Intraflagellar Transport Machinery Research Articles

Architecture of RabL2-associated complexes at the ciliary base: A structural modeling perspective: Deciphering the structural organization of ciliary RabL2 complexes.

Cilia are slender, micrometer-long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so-called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N-terminal domain of CEP43 forms a homo-dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N-terminal domain of CEP43 from the C-terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2-CEP19 associates with the IFT-B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT-B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled-coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.

Quantitative proteomics reveals insights into the assembly of IFT trains and ciliary assembly.

Intraflagellar transport (IFT) is required for ciliary assembly. The IFT machinery comprises the IFT motors kinesin-2 and IFT dynein plus IFT-A and IFT-B complexes, which assemble into IFT trains in cilia. To gain mechanistic understanding of IFT and ciliary assembly, here, we performed an absolute quantification of IFT machinery in Chlamydomonas reinhardtii cilium. There are ∼756, ∼532, ∼276 and ∼350 molecules of IFT-B, IFT-A, IFT dynein and kinesin-2, respectively, per cilium. The amount of IFT-B is sufficient to sustain rapid ciliary growth in terms of tubulin delivery. The stoichiometric ratio of IFT-B:IFT-A:dynein is ∼3:2:1 whereas the IFT-B:IFT-A ratio in an IFT dynein mutant is 2:1, suggesting that there is a plastic interaction between IFT-A and IFT-B that can be influenced by IFT dynein. Considering diffusion of kinesin-2 during retrograde IFT, it is estimated that one kinesin-2 molecule drives eight molecules of IFT-B during anterograde IFT. These data provide new insights into the assembly of IFT trains and ciliary assembly.

1H, 13C, and 15N resonance assignments and solution structure of the N-terminal divergent calponin homology (NN-CH) domain of human intraflagellar transport protein 54.

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.

The cilium like region of the Drosophila bifurca spermatocyte: Elongation of a giant axoneme without intraflagellar transport

AbstractThe growth of the ciliary axonemes mainly depends on the evolutionary conserved intraflagellar transport (IFT) machinery. However, insect spermatocytes are characterized by cilium‐like regions (CLRs) that elongate in the absence of IFT. It is generally believed that the dynamics of these structures relies on the free diffusion of soluble tubulin from the cytoplasm. However, this passive process could allow the elongation of short ciliary axonemes, but it is unclear whether simple diffusion of tubulin molecules can ensure the correct assembly of elongated ciliary structures. To decipher this point we analyzed the assembly of the CLRs held by the primary spermatocytes of Drosophila bifurca. These ciliary structures consist of a very elongated axoneme that grows without IFT and, therefore, could represent a good model in which to evaluate the role played by the free diffusion of soluble tubulin. The observation of wavy microtubules in the axonemal lumen of fully elongated CLRs of D. bifurca may be consistent with the diffusion of tubulin within the axonemal lumen. Progressive consumption of soluble tubulin used for axoneme growth at the apical tip of the CLRs could result in a gradient sufficient to move tubulin from the cytoplasm to the apical end of the forming ciliary structure. When the axoneme reaches its full length, tubulin molecules are not drawn to the tip of the CLRs and accumulate at the base of the axoneme, where its concentration may exceed the threshold need for microtubule polymerization. The presence of γ‐TuRCs at the proximal ends of the supernumerary microtubules could enhance their nucleation.

ANKS1A-Deficiency Aberrantly Increases the Entry of the Protein Transport Machinery into the Ependymal Cilia.

In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.

Paralog-specific TTC30 regulation of Sonic hedgehog signaling

The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A–IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly.

The IFT81-IFT74 complex acts as an unconventional RabL2 GTPase-activating protein during intraflagellar transport.

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.

Compound heterozygous IFT81 variations in a skeletal ciliopathy patient cause Bardet-Biedl syndrome-like ciliary defects.

