Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Rescue of cone and rod photoreceptor function in a CDHR1-model of age-related retinal degeneration.

Age-related macular degeneration (AMD) is the most common cause of untreatable blindness in the developed world. Recently, CDHR1 has been identified as the cause of a subset of AMD that has the appearance of the "dry" form, or geographic atrophy. Biallelic variants in CDHR1-a specialized protocadherin highly expressed in cone and rod photoreceptors-result in blindness from shortened photoreceptor outer segments and progressive photoreceptor cell death. Here we demonstrate long-term morphological, ultrastructural, functional, and behavioral rescue following CDHR1 gene therapy in a relevant murine model, sustained to 23-months after injection. This represents the first demonstration of rescue of a monogenic cadherinopathy in vivo. Moreover, the durability of CDHR1 gene therapy seems to be near complete-with morphological findings of the rescued retina not obviously different from wildtype throughout the lifespan of the mouse model. A follow-on clinical trial in patients with CDHR1-associated retinal degeneration is warranted. Hypomorphic CDHR1 variants may mimic advanced dry AMD. Accurate clinical classification is now critical, as their pathogenesis and treatment are distinct.

SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules.

The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients.

AMP-activated protein kinase is a key regulator of acute neurovascular permeability.

Many neuronal and retinal disorders are associated with pathological hyperpermeability of the microvasculature. We have used explants of rodent retinae to study acute neurovascular permeability, signal transduction and the role of AMP-activated protein kinase (AMPK). Following stimulation with either vascular endothelial growth factor (VEGF-A) or bradykinin (BK), AMPK was rapidly and strongly phosphorylated and acted as a key mediator of permeability downstream of Ca2+. Accordingly, AMPK agonists potently induced acute retinal vascular leakage. AMPK activation led to phosphorylation of endothelial nitric oxide synthase (eNOS, also known as NOS3), which in turn increased VE-cadherin (CDH5) phosphorylation on Y685. In parallel, AMPK also mediated phosphorylation of p38 MAP kinases (hereafter p38) and HSP27 (HSPB1), indicating that it regulated paracellular junctions and cellular contractility, both previously associated with endothelial permeability. Endothelial AMPK provided a missing link in neurovascular permeability, connecting Ca2+ transients to the activation of eNOS and p38, irrespective of the permeability-inducing factor used. Collectively, we find that, due to its compatibility with small molecule antagonists and agonists, as well as siRNA, the ex vivo retina model constitutes a reliable tool to identify and study regulators and mechanisms of acute neurovascular permeability.

Symmetric arrangement of mitochondria:plasma membrane contacts between adjacent photoreceptor cells regulated by Opa1.

Mitochondria are known to play an essential role in photoreceptor function and survival that enables normal vision. Within photoreceptors, mitochondria are elongated and extend most of the inner-segment length, where they supply energy for protein synthesis and the phototransduction machinery in the outer segment, as well as acting as a calcium store. Here, we examined the arrangement of the mitochondria within the inner segment in detail using three-dimensional (3D) electron microscopy techniques and show they are tethered to the plasma membrane in a highly specialized arrangement. Remarkably, mitochondria and their cristae openings align with those of neighboring inner segments. The pathway by which photoreceptors meet their high energy demands is not fully understood. We propose this to be a mechanism to share metabolites and assist in maintaining homeostasis across the photoreceptor cell layer. In the extracellular space between photoreceptors, Müller glial processes were identified. Due to the often close proximity to the inner-segment mitochondria, they may, too, play a role in the inner-segment mitochondrial arrangement as well as metabolite shuttling. OPA1 is an important factor in mitochondrial homeostasis, including cristae remodeling; therefore, we examined the photoreceptors of a heterozygous Opa1 knockout mouse model. The cristae structure in the Opa1+/- photoreceptors was not greatly affected, but the mitochondria were enlarged and had reduced alignment to neighboring inner-segment mitochondria. This indicates the importance of key regulators in maintaining this specialized photoreceptor mitochondrial arrangement.

