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Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cilia are microtubule-based protrusions on the surface of most eukaryotic cells. They are found in most, if not all, vertebrate organs. Prominent cilia form in sensory structures, the eye, the ear, and the nose, where they are crucial for the detection of environmental stimuli, such as light and odors. Cilia are also involved in developmental processes, including left-right asymmetry formation, limb morphogenesis, and the patterning of neurons in the neural tube. Some cilia, such as those found in nephric ducts, are thought to have mechanosensory roles. Zebrafish proved very useful in genetic analysis and imaging of cilia-related processes, and in the modeling of mechanisms behind human cilia abnormalities, known as ciliopathies. A number of zebrafish defects resemble those seen in human ciliopathies. Forward and reverse genetic strategies generated a wide range of cilia mutants in zebrafish, which can be studied using sophisticated genetic and imaging approaches. In this chapter, we provide a set of protocols to examine cilia morphology, motility, and cilia-related defects in a variety of organs, focusing on the embryo and early postembryonic development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4(T804M) mutant. PGE2 synthesis enzyme cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates the cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis.

Cilia are microtubule-based, hair-like organelles involved in sensory function or motility, playing critical roles in many physiological processes such as reproduction, organ development, and sensory perception. In insects, cilia are restricted to certain sensory neurons and sperms, being important for chemical and mechanical sensing, and fertility. Although great progress has been made regarding the mechanism of cilia assembly, the formation of insect cilia remains poorly understand, even in the insect model organism Drosophila. Intraflagellar transport (IFT) is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia. Here we investigated the role of IFT52, a core component of IFT-B, in cilia/flagellar formation in Drosophila. We show that Drosophila IFT52 is distributed along the sensory neuronal cilia, and is essential for sensory cilia formation. Deletion of Ift52 results in severe defects in cilia-related sensory behaviors. It should be noted that IFT52 is not detected in spermatocyte cilia or sperm flagella of Drosophila. Accordingly, ift52 mutants can produce sperms with normal motility, supporting a dispensable role of IFT in Drosophila sperm flagella formation. Altogether, IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects., (© 2022 The Authors. Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

The primary cilium compartmentalizes a tiny fraction of the cell surface and volume, yet many proteins are highly enriched in this area and so efficient mechanisms are necessary to concentrate them in the ciliary compartment. Here we review mechanisms that are thought to deliver protein cargo to the base of cilia and are likely to interact with ciliary gating mechanisms. Given the immense variety of ciliary cytosolic and transmembrane proteins, it is almost certain that multiple, albeit frequently interconnected, pathways mediate this process. It is also clear that none of these pathways is fully understood at the present time. Mechanisms that are discussed below facilitate ciliary localization of structural and signaling molecules, which include receptors, G-proteins, ion channels, and enzymes. These mechanisms form a basis for every aspect of cilia function in early embryonic patterning, organ morphogenesis, sensory perception and elsewhere.

Cilia are protrusions on the surface of cells. They are frequently motile and function to propel cells in an aqueous environment or to generate fluid flow. Equally important is the role of immotile cilia in detecting environmental changes or in sensing extracellular signals. The structure of cilia is supported by microtubules, and their formation requires microtubule-dependent motors, kinesins, which are thought to transport both structural and signaling ciliary proteins from the cell body into the distal portion of the ciliary shaft. In multicellular organisms, multiple kinesins are known to drive ciliary transport, and frequently cilia of a single cell type require more than one kinesin for their formation and function. In addition to kinesin-2 family motors, which function in cilia of all species investigated so far, kinesins from other families contribute to the transport of signaling proteins in a tissue-specific manner. It is becoming increasingly obvious that functional relationships between ciliary kinesins are complex, and a good understanding of these relationships is essential to comprehend the basis of biological processes as diverse as olfaction, vision, and embryonic development.

The differentiation of cilia is mediated by kinesin-driven transport. As the function of kinesins in vertebrate ciliogenesis is poorly characterized, we decided to determine the role of kinesin-2 family motors--heterotrimeric kinesin-II and the homodimeric Kif17 kinesin--in zebrafish cilia. We report that kif17 is largely dispensable for ciliogenesis; kif17 homozygous mutant animals are viable and display subtle morphological defects of olfactory cilia only. In contrast to that, the kif3b gene, encoding a heterotrimeric kinesin subunit, is necessary for cilia differentiation in most tissues, although exceptions exist, and include photoreceptors and a subset of hair cells. Cilia of these cell types persist even in kif3b/kif17 double mutants. Although we have not observed a functional redundancy of kif3b and kif17, kif17 is able to substitute for kif3b in some cilia. In contrast to kif3b/kif17 double mutants, simultaneous interference with kif3b and kif3c leads to the complete loss of photoreceptor and hair cell cilia, revealing redundancy of function. This is in agreement with the idea that Kif3b and Kif3c motor subunits form complexes with Kif3a, but not with each other. Interestingly, kif3b mutant photoreceptor cilia differentiate with a delay, suggesting that kif3c, although redundant with kif3b at later stages of differentiation, is not active early in photoreceptor ciliogenesis. Consistent with that, the overexpression of kif3c in kif3b mutants rescues early photoreceptor cilia defects. These data reveal unexpected diversity of functional relationships between vertebrate ciliary kinesins, and show that the repertoire of kinesin motors changes in some cilia during their differentiation.

