The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet–Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function.
DYF-13, originally identified in C. elegans within a collection of dye-filling chemosensory mutants, is one of several proteins that have been classified as putatively involved in intraflagellar transport (IFT), the bidirectional movement of protein complexes along cilia and flagella, and specifically in anterograde IFT. Although genetic studies have highlighted a fundamental role of DYF-13 in nematode sensory cilium and trypanosome flagellum biogenesis, biochemical studies on DYF-13 have lagged behind. Here, we show that in Trypanosoma brucei the ortholog to DYF13, PIFTC3, participates in a macromolecular complex of approximately 660 kDa. Mass spectroscopy of affinity purified PIFTC3 revealed several components of IFT complex B as well as orthologs of putative IFT factors DYF-1, DYF-3, DYF-11/Elipsa, and IFTA-2. DYF-11 was further analyzed and shown to be concentrated near the basal bodies and in the flagellum, and to be required for flagellum elongation. In addition, by coimunoprecipitation we detected an interaction between DYF-13 and IFT122, a component of IFT complex A, which is required for retrograde transport. Thus, our biochemical analysis supports the model, proposed by genetic analysis in C. elegans, that the trypanosome ortholog of DYF-13 plays a central role in the IFT mechanism.
Flagellum; PIFTC3; DYF-13; DYF-11; IFT-B; Trypanosoma brucei
The bidirectional movement of intraflagellar transport (IFT) particles, which are composed of motors, IFT-A and IFT-B subcomplexes, and cargos, is required for cilia biogenesis and signaling 1, 2. A successful IFT cycle depends on the massive IFT particle to be properly assembled at the ciliary base and turned around from anterograde to retrograde transport at the ciliary tip. However, how IFT assembly and turnaround are regulated in vivo remains elusive. From a whole-genome mutagenesis screen in C. elegans, we identified two hypomorphic mutations in dyf-2 and bbs-1 as the only mutants showing normal anterograde IFT transport but defective IFT turnaround at the ciliary tip. Further analyses revealed that the BBSome 3, 4, a group of conserved proteins affected in human Bardet-Biedl syndrome (BBS) 5, assembles IFT complexes at the ciliary base, then binds to anterograde IFT particle in a DYF-2- (an ortholog of human WDR19) and BBS-1-dependent manner, and lastly reaches the ciliary tip to regulate proper IFT recycling. Our results unravel the BBSome as the key player regulating IFT assembly and turnaround in cilia.
Sensory cilium biogenesis within Caenorhabditis elegans neurons depends on the kinesin-2–dependent intraflagellar transport (IFT) of ciliary precursors associated with IFT particles to the axoneme tip. Here we analyzed the molecular organization of the IFT machinery by comparing the in vivo transport and phenotypic profiles of multiple proteins involved in IFT and ciliogenesis. Based on their motility in wild-type and bbs (Bardet-Biedl syndrome) mutants, IFT proteins were classified into groups with similar transport profiles that we refer to as “modules.” We also analyzed the distribution and transport of fluorescent IFT particles in multiple known ciliary mutants and 49 new ciliary mutants. Most of the latter mutants were snip-SNP mapped and one, namely dyf-14(ks69), was cloned and found to encode a conserved protein essential for ciliogenesis. The products of these ciliogenesis genes could also be assigned to the aforementioned set of modules or to specific aspects of ciliogenesis, based on IFT particle dynamics and ciliary mutant phenotypes. Although binding assays would be required to confirm direct physical interactions, the results are consistent with the hypothesis that the C. elegans IFT machinery has a modular design, consisting of modules IFT-subcomplex A, IFT-subcomplex B, and a BBS protein complex, in addition to motor and cargo modules, with each module contributing to distinct functional aspects of IFT or ciliogenesis.
DYF-1 is a highly conserved protein. Our results demonstrate that DYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.
DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1–depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.
Conserved intraflagellar transport (IFT) particle proteins and IFT-associated motors are needed to assemble most eukaryotic cilia and flagella. Proteins in an IFT-A subcomplex are generally required for dynein-driven retrograde IFT, from the ciliary tip to the base. We describe novel structural and functional roles for IFT-A proteins in chordotonal organs, insect mechanosensory organs with cilia that are both sensory and motile.
