Intraflagellar Transport Motor Research Articles

IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium.

Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this 'open' system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a 'semi-open' system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.

Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery.

Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play critical roles in motility, sensory reception, and signal transduction in virtually all eukaryotic cells. During IFT, anterograde kinesin-2 and retrograde IFT dynein motors drive the bidirectional transport of IFT trains that deliver cargo, for example, axoneme precursors such as tubulins as well as molecules of the signal transduction machinery, to their site of assembly within the cilium. Following its discovery in Chlamydomonas, IFT has emerged as a powerful model system for studying general principles of motor-dependent cargo transport and we now appreciate the diversity that exists in the mechanism of IFT within cilia of different cell types. The absence of heterotrimeric kinesin-2 function, for example, causes a complete loss of both IFT and cilia in Chlamydomonas, but following its loss in Caenorhabditis elegans, where its primary function is loading the IFT machinery into cilia, homodimeric kinesin-2-driven IFT persists and assembles a full-length cilium. Generally, heterotrimeric kinesin-2 and IFT dynein motors are thought to play widespread roles as core IFT motors, whereas homodimeric kinesin-2 motors are accessory motors that mediate different functions in a broad range of cilia, in some cases contributing to axoneme assembly or the delivery of signaling molecules but in many other cases their ciliary functions, if any, remain unknown. In this review, we focus on mechanisms of motor action, motor cooperation, and motor-dependent cargo delivery during IFT.

Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements.

Cilia are multi-functional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multi-motor assemblies with kinesin-II. Combining these data with insights from single-particle electron microscopy, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2’s mechanical “linker” and “stalk” domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2’s recycling activity during IFT.

Cos2/Kif7 and Osm-3/Kif17 regulate onset of outer segment development in zebrafish photoreceptors through distinct mechanisms

Zebrafish morphants of osm-3/kif17, a kinesin-2 family member and intraflagellar transport motor, have photoreceptor outer segments that are dramatically reduced in number and size. However, two genetic mutant lines, osm-3/kif17sa0119 and osm-3/kif17sa18340, reportedly lack any observable morphological outer segment defects. In this work, we use TALENs to generate an independent allele, osm-3/kif17mw405, and show that both osm-3/kif17sa0119 and osm-3/kif17mw405 have an outer segment developmental delay in both size and density that is fully recovered by 6 days post-fertilization. Additionally, we use CRISPRs to generate cos2/kif7mw406, a mutation in the kinesin-4 family member cos2/kif7 that has been implicated in controlling ciliary architecture and Hedgehog signaling to test whether it may be functioning redundantly with osm-3/kif17. We show that cos2/kif7mw406 has an outer segment developmental delay similar to the osm-3/kif17 mutants. Using a three-dimensional mathematical model of outer segments, we show that while cos2/kif7mw406 and osm-3/kif17mw405 outer segments are smaller throughout the first 6 days of development, the volumetric rates of outer segment morphogenesis are not different among wild-type, cos2/kif7mw406, and osm-3/kif17mw405 after 60hpf. Instead, our model suggests that cos2/kif7mw406 and osm-3/kif17mw405 impact outer segment morphogenesis through upstream events that that are different for each motor. In the case of cos2/kif7mw406 mutants, we show that early defects in Hedgehog signaling lead to a general, non-photoreceptor-specific delay of retinal neurogenesis, which in turn causes the secondary phenotype of delayed outer segment morphogenesis. In contrast, the osm-3/kif17mw405 outer segment morphogenesis delays are linked specifically to initial disc morphogenesis of photoreceptors rather than an upstream event. Further, we show that osm-3/kif17 mutant mice also exhibit a similarly delayed outer segment development, suggesting a role for osm-3/kif17 in early outer segment development that is conserved across species. In conclusion, we show that both osm-3/kif17 and cos2/kif7 have comparable outer segment developmental delays, although through independent mechanisms.

Protein arginine methyltransferases interact with intraflagellar transport particles and change location during flagellar growth and resorption.

Changes in protein by posttranslational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMT 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature-sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.

Disruption of kif3a results in defective osteoblastic differentiation in dental mesenchymal stem/precursor cells via the Wnt signaling pathway.