Cilia are required for the development and function of many organs. Efficient transport of protein cargo along ciliary axoneme is necessary to sustain these processes. Despite its importance, the mode of interaction between the intraflagellar ciliary transport (IFT) mechanism and its cargo proteins remains poorly understood. Our studies demonstrate that IFT particle components, and a Meckel-Gruber syndrome 1 (MKS1)-related, B9 domain protein, B9d2, bind each other and contribute to the ciliary localization of Inversin (Nephrocystin 2). B9d2, Inversin, and Nephrocystin 5 support, in turn, the transport of a cargo protein, Opsin, but not another photoreceptor ciliary transmembrane protein, Peripherin. Interestingly, the components of this mechanism also contribute to the formation of planar cell polarity in mechanosensory epithelia. These studies reveal a molecular mechanism that mediates the transport of selected ciliary cargos and is of fundamental importance for the differentiation and survival of sensory cells.

The cilium, a previously little studied cell surface protrusion, has emerged as an important organelle in vertebrate cells. This tiny structure is essential for normal embryonic development, including the formation of left-right asymmetry, limb morphogenesis, and the differentiation of sensory cells. In the adult, cilia also function in a variety of processes, such as the survival of photoreceptor cells, and the homeostasis in several tissues, including the epithelia of nephric ducts. Human ciliary malfunction is associated with situs inversus, kidney cysts, polydactyly, blindness, mental retardation, obesity, and many other abnormalities. The genetic accessibility and optical transparency of the zebrafish make it an excellent vertebrate model system to study cilia biology. In this chapter, we describe the morphology and distribution of cilia in zebrafish embryonic and larval organs. We also provide essential protocols to analyze cilia formation and function., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The formation and function of cilia involves the movement of intraflagellar transport (IFT) particles underneath the ciliary membrane, along axonemal microtubules. Although this process has been studied extensively, its molecular basis remains incompletely understood. For example, it is unknown how the IFT particle interacts with transmembrane proteins. To study the IFT particle further, we examined elipsa, a locus characterized by mutations that cause particularly early ciliogenesis defects in zebrafish. We show here that elipsa encodes a coiled-coil polypeptide that localizes to cilia. Elipsa protein binds to Ift20, a component of IFT particles, and Elipsa homologue in Caenorhabditis elegans, DYF-11, translocates in sensory cilia, similarly to the IFT particle. This indicates that Elipsa is an IFT particle polypeptide. In the context of zebrafish embryogenesis, Elipsa interacts genetically with Rabaptin5, a well-studied regulator of endocytosis, which in turn interacts with Rab8, a small GTPase, known to localize to cilia. We show that Rabaptin5 binds to both Elipsa and Rab8, suggesting that these proteins provide a bridging mechanism between the IFT particle and protein complexes that assemble at the ciliary membrane.

Cilia play diverse roles in vertebrate and invertebrate sensory neurons. We show that a mutation of the zebrafish oval (ovl) locus affects a component of the ciliary transport (IFT) mechanism, the IFT88 polypeptide. In mutant retina, cilia are generated but not maintained, producing the absence of photoreceptor outer segments. A loss of cilia also occurs in auditory hair cells and olfactory sensory neurons. In all three sense organs, cilia defects are followed by degeneration of sensory cells. Similar phenotypes are induced by the absence of the IFT complex B polypeptides, ift52 and ift57, but not by the loss of complex A protein, ift140. The degeneration of mutant photoreceptor cells is caused, at least partially, by the ectopic accumulation of opsins. These studies reveal an essential role for IFT genes in vertebrate sensory neurons and implicate the molecular components of intraflagellar transport in degenerative disorders of these cells.

Primary cilia, microtubule-based protrusions present on the surface of most mammalian cells, function as sensory organelles that monitor extracellular signals and transduce them into intracellular biochemical responses. There is renewed research interest in primary cilia due to their essential roles in development, tissue homeostasis, and human diseases. Primary cilia dysfunction causes a large spectrum of human diseases, collectively known as ciliopathies. Despite significant advances in our understanding of primary cilia, there are still no effective agents for treating ciliopathies. Primary ciliogenesis is a highly ordered process involving membrane trafficking, basal body maturation, vesicle docking and fusion, transition zone assembly, and axoneme extension, in which actin and microtubule networks play critical and multiple roles. Actin and microtubule network architecture, isotropy, and dynamics are tightly controlled by cytoskeleton-associated proteins, a growing number of which are now recognized as responsible for cilium formation and maintenance. Here we summarize the roles of actin and microtubules and their associated proteins in primary ciliogenesis and maintenance. In doing so, we highlight that targeting cytoskeleton-associated proteins may be a promising therapeutic strategy for the treatment of ciliopathies., (© 2022 Wiley Periodicals LLC.)

Cellular processes are highly dependent on a dynamic proteome that undergoes structural and functional rearrangements to allow swift conversion between different cellular states. By inducing proteasomal degradation of inhibitory or stimulating factors, ubiquitylation is particularly well suited to trigger such transitions. One prominent example is the remodelling of the centrosome upon cell cycle exit, which is required for the formation of primary cilia - antenna-like structures on the surface of most cells that act as integrative hubs for various extracellular signals. Over the last decade, many reports on ubiquitin-related events involved in the regulation of ciliogenesis have emerged. Very often, these processes are considered to be initiated ad hoc, that is, directly before its effect on cilia biogenesis becomes evident. While such a temporal restriction may hold true for the majority of events, there is evidence that some of them are initiated earlier during the cell cycle. Here, we provide an overview of ubiquitin-dependent processes in ciliogenesis and discuss available data that indicate such an early onset of proteolytic regulation within preceding cell cycle stages., (© 2022 The Authors. BioEssays published by Wiley Periodicals LLC.)

The primary cilium is a sensory organelle that is vital in regulating several signalling pathways. Unlike most organelles cilia are open to the rest of the cell, not enclosed by membranes. The distinct protein composition is crucial to the function of cilia and many signalling proteins and receptors are specifically concentrated within distinct compartments. To maintain this composition, a mechanism is required to deliver proteins to the cilium whilst another must counter the entropic tendency of proteins to distribute throughout the cell. The combination of the two mechanisms should result in the concentration of ciliary proteins to the cilium. In this review we will look at different cellular mechanisms that play a role in maintaining the distinct composition of cilia, including regulation of ciliary access and trafficking of ciliary proteins to, from and within the cilium.

