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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.

This data includes XL/MS and structural models for the Tetrahymena motile cilia interactome.

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 ofTetrahymena 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 inXenopusmulticiliated 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.

Ependymal cells, lining brain ventricular walls, display tufts of cilia that beat in concert promoting laminar Cerebrospinal fluid (CSF) flow within brain ventricles. The ciliary axonemes of multiciliated ependymal cells display a 9+2 microtubule array common to motile cilia. Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how Kinesin motors contribute to cilia motility. Here, we define the function of Kinesin family member 6 (Kif6) using a mutation that lacks a highly conserved C-terminal tail domain (Kif6p.G555fs) and which displays progressive hydrocephalus in mice. An analogous mutation was isolated in a proband displaying macrocephaly, hypotonia, and seizures implicating an evolutionarily conserved function for Kif6 in neurodevelopment. We find that loss of Kif6 function caused decreased ependymal cilia motility and subsequently decreased fluid flow on the surface of brain ventricular walls. Kif6 protein was localized at ependymal cilia and displayed processive motor movement (676 nm/s) on microtubulesin vitro. Loss of the Kif6 C-terminal tail domain did not affect the initial ciliogenesisin vivo, but did result in defects in cilia orientation, the formation of robust apical actin networks, and stabilization of basal bodies at the apical surface. This suggests a novel role for the Kif6 motor in maintenance of ciliary homeostasis of ependymal cells.Summary statementWe found that Kif6 is localized to the axonemes of ependymal cells. In vitro analysis shows that Kif6 moves on microtubules and that its loss mice decrease cilia motility and cilia-driven flow, resulting in hydrocephalus.

Animals are shaped through the movement of large cellular collectives. Such morphogenetic processes require cadherin-based cell adhesion to maintain tissue cohesion and planar cell polarity to coordinate movement. Despite a vast literature surrounding cadherin-based adhesion and planar cell polarity it is unclear how these molecular networks interface. Here we investigate the relationship between cadherins and planar cell polarity during gastrulation cell movements inXenopus laevis. We first assessed the bulk cadherin localization and found that cadherins were enriched at a specific subset of morphogenetically active cell-cell junctions. We then found that disruption of planar cell polarity desynchronized temporal cadherin and actin dynamics. Next, using super-resolution time-lapse miscopy and quantitative image analysis we were able to measure the lifespan and size of individual cadherin clusters. Finally, we show that planar cell polarity not only controls the size of cadherin clusters but, more interestingly, regulates cluster stability. These results reveal an intriguing link between two essential cellular properties, adhesion and planar polarity, and provide insight into the molecular control of morphogenetic cell movements.HighlightsPlanar cell polarity is required for cadherin spatiotemporal dynamics.Super-resolution imaging reveals the spatiotemporal patterns of cadherin clustering.Cadherin cluster lifespan and size are highly heterogenous.Planar cell polarity controls cadherin cluster stability and size.

Convergent extension (CE) is an evolutionarily conserved developmental process whereby polarized collective cell movements drive the elongation of tissues or organs, and defects in CE are associated with multiple human birth defects. Here, we combined embryology with cell biology and newly developed biomechanical tools to interrogate the link between cell behavior and tissue-scale CE. We found that unique patterns of polarized cortex tension are required to resolve individual cell intercalation events, and this in turn maintains normal cell packing configurations that favor the planar propagation of the cellular forces across the tissue, and thus normal CE. Our data suggest that planar polarized force propagation plays a critical role in the propagation of cell behaviors via cellular mechanoreciprocity, underpinning the propagation of tissue-scale CE.

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 theprogesterone 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.

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. Recent data from mice and humans with neural tube defects suggest that key genes required for neural tube closure can exert non-cell autonomous effects on cell behavior, but the nature of these effects remains obscure. Here, we coupled tissue-scale, high-resolution time-lapse imaging of the closing neural tube of Xenopus to multivariate regression modeling, and we show that medial actin accumulation drives apical constriction non-autonomously in neighborhoods of cells, rather than solely in individual cells. To further explore this effect, we examined mosaic crispant embryos and identified both autonomous and non-autonomous effects of the apical constriction protein Shroom3.

