In vertebrate Hedgehog (Hh) signaling, the precise output of the final effectors, GLI (glioma-associated oncogene) transcription factors, depends on the primary cilium. Upon pathway initiation, generating the precise levels of the activator form of GLI depends on its concentration at the cilium tip. The mechanisms underlying this critical step in Hh signaling are unclear. We developed an assay to visualize GLI2, the primary GLI activator isoform, at single-particle resolution within the cilium. We found that GLI2 is a cargo of intraflagellar transport (IFT) machinery. Anterograde-biased IFT loading of GLI2 in a restricted time window following pathway activation results in the tip accumulation of GLI2. Unexpectedly, we found that the conserved Hh regulator KIF7, a nonmotile kinesin, is important for the temporal control of IFT-mediated GLI2 transport and retention of GLI2 at the cilium tip. Our findings underscore a design principle where a cilia-specific cytoskeletal transport system and an Hh pathway-specific cytoskeletal protein collaboratively regulate GLI2 trafficking for Hh signaling.

Progressive loss of motor neurons is the hallmark of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain incompletely understood. In this study, we investigate the effects of C21ORF2 mutations, a gene recently linked to ALS, and find that primary cilia are dysfunctional. Human patient-derived mutant C21ORF2 motor neurons have a reduced ciliary frequency and length. We report that C21ORF2 is located at the basal body of the primary cilium, and mutations associated with ALS alter this localization. Furthermore, we show that a reduction of C21ORF2 levels in cell lines and motor neurons is sufficient to cause fewer primary cilia and reduced cilial length. This ciliary dysfunction leads to defective downstream sonic hedgehog signalling and reduces the expression of cellular retinoic acid binding protein 1 (CRABP1), a protein involved in motor neuron maintenance and survival. In a compartmentalized co-culture system of motor neurons and muscle cells, these ciliary defects were associated with a reduced ability of neuromuscular junction formation. Interestingly, these cilia defects are seemingly not restricted to C21ORF2 ALS, as we also observed perturbed primary cilia in cultured motor neurons and post-mortem motor cortex from patients with the most common genetic subtype of ALS caused by repeat expansions in the C9ORF72 gene. Finally, overexpression of C21ORF2 in mutant C21ORF2 motor neurons rescued the ciliary frequency and length, CRAPBP1 expression and neuromuscular junction formation, confirming the importance of primary cilia for motor neuron function. These results point towards primary cilia dysfunction contributing to motor neuron degeneration in ALS and open new avenues for further research and interventions for this as yet untreatable disease.

Aberrations in primary cilia expression and intraflagellar transport (IFT) protein function have been implicated in tumourigenesis. This study explores the relationship between the alteration of primary cilia and tumourigenesis by investigating primary cilia expression and the role of IFT20 in regulating matrix metalloproteinase 9 (MMP-9) expression in oral squamous cell carcinoma (OSCC) cell lines.

The frequency and length of primary cilia were determined in OKF6-TERT2 cells, HSC-2 cells, and HSC-3 cells using immunofluorescence. Additionally, primary cilia presence in non-proliferating OSCC cells was examined. OSCC cells were treated with either small interfering RNA (siRNA) negative control or siRNA targeting IFT20 for functional analysis. mRNA expression levels of IFT20 and MMP-9 were quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Results showed that HSC-2 cells exhibit abundant primary cilia when cultured in low serum media (2% serum) for 48 h, followed by serum starvation for over 72 h. No significant changes in cilia expression were observed in HSC-3 cells compared to OKF6-TERT2 cells. Ciliated cells were found in non-proliferating HSC-2 and HSC-3 cells. OSCC cells showed longer cilia than OKF6-TERT2 cells, indicating ciliary abnormalities. Changes in ciliation and cilium length of OSCC cells were accompanied by increased expression of IFT20, an intraflagellar transport protein crucial for the primary cilia assembly. However, IFT20 knockdown did not affect MMP-9 at the mRNA level in these cells.