Primary cilia have been found to function as mechanosensors in low-magnitude high-frequency vibration (LMHFV)-induced osteogenesis. The PGE2 also regulates bone homeostasis and mechanical osteogenesis through its receptor EP4 signaling, but its involvement in LMHFV-induced or in primary cilia-induced osteogenesis has not been investigated. We hypothesized that LMHFV stimulates osteoblast (OB) differentiation by activating the COX2-PGE2-EP pathway in a manner dependent on primary cilia and that primary cilia are also affected by the PGE2 pathway. In this study, through western blot analysis, RNA interference, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cytochemical staining, we observed that COX2, mPGES-1, and PGE2 levels were markedly elevated in cells treated with LMHFV and were greatly decreased in LMHFV-treated cells following IFT88 silencing. EP4 expression was significantly increased in OBs following LMHFV treatment, but IFT88 silencing significantly blocked this increase. EP4 localized to the bases of primary cilia. LMHFV reduced the length and abundance of primary cilia, but the cells could self-repair their primary cilia after mechanical damage. EP4 antagonism significantly blocked the LMHFV-induced increase in IFT88 expression and blocked the recovery of primary cilia length and the proportion of cells with primary cilia. In addition, COX2 or EP4 antagonism disrupted LMHFV-induced osteogenesis. These results demonstrate the integration of and crosstalk between primary cilia and the COX2-PGE2-EP4 signaling pathway under mechanical stimulation., (© 2020 Wiley Periodicals LLC.)

Intraflagellar transport (IFT) represents a bidirectional dynamic process that carries cargo essential for cilia building and the maintenance of ciliary function, which is important for the locomotion of single cells, intracellular and intercellular signalling transduction. Accumulated evidence has revealed that defects in IFT cause several clinical disorders. Here, we determined the role of IFT80, an IFT-B protein that is mutated in Jeune asphyxiating thoracic dystrophy. Using the RNAi method in the ciliate Paramecium as model, we found that loss of IFT80 prevents cilia biogenesis and causes strong cell lethality. A specific antibody against IFT80 was also prepared in our study, which labelled IFT80 in cilia of Paramecium. GFP fusion experiments were performed to illustrate the dynamic movement of IFT-A and IFT-B proteins in cilia of Paramecium; then, we found that the depletion of IFT80 in cells prevents IFT-A and IFT-B proteins from entering the cilia. Our results showed the distribution change of other IFT proteins in cells that were depleted of IFT80, and we discuss the possible roles of IFT80 in Paramecium., (© 2020 International Society of Protistologists.)

Primary cilia are nonmotile sensory organelles found on the surface of almost all kidney tubule epithelial cells. Being exposed to the tubular lumen, primary cilia are thought to be chemo- and mechanosensors of luminal composition and flux, respectively. We hypothesized that, Na + transport and primary cilia exist in a sensory functional connection in mature renal tubule epithelial cells. Our results demonstrate that primary cilium length is reduced in mineralocorticoid receptor (MR) knockout (KO) mice in a cell autonomous manner along the aldosterone-sensitive distal nephron (ADSN) compared with wild type (as µm ± SEM; 3.1 ± 0.2 vs 4.0 ± 0.1). In mouse cortical collecting duct (mCCD) cl1 cells, which are a model of collecting duct (CD) principal cells, changes in Na + transport intensity were found to mediate primary cilium length in response to aldosterone (as µm ± SEM: control: 2.7 ± 0.9 vs aldosterone treated: 3.8 ± 0.8). Cilium length was positively correlated with the availability of IFT88, a major intraflagellar anterograde transport complex B component, which is stabilized in response to exposure to aldosterone treatment. This suggests that the abundance of IFT88 is a regulated, rate limiting factor in the elongation of primary cilia. As previously observed in vivo, aldosterone treatment increased cell volume of cultured CD principal cells. Knockdown of IFT88 prevents ciliogenesis and inhibits the adaptive increase in cell size that was observed in response to aldosterone treatment. In conclusion, our results reveal a functional connection between Na + transport, primary cilia, and cell size, which may play a key role in the morphological and functional adaptation of the CD to sustained changes in active Na + reabsorption due to variations in aldosterone secretion., (© 2019 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)

Cluap1/IFT38 is a ciliary protein that belongs to the IFT-B complex and is required for ciliogenesis. In this study, we have examined the behaviors of Cluap1 protein in nonciliated and ciliated cells. In proliferating cells, Cluap1 is located at the distal appendage of the mother centriole. When cells are induced to form cilia, Cluap1 is found in a novel noncentriolar compartment, the cytoplasmic IFT spot, which mainly exists once in a cell. Other IFT-B proteins such as IFT46 and IFT88 are colocalized in this spot. The cytoplasmic IFT spot is present in mouse embryonic fibroblasts (MEFs) but is absent in ciliogenesis-defective MEFs lacking Cluap1, Kif3a or Odf2. The cytoplasmic IFT spot is also found in mouse embryos but is absent in the Cluap1 mutant embryo. When MEFs are induced to form cilia, the cytoplasmic IFT spot appears at an early step of ciliogenesis but starts to disappear when ciliogenesis is mostly completed. These results suggest that IFT-B proteins such as Cluap1 accumulate in a previously undescribed cytoplasmic compartment during ciliogenesis., (© 2019 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Primary cilia have specific proteins on their membrane to fulfill their sensory functions. Preservation of the specific protein composition of cilia relies on the barrier function of the transition zone (TZ) located at the ciliary base. Defects in cilia and the TZ cause ciliopathies, which have diverse clinical manifestations, including Meckel syndrome (MKS). Many of the proteins mutated in individuals with MKS are known to constitute the MKS module of the TZ. Although TXNDC15 (also known as MKS14) is a thioredoxin-related transmembrane protein that is localized mainly in the endoplasmic reticulum (ER) and is mutated in individuals with MKS, its role at the TZ or within cilia has not been characterized. Here, we show that TXNDC15-knockout cells have defects in MKS module assembly and in ciliary membrane protein localization. These defects in TXNDC15-knockout cells were not rescued by exogenous expression of any of the TXNDC15 constructs with MKS variations in the thioredoxin domain. Furthermore, TXNDC15 with mutations of two cysteine residues within the thioredoxin domain failed to rescue defects in TXNDC15-knockout cells, suggesting that TXNDC15 controls the TZ integrity from outside the TZ via its thioredoxin domain. [ABSTRACT FROM AUTHOR]

