Ciliary Base Research Articles

Dynein Dynamics in Vivo: A Single-Molecule View on its Function in Intraflagellar Transport

IFT-dynein (cytoplasmic dynein 2) co-operates with kinesin-2 motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). Relatively little is known about this dynein, while the related cytoplasmic dynein 1 has received a lot of attention of late. Here, we study the in vivo IFT-dynein dynamics at the ensemble and single-molecule level.To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors, expressed at endogenous levels, in the chemosensory cilia of living C. elegans. Fluorescence movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using custom kymograph-analysis software. To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, to track individual IFT-dynein motors in vivo.The data revealed that IFT-dynein moves in trains consisting tens of motor proteins. Retrograde trains are smaller but more frequent than anterograde (kinesin-driven) trains. Anterograde and retrograde IFT-dynein flux are equal along cilia, indicating that the cilium is a closed system for dynein. While the kinesin composition of a train varies along the track, the amount of dynein per train remains relatively constant. Remarkably, this does not result in directionality changes along the track, like in reported ‘tug-of-war’ motor systems, suggesting that retrograde and anterograde motor activities are carefully orchestrated in IFT.Single IFT-dynein measurements support the ensemble findings. Trajectories show distinct motility behavior: diffusion at the ciliary base, pauses, turns and directed motion. Pauses in retrograde or anterograde trajectories are never followed by a directional switch. Moreover, retrograde-to-anterograde turn events are rare.This combined ensemble and single-molecule approach has provided new insights into IFT-dynein transport dynamics in living organisms, shedding light on dynein function in general.

Three-Dimensional Structures Associated with Photoreceptor Cilia by Cryo-Electron Tomography

The outer segment of rod and cone photoreceptor cells is a modified primary cilium. Specialized machinery is needed to assemble and maintain its structure, and to carry out the transport and sorting of molecular components between the inner segment, where most biosynthesis occurs, and the outer segment, where those components are utilized for phototransduction. The narrow connection between the inner and outer segment, known as the connecting cilium, is the location of proteins encoded by genes implicated in inherited diseases known as ciliopathies. Its structural core is a nine + zero microtubule-based axoneme that emerges from a centriole termed the basal body complex. Conserved features of this organelle include the transition fibers and transition zone with a ciliary necklace studding the ciliary membrane. We used cryo-electron tomography to determine the structures of the second basal body (daughter centriole), the connecting cilium and associated machinery in wildtype mice. Recently we used computational sub-tomogram averaging to determine the three-dimensional structure of the basal bodies. The 3D density map reveals a tapered cylindrical structure composed of nine MT bundle from the plus (triplet) end to the minus (doublet) end of the centriole. Additional densities that represent non-tubulin proteins attached to the triplet were revealed, which presumably stabilize the entire basal body. In addition, we have observed transition fibers, located at the ciliary base, connecting to microtubule triplets. The transition zone connects the microtubule doublets of the axoneme to the membrane near the basal body and extends into the connecting cilium toward the outer segment. These structural features in wildtype cilia will help us to understand the normal mechanisms and functions of the many proteins essential for cilium function and photoreceptor survival, and shed light on the ciliopathies that arise from defects in those proteins.

Seeing Single Molecules, from Early Spectroscopy in Solids, to Super-Resolution Microscopy, to 3D Dynamics of Biomolecules in Cells

