The nexin–dynein regulatory complex (N-DRC) is implicated in the control of dynein activity as a structural component of the nexin link. This study identifies several new subunits of the N-DRC and demonstrates for the first time that it forms a discrete biochemical complex that maintains outer doublet integrity and regulates microtubule sliding.

Abstract. Previous studies of flagellar mutants have identified six axonemal polypeptides as components of a "dynein regulatory complex" (DRC). The DRC is thought to coordinate the activity of the multiple flagellar dyneins, but its location within the axoneme has been unknown Piperno et al., 1992). We have used improved chromatographic procedures (Kagami and Kamiya, 1992) and computer averaging of EM images (Mastronarde et al., 1992) three DRC mutants lack a crescent-shaped density above the second radial spoke, and pf3 axonemes lack additional structures adjacent to the crescent. We propose that the crescent corresponds in part to the location of the DRC, and that this structure is also directly associated with a subset of the inner dynein arms. This position is appropriate for a complex that is thought to mediate signals between the radial spokes and the dynein arms.

Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation of IC138 phosphorylation controls flagellar bending, we cloned the IC138 gene. IC138 encodes a novel protein with a calculated mass of 111 kDa and is predicted to form seven WD-repeats at the C terminus. IC138 maps near the BOP5 locus, and bop5-1 contains a point mutation resulting in a truncated IC138 lacking the C terminus, including the seventh WD-repeat. bop5-1 cells display wild-type flagellar beat frequency but swim slower than wild-type cells, suggesting that bop5-1 is altered in its ability to control flagellar waveform. Swimming speed is rescued in bop5-1 transformants containing the wild-type IC138, confirming that BOP5 encodes IC138. With the exception of the roadblock-related light chain, LC7b, all the other known components of the I1 complex, including the truncated IC138, are assembled in bop5-1 axonemes. Thus, the bop5-1 motility phenotype reveals a role for IC138 and LC7b in the control of flagellar bending. IC138 is hyperphosphorylated in paralyzed flagellar mutants lacking radial spoke and central pair components, further indicating a role for the radial spokes and central pair apparatus in control of IC138 phosphorylation and regulation of flagellar waveform. INTRODUCTIONOur goal is to determine the mechanisms that regulate ciliary and eukaryotic flagellar bending. Based on informative mutations in Chlamydomonas, and effective in vitro functional studies, a surprisingly complex array of different dynein motors is required for generation and control of normal ciliary and flagellar bending (Mitchell, 1994;Gibbons, 1995;Porter, 1996;Porter and Sale, 2000;DiBella and King, 2001;Kamiya, 2002). For example, the outer arm dyneins are homogeneous structures responsible for control of beat frequency and power required for movement (Satir et al., 1993;Brokaw, 1994;DiBella and King, 2001;Kamiya, 2002). The inner arm dyneins, however, are more complex, composed of at least seven different dynein subspecies precisely organized in a 96-nm repeat pattern along each doublet microtubule of the axoneme (Porter, 1996;Porter and Sale, 2000). Diverse data indicate the inner arm dyneins control the size and shape of the flagellar bend (Brokaw and Kamiya, 1987;Brokaw, 1994;Kamiya, 2002). The mechanism for control of flagellar waveform involves additional structures (e.g., radial spokes, central pair apparatus, and the dynein regulatory complex) and control of dynein phosphorylation (Porter and Sale, 2000;DiBella and King, 2001;Kamiya, 2002;Smith and Yang, 2004).The present study is focused on a single inner arm dynein, the I1 complex, also called the f-dynein (Goodenough et al., 1987;Piperno et al., 1990;Kagami and Kamiya, 1992;Porter et al., 1992). The I1 complex is a tripartite structure, or triad, located near the base of the S1 radial spoke, at the proximal end of the axonemal 96-nm repeat (Goodenough and Heuser, 1985;Piperno et al., 1990;Mastronar...

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°C, but cannot maintain them at 33°C. Although the Kinesin-2 complex is present at both 21 and 33°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. INTRODUCTIONCilia and flagella perform essential motile and sensory functions for a variety of eukaryotic organisms. In vertebrates, defects in ciliary and flagellar motility (primary ciliary dyskinesia or PCD) result in randomization of left/right body asymmetry during embryonic development, chronic respiratory disease, and male sterility (Ibañ ez-Tallon et al., 2003). Defects in the assembly of primary cilia or cilia-associated proteins have also been linked to polycystic kidney disease (PKD), retinal degeneration, hearing loss, and human obesity disorders . Studies in Chlamydomonas, Caenorhabditis elegans, and mice have demonstrated that the machinery required for ciliary assembly is highly conserved (Rosenbaum and Witman, 2002). In most cases, the assembly and maintenance of these organelles depends on a bidirectional, intraflagellar transport (IFT) system of large protein particles driven by two distinct microtubule motors, a heterotrimeric Kinesin-2 and a novel cytoplasmic dynein (cDhc1b or Dhc2; Cole, 2003). Formerly known as kinesin-II (Lawrence et al., 2004), Kinesin-2 is responsible for transport from the basal body region to the tip of the axoneme (anterograde IFT; Kozminski et al., 1995;Piperno and Mead, 1997;Cole et al., 1998), whereas the cDhc1b/LIC complex is required for transport from the flagellar tip to the basal body region (retrograde IFT; Pazour et al., 1999;Porter et al., 1999;Signor et al., 1999a;Wicks et al., 2000;Perrone et al., 2003;Schafer et al., 2003).The Kinesin-2 complex consists of two distinct kinesinrelated motor subunits and a third kinesin-associated protein (KAP). This complex was first described in sea urchin eggs and subsequently identified in the mouse, Chlamydomonas, C. elegans, Dros...

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. INTRODUCTIONCilia and flagella are microtubule-based organelles that play critical roles in the fertility and viability of eucaryotic organisms. Defects in components required for flagellar assembly or motility can have profound consequences. In vertebrates, these include birth defects, the development of left-right asymmetries, respiratory distress, polycystic kidney disease, and male sterility (Nonaka et al., 1998;Marszalek et al., 1999;Afzelius, 1999;Murcia et al., 2000;Pazour et al., 2000;Yoder et al., 2002). The polypeptide complexity of the organelles has made it challenging to analyze these structures in vertebrate tissues, but studies in model organisms such as Chlamydomonas and Caenorhabditis elegans have provided significant insights into the identity of components required for flagellar assembly and motility. Indeed, recent work has shown that both the assembly and maintenance of cilia and flagella are dependent on a bidirectional, intraflagellar transport system (IFT) (reviewed in Rosenbaum et al., 1999;Sloboda, 2002). Anterograde transport (from the cell body to the tip of the flagellum) requires a heterotrimeric kinesin (Kozminski et al., 1995;Piperno and Mead, 1997;Cole et al., 1998), whereas retrograde transport (from the flagellar tip to the cell body) depends on an unusual class of cytoplasmic dynein known as cDHC1b (Pazour et al., 1999;Porter et al., 1999;Signor et al., 1999a;Wicks et al., 2000). The heterotrimeric kinesins have been characterized in several organisms (Cole et al., 1993(Cole...