The anterograde intraflagellar transport motor protein, kif3a, regulates the integrity of primary cilia and various cellular functions, however, the role of kif3a in dental mesenchymal stem/precursor cell differentiation remains to be fully elucidated. In the present study, the expression of kif3a was knocked down in human dental follicle cells (hDFCs) and human dental pulp cells (hDPCs) using short hairpin RNA. The results of subsequent immunofluorescence revealed that knocking down kif3a resulted in the loss of primary cilia, which led to impairment of substantial mineralization and expression of the differentiation-associated markers, including alkaline phosphatase, Runt-related transcription factor 2, dentin matrix protein 1 and dentin sialophosphoprotein in the hDFCs and hDPCs. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses showed that the expression levels of Wnt3a-mediated active β-catenin and lymphoid enhancer-binding factor 1 were attenuated, whereas the expression of phosphorylated glycogen synthase kinase 3β was enhanced, in the kif3a-knockdown cells. In addition, exogenous Wnt3a partially rescued osteoblastic differentiation in the hDFCs and hDPCs. These results demonstrated that inhibition of kif3a in the hDFCs and hDPCs disrupted primary cilia formation and/or function, and indicated that kif3a is important in the differentiation of hDFCs and hDPCs through the Wnt pathway. These findings not only enhance current understanding of tooth development and diseases of tooth mineralization, but also indicate possible strategies to regulate mineralization during tooth repair and regeneration.

Kymographic Analysis of Transport in an Individual Neuronal Sensory Cilium in Caenorhabditis elegans.

Intraflagellar Transport (IFT) is driven by molecular motors that travel upon microtubule-based ciliary axonemes. In the single-celled alga Chlamydomonas reinhardtii, movement of a single anterograde IFT motor, heterotrimeric kinesin-II, is required to generate two identical motile flagella. The function of this canonical anterograde IFT motor is conserved among all eukaryotes, yet multicellular organisms can generate cilia of diverse structures and functions, ranging from simple threadlike non-motile primary cilia to the elaborate cilia that make up rod and cone photoreceptors in the retina. An emerging theme is that additional molecular motors modulate the canonical IFT machinery to give rise to differing ciliary morphologies. Therefore, a complete understanding of the trafficking of ciliary receptors, as well as the biogenesis, maintenance, specialization, and function of cilia, requires the characterization of motor molecules.Here, we describe in detail our method for measuring the motility of proteins in cilia or dendrites of C. elegans male-specific CEM ciliated sensory neurons using time-lapse microscopy and kymography of green fluorescent protein (GFP)-tagged motors, receptors, and cargos. We describe, as a specific example, OSM-3::GFP puncta moving in cilia, but also include (Fig. 1) with settings that have worked well for us measuring movement of heterotrimeric kinesin-II, IFT particles, and the polycystin TRP channel PKD-2.

Mechanisms of flagellum construction and maintenance

Cilia and flagella are cylindrical organelles that protrude at the surface of numerous eukaryotes including most human cells. They are composed, from base to tip, of the basal body (9 triplet microtubules), the transition zone (TZ, 9 doublet microtubules) and the axoneme (9 doublet microtubules ± 2 central microtubules). New flagellar sub-units are added at the distal tip by intraflagellar transport (IFT), a dynamic process where IFT motors drag IFT particles in both anterograde and retrograde directions. IFTs concentrate in a pool at the base of the flagellum, whose localization depends on the organism. In Trypanosomatidae, immunofluorescence assays reveal the association of this pool to the TZ, split apart from the cytoplasm by the transitional fibres. In Trypanosoma brucei, we undertook to investigate about the role of the TZ in flagellum formation and maintenance of IFT in mature flagella, by deciphering the role of RP2, a candidate protein located at the TFs [Stephan et al., Traffic 2007]. We constructed a strain impaired in RP2 production by tetracycline-inducible RNAi and obtained the expected phenotype: parasite growth was affected from the third day of induction. In parallel, diminution of IFT concentration at the flagellum base suggests that RP2 is involved in IFT recruitment during flagellum formation and/or RP2 takes part in a ciliary gate that hinders IFT leaking towards the cytoplasm. We are currently studying these hypotheses by live microscopy using fluorescent fusions of IFT proteins, RP2, and other ciliary gate candidates, dissecting the molecular structure and mechanisms involved in flagellum construction and homeostasis.

Somatic CRISPR-Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia.

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.

FLA8/KIF3B Phosphorylation Regulates Kinesin-II Interaction with IFT-B to Control IFT Entry and Turnaround

The assembly and maintenance of cilia depends on intraflagellar transport (IFT). Activated IFT motor kinesin-II enters the cilium with loaded IFT particles comprising IFT-A and IFT-B complexes. At the ciliary tip, kinesin-II becomes inactivated, and IFT particles are released. Moreover, the rate of IFT entry is dynamically regulated during cilium assembly. However, the regulatory mechanism of IFT entry and loading/unloading of IFT particles remains elusive. We show that the kinesin-II motor subunit FLA8, a homolog of KIF3B, is phosphorylated on the conserved S663 by a calcium-dependent kinase in Chlamydomonas. This phosphorylation disrupts the interaction between kinesin-II and IFT-B, inactivates kinesin-II and inhibits IFT entry, and is also required for IFT-B unloading at the ciliary tip. Furthermore, our data suggest that the IFT entry rate is controlled by regulation of the cellular level of phosphorylated FLA8. Therefore, FLA8 phosphorylation acts as a molecular switch to control IFT entry and turnaround.