The reduced mechanoreceptor potential A (rempA) locus of Drosophila encodes the IFT-A component IFT140. Chordotonal cilia are shortened in rempA mutants and an IFT-B protein accumulates in the mutant cilia, consistent with a defect in retrograde IFT. A functional REMPA-YFP fusion protein concentrates at the site of the ciliary dilation (CD), a highly structured axonemal inclusion of hitherto unknown composition and function. The CD is absent in rempA mutants, and REMPA-YFP is undetectable in the absence of another IFT-A protein, IFT122. In a mutant lacking the IFT dynein motor, the CD is disorganized and REMPA-YFP is mislocalized. A TRPV ion channel, required to generate sensory potentials and regulate ciliary motility, is normally localized in the cilia, proximal to the CD. This channel spreads into the distal part of the cilia in dynein mutants, and is undetectable in rempA mutants.
IFT-A proteins are located at and required by the ciliary dilation, which separates chordotonal cilia into functionally distinct zones. A requirement for IFT140 in stable TRPV channel expression also suggests that IFT-A proteins may mediate preciliary transport of some membrane proteins.
The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet–Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.
Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.
Intraflagellar transport (IFT) is a motility process operating between the ciliary/flagellar (interchangeable terms) membrane and microtubular axoneme of motile and sensory cilia. Multi-polypeptide IFT particles, composed of complexes A and B, carry flagellar precursors to their assembly site at the flagellar tip (anterograde) powered by kinesin, and turnover products from the tip back to the cytoplasm (retrograde) driven by cytoplasmic dynein. IFT is essential for the assembly and maintenance of almost all eukaryotic cilia and flagella, and mutations affecting either the IFT motors or the IFT particle polypeptides result in the inability to assemble normal flagella or in defects in the sensory functions of cilia.
We found that the IFT complex B polypeptide, IFT27, is a Rab-like small G protein. Reduction of the level of IFT27 by RNA interference reduces the levels of other complex A and B proteins suggesting this protein is instrumental in maintaining the stability of both IFT complexes. Furthermore, in addition to its role in flagellar assembly, IFT27 is unique among IFT polypeptides in that its partial knockdown results in defects in cytokinesis and elongation of the cell cycle and a more complete knockdown is lethal.
IFT27, a small G protein, is one of a growing number of flagellar proteins that are now known to have a role in cell cycle control.
Intraflagellar transport (IFT) is the bidirectional movement of IFT particles between the cell body and the distal tip of a flagellum. Organized into complexes A and B, IFT particles are composed of at least 18 proteins. The function of IFT proteins in flagellar assembly has been extensively investigated. However, much less is known about the molecular mechanism of how IFT is regulated.
We herein report the identification of a novel IFT particle protein, IFT25, in Chlamydomonas. Dephosphorylation assay revealed that IFT25 is a phosphoprotein. Biochemical analysis of temperature sensitive IFT mutants indicated that IFT25 is an IFT complex B subunit. In vitro binding assay confirmed that IFT25 binds to IFT27, a Rab-like small GTPase component of the IFT complex B. Immunofluorescence staining showed that IFT25 has a punctuate flagellar distribution as expected for an IFT protein, but displays a unique distribution pattern at the flagellar base. IFT25 co-localizes with IFT27 at the distal-most portion of basal bodies, probably the transition zones, and concentrates in the basal body region by partially overlapping with other IFT complex B subunits, such as IFT46. Sucrose density gradient centrifugation analysis demonstrated that, in flagella, the majority of IFT27 and IFT25 including both phosphorylated and non-phosphorylated forms are cosedimented with other complex B subunits in the 16S fractions. In contrast, in cell body, only a fraction of IFT25 and IFT27 is integrated into the preassembled complex B, and IFT25 detected in complex B is preferentially phosphorylated.
IFT25 is a phosphoprotein component of IFT particle complex B. IFT25 directly interacts with IFT27, and these two proteins likely form a subcomplex in vivo. We postulate that the association and disassociation between the subcomplex of IFT25 and IFT27 and complex B might be involved in the regulation of IFT.
The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure–frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a “canonical” IFT motor, whereas OSM-3 is an “accessory” IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.
