Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella

Intraflagellar transport (IFT) of particles along flagellar microtubules is required for the assembly and maintenance of eukaryotic flagella and cilia. In Chlamydomonas, anterograde and retrograde particles viewed by light microscopy average 0.12-microm and 0.06-microm diameter, respectively. Examination of IFT particle structure in growing flagella by electron microscopy revealed similar size aggregates composed of small particles linked to each other and to the membrane and microtubules. To determine the relationship between the number of particles and flagellar length, the rate and frequency of IFT particle movement was measured in nongrowing, growing, and shortening flagella. In all flagella, anterograde and retrograde IFT averaged 1.9 microm/s and 2.7 microm/s, respectively, but retrograde IFT was significantly slower in flagella shorter than 4 mum. The number of flagellar IFT particles was not fixed, but depended on flagellar length. Pauses in IFT particle entry into flagella suggest the presence of a periodic "gate" that permits up to 4 particles/s to enter a flagellum.     

The Microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii

Flagellar axonemes assemble and continuously turn over at the flagellar tip. The supply and removal of axonemal subunits at the tip are mediated by intraflagellar transport (IFT), a motility process essential for the assembly and maintenance of all eukaryotic flagella and cilia. IFT is characterized by the movement of large protein complexes (IFT particles) from the basal bodies to the flagellar tip by kinesin-II and from the tip back to the basal bodies by cytoplasmic dynein 1b. The IFT particles consist of approximately 16 polypeptides partitioned into two complexes, A and B, and associate with axonemal precursors/turn over products. The mechanisms by which IFT motor regulation and cargo loading/unloading occur at the flagellar tip are unknown. We identified a Chlamydomonas reinhardtii ortholog of the microtubule (MT) plus end-tracking protein EB1 [4] (CrEB1) and show here that CrEB1 localizes to the tip of flagella and to the proximal part of the basal bodies. Furthermore, we found that CrEB1 is depleted from flagella of the temperature-sensitive (ts) flagellar assembly-defective (fla) mutant fla11(ts) at the restrictive temperature. This depletion of CrEB1 is accompanied by a dramatic accumulation of IFT particle polypeptides near the flagellar tip.     

A Dynein Light Chain Is Essential for the Retrograde Particle Movement of Intraflagellar Transport (IFT)

Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an M_r 8,000 light chain termed LC8. The function of this chain is unknown, but it is highly conserved between a wide variety of organisms. We have identified deletion alleles of the gene (fla14) encoding this protein in Chlamydomonas reinhardtii. These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules. Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT) (Kozminski, K.G., P.L. Beech, and J.L. Rosenbaum. 1995. J. Cell Biol. 131:1517–1527). IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules. Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown. Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum. This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process. Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes (Cole, D.G., D.R. Deiner, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993–1008), confirming that these complexes are subunits of the rafts. Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT.     

Chlamydomonas IFT172 is encoded by FLA11, interacts with CrEB1, and regulates IFT at the flagellar tip

The transport of flagellar precursors and removal of turnover products from the flagellar tip is mediated by intraflagellar transport (IFT) , which is essential for both flagellar assembly and maintenance . Large groups of IFT particles are moved from the flagellar base to the tip by kinesin-2, and smaller groups are returned to the base by cytoplasmic dynein 1b. The IFT particles are composed of two protein complexes, A and B, comprising approximately 16-18 polypeptides. How cargo is unloaded from IFT particles, turnover products loaded, and active IFT motors exchanged at the tip is unknown. We previously showed that the Chlamydomonas microtubule end binding protein 1 (CrEB1) localizes to the flagellar tip and is depleted from the tips of the temperature-sensitive (ts) mutant fla11ts . We demonstrate here that FLA11 encodes IFT protein 172, a component of IFT complex B, and show that IFT172 interacts with CrEB1. Because fla11ts cells are defective in IFT particle turnaround at the tip, our results indicate that IFT172 is involved in regulating the transition between anterograde and retrograde IFT at the tip, perhaps by a mechanism involving CrEB1. Therefore, IFT172 is involved in the control of flagellar assembly/disassembly at the tip.     

Electron-tomographic analysis of intraflagellar transport particle trains in situ

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.     

Dissecting the molecular mechanisms of intraflagellar transport in chlamydomonas

BACKGROUND: The assembly and maintenance of eukaryotic cilia and flagella are mediated by intraflagellar transport (IFT), a bidirectional microtubule (MT)-based transport system. The IFT system consists of anterograde (kinesin-2) and retrograde (cDynein1b) motor complexes and IFT particles comprising two complexes, A and B. In the current model for IFT, kinesin-2 carries cDynein1b, IFT particles, and axonemal precursors from the flagellar base to the tip, and cDynein1b transports kinesin-2, IFT particles, and axonemal turnover products from the tip back to the base. Most of the components of the IFT system have been identified and characterized, but the mechanisms by which these different components are coordinated and regulated at the flagellar base and tip are unclear. RESULTS: Using a variety of Chlamydomonas mutants, we confirm that cDynein1b requires kinesin-2 for transport toward the tip and show that during retrograde IFT, kinesin-2 can exit the flagella independent of the cDynein1b light intermediate chain (LIC) and IFT particles. Furthermore, using biochemical approaches, we find that IFT complex B can associate with cDynein1b independent of complex A and cDynein1b LIC. Finally, using electron microscopy, we show that the IFT tip turnaround point most likely is localized distal to the plus end of the outer-doublet B MTs. CONCLUSION: Our results support a model for IFT in which tip turnaround involves (1) dissociation of IFT complexes A and B and release of inactive cDynein1b from complex B, (2) binding of complex A to active cDynein1b, and (3) reassociation of complex B with A prior to retrograde IFT.     

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.     