Obesity is a highly prevalent disorder with complex aetiology. Therefore, studying its associated cellular and molecular pathways may be aided by analysing genetic tractable diseases. In this context, the study of ciliopathies such as Bardet–Biedl syndrome has highlighted the relevance of primary cilia in obesity, both in the central nervous system and peripheral tissues. Based on our previous in vitro results supporting the role of a novel Bbs4-cilia-Fstl1 axis in adipocyte differentiation, we evaluated the in vivo relevance of the zebrafish orthologous genes fstl1a and fstl1b in primary cilia and adipose tissue development. Using a combination of knockdowns and a new fstl1a mutant line, we show that fstl1a promotes primary cilia formation in early embryos and participates in adipose tissue formation in larvae. We also show that fstl1b partially compensates for the loss of fstl1a. Moreover, in high fat diet, fstl1a depletion affects the expression of differentiation and mature adipocyte markers. These results agree with our previous in vitro data and provide further support for the role of FSTL1 as a regulator of adipose tissue formation. Dissecting the exact biological role of proteins such as FSTL1 will likely contribute to understand obesity onset and presentation. [ABSTRACT FROM AUTHOR]

Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left–right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process. [ABSTRACT FROM AUTHOR]

Errors in embryonic cardiac development are a leading cause of congenital heart defects (CHDs), including morphological abnormalities of the heart that are often detected after birth. In the past few decades, an emerging role for cilia in the pathogenesis of CHD has been identified, but this topic still largely remains an unexplored area. Mouse forward genetic screens and whole exome sequencing analysis of CHD patients have identified enrichment for de novo mutations in ciliary genes or non‐ciliary genes, which regulate cilia‐related pathways, linking cilia function to aberrant cardiac development. Key events in cardiac morphogenesis, including left–right asymmetric development of the heart, are dependent upon cilia function. Cilia dysfunction during left–right axis formation contributes to CHD as evidenced by the substantial proportion of heterotaxy patients displaying complex CHD. Cilia‐transduced signaling also regulates later events during heart development such as cardiac valve formation, outflow tract septation, ventricle development, and atrioventricular septa formation. In this review, we summarize the role of motile and non‐motile (primary cilia) in cardiac asymmetry establishment and later events during heart development. [ABSTRACT FROM AUTHOR]

Most mammalian cells have a single primary cilium that acts as a signalling hub in mediating cellular functions. However, little is known about the mechanisms that result in aberrant supernumerary primary cilia per cell. In this study, we re‐analysed a previously published whole‐genome siRNA‐based reverse genetic screen for genes mediating ciliogenesis to identify knockdowns that permit multi‐ciliation. We identified siRNA knockdowns that caused significant formation of supernumerary cilia, validated candidate hits in different cell‐lines and confirmed that RACGAP1, a component of the centralspindlin complex, was the strongest candidate hit at the whole‐genome level. Following loss of RACGAP1, mother centrioles were specified correctly prior to ciliogenesis and the cilia appeared normal. Live cell imaging revealed that increased cilia incidence was caused by cytokinesis failure which led to the formation of multinucleate cells with supernumerary cilia. This suggests that the signalling mechanisms for ciliogenesis are unable to identify supernumerary centrosomes and therefore allow ciliation of duplicated centrosomes as if they were in a new diploid daughter cell. These results, demonstrating that aberrant ciliogenesis is de‐coupled from cell cycle regulation, have functional implications in diseases marked by centrosomal amplification. [ABSTRACT FROM AUTHOR]

