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.     

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.     

Subunit interactions and organization of the Chlamydomonas reinhardtii intraflagellar transport complex A proteins

Chlamydomonas reinhardtii intraflagellar transport (IFT) particles can be biochemically resolved into two smaller assemblies, complexes A and B, that contain up to six and 15 protein subunits, respectively. We provide here the proteomic and immunological analyses that verify the identity of all six Chlamydomonas A proteins. Using sucrose density gradient centrifugation and antibody pulldowns, we show that all six A subunits are associated in a 16 S complex in both the cell bodies and flagella. A significant fraction of the cell body IFT43, however, exhibits a much slower sedimentation of ～2 S and is not associated with the IFT A complex. To identify interactions between the six A proteins, we combined exhaustive yeast-based two-hybrid analysis, heterologous recombinant protein expression in Escherichia coli, and analysis of the newly identified complex A mutants, ift121 and ift122. We show that IFT121 and IFT43 interact directly and provide evidence for additional interactions between IFT121 and IFT139, IFT121 and IFT122, IFT140 and IFT122, and IFT140 and IFT144. The mutant analysis further allows us to propose that a subset of complex A proteins, IFT144/140/122, can form a stable 12 S subcomplex that we refer to as the IFT A core. Based on these results, we propose a model for the spatial arrangement of the six IFT A components.     

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.     

Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.     

The FLA3 KAP subunit is required for localization of kinesin-2 to the site of flagellar assembly and processive anterograde intraflagellar transport

Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21 degrees C, but cannot maintain them at 33 degrees C. Although the Kinesin-2 complex is present at both 21 and 33 degrees C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.     

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.     

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.     

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.     

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.     

High-resolution crystal structure and in vivo function of a kinesin-2 homologue in Giardia intestinalis

A critical component of flagellar assembly, the kinesin-2 heterotrimeric complex powers the anterograde movement of proteinaceous rafts along the outer doublet of axonemes in intraflagellar transport (IFT). We present the first high-resolution structures of a kinesin-2 motor domain and an ATP hydrolysis-deficient motor domain mutant from the parasitic protist Giardia intestinalis. The high-resolution crystal structures of G. intestinalis wild-type kinesin-2 (GiKIN2a) motor domain, with its docked neck linker and the hydrolysis-deficient mutant GiKIN2aT104N were solved in a complex with ADP and Mg(2+) at 1.6 and 1.8 A resolutions, respectively. These high-resolution structures provide unique insight into the nucleotide coordination within the active site. G. intestinalis has eight flagella, and we demonstrate that both kinesin-2 homologues and IFT proteins localize to both cytoplasmic and membrane-bound regions of axonemes, with foci at cell body exit points and the distal flagellar tips. We demonstrate that the T104N mutation causes GiKIN2a to act as a rigor mutant in vitro. Overexpression of GiKIN2aT104N results in significant inhibition of flagellar assembly in the caudal, ventral, and posterolateral flagellar pairs. Thus we confirm the conserved evolutionary structure and functional role of kinesin-2 as the anterograde IFT motor in G. intestinalis.     

Direct Interactions of Intraflagellar Transport Complex B Proteins IFT88, IFT52, and IFT46

Intraflagellar transport (IFT) particles of Chlamydomonas reinhardtii contain two distinct protein complexes, A and B, composed of at least 6 and 15 protein subunits, respectively. As isolated from C. reinhardtii flagella, IFT complex B can be further reduced to a ∼500-kDa core that contains IFT88, 2× IFT81, 2× IFT74/72, IFT52, IFT46, IFT27, IFT25, and IFT22. In this study, yeast-based two-hybrid analysis was combined with bacterial coexpression to show that three of the core B subunits, IFT88, IFT52, and IFT46, interact directly with each other and, together, are capable of forming a ternary complex. Chemical cross-linking results support the IFT52-IFT88 interaction and provide additional evidence of an association between IFT27 and IFT81. With previous studies showing that IFT81 and IFT74/72 interact to form a (IFT81)₂(IFT74/72)₂ heterotetramer and that IFT27 and IFT25 form a heterodimer, the architecture of complex B is revealing itself. Last, electroporation of recombinant IFT46 was used to rescue flagellar assembly of a newly identified ift46 mutant and to monitor in vivo localization and movement of the IFT particles.     