Owing to their crucial roles in development and homeostasis, defects in cilia cause ciliopathies with diverse clinical manifestations. The intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes, mediates not only the intraciliary bidirectional trafficking but also import and export of ciliary proteins together with the kinesin-2 and dynein-2 motor complexes. The BBSome, containing eight subunits encoded by causative genes of Bardet-Biedl syndrome (BBS), connects the IFT machinery to ciliary membrane proteins to mediate their export from cilia. Although mutations in subunits of the IFT-A and dynein-2 complexes cause skeletal ciliopathies, mutations in some IFT-B subunits are also known to cause skeletal ciliopathies. We here show that compound heterozygous variations of an IFT-B subunit, IFT81, found in a patient with skeletal ciliopathy cause defects in its interactions with other IFT-B subunits, and in ciliogenesis and ciliary protein trafficking when one of the two variants was expressed in IFT81-knockout (KO) cells. Notably, we found that IFT81-KO cells expressing IFT81(Δ490-519), which lacks the binding site for the IFT25-IFT27 dimer, causes ciliary defects reminiscent of those found in BBS cells and those in IFT74-KO cells expressing a BBS variant of IFT74, which forms a heterodimer with IFT81. In addition, IFT81-KO cells expressing IFT81(Δ490-519) in combination with the other variant, IFT81 (L645*), which mimics the cellular conditions of the above skeletal ciliopathy patient, demonstrated essentially the same phenotype as those expressing only IFT81(Δ490-519). Thus, our data indicate that BBS-like defects can be caused by skeletal ciliopathy variants of IFT81.

Dynein-2-driven intraciliary retrograde trafficking indirectly requires multiple interactions of IFT54 in the IFT-B complex with the dynein-2 complex.

Within cilia, the dynein-2 complex needs to be transported as an anterograde cargo to achieve its role as a motor to drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. We previously showed that interactions of WDR60 and the DYNC2H1-DYNC2LI1 dimer of dynein-2 with multiple IFT-B subunits, including IFT54, are required for the trafficking of dynein-2 as an IFT cargo. However, specific deletion of the IFT54-binding site from WDR60 demonstrated only a minor effect on dynein-2 trafficking and function. We here show that the C-terminal coiled-coil region of IFT54, which participates in its interaction with the DYNC2H1-DYNC2LI1 dimer of dynein-2 and with IFT20 of the IFT-B complex, is essential for IFT-B function, and suggest that the IFT54 middle linker region between the N-terminal WDR60-binding region and the C-terminal coiled-coil is required for ciliary retrograde trafficking, probably by mediating the effective binding of IFT-B to the dynein-2 complex, and thereby ensuring dynein-2 loading onto the anterograde IFT trains. The results presented here agree with the notion predicted from the previous structural models that the dynein-2 loading onto the anterograde IFT train relies on intricate, multivalent interactions between the dynein-2 and IFT-B complexes.

Deciphering the role of cytoplasmic domain of Channelrhodopsin in modulation of the interactome and SUMOylome of Chlamydomonas reinhardtii

Translocation of channelrhodopsins (ChRs) is mediated by the intraflagellar transport (IFT) machinery. However, the functional role of the network involving photoreceptors, IFT and other proteins in controlling algal ciliary motility is still not fully delineated. In the current study, we have identified two important motifs at the C-terminus of ChR1, VXPX and LKNE. VXPX is a known ciliary targeting sequence in animals, and LKNE is a well-known SUMOylation motif. To the best of our knowledge, this study gives prima facie insight into the role of SUMOylation in Chlamydomonas. We prove that VMPS of ChR1 is important for interaction with GTPase CrARL11. We show that SUMO motifs are present in the C-terminus of putative ChR1s from green algae. Performing experiments with n-Ethylmaleimide (NEM) and Ubiquitin-like protease 1 (ULP-1), we show that SUMOylation may modulate ChR1 protein in Chlamydomonas. Experiments with 2D08, a known sumoylation blocker, increased the concentration of ChR1 protein. Finally, we show the endogenous SUMOylated proteins (SUMOylome) of C. reinhardtii, identified by using immunoprecipitation followed by nano-LC-MS/MS detection. This report establishes a link between evolutionarily conserved SUMOylation and ciliary machinery for the maintenance and functioning of cilia across the eukaryotes. Our enriched SUMOylome of C. reinhardtii comprehends the proteins related to ciliary development and photo-signaling, along with the orthologue(s) associated to human ciliopathies as SUMO targets.