The cilium evolved to provide the ancestral eukaryote with the ability to move and sense its environment. Acquiring these functions required the compartmentalization of a dynein‐based motility apparatus and signaling proteins within a discrete subcellular organelle contiguous with the cytosol. Here, we explore the potential molecular mechanisms for how the proximal‐most region of the cilium, termed transition zone (TZ), acts as a diffusion barrier for both membrane and soluble proteins and helps to ensure ciliary autonomy and homeostasis. These include a unique complement and spatial organization of proteins that span from the microtubule‐based axoneme to the ciliary membrane; a protein picket fence; a specialized lipid microdomain; differential membrane curvature and thickness; and lastly, a size‐selective molecular sieve. In addition, the TZ must be permissive for, and functionally integrates with, ciliary trafficking systems (including intraflagellar transport) that cross the barrier and make the ciliary compartment dynamic. The quest to understand the TZ continues and promises to not only illuminate essential aspects of human cell signaling, physiology, and development, but also to unravel how TZ dysfunction contributes to ciliopathies that affect multiple organ systems, including eyes, kidney, and brain. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) is the most common cause of hereditary blindness, and may occur in isolation as a non-syndromic condition or alongside other features in a syndromic presentation. Biallelic or monoallelic mutations in one of eight genes encoding pre-mRNA splicing factors are associated with nonsyndromic RP. The molecular mechanism of disease remains incompletely understood, limiting opportunities for targeted treatment. Here we use CRISPR and base edited PRPF6 and PRPF31 mutant cell lines, and publiclyavailable data from human PRPF31+/- patient derived retinal organoids and PRPF31 siRNA-treated organotypic retinal cultures to confirm an enrichment of differential splicing of microtubule, centrosomal, cilium and DNA damage response pathway genes in these cells. We show that genes with microtubule/centrosome/centriole/cilium gene ontology terms are enriched for weak 3' and 5' splice sites, and that subtle defects in spliceosome activity predominantly affect efficiency of splicing of these exons. We suggest that the primary defect in PRPF6 or PRPF31 mutant cells is microtubule and centrosomal defects, leading to defects in cilium and mitotic spindle stability, with the latter leading to DNA damage, triggering differential splicing of DNA damage response genes to activate this pathway. Finally, we expand understanding of "splicing factor RP" by investigating the function of TTLL3, one of the most statistically differentially expressed genes in PRPF6 and PRPF31 mutant cells. We identify that TTLL3 is the only tubulin glycylase expressed in the human retina, essential for monoglycylation of microtubules of the cilium, including the retinal photoreceptor cilium, to prevent cilium degeneration and retinal degeneration. Our preliminary data suggest that rescue of tubulin glycylation through overexpression of TTLL3 is sufficient to rescue cilium number in PRPF6 and PRPF31 mutant cells, suggesting that this defect underlies the cellular defect and may represent a potential target for therapeutic intervention in this group of disorders. [ABSTRACT FROM AUTHOR]

Scoliosis is a common spinal deformity that considerably affects the physical and psychological health of patients. Studies have shown that genetic factors play an important role in scoliosis. However, its etiopathogenesis remain unclear, partially because of the genetic heterogeneity of scoliosis and the lack of appropriate model systems. Recently, the development of efficient gene editing methods and high-throughput sequencing technology has made it possible to explore the underlying pathological mechanisms of scoliosis. Owing to their susceptibility for developing scoliosis and high genetic homology with human, zebrafish are increasingly being used as a model for scoliosis in developmental biology, genetics, and clinical medicine. Here, we summarize the recent advances in scoliosis research on zebrafish and discuss the prospects of using zebrafish as a scoliosis model. [ABSTRACT FROM AUTHOR]

Meiosis is essential for evolution and genetic diversity in almost all sexual eukaryotic organisms. The mechanisms of meiotic recombination, such as synapsis, have been extensively investigated. However, it is still unclear whether signals from the cytoplasm or even from outside of the cell can regulate the meiosis process. Cilia are microtubule-based structures that protrude from the cell surface and function as signaling hubs to sense extracellular signals. Here, we reported an unexpected and critical role of cilia during meiotic recombination. During gametogenesis of zebrafish, cilia were specifically present in the prophase stages of both primary spermatocytes and primary oocytes. By developing a germ cell-specific CRISPR/Cas9 system, we demonstrated that germ cell-specific depletion of ciliary genes resulted in compromised double-strand break repair, reduced crossover formation, and increased germ cell apoptosis. Our study reveals a previously undiscovered role for cilia during meiosis and suggests that extracellular signals may regulate meiotic recombination via this particular organelle. [ABSTRACT FROM AUTHOR]

Cilia are quasi-ubiquitous microtubule-based sensory organelles, which play vital roles in signal transduction during development and cell homeostasis. Dysfunction of cilia leads to a group of Mendelian disorders called ciliopathies, divided into different diagnoses according to clinical phenotype constellation and genetic causes. Joubert syndrome (JBTS) is a prototypical ciliopathy defined by a diagnostic cerebellar and brain stem malformation termed the "Molar Tooth Sign" (MTS), in addition to which patients display variable combinations of typical ciliopathy phenotypes such as retinal dystrophy, fibrocystic renal disease, polydactyly or skeletal dystrophy. Like most ciliopathies, JBTS is genetically highly heterogeneous with ∼40 associated genes. Zebrafish are widely used to model ciliopathies given the high conservation of ciliary genes and the variety of specialized cilia types similar to humans. In this review, we compare different existing JBTS zebrafish models with each other and describe their contributions to our understanding of JBTS pathomechanism. We find that retinal dystrophy, which is the most investigated ciliopathy phenotype in zebrafish ciliopathy models, is caused by distinct mechanisms according to the affected gene. Beyond this, differences in phenotypes in other organs observed between different JBTS-mutant models suggest tissue-specific roles for proteins implicated in JBTS. Unfortunately, the lack of systematic assessment of ciliopathy phenotypes in the mutants described in the literature currently limits the conclusions that can be drawn from these comparisons. In the future, the numerous existing JBTS zebrafish models represent a valuable resource that can be leveraged in order to gain further insights into ciliary function, pathomechanisms underlying ciliopathy phenotypes and to develop treatment strategies using small molecules. [ABSTRACT FROM AUTHOR]

The Abelson-helper integration site 1 (AHI1) gene encodes for a ciliary transition zone localizing protein that when mutated causes the human ciliopathy, Joubert syndrome. We prepared and examined neuronal cultures derived from male and female embryonic Ahi1+/+ and Ahi1-/- mice (littermates) and found that the distribution of ciliary melanin-concentrating hormone receptor-1 (MchR1) was significantly reduced in Ahi1-/- neurons; however, the total and surface expression of MchR1 on Ahi1-/- neurons was similar to controls (Ahi1+/+). This indicates that a pathway for MchR1 trafficking to the surface plasma membrane is intact, but the process of targeting MchR1 into cilia is impaired in Ahi1-deficient mouse neurons, indicating a role for Ahi1 in localizing MchR1 to the cilium. Mouse Ahi1-/- neurons that fail to accumulate MchR1 in the ciliary membrane have significant decreases in two downstream MchR1 signaling pathways [cAMP and extracellular signal-regulated kinase (Erk)] on MCH stimulation. These results suggest that the ciliary localization of MchR1 is necessary and critical for MchR1 signaling, with Ahi1 participating in regulating MchR1 localization to cilia, and further supporting cilia as critical signaling centers in neurons. [ABSTRACT FROM AUTHOR]