The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein

Many genes on the uni linkage group of Chlamydomonas affect the basal body/flagellar apparatus. Among these are five FLA genes, whose mutations cause temperature-sensitive defects in flagellar assembly. We present the molecular analysis of a gene which maps to fla10 and functionally rescues the fla10 phenotype. Nucleotide sequencing revealed that the gene encodes a kinesin-homologous protein, KHP1. The 87-kD predicted KHP1 protein, like kinesin heavy chain, has an amino- terminal motor domain, a central alpha-helical stalk, and a basic, globular carboxy-terminal tail. Comparison to other kinesin superfamily members indicated striking similarity (64% identity in motor domains) to a mouse gene, KIF3, expressed primarily in cerebellum. In synchronized cultures, the KHP1 mRNA accumulated after cell division, as did flagellar dynein mRNAs. KHP1 mRNA levels also increased following deflagellation. Polyclonal antibodies detected KHP1 protein in Western blots of purified flagella and axonemes. The protein was partially released from axonemes with ATP treatment, but not with AMP- PNP. Western blot analysis of axonemes from various motility mutants suggested that KHP1 is not a component of radial spokes, dynein arms, or the central pair complex. The quantity of KHP1 protein in axonemes of the mutant fla10-1 was markedly reduced, although no reduction was observed in two other uni linkage group mutants, fla9 and fla11. Furthermore, fla10-1 was rescued by transformation with KHP1 genomic DNA. These results indicate that KHP1 is the gene product of FLA10 and suggest a novel role for this kinesin-related protein in flagellar assembly and maintenance.     

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.     

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.     

The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140^RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.     

<Emphasis Type="Italic">Chlamydomonas fla</Emphasis> mutants reveal a link between deflagellation and intraflagellar transport

Background

Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT). Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT.

Results

fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33°C), as previously reported. We find that in standard media containing ~300 microM calcium, fla10 cells lose flagella by deflagellation at 33°C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants.

Conclusions

Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly is initiated. This active disassembly is distinct from the constitutive disassembly which plays a role in flagellar length control.

     

Localization of EB1, IFT polypeptides, and kinesin-2 in Chlamydomonas flagellar axonemes via immunogold scanning electron microscopy

Intraflagellar transport (IFT) refers to the bi-directional movement of particles and associated cargo along the axonemes of eukaryotic flagella and cilia. To provide a new perspective on the morphology of IFT particles, their association with the axoneme, and their composition, we have used immunogold localization coupled to detection via scanning electron microscopy. Here we co-localize in the Chlamydomonas flagellar axoneme polypeptides labeled with specific antibodies. Chlamydomonas EB1 localizes to the distal tip of the axoneme, as expected from previous immunofluorescent data (Pedersen et al. Curr Biol2003;13(22):1969-1974), thus demonstrating the utility of this approach. Using antibodies to IFT-related polypeptides, particles can be identified associated with the axoneme that fall into one of two classes: The first class is composed of IFT particles labeled with polyclonal antibodies to kinesin-2 and monoclonal antibodies to either IFT139 (an IFT complex A polypeptide) or IFT172 (a complex B polypeptide). The second class is comprised of particles that label with antibodies to IFT139 alone; thus, discrete particles are present associated with the axoneme that are composed only of complex A polypeptides. When IFT particles were purified by sucrose gradient ultracentrifugation, they appeared as more or less spherical aggregates of varying dimensions labeled with antibodies to IFT139 and to the motor protein kinesin-2. By contrast, isolated IFT particles that were labeled with IFT172 antibodies were not labeled with kinesin-2 antibodies. The data are discussed in terms of the total polypeptide composition of an IFT particle and the interaction of the particles with the motors that power IFT.     

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.     

IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism thought to be required for the assembly and maintenance of all eukaryotic cilia and flagella. Although IFT proteins are present in cells with sensory cilia, the organization of IFT protein complexes in those cells has not been analyzed. To determine whether the IFT complex is conserved in the sensory cilia of photo-receptors, we investigated protein interactions among four mammalian IFT proteins: IFT88/Polaris, IFT57/Hippi, IFT52/NGD5, and IFT20. We demonstrate that IFT proteins extracted from bovine photoreceptor outer segments, a modified sensory cilium, co-fractionate at approximately 17 S, similar to IFT proteins extracted from mouse testis. Using antibodies to IFT88 and IFT57, we demonstrate that all four IFT proteins co-immunoprecipitate from lysates of mouse testis, kidney, and retina. We also extended our analysis to interactions outside of the IFT complex and demonstrate an ATP-regulated co-immunoprecipitation of heterotrimeric kinesin II with the IFT complex. The internal architecture of the IFT complex was investigated using the yeast two-hybrid system. IFT20 exhibited a strong interaction with IFT57/Hippi and the kinesin II subunit, KIF3B. Our data indicate that all four mammalian IFT proteins are part of a highly conserved complex in multiple ciliated cell types. Furthermore, IFT20 appears to bridge kinesin II with the IFT complex.