Mutations in the Gene Encoding IFT Dynein Complex Component WDR34 Cause Jeune Asphyxiating Thoracic Dystrophy

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 invitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.

Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization inC. elegans

Primary cilia are ubiquitous sensory organelles that concentrate transmembrane signaling proteins essential for sensing environmental cues. Mislocalization of crucial ciliary signaling proteins, such as the tetrameric cyclic nucleotide-gated (CNG) channels, can lead to cellular dysfunction and disease. Although several cis- and trans-acting factors required for ciliary protein trafficking and localization have been identified, whether these mechanisms act in a protein- and cell-specific manner is largely unknown. Here, we show that CNG channel subunits can be localized to discrete ciliary compartments in individual sensory neurons in C. elegans, suggesting that channel composition is heterogeneous across the cilium. We demonstrate that ciliary localization of CNG channel subunits is interdependent on different channel subunits in specific cells, and identify sequences required for efficient ciliary targeting and localization of the TAX-2 CNGB and TAX-4 CNGA subunits. Using a candidate gene approach, we show that Inversin, transition zone proteins, intraflagellar transport motors and a MYND-domain protein are required to traffic and/or localize CNG channel subunits in both a cell- and channel subunit-specific manner. We further find that TAX-2 and TAX-4 are relatively immobile in specific sensory cilia subcompartments, suggesting that these proteins undergo minimal turnover in these domains in mature cilia. Our results uncover unexpected diversity in the mechanisms that traffic and localize CNG channel subunits to cilia both within and across cell types, highlighting the essential contribution of this process to cellular functions.

Single-Molecule Observation of IFT Turnover using a Photobleaching Gate

Intraflagellar transport (IFT) is required for the assembly and maintenance of eukaryotic cilia and flagella. In Chlamydomonas reinhardtii, IFT trains are transported by kinesin-2 to the flagellar distal tip and by dynein-1b back to the basal body. Remodeling of IFT trains and motor protein regulation are believed to occur at the flagellar tip complex. Due to the complex and crowded nature of the flagellar turnaround zones, IFT train and motor behavior at the tip remains unclear. To investigate the turnover of a single IFT train or a motor cluster, we introduced a high-power laser gate that continuously photobleaches incoming IFT trains and selectively allows a single unbleached train to enter the flagellum. We observed that anterograde IFT trains pause at tip for ∼4 s on average and split into multiple retrograde trains. In contrast, the KAP subunit of kinesin-2 releases from IFT trains at the tip after a shorter pausing period. Different from C. elegans IFT, in which KAP is recycled as a retrograde cargo, KAP in Chlmaydomonas returns to the basal body by diffusion with an average diffusion constant of 1.2 μm2/s. We also observed a concentration gradient for the KAP-GFP background that increases towards the tips of flagella. We anticipate our photobleaching gate assays to be a starting point to address the detailed mechanism of IFT regulation at turnaround zones.

Cytoplasmic dynein-2: from molecules to human diseases

The dynein motor protein family is involved in a wide variety of functions in eukaryotic cells. The axonemal dynein class and cytoplasmic dynein-1 subclass have been well characterized. However, the cytoplasmic dynein-2 subclass of the family has only recently begun to be understood. We describe the entire dynein family but focus on cytoplasmic dynein-2. Dynein-2 consists of a heavy, an intermediate, a light intermediate, and a light chain. The complex appears to function primarily as the retrograde motor for intraflagellar transport. This process is important for the formation and maintenance of cilia and flagella. Additionally, dynein-2 has roles in the control of ciliary length and in non-ciliary functions. Mutations in the human dynein-2 heavy chain lead to cilia-related diseases.