Membrane trafficking in the retinal pigment epithelium at a glance.

The retinal pigment epithelium (RPE) is a highly specialised pigmented monolayer sandwiched between the choroid and the photoreceptors in the retina. Key functions of the RPE include transport of nutrients to the neural retina, removal of waste products and water from the retina to the blood, recycling of retinal chromophores, absorption of scattered light and phagocytosis of the tips of the photoreceptor outer segments. These functions place a considerable membrane trafficking burden on the RPE. In this Cell Science at a Glance article and the accompanying poster, we focus on RPE-specific adaptations of trafficking pathways. We outline mechanisms underlying the polarised expression of membrane proteins, melanosome biogenesis and movement, and endocytic trafficking, as well as photoreceptor outer segment phagocytosis and degradation. We also briefly discuss theories of how dysfunction in trafficking pathways contributes to retinal disease.

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells.

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle.

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.

Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus.

Motile cilia move body fluids and gametes and the beating of cilia lining the airway epithelial surfaces ensures that they are kept clear and protected from inhaled pathogens and consequent respiratory infections. Dynein motor proteins provide mechanical force for cilia beating. Dynein mutations are a common cause of primary ciliary dyskinesia (PCD), an inherited condition characterized by deficient mucociliary clearance and chronic respiratory disease coupled with laterality disturbances and subfertility. Using next-generation sequencing, we detected mutations in the ciliary outer dynein arm (ODA) heavy chain gene DNAH9 in individuals from PCD clinics with situs inversus and in one case male infertility. DNAH9 and its partner heavy chain DNAH5 localize to type 2 ODAs of the distal cilium and in DNAH9-mutated nasal respiratory epithelial cilia we found a loss of DNAH9/DNAH5-containing type 2 ODAs that was restricted to the distal cilia region. This confers a reduced beating frequency with a subtle beating pattern defect affecting the motility of the distal cilia portion. 3D electron tomography ultrastructural studies confirmed regional loss of ODAs from the distal cilium, manifesting as either loss of whole ODA or partial loss of ODA volume. Paramecium DNAH9 knockdown confirms an evolutionarily conserved function for DNAH9 in cilia motility and ODA stability. We find that DNAH9 is widely expressed in the airways, despite DNAH9 mutations appearing to confer symptoms restricted to the upper respiratory tract. In summary, DNAH9 mutations reduce cilia function but some respiratory mucociliary clearance potential may be retained, widening the PCD disease spectrum.

Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia.

Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left-right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.

PCD Detect: enhancing ciliary features through image averaging and classification.

Primary ciliary dyskinesia (PCD) is an inherited disorder of the motile cilia. Early accurate diagnosis is important to help prevent lung damage in childhood and to preserve lung function. Confirmation of a diagnosis traditionally relied on assessment of ciliary ultrastructure by transmission electron microscopy (TEM); however, >50 known PCD genes have made the identification of biallelic mutations a viable alternative to confirm diagnosis. TEM and genotyping lack sensitivity, and research to improve accuracy of both is required. TEM can be challenging when a subtle or partial ciliary defect is present or affected cilia structures are difficult to identify due to poor contrast. Here, we demonstrate software to enhance TEM ciliary images and reduce background by averaging ciliary features. This includes an option to classify features into groups based on their appearance, to generate multiple averages when a nonhomogeneous abnormality is present. We validated this software on images taken from subjects with well-characterized PCD caused by variants in the outer dynein arm (ODA) heavy chain gene DNAH5. Examining more difficult to diagnose cases, we detected 1) regionally restricted absence of the ODAs away from the ciliary base, in a subject carrying mutations in DNAH9; 2) loss of the typically poorly contrasted inner dynein arms; and 3) sporadic absence of part of the central pair complex in subjects carrying mutations in HYDIN, including one case with an unverified genetic diagnosis. We show that this easy-to-use software can assist in detailing relationships between genotype and ultrastructural phenotype, improving diagnosis of PCD.

