Dynein Regulatory Complex Research Articles

A molecular genetic analysis of the interaction between the cytoplasmic dynein intermediate chain and the glued (dynactin) complex.

The microtubule motor cytoplasmic dynein performs multiple cellular functions; however, the regulation and targeting of the motor to different cargoes is not well understood. A biochemical interaction between the dynein intermediate chain subunit and the p150-Glued component of the dynein regulatory complex, dynactin, has supported the hypothesis that the intermediate chain is a key modulator of dynein attachment to cellular cargoes. In this report, we identify multiple intermediate chain polypeptides that cosediment with the 19S dynein complex and two differentially expressed transcripts derived from the single cytoplasmic dynein intermediate chain (Cdic) gene that differ in the 3' untranslated region sequence. These results support previous observations of multiple Cdic gene products that may contribute to the specialization of dynein function. Most significantly, we provide genetic evidence that the interaction between the dynein intermediate chain and p150-Glued is functionally relevant. We use a genomic Cdic transgene to show that extra copies of the dynein intermediate chain gene act to suppress the rough eye phenotype of the mutant Glued(1), a mutation in the p150-Glued subunit of dynactin. Furthermore, we show that the interaction between the dynein intermediate chain and p150-Glued is dependent on the dosage of the Cdic gene. This result suggests that the dynein intermediate chain may be a limiting component in the assembly of the dynein complex and that the regulation of the interaction between the dynein intermediate chain and dynactin is critical for dynein function.

Protein phosphatases PP1 and PP2A are located in distinct positions in the Chlamydomonas flagellar axoneme.

We postulated that microcystin-sensitive protein phosphatases are integral components of the Chlamydomonas flagellar axoneme, positioned to regulate inner arm dynein activity. To test this, we took a direct biochemical approach. Microcystin-Sepharose affinity purification revealed a prominent 35-kDa axonemal protein, predicted to be the catalytic subunit of type-1 protein phosphatase (PP1c). We cloned the Chlamydomonas PP1c and produced specific polyclonal peptide antibodies. Based on western blot analysis, the 35-kDa PP1c is anchored in the axoneme. Moreover, analysis of flagella and axonemes from mutant strains revealed that PP1c is primarily, but not exclusively, anchored in the central pair apparatus, associated with the C1 microtubule. Thus, PP1 is part of the central pair mechanism that controls flagellar motility. Two additional axonemal proteins of 62 and 37 kDa were also isolated using microcystin-Sepharose affinity. Based on direct peptide sequence and western blots, these proteins are the A- and C-subunits of type 2A protein phosphatase (PP2A). The axonemal PP2A is not one of the previously identified components of the central pair apparatus, outer arm dynein, inner arm dynein, dynein regulatory complex or the radial spokes. We postulate PP2A is anchored on the doublet microtubules, possibly in position to directly control inner arm dynein activity.

Rotation of the central pair microtubules in eukaryotic flagella.

SUMMARY It has been 20 years since the phenomenon of centralpair rotation in eukaryotic flagella was reported(Omoto, and Kung, 1979). At that time, a model wasproposed in which the central pair functions as adistributor to regulate dynein activity among the outerdoublet microtubules. The video evidence obtainedsince then and gathered together in this essay is con-sistent with this model. Geometric arguments indicatethat there must be circumferential and longitudinalregulation of shear forces to produce effective bendingmotion of an axoneme. Central pair microtubules areideally situated to perform this regulatory function.The regulation of outer doublet sliding by the centralpair, together with the rotation of the latter, where thisoccurs, may explain the wide diversity of two- andthree-dimensional flagellar and ciliary waveforms thatis found in organisms using the same basic 9 1 2structure. ACKNOWLEDGMENTS We thank Mike McLaughlin of Material Media Services, Washing-ton State University for converting 16-mm film to video format, andDenise A. Palmen (Technical Services, Washington State University)for editing the videos, assembling the QuickTime movies, and pro-ducing the jpeg images to use as figures. The research based onvideo images shown in this essay was first published by Kamiya(1982), Omoto and Witman (1981), Gibbons