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.

Cilia are multifunctional organelles that originated with the last eukaryotic common ancestor and play central roles in the life cycles of diverse organisms. The motile flagella that move single cells like sperm or unicellular organisms, the motile cilia on animal multiciliated cells that generate fluid flow in organs, and the immotile primary cilia that decorate nearly all cells in animals share many protein components in common, yet each also requires specialized proteins to perform their specialized functions. Despite a now-advanced understanding of how such proteins are transported within cilia, we still know very little about how they are transported from their sites of synthesis through the cytoplasm to the ciliary base. Here, we review the literature concerning this underappreciated topic in ciliary cell biology. We discuss both general mechanisms, as well as specific examples of motor-driven active transport and passive transport via diffusion-and-capture. We then provide deeper discussion of specific, illustrative examples, such as the diverse array of protein subunits that together comprise the intraflagellar transport (IFT) system and the multi-protein axonemal dynein motors that drive beating of motile cilia. We hope this Review will spur further work, shedding light not only on ciliogenesis and ciliary signaling, but also on intracellular transport in general.

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.

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.

Apical constriction refers to the active, actomyosin-driven process that reduces apical cell surface area in epithelial cells. Apical constriction is utilized in epithelial morphogenesis during embryonic development in multiple contexts, such as gastrulation, neural tube closure, and organogenesis. Defects in apical constriction can result in congenital birth defects, yet our understanding of the molecular control of apical constriction is relatively limited. To uncover new genetic regulators of apical constriction and gain mechanistic insight into the cell biology of this process, we need reliable assay systems that allow real-time observation and quantification of apical constriction as it occurs and permit gain- and loss-of-function analyses to explore gene function and interaction during apical constriction. In this chapter, we describe using the early Xenopus embryo as an assay system to investigate molecular mechanisms involved in apical constriction during both gastrulation and neurulation.

SummaryConvergent extension (CE) is a collective cell movement required for embryonic axis elongation, neural tube closure, and kidney tubule elongation. During CE, iterative cell intercalations drive the elongation of a tissue 1. In order to move, cells reorganize their actin cytoskeleton; the implication of actin regulators during CE, then, is expected. Interestingly, two other groups of proteins have been shown to be required for this cell behavior: Planar Cell Polarity proteins (PCP)2-4 and Septins5, both of which interact with the actin cytoskeleton 6-14. Disruption of any of these systems have substantial overlap in terms of embryonic phenotypes, but how they interact to govern CE is largely unknown15-17. Here, we find that PCP proteins and Septins sequester actomyosin activity, create flows of actin across the anterior side of cells, and bundle actin into a node and cable system that displays a planar polarized gradient of stiffness.Key findings-PCP- and Septin-dependent actin node and cable structures form and become anteriorized over time-Actin cytoskeletal reorganization correlates with PCP protein polarization.-Actomyosin flows are directed to anterior PCP puncta and PCP movement relies on actin turnover.-Septins localize to anterior nodes and bundle actin filaments into anterior actin cable.-PCP- and Septin-dependent anterior actin cables increase local cellular stiffness and create a planar polarized stiffness gradient across the cell.

The design of an animal's body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development.

Convergent extension is an evolutionarily conserved collective cell movement that elongates the body axis of almost all animals and is required for the morphogenesis of several organ systems. Decades of study have revealed two distinct mechanisms of cell movement during CE, one based on cell crawling and the other on junction contraction. How these two behaviors collaborate during CE is not understood. Here, using quantitative live cell imaging we show that these two modes act both independently and in concert during CE, but that cell movement is more effective when the two modes are integrated via mechano-reciprocity. Based on these findings, we developed a novel computational model that for the first time treats crawling and contraction independently. This model not only confirmed the biomechanical efficacy of integrating the two modes, but also revealed for the first time how the two modes -and their integration- are influenced by cell adhesion. Finally, we use these new insights to further understand the complex CE phenotype resulting from loss of the C-cadherin interacting catenin Arvcf. These data are significant for providing new biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.