Physiological and Pathophysiological Aspects of Primary Cilia-A Literature Review with View on Functional and Structural Relationships in Cartilage.

Cilia are cellular organelles that project from the cell. They occur in nearly all non-hematopoietic tissues and have different functions in different tissues. In mesenchymal tissues primary cilia play a crucial role in the adequate morphogenesis during embryological development. In mature articular cartilage, primary cilia fulfil chemo- and mechanosensitive functions to adapt the cellular mechanisms on extracellular changes and thus, maintain tissue homeostasis and morphometry. Ciliary abnormalities in osteoarthritic cartilage could represent pathophysiological relationships between ciliary dysfunction and tissue deformation. Nevertheless, the molecular and pathophysiological relationships of 'Primary Cilia' (PC) in the context of osteoarthritis is not yet fully understood. The present review focuses on the current knowledge about PC and provide a short but not exhaustive overview of their role in cartilage.

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

Endothelial MAPKs Direct ICAM-1 Signaling to Divergent Inflammatory Functions.

Lymphocyte transendothelial migration (TEM) is critically dependent on intraendothelial signaling triggered by adhesion to ICAM-1. Here we show that endothelial MAPKs ERK, p38, and JNK mediate diapedesis-related and diapedesis-unrelated functions of ICAM-1 in cerebral and dermal microvascular endothelial cells (MVECs). All three MAPKs were activated by ICAM-1 engagement, either through lymphocyte adhesion or Ab-mediated clustering. MAPKs were involved in ICAM-1-dependent expression of TNF-α in cerebral and dermal MVECs, and CXCL8, CCL3, CCL4, VCAM-1, and cyclooxygenase 2 (COX-2) in cerebral MVECs. Endothelial JNK and to a much lesser degree p38 were the principal MAPKs involved in facilitating diapedesis of CD4+ lymphocytes across both types of MVECs, whereas ERK was additionally required for TEM across dermal MVECs. JNK activity was critical for ICAM-1-induced F-actin rearrangements. Furthermore, activation of endothelial ICAM-1/JNK led to phosphorylation of paxillin, its association with VE-cadherin, and internalization of the latter. Importantly ICAM-1-induced phosphorylation of paxillin was required for lymphocyte TEM and converged functionally with VE-cadherin phosphorylation. Taken together we conclude that during lymphocyte TEM, ICAM-1 signaling diverges into pathways regulating lymphocyte diapedesis, and other pathways modulating gene expression thereby contributing to the long-term inflammatory response of the endothelium.

Primary ciliary dyskinesia with normal ultrastructure: three-dimensional tomography detects absence of DNAH11.

In primary ciliary dyskinesia (PCD), motile ciliary dysfunction arises from ciliary defects usually confirmed by transmission electron microscopy (TEM). In 30% of patients, such as those with DNAH11 mutations, apparently normal ultrastructure makes diagnosis difficult. Genetic analysis supports diagnosis, but may not identify definitive causal variants. Electron tomography, an extension of TEM, produces three-dimensional ultrastructural ciliary models with superior resolution to TEM. Our hypothesis is that tomography using existing patient samples will enable visualisation of DNAH11-associated ultrastructural defects. Dual axis tomograms from araldite-embedded nasal cilia were collected in 13 PCD patients with normal ultrastructure (DNAH11 n=7, HYDIN n=2, CCDC65 n=3 and DRC1 n=1) and six healthy controls, then analysed using IMOD and Chimera software.DNAH11 protein is localised to the proximal ciliary region. Within this region, electron tomography indicated a deficiency of >25% of proximal outer dynein arm volume in all patients with DNAH11 mutations (n=7) compared to other patients with PCD and normal ultrastructure (n=6) and healthy controls (n=6). DNAH11 mutations cause a shared abnormality in ciliary ultrastructure previously undetectable by TEM. Advantageously, electron tomography can be used on existing diagnostic samples and establishes a structural abnormality where ultrastructural studies were previously normal.

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment.

The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that "zipper" up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.

CCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation.

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.