Mechanisms of ciliary motility: an update

Summary Ciliary motility is dependent upon dynein-driven sliding of microtubules. Attention has been focussed on the molecular biology and mechanochemistry of dynein, via techniques involving mutant analysis and in vitro motility measurements. In Chlamydomonas, Tetrahymena and Paramecium, the best studied protistan genera, the outer dynein arm is a compacted three-headed heterotrimeric bouquet. Every bouquet is identical, while each ATPase head of the bouquet is coded by a different dynein heavy (H) chain gene. One H chain (Paramecium α) binds a 29 kDa regulatory light chain whose phosphorylation increases the velocity of sliding of the dynein, which in turn increases ciliary beat frequency. At least seven different H chains within a 96 nm spoke group repeat form the inner dynein arms. The structural organization of the inner arms is complex, and several models have been proposed. The inner dynein arms evidently control bend formation. Their activity also changes upon phosphorylation or dephosphorylation, the signal for which is transduced via the spoke-central sheath complex and a dynein-regulatory complex that lies adjacent to the arms on the doublet. How inner and outer arms operate coordinately is not understood. The outer dynein arms have a strong attachment and force-producing phase that is very short (1%) compared to their cycle time. Only a few dynein steps probably occur per doublet during an effective stroke, which suggests that arm activity on an active doublet is stochastic. The orientation of the central pair within the axoneme determines the plane of bending, probably by dividing the doublets into active and inactive half axonemes, according to a switch point model. Computer reconstructions and analytical techniques such as finite element analysis are useful tools of exploring this model. Although many aspects of the mechanism of bend production and propagation still remain either undefined or controversial, a general outline is beginning to emerge.

The light chain p28 associates with a subset of inner dynein arm heavy chains in Chlamydomonas axonemes.

We show here that I2 and I3 inner dynein arm heavy chains of Chlamydomonas axonemes are resolved into two classes: one class associated with the protein p28 and the other associated with the protein caltractin/centrin. We have determined the nucleotide sequence of the gene encoding p28, a light chain that, together with actin and caltractin/centrin, is associated with inner dynein arms I2 and I3 of Chlamydomonas axonemes. p28 is a novel protein with affinity for a subset of the inner dynein arm heavy chains, but with no apparent significant homologies to tubulin- or actin-binding proteins. An antiserum specific for p28 showed that p28 is present along the entire axoneme. The same antiserum coimmunoprecipitated p28, actin, and dynein heavy chains 2' and 2. In contrast, an anti-caltractin/centrin antiserum coimmunoprecipitated caltractin/centrin, actin, and the heavy chains 2, 3, and 3'. It is likely that the dynein heavy chain 2 associated with p28, referred to as 2A, is a different polypeptide from dynein heavy chain 2 bound to caltractin/centrin, referred to as 2B. The complex formed by heavy chain 2B, actin, and caltractin/centrin is preferentially extracted by exposure to Nonidet P-40 and is missing in mutants lacking components 1 and 2 of the dynein regulatory complex.

Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase.

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild-type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke-deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP-mediated mechanism.

Components of a "dynein regulatory complex" are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella.

Previous studies of flagellar mutants have identified six axonemal polypeptides as components of a "dynein regulatory complex" (DRC). The DRC is though to coordinate the activity of the multiple flagellar dyneins, but its location within the axoneme has been unknown (Huang et al., 1982; Piperno et al., 1992). We have used improved chromatographic procedures (Kagami and Kamiya, 1992) and computer averaging of EM images (Mastronarde et al., 1992) to analyze the relationship between the DRC and the dynein arms. Our results suggest that some of the DRC components are located at the base of the second radial spoke in close association with the inner dynein arms. (a) Averages of axoneme cross-sections indicate that inner arm structures are significantly reduced in three DRC mutants (pf3 < pf2 < sup-pf-3 < wt). (b) These defects are more pronounced in distal/medial regions of the axoneme than in proximal regions. (c) Analysis of flagellar extracts by fast protein liquid chromatography and SDS-PAGE indicates that a specific dynein I2 isoform is missing in pf3 and reduced in pf2 and sup-pf-3. Comparison with ida4 and pf3ida4 extracts reveals that this isoform differs from those missing in ida4. (d) When viewed in longitudinal section, all 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.