More than 25 years ago, low temperature experiments aimed at establishing the ultimate limits to optical storage in solids led to the first optical detection and spectroscopy of a single molecule in the condensed phase. At this unexplored ultimate limit, many surprises occurred where single molecules showed both spontaneous changes (blinking) and light-driven control of emission, properties that were also observed in 1997 at room temperature with single green fluorescent protein variants. In 2006, PALM and subsequent approaches showed that the optical diffraction limit of ∼200 nm can be circumvented with single molecules to achieve super-resolution fluorescence microscopy with relatively nonperturbative visible light. Super-resolution microscopy has opened up a new frontier in which biological structures and behavior can be observed in fixed and live cells with resolutions down to 20-40 nm and below. Current methods development research addresses ways to extract more information from each single molecule such as 3D position and orientation, and both of these can be obtained by proper point-spread function engineering of a wide-field microscope. It is worth noting that in spite of all the current focus on super-resolution, even in the “conventional” low concentration, single-molecule tracking regime where the motions of individual biomolecules are recorded rather than the shapes of extended structures, much can still be learned about biological processes. For example, my laboratory has explored the motions of single Smoothened proteins in the primary cilium, providing evidence of binding sites at the ciliary base whose affinity is modulated by Hedgehog pathway activation. Using 3D precision tracking at high speed, correlations in the motions of pairs of DNA loci in the yeast nucleus highlight the complexity of the nuclear environment through the appearance of subdiffusive motion.

Recombinant Reconstitution and Purification of the IFT-B Core Complex from Chlamydomonas reinhardtii.

Eukaryotic cilia and flagella are assembled and maintained by intraflagellar transport (IFT), the bidirectional transport of proteins between the ciliary base and tip. IFT is mediated by the multi-subunit IFT complex, which simultaneously binds cargo proteins and the ciliary motors. So far 22 subunits of the IFT complex have been identified, but insights into the biochemical architecture and especially the three-dimensional structure of this machinery are only starting to emerge because of difficulties in obtaining homogeneous material suitable for structural analysis. Here, we describe a protocol for the purification and reconstitution of a complex containing nine Chlamydomonas reinhardtii IFT proteins, commonly known as the IFT-B core complex. In our hands, this protocol routinely yields several milligrams of pure complex suitable for structural analysis by X-ray crystallography and single-particle cryo-electron microscopy.

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry.

Ciliary Phosphoinositide Regulates Ciliary Protein Trafficking in Drosophila.

Cilia are highly specialized antennae-like cellular organelles. Inositol polyphosphate 5-phosphatase E (INPP5E) converts PI(4,5)P2 into PI4P and is required for proper ciliary function. Although Inpp5e mutations are associated with ciliopathies in humans and mice, the precise molecular role INPP5E plays in cilia remains unclear. Here, we report that Drosophila INPP5E (dINPP5E) regulates ciliary protein trafficking by controlling the phosphoinositide composition of ciliary membranes. Mutations in dInpp5e lead to hearing deficits due to the mislocalization of dTULP and mechanotransduction channels, Inactive and NOMPC, in chordotonal cilia. Both loss of dINPP5E and ectopic expression of the phosphatidylinositol-4-phosphate 5-kinase Skittles increase PI(4,5)P2 levels in the ciliary base. The fact that Skittles expression phenocopies the dInpp5e mutants confirms a central role for PI(4,5)P2 in the regulation of dTULP, Inactive, and NOMPC localization. These data suggest that the spatial localization and levels of PI(4,5)P2 in ciliary membranes are important regulators of ciliary trafficking and function.

Prion protein localizes at the ciliary base during neural and cardiovascular development, and its depletion affects α-tubulin post-translational modifications.

Although conversion of the cellular form of the prion protein (PrPC) into a misfolded isoform is the underlying cause of prion diseases, understanding PrPC physiological functions has remained challenging. PrPC depletion or overexpression alters the proliferation and differentiation properties of various types of stem and progenitor cells in vitro by unknown mechanisms. Such involvement remains uncertain in vivo in the absence of any drastic phenotype of mice lacking PrPC. Here, we report PrPC enrichment at the base of the primary cilium in stem and progenitor cells from the central nervous system and cardiovascular system of developing mouse embryos. PrPC depletion in a neuroepithelial cell line dramatically altered key cilium-dependent processes, such as Sonic hedgehog signalling and α-tubulin post-translational modifications. These processes were also affected over a limited time window in PrPC–ablated embryos. Thus, our study reveals PrPC as a potential actor in the developmental regulation of microtubule dynamics and ciliary functions.

KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome.

BackgroundJoubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures.ResultsUsing autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556-/- null mice possess a Joubert syndrome-associated brain-restricted phenotype. Functional studies in Caenorhabditis elegans nematodes and cultured human cells support a conserved ciliary role for KIAA0556 linked to microtubule regulation. First, nematode KIAA0556 is expressed almost exclusively in ciliated cells, and the worm and human KIAA0556 proteins are enriched at the ciliary base. Second, C. elegans KIAA0056 regulates ciliary A-tubule number and genetically interacts with an ARL13B (JBTS8) orthologue to control cilium integrity. Third, human KIAA0556 binds to microtubules in vitro and appears to stabilise microtubule networks when overexpressed. Finally, human KIAA0556 biochemically interacts with ciliary proteins and p60/p80 katanins. The latter form a microtubule-severing enzyme complex that regulates microtubule dynamics as well as ciliary functions.ConclusionsWe have identified KIAA0556 as a novel microtubule-associated ciliary base protein mutated in Joubert syndrome. Consistent with the mild patient phenotype, our nematode, mice and human cell data support the notion that KIAA0556 has a relatively subtle and variable cilia-related function, which we propose is related to microtubule regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0858-z) contains supplementary material, which is available to authorized users.

TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubertsyndrome.

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.

Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.

Intracellular transport depends on cooperation between distinct motor proteins. Two anterograde intraflagellar transport (IFT) motors, heterotrimeric kinesin-II and homodimeric OSM-3, cooperate to move cargo along Caenorhabditis elegans cilia. Here, using quantitative fluorescence microscopy, with single-molecule sensitivity, of IFT in living strains containing single-copy transgenes encoding fluorescent IFT proteins, we show that kinesin-II transports IFT trains through the ciliary base and transition zone to a 'handover zone' on the proximal axoneme. There, OSM-3 gradually replaces kinesin-II, yielding velocity profiles inconsistent with in vitro motility assays, and then drives transport to the ciliary tip. Dissociated kinesin-II motors undergo rapid turnaround and recycling to the ciliary base, whereas OSM-3 is recycled mainly to the handover zone. This reveals a functional differentiation in which the slower, less processive kinesin-II imports IFT trains into the cilium and OSM-3 drives their long-range transport, thereby optimizing cargo delivery.

Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone.

The transition zone (TZ) of primary cilia serves as a diffusion barrier to regulate ciliogenesis and receptor localization for key signaling events such as sonic hedgehog signaling. Its gating mechanism is poorly understood due to the tiny volume accommodating a large number of ciliopathy-associated molecules. Here we performed stimulated emission depletion (STED) imaging of collective samples and recreated superresolved relative localizations of eight representative species of ciliary proteins using position averages and overlapped with representative electron microscopy (EM) images, defining an architectural foundation at the ciliary base. Upon this framework, transmembrane proteins TMEM67 and TCTN2 were accumulated at the same axial level as MKS1 and RPGRIP1L, suggesting that their regulation roles for tissue-specific ciliogenesis occur at a specific level of the TZ. CEP290 is surprisingly localized at a different axial level bridging the basal body (BB) and other TZ proteins. Upon this molecular architecture, two reservoirs of intraflagellar transport (IFT) particles, correlating with phases of ciliary growth, are present: one colocalized with the transition fibers (TFs) while the other situated beyond the distal edge of the TZ. Together, our results reveal an unprecedented structural framework of the TZ, facilitating our understanding in molecular screening and assembly at the ciliary base.

Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling

Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling.