This study reveals the differences in primary cilia expression among OSCC cells. Furthermore, the increased abundance and elongation of primary cilia in OSCC cells are accompanied by elevated expression of IFT20. Nonetheless, IFT20 did not affect MMP-9 mRNA expression in OSCC cells.

Intraflagellar transport 25 is a component of the intraflagellar transport 25-B complex. In mice, even though this intraflagellar transport component is not required for cilia formation in somatic cells, it is essential for sperm formation. However, the intracellular localization of this protein in male germ cells is not known given no reliable antibodies are available for histologic studies, and the dynamic trafficking in the developing sperm flagella is not clear. To examine localization of the protein in male germ cells and further investigate the mechanism of intraflagellar transport in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse intraflagellar transport 25-green fluorescent protein knock-in mouse model using the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 system, with the mouse intraflagellar transport 25 protein fused with a green fluorescent protein tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-intraflagellar transport 25 and anti-green fluorescent protein antibodies showed that the intraflagellar transport 25-green fluorescent protein fusion protein was highly abundant only in the testis, which is consistent with the endogenous intraflagellar transport 25 protein. Examination of localization of the intraflagellar transport 25-green fluorescent protein in isolated germ cells revealed that the fusion protein was present in the cytoplasm of spermatocytes and round spermatids and a strong signal was present in the developing sperm flagellar. The homozygous knock-in mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of intraflagellar transport 25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching. Kymograph and fluorescence recovery after photobleaching analyses demonstrate the transport of intraflagellar transport 25-green fluorescent protein within the developing tail demonstrate no apparent preference for trafficking toward and away from the cell body. The speed of trafficking depends on the stage of sperm development, ranging from highly mobile unrestricted diffusion initially, mobile punctate structures in developing sperm, and immobile punctate structures in mature sperm. Our studies demonstrate that mouse intraflagellar transport 25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.

The ventral body wall (VBW) that encloses the thoracic and abdominal cavities arises by extensive cell movements and morphogenetic changes during embryonic development. These morphogenetic processes include embryonic folding generating the primary body wall; the initial ventral cover of the embryo, followed by directed mesodermal cell migrations, contributing to the secondary body wall. Clinical anomalies in VBW development affect approximately 1 in 3000 live births. However, the cell interactions and critical cellular behaviors that control VBW development remain little understood. Here, we describe the embryonic origins of the VBW, the cellular and morphogenetic processes, and key genes, that are essential for VBW development. We also provide a clinical overview of VBW anomalies, together with environmental and genetic influences, and discuss the insight gained from over 70 mouse models that exhibit VBW defects, and their relevance, with respect to human pathology. In doing so we propose a phenotypic framework for researchers in the field which takes into account the clinical picture. We also highlight cases where there is a current paucity of mouse models for particular clinical defects and key gaps in knowledge about embryonic VBW development that need to be addressed to further understand mechanisms of human VBW pathologies.

Primary cilia are sensory organelles that regulate various signaling pathways. When microtubules are compared to a highway, motor proteins carry and transport cargo proteins, which are tuned by post-translational modifications, such as acetylation. However, the role of acetylation in primary cilia regulation remains unclear. In this study, histone K (lysine) acetyltransferase 2 B (Kat2b) was identified as a novel regulator of primary cilia. Kat2b, which mainly regulates transcription as a p300/CBP associated factor, is localized to the cytosol, centrosome, and cilium basal body. In addition, basal Kat2b expression gradually increased during ciliogenesis. Kat2b regulates the rate of cilia assembly, particularly in the early stages, and loss of Kat2b reduces the recruitment of intraflagellar transport (IFT) components to the ciliary axoneme and impairs Hedgehog (Hh) signaling activation. In addition, Kat2b-knockout mice showed mild abnormalities and ciliary IFT defects in the kidneys. These results establish a link between acetylation regulated by Kat2b and its relevance to ciliary assembly and function.