Motile cilia are miniature, whip-like organelles whose beating generates a directional fluid flow. The flow generated by ciliated epithelia is a subject of great interest, as defective ciliary motility results in severe human diseases called motile ciliopathies. Despite the abundance of motile cilia in diverse organs including the nervous system, their role in organ development and homeostasis remains poorly understood. Recently, much progress has been made regarding the identity of motile ciliated cells and the role of motilecilia- mediated flow in the development and physiology of the nervous system. In this review, we will discuss these recent advances from sensory organs, specifically the nose and the ear, to the spinal cord and brain ventricles. [ABSTRACT FROM AUTHOR]

Polydactyly is one of the most frequent inherited defects of the limbs characterized by supernumerary digits and high‐genetic heterogeneity. Among the many genes involved, either in isolated or syndromic forms, eight have been implicated in postaxial polydactyly (PAP). Among those, IQCE has been recently identified in a single consanguineous family. Using whole‐exome sequencing in patients with uncharacterized ciliopathies, including PAP, we identified three families with biallelic pathogenic variations in IQCE. Interestingly, the c.895_904del (p.Val301Serfs*8) was found in all families without sharing a common haplotype, suggesting a recurrent mechanism. Moreover, in two families, the systemic phenotype could be explained by additional pathogenic variants in known genes (TULP1, ATP6V1B1). RNA expression analysis on patients' fibroblasts confirms that the dysfunction of IQCE leads to the dysregulation of genes associated with the hedgehog‐signaling pathway, and zebrafish experiments demonstrate a full spectrum of phenotypes linked to defective cilia: Body curvature, kidney cysts, left–right asymmetry, misdirected cilia in the pronephric duct, and retinal defects. In conclusion, we identified three additional families confirming IQCE as a nonsyndromic PAP gene. Our data emphasize the importance of taking into account the complete set of variations of each individual, as each clinical presentation could finally be explained by multiple genes. [ABSTRACT FROM AUTHOR]

Cilia are complex organelles involved in a broad array of functions in eukaryotic organisms. Nematodes employ cilia for environmental sensing, which shapes developmental decisions and influences morphologically plastic traits and adaptive behaviours. Here, we assess the role of cilia in the nematode Pristionchus pacificus, and determine their importance in regulating the developmentally plastic mouth-form decision in addition to predatory feeding and self-recognition behaviours, all of which are not present in Caenorhabditis elegans. An analysis of a multitude of cilia-related mutants including representatives of the six protein subcomplexes required in intraflagellar transport (IFT) plus the regulatory factor X transcription factor daf-19 revealed that cilia are essential for processing the external cues influencing the mouth-form decision and for the efficient detection of prey. Surprisingly, we observed that loss-of-function mutations in the different IFT components resulted in contrasting mouth-form phenotypes and different degrees of predation deficiencies. This observation supports the idea that perturbing different IFT subcomplexes has different effects on signalling downstream of the cilium. Finally, selfrecognition was maintained in the cilia deficient mutants tested, indicating that the mechanisms triggering self-recognition in P. pacificus may not require the presence of fully functional cilia. [ABSTRACT FROM AUTHOR]

Abstrct: Background: Cilia are essential for morphogenesis and maintenance of many tissues. Loss‐of‐function of cilia in early Zebrafish development causes a range of vascular defects, including cerebral hemorrhage and reduced arterial vascular mural cell coverage. In contrast, loss of endothelial cilia in mice has little effect on vascular development. We therefore used a conditional rescue approach to induce endothelial cilia ablation after early embryonic development and examined the effect on vascular development and mural cell development in postembryonic, juvenile, and adult Zebrafish. Results: ift54(elipsa)‐mutant Zebrafish are unable to form cilia. We rescued cilia formation and ameliorated the phenotype of ift54 mutants using a novel Tg(ubi:loxP‐ift54‐loxP‐myr‐mcherry,myl7:EGFP)sh488 transgene expressing wild‐type ift54 flanked by recombinase sites, then used a Tg(kdrl:cre)s898 transgene to induce endothelial‐specific inactivation of ift54 at postembryonic ages. Fish without endothelial ift54 function could survive to adulthood and exhibited no vascular defects. Endothelial inactivation of ift54 did not affect development of tagln‐positive vascular mural cells around either the aorta or the caudal fin vessels, or formation of vessels after tail fin resection in adult animals. Conclusions: Endothelial cilia are not essential for development and remodeling of the vasculature in juvenile and adult Zebrafish when inactivated after embryogenesis. Key Findings: We successfully rescued homozygous ift54 mutants using a rescue trangene expressing wild type ift54, which partially recovered cilia formation and function.Endothelial specific inactivation of ift54 (required for cilia formation) did not affect endothelial patterning in juvenile and adult zebrafish.Endothelial ift54 inactivation had no effect on mural cell coverage in juveniles and adult zebrafish. [ABSTRACT FROM AUTHOR]

