A novel dynein light intermediate chain colocalizes with the retrograde motor for intraflagellar transport at sites of axoneme assembly in chlamydomonas and Mammalian cells

The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms.     

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.     

The DHC1b (DHC2) Isoform of Cytoplasmic Dynein Is Required for Flagellar Assembly

Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993–1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is 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.     

Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo.     

Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans.

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.     

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.     

IFT54 directly interacts with kinesin‐II and IFT dynein to regulate anterograde intraflagellar transport

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^Δ342–356 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.     

Dyneins across eukaryotes: a comparative genomic analysis

Dyneins are large minus-end-directed microtubule motors. Each dynein contains at least one dynein heavy chain (DHC) and a variable number of intermediate chains (IC), light intermediate chains (LIC) and light chains (LC). Here, we used genome sequence data from 24 diverse eukaryotes to assess the distribution of DHCs, ICs, LICs and LCs across Eukaryota. Phylogenetic inference identified nine DHC families (two cytoplasmic and seven axonemal) and six IC families (one cytoplasmic). We confirm that dyneins have been lost from higher plants and show that this is most likely because of a single loss of cytoplasmic dynein 1 from the ancestor of Rhodophyta and Viridiplantae, followed by lineage-specific losses of other families. Independent losses in Entamoeba mean that at least three extant eukaryotic lineages are entirely devoid of dyneins. Cytoplasmic dynein 2 is associated with intraflagellar transport (IFT), but in two chromalveolate organisms, we find an IFT footprint without the retrograde motor. The distribution of one family of outer-arm dyneins accounts for 2-headed or 3-headed outer-arm ultrastructures observed in different organisms. One diatom species builds motile axonemes without any inner-arm dyneins (IAD), and the unexpected conservation of IAD I1 in non-flagellate algae and LC8 (DYNLL1/2) in all lineages reveals a surprising fluidity to dynein function.     

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.     

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.     

A role for Tctex-1 (DYNLT1) in controlling primary cilium length

The microtubule motor complex cytoplasmic dynein is known to be involved in multiple processes including endomembrane organization and trafficking, mitosis, and microtubule organization. The majority of studies of cytoplasmic dynein have focused on the form of the motor that is built around the dynein-1 heavy chain. A second isoform, dynein heavy chain-2, and its specifically associated light intermediate chain, LIC3 (D2LIC), are known to be involved in the formation and function of primary cilia. We have used RNAi in human epithelial cells to define the cytoplasmic dynein subunits that function with dynein heavy chain 2 in primary cilia. We identify the dynein light chain Tctex-1 as a key modulator of cilia length control; depletion of Tctex-1 results in longer cilia as defined by both acetylated tubulin labeling of the axoneme and Rab8a labeling of the cilia membrane. Suppression of dynein heavy chain-2 causes concomitant loss of Tctex-1 and this correlates with an increase in cilia length. Compared to individual depletions, double siRNA depletion of DHC2 and Tctex-1 causes an even greater increase in cilia length. Our data show that Tctex-1 is a key regulator of cilia length and most likely functions as part of dynein-2.     

An InCytes from MBC Selection: Specificity of Cytoplasmic Dynein Subunits in Discrete Membrane-trafficking Steps

The cytoplasmic dynein motor complex is known to exist in multiple forms, but few specific functions have been assigned to individual subunits. A key limitation in the analysis of dynein in intact mammalian cells has been the reliance on gross perturbation of dynein function, e.g., inhibitory antibodies, depolymerization of the entire microtubule network, or the use of expression of dominant negative proteins that inhibit dynein indirectly. Here, we have used RNAi and automated image analysis to define roles for dynein subunits in distinct membrane-trafficking processes. Depletion of a specific subset of dynein subunits, notably LIC1 (DYNC1LI1) but not LIC2 (DYNC1LI2), recapitulates a direct block of ER export, revealing that dynein is required to maintain the steady-state composition of the Golgi, through ongoing ER-to-Golgi transport. Suppression of LIC2 but not of LIC1 results in a defect in recycling endosome distribution and cytokinesis. Biochemical analyses also define the role of each subunit in stabilization of the dynein complex; notably, suppression of DHC1 or IC2 results in concomitant loss of Tctex1. Our data demonstrate that LIC1 and LIC2 define distinct dynein complexes that function at the Golgi versus recycling endosomes, respectively, suggesting that functional populations of dynein mediate discrete intracellular trafficking pathways.     

An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9

Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.     

Evidence for cooperative interactions between the two motor domains of cytoplasmic dynein.

Cytoplasmic dynein is a force-transducing ATPase that powers the movement of cellular cargoes along microtubules. Two identical heavy chain polypeptides (> 500 kDa) of the cytoplasmic dynein complex contain motor domains that possess the ATPase and microtubule-binding activities required for force production [1]. It is of great interest to determine whether both heavy chains (DHCs) in the dynein complex are required for progression of the mechanochemical cycle and motility, as observed for other dimeric motors. We have used transgenic constructs to investigate cooperative interactions between the two motor domains of the Drosophila cytoplasmic dynein complex. We show that 138 kDa and 180 kDa amino-terminal fragments of DHC can assemble with full-length DHC to form heterodimeric complexes containing only a single motor domain. The single-headed dynein complexes can bind and hydrolyze ATP, yet do not show the ATP-induced detachment from microtubules that is characteristic of wild-type homodimeric dynein. These results suggest that cooperative interactions between the monomeric units of the dimer are required for efficient ATP-induced detachment of dynein and unidirectional movement along the microtubule.     

Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations

Cytoplasmic dynein, a minus-end-directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of a Caenorhabditis elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of Saccharomyces cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems.     

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.     

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.