Intraflagellar transport (IFT) is a conserved mechanism for cilia formation. Twenty-two IFT components form the IFT-A complex (six components) and IFT-B complex (sixteen components). Driven by kinesin and dynein motor proteins, these IFT complexes are involved in the trafficking of proteins needed for cilia assembly by anterograde transport and retrograde transport. IFT core components also associate with other proteins for cilia formation. Mutations in IFT core components result in ciliogenesis defects and human diseases, including male infertility. Sperm flagella are specialized motile cilia that not only have core axoneme structure but also possess accessory structures. IFT is required to assemble these structures to form functional sperm. This summary highlights the regulatory roles of specific IFT proteins in spermatogenesis. A deeper understanding of IFT-related mechanisms can shed light on the etiology and pathophysiology of certain male infertility cases, as well as provide insights for the development of novel male contraceptives.

In humans, inositol polyphosphate-5-phosphatase e (INPP5E) mutations cause retinal degeneration as part of Joubert and MORM syndromes and can also cause non-syndromic blindness. In mice, mutations cause a spectrum of brain, kidney, and other anomalies and prevent the formation of photoreceptor outer segments. To further explore the function of Inpp5e in photoreceptors, we generated conditional and inducible knockouts of mouse Inpp5e where the gene was deleted either during outer segment formation or after outer segments were fully formed. In both cases, the loss of Inpp5e led to severe defects in photoreceptor outer segment morphology and ultimately photoreceptor cell loss. The primary morphological defect consisted of outer segment shortening and reduction in the number of newly forming discs at the outer segment base. This was accompanied by structural abnormalities of the Golgi apparatus, mislocalized rhodopsin, and an accumulation of extracellular vesicles. In addition, knockout cells showed a reduction in the size and prevalence of the actin network at the site of new disc morphogenesis and the occasional formation of membrane whorls instead of discs in a subset of cells. Together, these data demonstrate that Inpp5e plays a critical role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

Calibrated transcriptional outputs in cellular signaling require fine regulation of transcription factor activity. In vertebrate Hedgehog (Hh) signaling, the precise output of the final effectors, the GLI (Glioma-associated-oncogene) transcription factors, depends on the primary cilium. In particular, the formation of the activator form of GLI upon pathway initiation requires its concentration at the distal cilium tip. However, the mechanisms underlying this critical step in Hh signaling are unclear. We developed a real-time imaging assay to visualize GLI2, the primary GLI activator isoform, at single particle resolution within the cilium. We observed that GLI2 is a cargo of Intraflagellar Transport (IFT) machinery and is transported with anterograde bias during a restricted time window following pathway activation. Our findings position IFT as a crucial mediator of transcription factor transport within the cilium for vertebrate Hh signaling, in addition to IFT's well-established role in ciliogenesis. Surprisingly, a conserved Hh pathway regulator, the atypical non-motile kinesin KIF7, is critical for the temporal control of GLI2 transport by IFT and the spatial control of GLI2 localization at the cilium tip. This discovery underscores the collaborative role of a motile and a non-motile cilium-specific cytoskeletal system in determining the transcriptional output during Hh signaling.

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods-liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

Primary cilia, serving as the central hub for cellular signal transduction, possess the remarkable ability to translate diverse extracellular signals, both chemical and mechanical, into intracellular responses. Their ubiquitous presence in the reproductive system underscores their pivotal roles in various cellular processes including development, differentiation, and migration. Emerging evidence suggests primary cilia as key players in reproductive physiology and associated pathologies. Notably, primary cilia have been identified in granulosa cells within mouse ovaries and uterine stromal cells, and perturbations in their structure and function have been implicated in a spectrum of reproductive dysfunctions and ciliary-related diseases. Furthermore, disruptions in primary cilia-mediated signal transduction pathways under pathological conditions exacerbate the onset and progression of reproductive disorders. This review provides a comprehensive overview of current research progress on primary cilia and their associated signaling pathways in reproductive physiology and diseases, with the aim of furnishing theoretical groundwork for the prevention and management of primary cilia-related structural and functional abnormalities contributing to reproductive system pathologies.