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.     

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella

Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.     

Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits

Required for the assembly and maintenance of eukaryotic cilia and flagella, intraflagellar transport (IFT) consists of the bidirectional movement of large protein particles between the base and the distal tip of the organelle. Anterograde movement of particles away from the cell body is mediated by kinesin-2, whereas retrograde movement away from the flagellar tip is powered by cytoplasmic dynein 1b/2. IFT particles contain multiple copies of two distinct protein complexes, A and B, which contain at least 6 and 11 protein subunits, respectively. In this study, we have used increased ionic strength to remove four peripheral subunits from the IFT complex B of Chlamydomonas reinhardtii, revealing a 500-kDa core that contains IFT88, IFT81, IFT74/72, IFT52, IFT46, and IFT27. This result demonstrates that the complex B subunits, IFT172, IFT80, IFT57, and IFT20 are not required for the core subunits to stay associated. Chemical cross-linking of the complex B core resulted in multiple IFT81-74/72 products. Yeast-based two-hybrid and three-hybrid analyses were then used to show that IFT81 and IFT74/72 directly interact to form a higher order oligomer consistent with a tetrameric complex. Similar analysis of the vertebrate IFT81 and IFT74/72 homologues revealed that this interaction has been evolutionarily conserved. We hypothesize that these proteins form a tetrameric complex, (IFT81)2(IFT74/72)2, which serves as a scaffold for the formation of the intact IFT complex B.     

The intraflagellar transport machinery of Chlamydomonas reinhardtii

First discovered in the green alga, Chlamydomonas , intraflagellar transport (IFT) is the bidirectional movement of protein particles along the length of eukaryotic cilia and flagella. Composed of ∼16 different proteins, IFT particles are moved out to the distal tip of the organelle by kinesin-II and are brought back to the cell body by cytoplasmic dynein 1b. Mutant analysis of the IFT motor and particle proteins using diverse organisms has revealed a conserved and essential role for IFT in the assembly and maintenance of cilia and flagella. IFT is thought to mediate this assembly through the delivery of axonemal precursors out to the distal tip of the growing organelle. Consistent with this model, the IFT particle proteins are rich in protein–protein binding motifs, suggesting that the particles may act as scaffolds for the binding of multiple cargoes. With most of the IFT proteins now identified at the level of the gene, this review will briefly examine both the structure and function of the IFT machinery of Chlamydomonas reinhardtii .     

Protein transport in growing and steady‐state cilia

Cilia and eukaryotic flagella are threadlike cell extensions with motile and sensory functions. Their assembly requires intraflagellar transport (IFT), a bidirectional motor‐driven transport of protein carriers along the axonemal microtubules. IFT moves ample amounts of structural proteins including tubulin into growing cilia likely explaining its critical role for assembly. IFT continues in non‐growing cilia contributing to a variety of processes ranging from axonemal maintenance and the export of non‐ciliary proteins to cell locomotion and ciliary signaling. Here, we discuss recent data on cues regulating the type, amount and timing of cargo transported by IFT. A regulation of IFT‐cargo interactions is critical to establish, maintain and adjust ciliary length, protein composition and function.     

Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella

Intraflagellar transport (IFT), which is the bidirectional movement of particles within flagella, is required for flagellar assembly. IFT particles are composed of approximately 16 proteins, which are organized into complexes A and B. We have cloned Chlamydomonas reinhardtii and mouse IFT46, and show that IFT46 is a highly conserved complex B protein in both organisms. A C. reinhardtii insertional mutant null for IFT46 has short, paralyzed flagella lacking dynein arms and with central pair defects. The mutant has greatly reduced levels of most complex B proteins, indicating that IFT46 is necessary for complex B stability. A partial suppressor mutation restores flagellar length to the ift46 mutant. IFT46 is still absent, but levels of the other IFT particle proteins are largely restored, indicating that complex B is stabilized in the suppressed strain. Axonemal ultrastructure is restored, except that the outer arms are still missing, although outer arm subunits are present in the cytoplasm. Thus, IFT46 is specifically required for transporting outer arms into the flagellum.     

The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

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.     

Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly.     

Regulation of flagellar length in Chlamydomonas

Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length.     

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.     

Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles.

Intraflagellar transport (IFT) is a motility in which particles composed of at least 17 polypeptides move underneath the flagellar membrane. Anterograde (outward) and retrograde (inward) movements of these IFT particles are mediated by FLA10 kinesin-II and cytoplasmic dynein DHC1b, respectively. Mutations affecting IFT particle polypeptides or motors result in the inability to assemble flagella. IFT particles and the motors moving them are located principally around the basal bodies as well as in the flagella. Here, we clone the cDNA encoding one of the IFT particle proteins, IFT52, and show by immunofluorescence that while some IFT52 is in the flagella, the majority is found in two horseshoe-shaped rings around the basal bodies. Immunoelectron microscopy indicates that IFT52 is associated with the periphery of the transitional fibers, which extend from the distal portion of the basal body to the cell membrane and demarcate the entrance to the flagellar compartment. This localization suggests that the transitional fibers form a docking complex for the IFT particles destined for the flagellum. Finally, the flagellaless mutant bld1 completely lacks IFT52 due to a deletion in the gene encoding IFT52.     

Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control

BACKGROUND: Intraflagellar transport (IFT) is a motility process operating between the ciliary/flagellar (interchangeable terms) membrane and the microtubular axoneme of motile and sensory cilia. Multipolypeptide 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. RESULTS: 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 that 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. CONCLUSION: 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.     

Chlamydomonas Kinesin-II–dependent Intraflagellar Transport (IFT): IFT Particles Contain Proteins Required for Ciliary Assembly in Caenorhabditis elegans Sensory Neurons

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979–992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.