We cloned a Tetrahymena thermophila gene, IFT52, encoding
a homolog of the Chlamydomonas intraflagellar transport protein,
IFT52. Disruption of IFT52 led to loss of cilia and incomplete
cytokinesis, a phenotype indistinguishable from that of mutants lacking
kinesin-II, a known ciliary assembly transporter. The cytokinesis failures
seem to result from lack of cell movement rather than from direct involvement
of ciliary assembly pathway components in cytokinesis. Spontaneous partial
suppressors of the IFT52 null mutants occurred, which assembled cilia
at high cell density and resorbed cilia at low cell density. The stimulating
effect of high cell density on cilia formation is based on the creation of
pericellular hypoxia. Thus, at least under certain conditions, ciliary
assembly is affected by an extracellular signal and the Ift52p function may be
integrated into signaling pathways that regulate ciliogenesis.
In most cilia, the axoneme can be subdivided into three segments: proximal (the transition zone), middle (with outer doublet microtubules), and distal (with singlet extensions of outer doublet microtubules). How the functionally distinct segments of the axoneme are assembled and maintained is not well understood. DYF-1 is a highly conserved ciliary protein containing tetratricopeptide repeats. In Caenorhabditis elegans, DYF-1 is specifically needed for assembly of the distal segment (G. Ou, O. E. Blacque, J. J. Snow, M. R. Leroux, and J. M. Scholey. Nature. 436:583-587, 2005). We show that Tetrahymena cells lacking an ortholog of DYF-1, Dyf1p, can assemble only extremely short axoneme remnants that have structural defects of diverse natures, including the absence of central pair and outer doublet microtubules and incomplete or absent B tubules on the outer microtubules. Thus, in Tetrahymena, DYF-1 is needed for either assembly or stability of the entire axoneme. Our observations support the conserved function for DYF-1 in axoneme assembly or stability but also show that the consequences of loss of DYF-1 for axoneme segments are organism specific.
Cilia intraflagellar transport and ciliogenesis are regulated by two small GTPases that maintain binding between IFT subcomplexes.
Intraflagellar transport (IFT) machinery mediates the bidirectional movement of cargos that are required for the assembly and maintenance of cilia. However, little is known about how IFT is regulated in vivo. In this study, we show that the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor–like protein 13 (ARL-13) encoded by the Caenorhabditis elegans homologue of the human Joubert syndrome causal gene ARL13B, localizes exclusively to the doublet segment of the cilium. arl-13 mutants have shortened cilia with various ultrastructural deformities and a disrupted association between IFT subcomplexes A and B. Intriguingly, depletion of ARL-3, another ciliary small GTPase, partially suppresses ciliogenesis defects in arl-13 mutants by indirectly restoring binding between IFT subcomplexes A and B. Rescue of arl-13 mutants by ARL-3 depletion is mediated by an HDAC6 deacetylase-dependent pathway. Thus, we propose that two conserved small GTPases, ARL-13 and ARL-3, coordinate to regulate IFT and that perturbing this balance results in cilia deformation.
Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3–4 and 7–8, suggesting the existence of specific IFT tracks. Rapid (>3 μm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.
Most eukaryotic cells have a primary cilium which acts as a sensory organelle1. Cilia are assembled by intraflagellar transport (IFT), a process mediated by multimeric IFT particles and molecular motors2. Here we show that lymphoid and myeloid cells, which lack primary cilia, express IFT proteins. IFT20, an IFT component essential for ciliary assembly3,4, was found to colocalize with both the MTOC and Golgi and post-Golgi compartments in T-lymphocytes. In antigen-specific conjugates, IFT20 translocated to the immune synapse (IS). IFT20 knockdown resulted in impaired TCR/CD3 clustering and signaling at the IS due to defective polarized recycling. Moreover, IFT20 was required for the inducible assembly of a complex with other IFT components (IFT57, IFT88) and the TCR. The results identify IFT20 as a novel regulator of IS assembly in T-cells and provide the first evidence that IFT is implicated in membrane trafficking in cells lacking primary cilia, thereby opening a new perspective on IFT function beyond its role in ciliogenesis.
IFT proteins are differentially localized in photoreceptor cilia, including within the inner segment, and some are shown to function in trafficking in nonciliated retinal neurons.