Analysis of KIF17 distal tip trafficking in zebrafish cone photoreceptors

Multiple proteins are targeted to photoreceptor outer segments (OSs) where they function in phototransduction. Intraflagellar transport (IFT), a highly conserved bidirectional transport pathway occurring along the microtubules of the ciliary axoneme has been implicated in OS trafficking. The canonical anterograde motor for IFT is the heterotrimeric kinesin II or KIF3 complex. Previous work from our laboratory has demonstrated a role for an additional kinesin 2 family motor, the homodimeric KIF17. To gain a better understanding of KIF17 function in photoreceptor OS we utilized transgenic zebrafish expressing zfKIF17-GFP to assess the localization and dynamics of zfKIF17. Our data indicate that both endogenous KIF17 and KIF17-GFP are associated with the axoneme of zebrafish cones at both early (5dpf) and late (21 dfp) stages of development. Strikingly, KIF17-GFP accumulates at the OS distal tip in a phenomenon referred to as “tipping”. Tipping occurs in the large majority of photoreceptors and also occurs when mammalian KIF17-mCherry is expressed in ciliated epithelial cells in culture. In some cases KIF17-GFP is shed with the OS tip as part of the disc shedding process. We have also found that KIF17-GFP moves within the OS at rates consistent with those observed for IFT and other kinesins.

Dissecting the Sequential Assembly and Localization of Intraflagellar Transport Particle Complex B in Chlamydomonas

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.

Kinesin motors and primary cilia

Cilia and flagella play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in ciliary assembly and/or function can lead to a range of human diseases, collectively known as the ciliopathies, including polycystic kidney, liver and pancreatic diseases, sterility, obesity, situs inversus, hydrocephalus and retinal degeneration. A basic understanding of how cilia form and function is essential for deciphering ciliopathies and generating therapeutic treatments. The cilium is a unique compartment that contains a distinct complement of protein and lipid. However, the molecular mechanisms by which soluble and membrane protein components are targeted to and trafficked into the cilium are not well understood. Cilia are generated and maintained by IFT (intraflagellar transport) in which IFT cargoes are transported along axonemal microtubules by kinesin and dynein motors. A variety of genetic, biochemical and cell biological approaches has established the heterotrimeric kinesin-2 motor as the 'core' IFT motor, whereas other members of the kinesin-2, kinesin-3 and kinesin-4 families function as 'accessory' motors for the transport of specific cargoes in diverse cell types. Motors of the kinesin-9 and kinesin-13 families play a non-IFT role in regulating ciliary beating or axonemal length, respectively. Entry of kinesin motors and their cargoes into the ciliary compartment requires components of the nuclear import machinery, specifically importin-β2 (transportin-1) and Ran-GTP (Ran bound to GTP), suggesting that similar mechanisms may regulate entry into the nuclear and ciliary compartments.

Probing the role of IFT particle complex A and B in flagellar entry and exit of IFT-dynein in Chlamydomonas

Mediating the transport of flagellar precursors and removal of turnover products, intraflagellar transport (IFT) is required for flagella assembly and maintenance. The IFT apparatus is composed of the anterograde IFT motor kinesin II, the retrograde IFT motor IFT-dynein, and IFT particles containing two complexes, A and B. In order to have a balanced two-way transportation, IFT-dynein has to be carried into flagella and transported to the flagellar tip by kinesin II, where it is activated to drive the retrograde IFT back to the flagellar base. In this study, we investigated the role of complex A and complex B in the flagellar entry and exit of IFT-dynein. We showed that regardless of the amount of complex A, IFT-dynein accumulated proportionally to the amount of complex B in the flagella of fla15/ift144 and fla17-1/ift139, two complex A temperature-sensitive mutants. Complex A was depleted from both cellular and flagellar compartments in fla15/ift144 mutant. However, in fla17-1/ift139 mutant, the flagellar level of complex A was at the wild-type level, which was in radical contrast to the significantly reduced cellular amount of complex A. These results support that complex A is not required for the flagellar entry of IFT-dynein, but might be essential for the lagellar exit of IFT-dynein. Additionally, we confirmed the essential role of IFT172, a complex B subunit, in the flagellar entry of IFT-dynein. These results indicate that complexes A and B play complementary but distinct roles for IFT-dynein, with complex B carrying IFT-dynein into the flagella while complex A mediates the flagellar exit of IFT-dynein.

Kinesin-3 KLP-6 Regulates Intraflagellar Transport in Male-Specific Cilia of Caenorhabditis elegans

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 they 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. 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 shows partial uncoupling of the IFT motors of the kinesin-2 family, kinesin-II and OSM-3/KIF17, with a portion of OSM-3 moving independently of the 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 that 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.

Motor Coordination via a Tug-of-War Mechanism Drives Bidirectional Vesicle Transport

The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High-resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility via automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of LysoTracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement, and pauses. Quantitative analysis indicates that vesicles copurify with a low number of stably bound motors: one to five dynein and one to four kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly bound motors and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.