Interaction of heterotrimeric kinesin-II with IFT-B-connecting tetramer is crucial for ciliogenesis.
ABSTRACT: Intraflagellar transport (IFT) is crucial for the assembly and maintenance of cilia and is mediated by IFT particles containing IFT-A and IFT-B complexes. IFT-B powered by heterotrimeric kinesin-II and IFT-A powered by the dynein-2 complex are responsible for anterograde and retrograde protein trafficking, respectively. However, little is known about the molecular basis of the trafficking of these IFT particles regulated by kinesin and dynein motors. Using the visible immunoprecipitation assay, we identified in this study a three-to-four protein interaction involving the kinesin-II trimer KIF3A-KIF3B-KAP3 and the IFT-B-connecting tetramer IFT38-IFT52-IFT57-IFT88; among the kinesin-II subunits, KIF3B contributed mainly to IFT-B binding. Furthermore, we showed that the ciliogenesis defect of KIF3B-knockout cells can be rescued by the exogenous expression of wild-type KIF3B but not by that of its mutant compromised with respect to IFT-B binding. Thus, interaction of heterotrimeric kinesin-II with the IFT-B-connecting tetramer is crucial for ciliogenesis via the powering of IFT particles to move in the anterograde direction.
Project description:The trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in cilium assembly and IFT. We used a chemical-genetic approach to generate an inhibitable KIF3A/KIF3B/KAP kinesin-2 motor (i3A/i3B) that is capable of rescuing wild-type (WT) motor function for cilium assembly and Hedgehog signaling in Kif3a/Kif3b double-knockout cells. We demonstrate that KIF3A/KIF3B function is required not just for cilium assembly but also for cilium maintenance, as inhibition of i3A/i3B blocks IFT within 2 min and leads to a complete loss of primary cilia within 8 h. In contrast, inhibition of dynein-2 has no effect on cilium maintenance within the same time frame. The kinetics of cilia loss indicate that two processes contribute to ciliary disassembly in response to cessation of anterograde IFT: a slow shortening that is steady over time and a rapid deciliation that occurs with stochastic onset. We also demonstrate that the kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells or delay the loss of assembled cilia upon i3A/i3B inhibition. These results demonstrate that KIF3A/KIF3B/KAP is the sole and essential motor for cilium assembly and maintenance in mammalian cells. These findings highlight differences in how kinesin-2 motors were adapted for cilium assembly and IFT function across species.
2019-01-01 | S-EPMC6445692 | BioStudies
Project description:The intraflagellar transport (IFT) machinery consists of the anterograde motor kinesin-II, the retrograde motor IFT dynein and the IFT-A and -B complexes. However, the interaction among IFT motors and IFT complexes during IFT remains elusive. Here, we show that the IFT-B protein IFT54 interacts with both kinesin-II and IFT dynein and regulates anterograde IFT. Deletion of residues 342-356 of Chlamydomonas IFT54 resulted in diminished anterograde traffic of IFT and accumulation of IFT motors and complexes in the proximal region of cilia. IFT54 directly interacted with kinesin-II and this interaction was strengthened for the IFT54?342-356 mutant in vitro and in vivo. The deletion of residues 261-275 of IFT54 reduced ciliary entry and anterograde traffic of IFT dynein with accumulation of IFT complexes near the ciliary tip. IFT54 directly interacted with IFT dynein subunit D1bLIC and deletion of residues 261-275 reduced this interaction. The interactions between IFT54 and the IFT motors were also observed in mammalian cells. Our data indicate a central role for IFT54 in binding the IFT motors during anterograde IFT.
Project description:Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2-driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT-dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.
Project description:Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.
Project description:The differentiation of cilia is mediated by kinesin-driven transport. As the function of kinesins in vertebrate ciliogenesis is poorly characterized, we decided to determine the role of kinesin-2 family motors--heterotrimeric kinesin-II and the homodimeric Kif17 kinesin--in zebrafish cilia. We report that kif17 is largely dispensable for ciliogenesis; kif17 homozygous mutant animals are viable and display subtle morphological defects of olfactory cilia only. In contrast to that, the kif3b gene, encoding a heterotrimeric kinesin subunit, is necessary for cilia differentiation in most tissues, although exceptions exist, and include photoreceptors and a subset of hair cells. Cilia of these cell types persist even in kif3b/kif17 double mutants. Although we have not observed a functional redundancy of kif3b and kif17, kif17 is able to substitute for kif3b in some cilia. In contrast to kif3b/kif17 double mutants, simultaneous interference with kif3b and kif3c leads to the complete loss of photoreceptor and hair cell cilia, revealing redundancy of function. This is in agreement with the idea that Kif3b and Kif3c motor subunits form complexes with Kif3a, but not with each other. Interestingly, kif3b mutant photoreceptor cilia differentiate with a delay, suggesting that kif3c, although redundant with kif3b at later stages of differentiation, is not active early in photoreceptor ciliogenesis. Consistent with that, the overexpression of kif3c in kif3b mutants rescues early photoreceptor cilia defects. These data reveal unexpected diversity of functional relationships between vertebrate ciliary kinesins, and show that the repertoire of kinesin motors changes in some cilia during their differentiation.
Project description:The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 microm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope-based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at approximately 1.1 microm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end-directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.
Project description:Heterodimeric motor organization of kinesin-II is essential for its function in anterograde IFT in ciliogenesis. However, the underlying mechanism is not well understood. In addition, the anterograde IFT velocity varies significantly in different organisms, but how this velocity affects ciliary length is not clear. We show that in Chlamydomonas motors are only stable as heterodimers in vivo, which is likely the key factor for the requirement of a heterodimer for IFT. Second, chimeric CrKinesin-II with human kinesin-II motor domains functioned in vitro and in vivo, leading to a ~ 2.8 fold reduced anterograde IFT velocity and a similar fold reduction in IFT injection rate that supposedly correlates with ciliary assembly activity. However, the ciliary length was only mildly reduced (~15%). Modeling analysis suggests a nonlinear scaling relationship between IFT velocity and ciliary length that can be accounted for by limitation of the motors and/or its ciliary cargoes, e.g. tubulin.
Project description:Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.
Project description:The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.
Project description:Intraflagellar transport (IFT) is a process required for flagella and cilia assembly that describes the dynein and kinesin mediated movement of particles along axonemes that consists of an A and a B complex, defects in which disrupt retrograde and anterograde transport, respectively. Herein, we describe a novel Caenorhabditis elegans gene, xbx-1, that is required for retrograde IFT and shares homology with a mammalian dynein light intermediate chain (D2LIC). xbx-1 expression in ciliated sensory neurons is regulated by the transcription factor DAF-19, as demonstrated previously for genes encoding IFT complex B proteins. XBX-1 localizes to the base of the cilia and undergoes anterograde and retrograde movement along the axoneme. Disruption of xbx-1 results in cilia defects and causes behavioral abnormalities observed in other cilia mutants. Analysis of cilia in xbx-1 mutants reveals that they are shortened and have a bulb like structure in which IFT proteins accumulate. The role of XBX-1 in IFT was further confirmed by analyzing the effect that other IFT mutations have on XBX-1 localization and movement. In contrast to other IFT proteins, retrograde XBX-1 movement was detected in complex A mutants. Our results suggest that the DLIC protein XBX-1 functions together with the CHE-3 dynein in retrograde IFT, downstream of the complex A proteins.