The cilium, a pivotal organelle crucial for cell signaling and proper cell function, relies on meticulous macromolecular transport from the cytoplasm for its formation and maintenance. While the intraflagellar transport (IFT) pathway has traditionally been the focus of extensive study concerning ciliogenesis and ciliary maintenance, recent research highlights a complementary and alternative mechanism-vesicle-assisted transport (VAT) in cytoplasm to cilium trafficking. Despite its potential significance, the VAT pathway remains largely uncharacterized. This review explores recent studies providing evidence for the dynamics of vesicle-related diffusion and transport within the live primary cilium, employing high-speed super-resolution light microscopy. Additionally, we analyze the spatial distribution of vesicles in the cilium, mainly relying on electron microscopy data. By scrutinizing the VAT pathways that facilitate cargo transport into the cilium, with a specific emphasis on recent advancements and imaging data, our objective is to synthesize a comprehensive model of ciliary transport through the integration of IFT-VAT mechanisms.

Approximately a quarter of Retinitis Pigmentosa (RP) is caused by mutations in transport-related genes in cilia. IFT27 (Intraflagellar Transport 27), a core component of the ciliary intraflagellar transport (IFT) system, has been implicated as a significant pathogenic gene in RP. The pathogenic mechanisms and subsequent pathology related to IFT27 mutations in RP are largely obscure. Here, we utilized TALEN technology to create an ift27 knockout (ift27-/-) zebrafish model. Electroretinography (ERG) detection showed impaired vision in this model. Histopathological examinations disclosed that ift27 mutations cause progressive degeneration of photoreceptors in zebrafish, and this degeneration was late-onset. Immunofluorescence labeling of outer segments showed that rods degenerated before cones, aligning with the conventional characterization of RP. In cultured human retinal pigment epithelial cells, we found that IFT27 was involved in maintaining ciliary morphology. Furthermore, decreased IFT27 expression resulted in the inhibition of the Hedgehog (Hh) signaling pathway, including decreased expression of key factors in the Hh pathway and abnormal localization of the ciliary mediator Gli2. In summary, we generated an ift27-/- zebrafish line with retinal degeneration which mimicked the symptoms of RP patients, highlighting IFT27's integral role in the long-term maintenance of cilia via the Hh signaling pathway. This work may furnish new insights into the treatment or delay of RP caused by IFT27 mutations.

Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.

Ciliary dysfunction causes a large group of developmental and degenerative human diseases known as ciliopathies. These diseases reflect the critical roles that cilia play in sensing the environment and in force generation for motility. Sensory functions include our senses of vision and olfaction. In addition, primary and motile cilia throughout our body monitor the environment allowing cells to coordinate their biology with the cells around them. This coordination is critical to organ development and maintenance, and ciliary dysfunction causes diverse structural birth defects and degenerative diseases. Defects in motility cause lung disease due to the failure of mucociliary clearance, male infertility due to the failure of sperm motility and the ability of sperm to move through the efferent ducts, and disturbances of the left-right axis due to a failure of nodal cilia to establish proper left-right cues.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery, which is followed by initiation of IFT. We reconstituted and purified RabL2 with CEP19 or IFT proteins to show that a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis rate of RabL2. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 was mapped to a 70-amino-acid-long coiled-coil region of IFT81/74. We present structural models for RabL2-containing IFT complexes that we validate in vitro and in cellulo and demonstrate that Chlamydomonas IFT81/74 enhances GTP hydrolysis of human RabL2, suggesting an ancient evolutionarily conserved activity. Our results provide an architectural understanding of how RabL2 is incorporated into the IFT complex and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.