X-linked retinitis pigmentosa 2 (XLRP2) patients and Rp2 null mice exhibit severe cone photoreceptor degeneration. However, due to the paucity of cones in mammalian model systems, it is not clear how cones respond to the loss of RP2. Here we have used the Nrl-/- mice, which develop a rodless and short wavelength (S) opsin-containing cone-only retina, to generate Rp2 null:: Nrl-/- double knock out (Rp2 -DKO) mice. We found that the ciliary axoneme and the outer segments (OSs) of the cones were significantly longer with disorganized membrane infoldings as compared to the Nrl-/- mice. Additionally, we found misregulation in the expression of the genes related to ophthalmic disease, cell trafficking, and stress-response in the Rp2 -DKO mice prior to the onset of cone degeneration. Surprisingly, the loss of RP2 did not affect progressive photoreceptor dysfunction of the Nrl-/- mice and the trafficking of S opsin. Our data suggest that RP2 is a negative regulator of cone OS length but does not affect S-opsin trafficking and S-cone function. Our studies also provide a cone-only platform to design cone-targeted therapeutic strategies for X-linked RP2. [ABSTRACT FROM AUTHOR]

Cilia and flagella are microtubule‐based antenna which are highly conserved among eukaryotes. In vertebrates, primary and motile cilia have evolved to exert several key functions during development and tissue homoeostasis. Ciliary dysfunction in humans causes a highly heterogeneous group of diseases called ciliopathies, a class of genetic multisystemic disorders primarily affecting kidney, skeleton, retina, lung and the central nervous system. Among key ciliary proteins, kinesin family members (KIF) are microtubule‐interacting proteins involved in many diverse cellular functions, including transport of cargo (organelles, proteins and lipids) along microtubules and regulating the dynamics of cytoplasmic and spindle microtubules through their depolymerising activity. Many KIFs are also involved in diverse ciliary functions including assembly/disassembly, motility and signalling. We here review these ciliary kinesins in vertebrates and focus on their involvement in ciliopathy‐related disorders. Review: Cilia are highly conserved, microtubule‐based organelles subdivided into motile and non‐motile/primary cilia, both playing key roles during development and tissue homoeostasis. This review summarises the ciliary functions of the Kinesin superfamily (KIFs) of microtubule‐based motors. Initially implicated in ciliary assembly, KIFs were later shown to be involved in many other key ciliary functions, including cargo transport, signalling (Hedgehog) and the regulation of cilia length and disassembly. [ABSTRACT FROM AUTHOR]

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates. [ABSTRACT FROM AUTHOR]

Background: KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. Results: Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. Conclusions: Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding. [ABSTRACT FROM AUTHOR]

Abstract: Primary cilium structure and function relies on control of ciliary membrane homeostasis, regulated by membrane trafficking processes that deliver and retrieve ciliary components at the periciliary membrane. However, the molecular mechanisms controlling ciliary membrane establishment and maintenance, especially in relation to endocytosis, remain poorly understood. Here, using Caenorhabditis elegans, we describe closely linked functions for early endosome (EE) maturation factors RABS‐5 (Rabenosyn‐5) and VPS‐45 (VPS45) in regulating cilium length and morphology, ciliary and periciliary membrane volume, and ciliary signalling‐related sensory behaviour. We demonstrate that RABS‐5 and VPS‐45 control periciliary vesicle number and levels of select EE/endocytic markers (WDFY‐2, CAV‐1) and the ciliopathy membrane receptor PKD‐2 (polycystin‐2). Moreover, we show that CAV‐1 (caveolin‐1) also controls PKD‐2 ciliary levels and associated sensory behaviour. These data link RABS‐5 and VPS‐45 ciliary functions to the processing of periciliary‐derived endocytic vesicles and regulation of ciliary membrane homeostasis. Our findings also provide insight into the regulation of PKD‐2 ciliary levels via integrated endosomal sorting and CAV‐1‐mediated endocytosis. [ABSTRACT FROM AUTHOR]

Abstract: Congenital malformations affecting the neural tube can present as isolated malformations or occur in association with other developmental abnormalities and syndromes. Using high‐resolution copy number screening in 66 fetuses with neural tube defects, we identified six fetuses with likely pathogenic mutations, three aneuploidies (one trisomy 13 and two trisomy 18) and three deletions previously reported in NTDs (one 22q11.2 deletion and two 1p36 deletions) corresponding to 9% of the cohort. In addition, we identified five rare deletions and two duplications of uncertain significance including a rare intragenic heterozygous in‐frame WDR63 deletion in a fetus with occipital encephalocele. Whole genome sequencing verified the deletion and excluded known pathogenic variants. The deletion spans exons 14–17 resulting in the expression of a protein missing the third and fourth WD‐repeat domains. These findings were supported by CRISPR/Cas9‐mediated somatic deletions in zebrafish. Injection of two different sgRNA‐pairs targeting relevant intronic regions resulted in a deletion mimicking the human deletion and a concomitant increase of abnormal embryos with body and brain malformations (41%, n = 161 and 62%, n = 224, respectively), including a sac‐like brain protrusion (7% and 9%, P < 0.01). Similar results were seen with overexpression of RNA encoding the deleted variant in zebrafish (total abnormal; 46%, n = 255, P < 0.001) compared with the overexpression of an equivalent amount of wild‐type RNA (total abnormal; 3%, n = 177). We predict the in‐frame WDR63 deletion to result in a dominant negative or gain‐of‐function form of WDR63. These are the first findings supporting a role for WDR63 in encephalocele formation. [ABSTRACT FROM AUTHOR]

The primary cilium is a sensory organelle that is vital in regulating several signalling pathways. Unlike most organelles cilia are open to the rest of the cell, not enclosed by membranes. The distinct protein composition is crucial to the function of cilia and many signalling proteins and receptors are specifically concentrated within distinct compartments. To maintain this composition, a mechanism is required to deliver proteins to the cilium whilst another must counter the entropic tendency of proteins to distribute throughout the cell. The combination of the two mechanisms should result in the concentration of ciliary proteins to the cilium. In this review we will look at different cellular mechanisms that play a role in maintaining the distinct composition of cilia, including regulation of ciliary access and trafficking of ciliary proteins to, from and within the cilium. [ABSTRACT FROM AUTHOR]