Coordination of TGFβ/BMP signaling is associated with the primary cilium

We previously showed that canonical TGFβ signaling is regulated in part by the primary cilium, and that ciliary TGFβ signaling is upregulated in stem cells differentiating into cardiomyocytes [1]. Ciliary signaling was shown to be associated with clathrin-dependent endocytosis at the ciliary pocket for activation of SMAD2/3 transcription factors that associate with and promote SMAD4 translocation to the nucleus for target gene expression. Here we investigated whether other receptor types of the TGFβ/BMP superfamily are associated with the primary cilium and whether ciliary TGFβ/BMP signaling regulates the commitment of stem cells to different lineages. Using retinal pigment epithelium cells, we demonstrate that multiple receptor systems within the TGFβ/BMP superfamily localize to the cilium and the ciliary pocket region, including TGFβ receptors I and II (TGF-RI/II), BMP receptors I and II (BMP-RI/II) as well as two isoforms of Activin II receptors (AcRIIa/b) that can be activated by their corresponding ligands to phosphorylate SMAD2/3, SMAD1/5, ERK1/2, AKT and TAK1 at the ciliary base. Further, knockdown of the feedback inhibitor of SMAD signaling, SMURF1, leads to increased SMAD1/5 phosphorylation at the ciliary base, indicating a major role of the primary cilium in balancing the cellular level of TGFβ/BMP signaling to control cellular processes during development and in tissue homeostasis. Indeed, the level of ciliary TGFβ/BMP signaling was shown to be associated with the ability to commit stem cells to either neurogenesis or cardiomyogenesis, such that downregulation of ciliary signaling promotes neurogenesis and inhibits cardiomyogenesis. Current studies focus on the mechanisms for targeting of TGFβ/BMP superfamily receptors to the primary cilium, trafficking and activation of the receptors within the ciliary compartment, and how these processes contribute to differential cross-talking with other signaling pathways in the cilium and at the pocket region to control cellular processes during development.

Nucleoporins but not septins at the transition zone of cilia in Paramecium and Tetrahymena

The transition zone (TZ) of locomotory cilia participates in docking young basal bodies to the surface, stabilizes the basal body-membrane connection, forms a diffusion barrier and modulates the intraciliary transport. In vertebrate ciliated cells, nucleoporins and septins were suggested to be present in the TZ of locomotory and primary cilia. Here we present data suggesting the presence of nucleoporins, at both nuclear pores and ciliary TZ, in Tetrahymena and Paramecium. Using the monoclonal 414, specific to FG-containing nucleoporins, and anti-tubulin antibodies, we detected labeling at the distal part of basal bodies and as a spotted pattern around nuclei. In previous studies on Tetrahymena, GFP-tagged septins (Sep1, Sep2 and Sep3) localized to mitochondrial/ER compartments but neither to basal bodies, cilia nor nuclei. Tetrahymena cells with knocked out septins display disrupted nuclear (Mac and Mic) membranes, abnormal mitochondria, but unaffected ciliary TZ. Similarly, in Paramecium, silencing of the SEP2 gene revealed affected nuclear membranes and mitochondria but normal TZ, and the mAb 414 and DAPI showed a normal labeling of the ciliary base, but patches of missing nuclear pores in the macronucleus. Our studies support the conclusion that some nucleoporins are present in both nuclear pores and ciliary TZ. In addition, the nuclear pore proteins seem to interact with septins, yet the lack of effect of septin silencing on the localization of the mAb 414 in the ciliary TZ suggests that nucleoporins may have different binding partners in nuclear pores and ciliary TZ.

Mechanical Properties of a Primary Cilium As Measured by Resonant Oscillation

Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established ciliary hypothesis states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1) the primary cilium is not a simple cantilevered beam; 2) the base of the cilium may be modeled as a nonlinear rotatory spring, with the linear spring constant k of the cilium base calculated to be (4.6 ± 0.62) × 10−12 N/rad and nonlinear spring constant α to be (−1 ± 0.34) × 10−10 N/rad2; and 3) the ciliary base may be an essential regulator of mechanotransduction signaling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia—anatomically similar structures with very different physiological functions.