Mutations in SHH and several other genes encoding components of the Hedgehog signaling pathway have been associated with holoprosencephaly syndromes, with craniofacial anomalies ranging in severity from cyclopia to facial cleft to midfacial and mandibular hypoplasia. Studies in animal models have revealed that SHH signaling plays crucial roles at multiple stages of craniofacial morphogenesis, from cranial neural crest cell survival to growth and patterning of the facial primordia to organogenesis of the palate, mandible, tongue, tooth, and taste bud formation and homeostasis. This article provides a summary of the major findings in studies of the roles of SHH signaling in craniofacial development, with emphasis on recent advances in the understanding of the molecular and cellular mechanisms regulating the SHH signaling pathway activity and those involving SHH signaling in the formation and patterning of craniofacial structures.

Members of the NSL histone acetyltransferase complex are involved in multiorgan developmental syndromes. While the NSL complex is known for its importance in early development, its role in fully differentiated cells remains enigmatic. Using a kidney-specific model, we discovered that deletion of NSL complex members KANSL2 or KANSL3 in postmitotic podocytes led to catastrophic kidney dysfunction. Systematic comparison of two primary differentiated cell types reveals the NSL complex as a master regulator of intraciliary transport genes in both dividing and nondividing cells. NSL complex ablation led to loss of cilia and impaired sonic hedgehog pathway in ciliated fibroblasts. By contrast, nonciliated podocytes responded with altered microtubule dynamics and obliterated podocyte functions. Finally, overexpression of wild-type but not a double zinc finger (ZF-ZF) domain mutant of KANSL2 rescued the transcriptional defects, revealing a critical function of this domain in NSL complex assembly and function. Thus, the NSL complex exhibits bifurcation of functions to enable diversity of specialized outcomes in differentiated cells.

Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway, indispensable for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. Murine Rab-like 2 (Rabl2) GTPase resembles Chlamydomonas Arf-like 3 (ARL3) GTPase in promoting outward TZ passage of the signaling protein cargo-laden BBSome. During this process, ARL3 binds to and recruits the retrograde IFT train-dissociated BBSome as its effector to diffuse through the TZ for ciliary retrieval, while how RABL2 and ARL3 cross talk in this event remains uncertain. Here, we report that Chlamydomonas RABL2 in a GTP-bound form (RABL2GTP) cycles through cilia via IFT as an IFT-B1 cargo, dissociates from retrograde IFT trains at a ciliary region right above the TZ, and converts to RABL2GDP for activating ARL3GDP as an ARL3 guanine nucleotide exchange factor. This confers ARL3GTP to detach from the ciliary membrane and become available for binding and recruiting the phospholipase D (PLD)-laden BBSome, autonomous of retrograde IFT association, to diffuse through the TZ for ciliary retrieval. Afterward, RABL2GDP exits cilia by being bound to the ARL3GTP/BBSome entity as a BBSome cargo. Our data identify ciliary signaling proteins exported from cilia via the RABL2-ARL3 cascade-mediated outward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defect-induced Bardet-Biedl syndrome caused by RABL2 mutations in humans could be well explained in a mutation-specific manner, providing us with a mechanistic understanding behind the outward BBSome TZ passage required for proper ciliary signaling.

The cytoplasmic droplet is a conserved dilated area of cytoplasm situated at the neck of the sperm flagellum. Viewed as residual cytoplasm inherited from late spermatids, the cytoplasmic droplet contains numerous saccular elements as its key content. However, the origin of these saccules and the function of the cytoplasmic droplet have long been speculative. Here, we identify the molecular origin of these cytoplasmic droplet components by uncovering a vesicle pathway essential for formation and sequestration of saccules within the cytoplasmic droplet. This process is governed by a transmembrane protein SYPL1 and its interaction with VAMP3. Genetic ablation of SYPL1 in mice reveals that SYPL1 dictates the formation and accumulation of saccular elements in the forming cytoplasmic droplet. Derived from the Golgi, SYPL1 vesicles are critical for segregation of key metabolic enzymes within the forming cytoplasmic droplet of late spermatids and epididymal sperm, which are required for sperm development and male fertility. Our results uncover a mechanism to actively form and segregate saccules within the cytoplasmic droplet to promote sperm fertility.

The BBSome is an octameric protein complex that regulates ciliary transport and signaling. Mutations in BBSome subunits are closely associated with ciliary defects and lead to ciliopathies, notably Bardet-Biedl syndrome. Over the past few years, there has been significant progress in elucidating the molecular organization and functions of the BBSome complex. An improved understanding of BBSome-mediated biological events and molecular mechanisms is expected to help advance the development of diagnostic and therapeutic approaches for BBSome-related diseases. Here, we review the current literature on the structural assembly, transport regulation, and molecular functions of the BBSome, emphasizing its roles in cilium-related processes. We also provide perspectives on the pathological role of the BBSome in ciliopathies as well as how these can be exploited for therapeutic benefit.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBD), indicating important roles for cilia in development. Here we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140 , an intraflagellar transport protein regulating ciliogenesis. Ift140 deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula, randomized heart looping, congenital heart defects (CHD), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAG-Cre deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD was not observed with four Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathy.

Certain ciliary transmembrane and membrane-tethered signaling proteins migrate from the ciliary tip to base via retrograde intraflagellar transport (IFT), essential for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. During this process, the BBSome functions as an adaptor between retrograde IFT trains and these signaling protein cargoes. The Arf-like 13 (ARL13) small GTPase resembles ARL6/BBS3 in facilitating these signaling cargoes to couple with the BBSome at the ciliary tip prior to loading onto retrograde IFT trains for transporting towards the ciliary base, while the molecular basis for how this intricate coupling event happens remains elusive. Here, we report that Chlamydomonas ARL13 only in a GTP-bound form (ARL13GTP) anchors to the membrane for diffusing into cilia. Upon entering cilia, ARL13 undergoes GTPase cycle for shuttling between the ciliary membrane (ARL13GTP) and matrix (ARL13GDP). To achieve this goal, the ciliary membrane-anchored BBS3GTP binds the ciliary matrix-residing ARL13GDP to activate the latter as an ARL13 guanine nucleotide exchange factor. At the ciliary tip, ARL13GTP recruits the ciliary matrix-residing and post-remodeled BBSome as an ARL13 effector to anchor to the ciliary membrane. This makes the BBSome spatiotemporally become available for the ciliary membrane-tethered phospholipase D (PLD) to couple with. Afterward, ARL13GTP hydrolyzes GTP for releasing the PLD-laden BBSome to load onto retrograde IFT trains. According to this model, hedgehog signaling defects associated with ARL13b and BBS3 mutations in humans could be satisfactorily explained, providing us a mechanistic understanding behind BBSome-cargo coupling required for proper ciliary signaling.

Episcopic imaging using techniques such as High Resolution Episcopic Microscopy (HREM) and its variants, allows biological samples to be visualized in three dimensions over a large field of view. Quantitative analysis of episcopic image data is undertaken using a range of methods. In this systematic review, we look at trends in quantitative analysis of episcopic images and discuss avenues for further research. Papers published between 2011 and 2022 were analyzed for details about quantitative analysis approaches, methods of image annotation and choice of image processing software. It is shown that quantitative processing is becoming more common in episcopic microscopy and that manual annotation is the predominant method of image analysis. Our meta-analysis highlights where tools and methods require further development in this field, and we discuss what this means for the future of quantitative episcopic imaging, as well as how annotation and quantification may be automated and standardized across the field.

Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

Primary cilia, microtubule-based hair-like structures protruding from most cells, contain membranes enriched in signaling molecules and function as sensory and regulatory organelles critical for development and tissue homeostasis. Intraflagellar transport (IFT), cilia-specific bidirectional transport, is required for the assembly, maintenance, and function of cilia. BBSome, the coat complex, acts as the adaptor between the IFT complex and membrane proteins and is therefore essential for establishing the specific compartmentalization of signaling molecules in the cilia. Recent findings have revealed that three ciliary Rab-like small GTPases, IFT27, IFT22, and Rabl2, play critical regulatory roles in ciliary BBSome transport. In this review, we provide an overview of these three Rab-like small GTPases and their relationship with BBSome.

Cilia are hair-like structures that project from the surface of cells. In vertebrates, most cells have an immotile primary cilium that mediates cell signaling, and some specialized cells assemble one or multiple cilia that are motile and beat synchronously to move fluids in one direction. Gene mutations that alter cilia structure or function cause a broad spectrum of disorders termed ciliopathies that impact virtually every system in the body. A wide range of birth defects associated with ciliopathies underscores critical functions for cilia during embryonic development. In many cases, the mechanisms underlying cilia functions during development and disease remain poorly understood. This review describes different types of cilia in vertebrate embryos and discusses recent research results from diverse model systems that provide novel insights into how cilia form and function during embryo development. The work discussed here not only expands our understanding of in vivo cilia biology, but also opens new questions about cilia and their roles in establishing healthy embryos.

The intraflagellar transport (IFT) machinery mediates the import and export of ciliary proteins across the ciliary gate, as well as bidirectional protein trafficking within cilia. In addition to ciliary anterograde protein trafficking, the IFT-B complex participates in the export of membrane proteins together with the BBSome, which consists of eight subunits encoded by the causative genes of Bardet-Biedl syndrome (BBS). The IFT25-IFT27/BBS19 dimer in the IFT-B complex constitutes its interface with the BBSome. We show here that IFT25-IFT27 and the RABL2 GTPase bind the IFT74/BBS22-IFT81 dimer of the IFT-B complex in a mutually exclusive manner. Cells expressing GTP-locked RABL2 [RABL2(Q80L)], but not wild-type RABL2, phenocopied IFT27-knockout cells, that is, they demonstrated BBS-associated ciliary defects, including accumulation of LZTFL1/BBS17 and the BBSome within cilia and the suppression of export of the ciliary GPCRs GPR161 and Smoothened. RABL2(Q80L) enters cilia in a manner dependent on the basal body protein CEP19, but its entry into cilia is not necessary for causing BBS-associated ciliary defects. These observations suggest that GTP-bound RABL2 is likely to be required for recruitment of the IFT-B complex to the ciliary base, where it is replaced with IFT25-IFT27.

Ciliogenesis-associated kinase 1 (CILK1) is a ciliary gene that localizes in primary cilia and regulates ciliary transport. Mutations in CILK1 cause various ciliopathies. However, the pathogenesis of CILK1-deficient kidney disease is unknown.

To examine whether CILK1 deficiency causes PKD accompanied by abnormal cilia, we generated mice with deletion of Cilk1 in cells of the renal collecting duct. A yeast two-hybrid system and coimmunoprecipitation (co-IP) were used to identify a novel regulator, kinesin light chain-3 (KLC3), of ciliary trafficking and cyst progression in the Cilk1-deficient model. Immunocytochemistry and co-IP were used to examine the effect of KLC3 on ciliary trafficking of the IFT-B complex and EGFR. We evaluated the effects of these genes on ciliary trafficking and cyst progression by modulating CILK1 and KLC3 expression levels.

CILK1 deficiency leads to PKD accompanied by abnormal ciliary trafficking. KLC3 interacts with CILK1 at cilia bases and is increased in cyst-lining cells of CILK1-deficient mice. KLC3 overexpression promotes ciliary recruitment of IFT-B and EGFR in the CILK1 deficiency condition, which contributes to the ciliary defect in cystogenesis. Reduction in KLC3 rescued the ciliary defects and inhibited cyst progression caused by CILK1 deficiency.

