dependent polarized mechanics in the cortex and cytoplasm of single cells during convergent extension

Understanding biomechanics of biological systems is crucial for unraveling complex processes like tissue morphogenesis. However, current methods for studying cellular mechanics in vivo are limited by the need for specialized equipment and often provide limited spatiotemporal resolution. Here we introduce two new techniques, Tension by Transverse Fluctuation (TFlux) and in vivo microrheology, that overcome these limitations. They both offer time-resolved, subcellular biomechanical analysis using only fluorescent reporters and widely available microscopes. Employing these two techniques, we have revealed a planar cell polarity (PCP)-dependent mechanical gradient both in the cell cortex and the cytoplasm of individual cells engaged in convergent extension. Importantly, the non-invasive nature of these methods holds great promise for its application for uncovering subcellular mechanical variations across a wide array of biological contexts. SummaryNon-invasive imaging-based techniques providing time-resolved biomechanical analysis at subcellular scales in developing vertebrate embryos.

developmental 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

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↗

bioRxiv2022

Iterative remodeling of the mouse uterus requires Hedgehog signaling

The adult uterus regenerates in the human during the menstrual cycle, and remodels in the mouse during the estrous cycle. Decades of work has demonstrated that this process is controlled by cycling steroid hormones, estrogen and progesterone. However, downstream signaling pathways that link hormonal action to this regeneration and remodeling are yet to be identified in the cycling uterus. We set out to identify these pathways, with the overarching hypothesis that developmental signaling pathways are redeployed in the adult uterus to control remodeling in the mouse. We were surprised to find that the majority of Hedgehog signaling components were transcriptionally co-regulated throughout the estrous cycle. To test the role of Hh signaling in cyclical uterine remodeling, we conditionally knocked out the major activator of the pathway, smoothened (Smo) using the progesterone receptor cre (PR-Cre). In the absence of Hh signaling, the uterus no longer remodels throughout the estrous cycle. We also show that the smooth muscle fibers of the uterus are significantly larger in the conditional knockouts compared to the controls suggesting hypertrophy of the smooth muscle. Our findings support the possibility that this smooth muscle homeostasis may underlie important aspects of uterine function such as contractility during late-stage pregnancy or the development of uterine smooth muscle tumors.

developmental biology↗

bioRxiv2022

The RhoGEF protein Plekhg5 regulates medioapical actomyosin dynamics of apical constriction during Xenopus gastrulation

Apical constriction results in apical surface reduction in epithelial cells and is a widely used mechanism for epithelial morphogenesis during embryo development. Both medioapical and junctional actomyosin remodeling are involved in apical constriction, but the deployment of medial versus junctional actomyosin in specific developmental processes has not been fully described. Additionally, genetic regulation of actomyosin dynamics during apical constriction is poorly understood in vertebrate systems. In this study, we investigate actomyosin dynamics and their regulation by the RhoGEF protein Plekhg5 in Xenopus bottle cells. Using live imaging and quantitative image analysis, we show that bottle cells assume different shapes, with rounding bottle cells constricting earlier in small clusters followed by fusiform bottle cells forming between the clusters. Though both medioapical and junctional actomyosin accumulate as surface area decreases, apical constriction is better correlated with medioapical actomyosin localization, which may promote formation of microvilli in the apical membrane. Knockdown of plekhg5 disrupts both medioapical actomyosin activity and apical constriction but does not affect initial F-actin dynamics. Taken together, our study reveals distinct cell morphologies, uncovers actomyosin behaviors, and demonstrates the crucial role of a RhoGEF protein in controlling actomyosin dynamics during apical constriction of bottle cells in Xenopus gastrulation.

developmental biology↗

bioRxiv2022

Ordered deployment of distinct ciliary beating machines in growing axonemes of vertebrate multiciliated cells

The beating of motile cilia requires to coordinated action of diverse machineries that include not only the axonemal dynein arms, but also the central apparatus, the radial spokes, and the microtubule inner proteins. These machines exhibit complex radial and proximodistal patterns in mature axonemes, but little is known about the interplay between them during motile ciliogenesis. Here, we describe and quantify the relative rates of axonemal deployment for these diverse cilia beating machineries during the final stages of differentiation of Xenopus epidermal multiciliated cells.

developmental biology↗

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↗

bioRxiv2022

In vivo imaging and regression analysis reveal cell-non-autonomous functions of Shroom3 during neural tube closure.

During neural tube closure, neural ectoderm cells constrict their apical surfaces to bend and fold the tissue into a tube that will become the central nervous system. These cells are physically interconnected via N-cadherin, and mutation of critical genes within relatively small numbers of cells can result in neural tube closure defects due to non-cell autonomous cell behavior defects. Despite this finding, we have a poor understanding of how neuroepithelial cells interact during apical constriction. In our previous paper, we introduced an imaging and analysis paradigm for tracking and quantifying apical constriction during neural tube closure, while also using CRISPR/Cas9 to generate mosaic loss of function of the apical constriction gene shroom3. Here we analyze the behaviors of cells along the mosaic interface of our shroom3 crispant clones, and find that Shroom3 non-cell autonomously regulates apical constriction and N-cadherin localization. Control cells along the interface constrict less, while shroom3 crispant cells along the interface constrict more. Finally, we construct a partial least squares regression (PLSR) model to estimate how both autonomous and non-cell autonomous dynamics of actin and N-cadherin affect apical surface area in both control and shroom3 crispant cells. Overall, our results demonstrate a previously unidentified non-cell autonomous role for Shroom3 in neural tube closure.

developmental biology↗