Multiple interactions of the dynein-2 complex with the IFT-B complex are required for effective intraflagellar transport.

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60 (also known as DYNC2I1) and the DYNC2H1-DYNC2LI1 dimer from dynein-2, and IFT54 (also known as TRAF3IP1) and IFT57 from IFT-B contribute to the dynein-2-IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that N-terminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection.

Molecular characterization of phototropin signalosome regulating photophysiological processes in green algae.

Phototropin is a blue light-sensing photoreceptor, comprises two LOV domains that non-covalently bind FMN as chromophore and a serine-threonine kinase domain at its C-terminus. Phototropin is well established in higher plants and aquatic algae. However, the photoreceptor biology of the evolutionary important alga has not yet been established. We characterized the phototropin LOV domain from evolutionary important algae and compared the changes with the aquatic green algae, Chlamydomonas reinhardtii phototropin LOV domain. The intraflagellar transport (IFT) machinery responsible for bidirectional movement of proteins inwithin cilia or flagella is involved in trafficking of Chlamydomonas phototropin (CrPhot). Through immunoblotting and immunolocalization experiments with a variety of IFT mutants, we investigated the molecular details of phototropin trafficking. The CrPhot knock-out strains showed reduced photomotility, decrease level of other predicted interacting proteins like ChRs and, 14-3-3 as compared to that of the wild type strain. Various approaches like protein profiling, expression profile (qRT-PCR), and proteomics were applied to delineate its role in light-mediated physiology in C. reinhardtii. We studied the photochemistry, oligomerization, and photodynamic behaviour of LOV1 domain of a novel terrestrial algal phototropin using UV-visible, fluorescence, CD, atomic force microscopy, and time-resolved fluorescence spectroscopy techniques. Our findings highlight the unique characteristics of LOV domain in terms of very long dark recovery time. We identified the key residues responsible for this recovery kinetics in novel LOV1 protein. Different variants of LOV1 protein were produced and photochemistry was compared with the respect to the wild type protein. We proposed optogenetic potential of the engineered LOV domain in modulation of diverse cellular signalling pathways such as ciliogenesis, cell cycle, and apoptosis.

Drosophila transition fibers are essential for IFT-dependent ciliary elongation but not basal body docking and ciliary budding

Cilia are highly conserved organelles critical for animal development and perception. Dysfunction of cilia has been linked to a wide spectrum of human genetic diseases, termed ciliopathies.1,2 Transition fibers (TFs) are striking ciliary base structures essential for cilia assembly. Vertebrates' TFs that originate from centriole distal appendages (DAs) mediate basal body docking to ciliary vesicles to initiate ciliogenesis and regulate the entry of ciliary proteins for axoneme assembly via intraflagellar transport (IFT) machinery.3 Although no distal appendages can be observed on Drosophila centrioles,4,5 three key TF proteins, FBF1, CEP164, and CEP89, have obvious homologs in Drosophila. We aimed to compare their functions with their mammalian counterparts in Drosophila ciliogenesis. Here, we show that all three proteins are localized like TF proteins at the ciliary base in both sensory neurons and spermatocytes, the only two types of ciliated cells in flies. Fbf1 and Cep89 are essential for the formation of IFT-dependent neuronal cilia, but Cep164 is dispensable for ciliogenesis in flies. Strikingly, none are required for basal body docking and transition zone (TZ) assembly in IFT-dependent neuronal cilia or IFT-independent spermatocyte cilia. Furthermore, we demonstrate that Unc is essential to recruit all three TF proteins and establish a hierarchical order, with Cep89 acting on Fbf1. Collectively, our results not only demonstrate that TF proteins are required for IFT-dependent ciliogenesis in Drosophila, in agreement with an evolutionarily conserved function of these proteins in regulating ciliary protein entry, but also that the basal body docking function of TFs has diverged during evolution.