Our findings suggest that CILK1 deficiency in renal collecting ducts leads to PKD and promotes ciliary trafficking via increased KLC3.

Defects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.

The primary cilium is a highly specialized and evolutionary conserved organelle in eukaryotes that plays a significant role in cell signaling and trafficking. Over the past few decades tremendous progress has been made in understanding the physiology of cilia and the underlying pathomechanisms of various ciliopathies. Syndromic ciliopathies consist of a group of disorders caused by ciliary dysfunction or abnormal ciliogenesis. These disorders have multiorgan involvement in addition to retinal degeneration underscoring the ubiquitous distribution of primary cilia in different cell types. Genotype-phenotype correlation is often challenging due to the allelic heterogeneity and pleiotropy of these disorders. In this review, we discuss the clinical and genetic features of syndromic ciliopathies with a focus on Bardet-Biedl syndrome (BBS) as a representative disorder. We discuss the structure and function of primary cilia and their role in retinal photoreceptors. We describe the progress made thus far in understanding the functional and genetic characterization including expression quantitative trait locus (eQTL) analysis of BBS genes. In the future directions section, we discuss the emerging technologies, such as gene therapy, as well as anticipated challenges and their implications in therapeutic development for ciliopathies.

The IFT-B complex mediates ciliary anterograde protein trafficking and membrane protein export together with the BBSome. Bardet-Biedl syndrome (BBS) is caused by mutations in not only all BBSome subunits, but also in some IFT-B subunits, including IFT74/BBS22 and IFT27/BBS19, which form heterodimers with IFT81 and IFT25, respectively. We found that the IFT25-IFT27 dimer bind the C-terminal region of the IFT74-IFT81 dimer, and that the IFT25-IFT27-binding region encompasses the region deleted in the BBS variants of IFT74. In addition, we found that the missense BBS variants of IFT27 are impaired in IFT74-IFT81 binding, and are unable to rescue the BBS-like phenotypes of IFT27-knockout cells. Furthermore, the BBS variants of IFT74 rescued the ciliogenesis defect of IFT74-knockout cells, but the rescued cells demonstrated BBS-like abnormal phenotypes. Taken together, we conclude that the impaired interaction between IFT74-IFT81 and IFT25-IFT27 causes the BBS-associated ciliary defects.

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits and demonstrate an accumulation of 'coat-less' vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

Primary cilia are dynamic compartments that regulate multiple aspects of cellular signaling. The production, maintenance, and function of cilia involve more than 1000 genes in mammals, and their mutations disrupt the ciliary signaling which manifests in a plethora of pathological conditions-the ciliopathies. Skeletal ciliopathies are genetic disorders affecting the development and homeostasis of the skeleton, and encompass a broad spectrum of pathologies ranging from isolated polydactyly to lethal syndromic dysplasias. The recent advances in forward genetics allowed for the identification of novel regulators of skeletogenesis, and revealed a growing list of ciliary proteins that are critical for signaling pathways implicated in bone physiology. Among these, a group of protein kinases involved in cilia assembly, maintenance, signaling, and disassembly has emerged. In this review, we summarize the functions of cilia kinases in skeletal development and disease, and discuss the available and upcoming treatment options.

The Hedgehog (Hh) signaling is one of the essential signaling pathways during embryogenesis and in adults. Hh signal transduction relies on primary cilium, a specialized cell surface organelle viewed as the hub of cell signaling. Protein kinase A (PKA) has been recognized as a potent negative regulator of the Hh pathway, raising the question of how such a ubiquitous kinase specifically regulates one signaling pathway. We reviewed recent genetic, molecular and biochemical studies that have advanced our mechanistic understanding of PKA's role in Hh signaling in vertebrates, focusing on the compartmentalized PKA at the centrosome and in the primary cilium. We outlined the recently developed genetic and optical tools that can be harvested to study PKA activities during the course of Hh signal transduction.