The assembly and maintenance of cilia require intraflagellar transport (IFT), a process mediated by molecular motors and IFT particles. Although IFT is a focus of current intense research, the spatial distribution of individual IFT proteins remains elusive. In this study, we analyzed the subcellular localization of IFT proteins in retinal cells by high resolution immunofluorescence and immunoelectron microscopy. We report that IFT proteins are differentially localized in subcompartments of photoreceptor cilia and in defined periciliary target domains for cytoplasmic transport, where they are associated with transport vesicles. IFT20 is not in the IFT core complex in photoreceptor cilia but accompanies Golgi-based sorting and vesicle trafficking of ciliary cargo. Moreover, we identify a nonciliary IFT system containing a subset of IFT proteins in dendrites of retinal neurons. Collectively, we provide evidence to implicate the differential composition of IFT systems in cells with and without primary cilia, thereby supporting new functions for IFT beyond its well-established role in cilia.
Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.
We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.
We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.
Ultrastructural study of Chlamydomonas cilia shows that anterograde IFT particles form trains that are long and narrow, while retrograde IFT form short, compact particle trains.
Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.
Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated if two C. elegans α- and β-tubulin isotypes, identified via mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules (MTs). One isotype, TBB-4, assembles into MTs at the tips of the axoneme core and distal segments, where the MT tip-tracker, EB1, is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, FRAP analysis and modeling suggest that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady state length.
A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.
Chlamydomonas; temperature-sensitive mutants; intraflagellar particles; video microscopy; retrograde transport
Cilia are cellular sensory organelles whose integrity of structure and function are important to human health . All cilia are assembled and maintained by kinesin-2 motors in a process termed intraflagellar transport (IFT), but exhibit great variety of morphology and function. This diversity is proposed to be conferred by cell-specific modulation of the core IFT by additional factors, but examples of such IFT modulators are limited [2–4]. Here we demonstrate that the cell-specific kinesin-3 KLP-6 acts as a modulator of both IFT dynamics and length in the cephalic male (CEM) cilia of Caenorhabditis elegans. Live imaging of GFP-tagged kinesins in CEM cilia showed partial uncoupling of the IFT motors of kinesin-2 family, kinesin-II and OSM-3/KIF17, with a portion of OSM-3 moving independently of IFT complex. KLP-6 moves independently of the kinesin-2 motors and acts to reduce the velocity of OSM-3 and IFT. Additionally, kinesin-II mutants display a novel CEM cilia elongation phenotype, which is partially dependent on OSM-3 and KLP-6. Our observations illustrate modulation of the general kinesin-2-driven IFT process by a cell-specific kinesin-3 in cilia of C. elegans male neurons.
IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the Intraflagellar Transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5–13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.
IFT172; Slb; cilia; node; nodal; MHB boundary
The planar cell polarity effector Fuz is required for normal particle dynamics of the intraflagellar transport system, specifically in the retrograde transport of proteins.
Cilia play key roles in development and homeostasis, and defects in cilia structure or function lead to an array of human diseases. Ciliogenesis is accomplished by the intraflagellar transport (IFT) system, a set of proteins governing bidirectional transport of cargoes within ciliary axonemes. In this paper, we present a novel platform for in vivo analysis of vertebrate IFT dynamics. Using this platform, we show that the planar cell polarity (PCP) effector Fuz was required for normal IFT dynamics in vertebrate cilia, the first evidence directly linking PCP to the core machinery of ciliogenesis. Further, we show that Fuz played a specific role in trafficking of retrograde, but not anterograde, IFT proteins. These data place Fuz in the small group of known IFT effectors outside the core machinery and, additionally, identify Fuz as a novel cytoplasmic effector that differentiates between the retrograde and anterograde IFT complexes.
Intraflagellar transport (IFT), the motor-dependent movement of IFT particles along the axoneme, is critical for the assembly, maintenance, and function of motile and sensory cilia, and, consequently, this process underlies ciliary motility, cilium-based signaling, and ciliopathies. Here, I present my perspective on IFT as a model system for studying motor-driven cargo transport. I review evidence that kinesin-2 motors physically transport IFT particles as cargo and hypothesize that several accessory kinesins confer cilia-specific functions by augmenting the action of the two core IFT motors, kinesin-2 and dynein 1b, which assemble the cilium foundation.