DYF-5/MAK–dependent phosphorylation promotes ciliary tubulin unloading

Cilia are microtubule-based organelles that power cell motility and regulate sensation and signaling, and abnormal ciliary structure and function cause various ciliopathies. Cilium formation and maintenance requires intraflagellar transport (IFT), during which the kinesin-2 family motor proteins ferry IFT particles carrying axonemal precursors such as tubulins into cilia. Tubulin dimers are loaded to IFT machinery through an interaction between tubulin and the IFT-74/81 module; however, little is known of how tubulins are unloaded when arriving at the ciliary tip. Here, we show that the ciliary kinase DYF-5/MAK phosphorylates multiple sites within the tubulin-binding module of IFT-74, reducing the tubulin-binding affinity of IFT-74/81 approximately sixfold. Ablation or constitutive activation of IFT-74 phosphorylation abnormally elongates or shortens sensory cilia in Caenorhabditis elegans neurons. We propose that DYF-5/MAK-dependent phosphorylation plays a fundamental role in ciliogenesis by regulating tubulin unloading.

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase‐4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.

Male infertility-associated Ccdc108 regulates multiciliogenesis via the intraflagellar transport machinery.

Motile cilia on the cell surface generate movement and directional fluid flow that is crucial for various biological processes. Dysfunction of these cilia causes human diseases such as sinopulmonary disease and infertility. Here, we show that Ccdc108, a protein linked to male infertility, has an evolutionarily conserved requirement in motile multiciliation. Using Xenopus laevis embryos, Ccdc108 is shown to be required for the migration and docking of basal bodies to the apical membrane in epidermal multiciliated cells (MCCs). We demonstrate that Ccdc108 interacts with the IFT‐B complex, and the ciliation requirement for Ift74 overlaps with Ccdc108 in MCCs. Both Ccdc108 and IFT‐B proteins localize to migrating centrioles, basal bodies, and cilia in MCCs. Importantly, Ccdc108 governs the centriolar recruitment of IFT while IFT licenses the targeting of Ccdc108 to the cilium. Moreover, Ccdc108 is required for the centriolar recruitment of Drg1 and activated RhoA, factors that help establish the apical actin network in MCCs. Together, our studies indicate that Ccdc108 and IFT‐B complex components cooperate in multiciliogenesis.

Large deletion of Wdr19 in developing renal tubules disrupts primary ciliogenesis, leading to polycystic kidney disease in mice.

WD repeat domain 19 (Wdr19) is a major component of the intraflagellar transport (IFT) machinery, which is involved in the function of primary cilia. However, the effects of Wdr19 on primary cilia formation, cystogenesis, and polycystic kidney disease (PKD) progression remain unclear. To study these effects, we generated three lines of kidney-specific conditional knockout mice: Wdr19-knockout (Wdr19-KO, Wdr19f/- ::Cdh16-CreTg/0 ), Pkd1-knockout (Pkd1-KO, Pkd1f/- ::Cdh16-CreTg/0 ), and Wdr19/Pkd1-double knockout (Wdr19&Pkd1-dKO, Wdr19f/- ;Pkd1f/- ::Cdh16-CreTg/0 ) mice. Ultrastructural analysis using transmission electron microscopy (TEM) indicated that the primary cilia were almost absent at postnatal day 10 in Wdr19-KO mice compared with Pkd1-KO and wild-type (WT) mice. However, the primary cilia appeared structurally normal even if malfunctional in Pkd1-deficient cysts. The Pkd1-KO mice had the most severe PKD progression, including the shortest lifespan (14 days) and the largest renal cysts, among the three knockout lines. Thus, the molecular mechanism of renal cystogenesis in Wdr19-KO mice (primary cilia abrogation) was different from that in Pkd1-KO mice (primary cilia malfunction). In summary, Wdr19 deficiency leads to primary cilia abrogation and renal cyst formation. Wdr19 is primarily proposed to participate in retrograde IFT and to be crucial for the construction of primary cilia, which are critical organelles for tubulogenesis in the developing kidneys. © 2022 The Pathological Society of Great Britain and Ireland.

Combinations of deletion and missense variations of the dynein-2 DYNC2LI1 subunit found in skeletal ciliopathies cause ciliary defects

Cilia play crucial roles in sensing and transducing extracellular signals. Bidirectional protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes, with the aid of kinesin-2 and dynein-2 motors. The dynein-2 complex drives retrograde trafficking of the IFT machinery after its transportation to the ciliary tip as an IFT cargo. Mutations in genes encoding the dynein-2-specific subunits (DYNC2H1, WDR60, WDR34, DYNC2LI1, and TCTEX1D2) are known to cause skeletal ciliopathies. We here demonstrate that several pathogenic variants of DYNC2LI1 are compromised regarding their ability to interact with DYNC2H1 and WDR60. When expressed in DYNC2LI1-knockout cells, deletion variants of DYNC2LI1 were unable to rescue the ciliary defects of these cells, whereas missense variants, as well as wild-type DYNC2LI1, restored the normal phenotype. DYNC2LI1-knockout cells coexpressing one pathogenic deletion variant together with wild-type DYNC2LI1 demonstrated a normal phenotype. In striking contrast, DYNC2LI1-knockout cells coexpressing the deletion variant in combination with a missense variant, which mimics the situation of cells of compound heterozygous ciliopathy individuals, demonstrated ciliary defects. Thus, DYNC2LI1 deletion variants found in individuals with skeletal ciliopathies cause ciliary defects when combined with a missense variant, which expressed on its own does not cause substantial defects.

Endoderm development requires centrioles to restrain p53-mediated apoptosis in the absence of ERK activity.

Centrioles comprise the heart of centrosomes, microtubule-organizing centers. To study the function of centrioles in lung and gut development, we genetically disrupted centrioles throughout the mouse endoderm. Surprisingly, removing centrioles from the endoderm did not disrupt intestinal growth or development but blocked lung branching. In the lung, acentriolar SOX2-expressing airway epithelial cells apoptosed. Loss of centrioles activated p53, and removing p53 restored survival of SOX2-expressing cells, lung branching, and mouse viability. To investigate how endodermal p53 activation specifically killed acentriolar SOX2-expressing cells, we assessed ERK, a prosurvival cue. ERK was active throughout the intestine and in the distal lung buds, correlating with tolerance to centriole loss. Pharmacologically inhibiting ERK activated apoptosis in acentriolar cells, revealing that ERK activity protects acentriolar cells from apoptosis. Therefore, centrioles are largely dispensable for endodermal growth and the spatial distribution of ERK activity in the endoderm shapes the developmental consequences of centriolar defects and p53 activation.

Ectocytosis prevents accumulation of ciliary cargo in C. elegans sensory neurons.

Cilia are sensory organelles protruding from cell surfaces. Release of extracellular vesicles (EVs) from cilia was previously observed in mammals, Chlamydomonas, and in male Caenorhabditis elegans. Using the EV marker TSP-6 (an ortholog of mammalian CD9) and other ciliary receptors, we show that EVs are formed from ciliated sensory neurons in C. elegans hermaphrodites. Release of EVs is observed from two ciliary locations: the cilia tip and/or periciliary membrane compartment (PCMC). Outward budding of EVs from the cilia tip leads to their release into the environment. EVs' budding from the PCMC is concomitantly phagocytosed by the associated glial cells. To maintain cilia composition, a tight regulation of cargo import and removal is achieved by the action of intra-flagellar transport (IFT). Unbalanced IFT due to cargo overexpression or mutations in the IFT machinery leads to local accumulation of ciliary proteins. Disposal of excess ciliary proteins via EVs reduces their local accumulation and exports them to the environment and/or to the glia associated to these ciliated neurons. We suggest that EV budding from cilia subcompartments acts as a safeguard mechanism to remove deleterious excess of ciliary material.