Assembly of IFT Trains at the Ciliary Base Depends on IFT74

Intraflagellar transport (IFT) moves IFT trains carrying cargoes from the cell body into the flagellum and from the flagellum back to the cell body. IFT trains are composed of complexes IFT-A and IFT-B and cargo adaptors such as the BBSome. The IFT-B core proteins IFT74 and IFT81 interact directly through central and C-terminal coiled-coil domains, and recently it was shown that the N termini of these proteins form a tubulin-binding module important for ciliogenesis. To investigate the function of IFT74 and its domains invivo, we have utilized Chlamydomonas reinhardtii ift74 mutants. In a null mutant, lack of IFT74 destabilized IFT-B, leading to flagella assembly failure. In this null background, expression of IFT74 lacking 130 amino acids (aa) of the charged N terminus stabilized IFT-B and promoted slow assembly of nearly full-length flagella. A further truncation (lacking aa 1-196, including part of coiled-coil 1) also stabilized IFT-B, but failure in IFT-A/IFT-B interaction within the pool at the base of the flagellum prevented entry of IFT-A into the flagellum and led to severely decreased IFT injection frequency and flagellar-assembly defects. Decreased IFT-A in these short flagella resulted in aggregates of stalled IFT-B in the flagella. We conclude that IFT74 is required to stabilize IFT-B; aa 197-641 are sufficient for this function invivo. The N terminus of IFT74 may be involved in, but is not required for, tubulin entry into flagella. It is required for association of IFT-A and IFT-B at the base of the flagellum and flagellar import of IFT-A.

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation.

The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension.

Image analysis of Caenorhabditis elegans ciliary transition zone structure, ultrastructure, molecular composition, and function.

The transition zone (TZ) at the ciliary base has emerged as an important regulator of the composition and functions of cilia, which are microtubule-based structures extending from the surfaces of most eukaryotic cells, serving motility, chemo-/mechano-/photosensation and developmental signaling roles. Possessing distinct ultrastructural features such as microtubule-membrane spanning Y-links, the ∼0.2-1.0-μm long TZ is thought to act as a gated cytosolic (size dependent) and membrane diffusion barrier that drives ciliary compartmentalization by preventing unregulated protein exchange between the cilium and the rest of the cell. Multiple proteins associated with ciliary diseases (ciliopathies) such as Meckel-Gruber syndrome (MKS) and nephronophthisis are specifically found in the TZ, and work from a number of model systems, including Chlamydomonas reinharditii, Caenorhabditis elegans and the mouse indicates TZ-gating and associated ciliogenic functions for a number of these proteins. Here we present a suite of assays for probing the structure, function, and molecular composition of the C. elegans TZ, with emphasis on TZ ultrastructure, diffusion barrier kinetics, MKS module assembly hierarchy, and TZ-dependent behaviors.

Quantification of IFT-Dynein Dynamics in C.elegans

Cytoplasmic dyneins are the main drivers of microtubule-based retrograde transport in eukaryotic cells. Cytoplasmic dynein 1 plays a role in retrograde intracellular transport and cell division, whereas cytoplasmic dynein 2, also known as IFT-dynein, co-operates with kinesin motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). While cytoplasmic dynein 1 has been the subject of many recent studies, relatively little is known about IFT-dynein. Here, we focus on the mechanism and dynamics of IFT-dynein: how does it behave in vivo at the ensemble and single-molecule level?To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors in the chemosensory cilia of living C. elegans. Transgene worms were generated using the Mos1-mediated single copy insertion (MosSCI) method to ensure endogenous motor expression levels. Time-lapse fluorescence movies showed that IFT-dynein moves in trains consisting of tens of motor proteins. The movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using in-house developed kymograph-analysis software. This analysis revealed that IFT-dynein train velocities and motor numbers are dynamic, changing along the cilium. Double-labeled constructs allowed us to look more closely into motor co-operation, determining the dynein:kinesin ratio at different positions in the cilium.To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, the tracking of individual IFT-dynein motors in vivo. Single-motor trajectories revealed distinct features of IFT-dynein motility: diffusive behavior at the ciliary base, pauses, turns, directed motion and switches between these behaviors. This combined ensemble and single-molecule approach has provided novel quantitative insight into IFT-dynein dynamics in living organisms, shedding light on the complex functioning of dynein motors in general.

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry.

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry.