A Novel Dynein Light Intermediate Chain Colocalizes with the Retrograde Motor for Intraflagellar Transport at Sites of Axoneme Assembly inChlamydomonasand Mammalian Cells

Abstract

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...

Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor

“…LC8 has been assumed to be part of the retrograde IFT motor complex because the absence of LC8 in Chlamydomonas results in defects in this process (Pazour et al, 1998); however, it was not observed to copurify with the DHC1b-D1bLIC complex (Perrone et al, 2003). We found that LC8 co-purified with FAP133 in sucrose gradients (Fig.…”

“…basal-body localization of DHC1b (Hou et al, 2004;Perrone et al, 2003). To determine whether the protein levels and subcellular localization of FAP133 are affected by the absence of specific subunits of the retrograde IFT dynein motor, we employed three Chlamydomonas null mutant strains; dhc1b, d1blic and fla14, which lack the DHC1b, D1bLIC and LC8 genes, respectively (Hou et al, 2004;Pazour et al, 1999;Pazour et al, 1998).…”

“…In C. elegans, this dynein is required for retrograde transport in chemosensory cilia (Signor et al, 1999), and the mammalian ortholog is enriched in ciliated epithelia and localizes at the connecting cilia in the retinal epithelium and in primary cilia of cultured cells (Mikami et al, 2002), indicating a conserved function in ciliary assembly. A LIC (D1bLIC) was identified first in mammals and subsequently in Chlamydomonas and C. elegans, as an integral component of the retrograde IFT dynein complex (Grissom et al, 2002;Hou et al, 2004;Perrone et al, 2003;Schafer et al, 2003). In Chlamydomonas a null mutation in D1bLIC also results in stumpy flagella with large accumulations of IFT particles (Hou et al, 2004).…”

Assembly of primary cilia

“…The DYNC2LI1 subunit was first identified in mammals and, subsequently, in Chlamydomonas and C. elegans (Grissom et al, 2002;Hou et al, 2004;Perrone et al, 2003;. Chlamydomonas mutants that are null in the gene encoding the DYNC2LI1 homolog (D1bLIC) display a phenotype similar to that of DHC1b null mutants in that the mutant cells assemble short flagella that are filled with IFT particles (Hou et al, 2004).…”

Biochemical Mapping of Interactions within the Intraflagellar Transport (IFT) B Core Complex

“…This process was first identified microscopically in the flagella of the single-celled green alga Chlamydomonas reinhardtii (8), and it was later shown by electron microscopy that "trains" of IFT particles move between the ciliary membrane and the underlying axonemal outer doublet microtubules (9). The driving force for this movement is provided by motor proteins that were identified as heterotrimeric kinesin II for anterograde transport (9 -11) and cytoplasmic dynein 1b for retrograde transport (12)(13)(14)(15). The IFT particles contain arrays of the so-called IFT complex that consists of two distinct subcomplexes, IFT-A and IFT-B, containing at least 6 and 14 subunits, respectively (10, 16 -22).…”