The bop2-1 mutation reveals radial asymmetry in the inner dynein arm region of Chlamydomonas reinhardtii.

Strains of Chlamydomonas reinhardtii with a mutant allele at the BOP2 locus swim slowly and have an abnormal flagellar waveform similar to previously identified strains with defects in the inner arm region. Double mutant strains with the bop2-1 allele and any of 17 different mutations that affect the dynein arm region swim more slowly than either parent, which suggests that the bop2-1 mutation does not affect solely the outer dynein arms, the I1 or ida4 inner dynein arms, or the dynein regulatory complex. Flagellar axonemes isolated from bop2-1 cells are missing a phosphorylated polypeptide of 152 kD. Electron microscopic analysis shows that bop2-1 axonemes are missing density in the inner dynein arm region. Surprisingly, two populations of images were observed in longitudinal sections of axonemes from the bop2-1 strain. In the 10 longitudinal axonemes examined, a portion of the dynein regulatory complex and a newly identified structure, the projection, are affected. In five of these 10 longitudinal axonemes examined, two lobes of the ida4 inner arm are also missing. By examining the cross-sectional images of wild-type and bop2-1 axonemes at each outer doublet position around the axoneme, we have determined that the bop2-1 mutation affects the assembly of inner arm region components in a doublet specific manner. Doublets 5, 6, and 8 have the most severe deficiency, doublet 9 has an intermediate phenotype, and doublets 2, 3, 4, and 7 have the least severe phenotype. The bop2-1 mutation provides the first evidence of radial asymmetry in the inner dynein arm region.

Mutations in the "dynein regulatory complex" alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes.

To understand mechanisms of regulation of dynein activity along and around the axoneme we further characterized the "dynein regulatory complex" (drc). The lack of some axonemal proteins, which together are referred to as drc, causes the suppression of flagellar paralysis of radial spoke and central pair mutants. The drc is also an adapter involved in the ATP-insensitive binding of I2 and I3 inner dynein arms to doublet microtubules. Evidence supporting these conclusions was obtained through analyses of five drc mutants: pf2, pf3, suppf3, suppf4, and suppf5. Axonemes from drc mutants lack part of I2 and I3 inner dynein arms as well as subsets of seven drc components (apparent molecular weight from 29,000 to 192,000). In the absence of ATP-Mg, dynein-depleted axonemes from the same mutants bind I2 and I3 inner arms at both ATP-sensitive and -insensitive sites. At ATP-insensitive sites, they bind I2 and I3 inner arms to an extent that depends on the drc defect. This evidence suggested to us that the drc forms one binding site for the I2 and I3 inner arms on the A part of doublet microtubules.

The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella.

We provide indirect evidence that six axonemal proteins here referred to as "dynein regulatory complex" (drc) are located in close proximity with the inner dynein arms I2 and I3. Subsets of drc subunits are missing from five second-site suppressors, pf2, pf3, suppf3, suppf4, and suppf5, that restore flagellar motility but not radial spoke structure of radial spoke mutants. The absence of drc components is correlated with a deficiency of all four heavy chains of inner arms I2 and I3 from axonemes of suppressors pf2, pf3, suppf3, and suppf5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, pf3, and suppf5. Recombinant strains carrying one of the mutations pf2, pf3, or suppf5 and the inner arm mutation ida4 are more defective for I2 inner arm heavy chains than the parent strains. This evidence indicates that at least one subunit of the drc affects the assembly of and interacts with the inner arms I2.