Cilia are microtubule-based organelles extending from a basal body at the surface of eukaryotic cells. Cilia regulate cell and fluid motility, sensation and developmental signaling, and ciliary defects cause human diseases (ciliopathies) affecting the formation and function of many tissues and organs. Over the past decade, various Rab and Rab-like membrane trafficking proteins have been shown to regulate cilia-related processes such as basal body maturation, ciliary axoneme extension, intraflagellar transport and ciliary signaling. In this review, we provide a comprehensive overview of Rab protein ciliary associations, drawing on findings from multiple model systems, including mammalian cell culture, mice, zebrafish, C. elegans, trypanosomes, and green algae. We also discuss several emerging mechanistic themes related to ciliary Rab cascades and functional redundancy. [ABSTRACT FROM AUTHOR]

The assembly of microtubule-based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin-2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C-terminal tail fragments of heterotrimeric kinesin-2 and α-tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin-treated microtubule and LC-ESI-MS/ MS characterization of the tail-fragment-associated tubulin identified an association between the tail domains and α- tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue-cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α-tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non-particulate form requiring kinesin-2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia. [ABSTRACT FROM AUTHOR]

Background: Retinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors. The primary cilium, as a signaling hub in the cell, may have a role during this process but its presence and localization during RGC generation, and its contribution to the process of cell differentiation, have not been previously assessed in vivo. Methods: In this work we analyzed the distribution of primary cilia in vivo using laser scanning confocal microscopy, as well as their main ultrastructural features by transmission electron microscopy, in the early stages of retinal histogenesis in the zebrafish, around the time of RGC generation and initial differentiation. In addition, we knocked-down ift88 and elipsa, two genes with an essential role in cilia generation and maintenance, a treatment that caused a general reduction in organelle size. The effect on retinal development and RGC differentiation was assessed by confocal microscopy of transgenic or immunolabeled embryos. Results: Our results show that retinal neuroepithelial cells have an apically-localized primary cilium usually protruding from the apical membrane. We also found a small proportion of sub-apical cilia, before and during the neurogenic period. This organelle was also present in an apical position in neuroblasts during apical process retraction and dendritogenesis, although between these stages cilia appeared highly dynamic regarding both presence and position. Disruption of cilia caused a decrease in the proliferation of retinal progenitors and a reduction of neural retina volume. In addition, retinal histogenesis was globally delayed albeit RGC layer formation was preferentially reduced with respect to the amacrine and photoreceptor cell layers. Conclusions: These results indicate that primary cilia exhibit a highly dynamic behavior during early retinal differentiation, and that they are required for the proliferation and survival of retinal progenitors, as well as for neuronal generation, particularly of RGCs. [ABSTRACT FROM AUTHOR]

Intraflagellar transport ( IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six 'peripheral' subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six 'peripheral' IFT-B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/ IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology ( CH) domain ( IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues. [ABSTRACT FROM AUTHOR]

Cilia and flagella play important roles in cell motility and cell signaling. These functions require that the cilium establishes and maintains a unique lipid and protein composition. Recent work indicates that a specialized region at the base of the cilium, the transition zone, serves as both a barrier to entry and a gate for passage of select components. For at least some cytosolic proteins, the barrier and gate functions are provided by a ciliary pore complex (CPC) that shares molecular and mechanistic properties with nuclear gating. Specifically, nucleoporins of the CPC limit the diffusional entry of cytosolic proteins in a size-dependent manner and enable the active transport of large molecules and complexes via targeting signals, importins, and the small G protein Ran. For membrane proteins, the septin protein SEPT2 is part of the barrier to entry whereas the gating function is carried out and/or regulated by proteins associated with ciliary diseases (ciliopathies) such as nephronophthisis, Meckel-Gruber syndrome and Joubert syndrome. Here, we discuss the evidence behind these models of ciliary gating as well as the similarities to and differences from nuclear gating. [ABSTRACT FROM AUTHOR]

Cilia are antenna-like extensions of the plasma membrane found in nearly all cell types. In the retina of the eye, photoreceptors develop unique sensory cilia. Not much was known about the mechanisms underlying the formation and function of photoreceptor cilia, largely because of technical limitations and the specific structural and functional modifications that cannot be modeled in vitro. With recent advances in microscopy techniques and molecular and biochemical approaches, we are now beginning to understand the molecular basis of photoreceptor ciliary architecture, ciliary function and its involvement in human diseases. Here, I will discuss the studies that have revealed new knowledge of how photoreceptor cilia regulate their identity and function while coping with high metabolic and trafficking demands associated with processing light signal. [ABSTRACT FROM AUTHOR]

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this 'ciliary gate' assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment. [ABSTRACT FROM AUTHOR]

Background: Otoconia are bio-crystals that couple mechanic forces to the sensory hair cells in the utricle and saccule, a process essential for us to sense linear acceleration and gravity for the purpose of maintaining bodily balance. In fish, structurally similar bio-crystals called otoliths mediate both balance and hearing. Otoconia abnormalities are common and can cause vertigo and imbalance in humans. However, the molecular etiology of these illnesses is unknown, as investigators have only begun to identify genes important for otoconia formation in recent years. Results: To date, in-depth studies of selected mouse otoconial proteins have been performed, and about 75 zebrafish genes have been identified to be important for otolith development. Conclusions: This review will summarize recent findings as well as compare otoconia and otolith development. It will provide an updated brief review of otoconial proteins along with an overview of the cells and cellular processes involved. While continued efforts are needed to thoroughly understand the molecular mechanisms underlying otoconia and otolith development, it is clear that the process involves a series of temporally and spatially specific events that are tightly coordinated by numerous proteins. Such knowledge will serve as the foundation to uncover the molecular causes of human otoconia-related disorders. Developmental Dynamics 244:239-253, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]