Chlamydomonas reinhardtii produces a profilin with unusual biochemical properties.

We report the characterization of a profilin orthologue from Chlamydomonas reinhardtii. CrPRF, probably the only profilin isoform, is present in both the cell body and flagella. Examination of vegetative and gametic cells by immunofluorescence microscopy using multiple fixation procedures also revealed enrichment of CrPRF at the anterior of the cell near the base of flagella and near the base of the fertilization tubule in mating type plus gametes. Purified, recombinant CrPRF binds to actin with a Kd value approximately 10(-7) and displaces nuclei in a live cell 'nuclear displacement' assay, consistent with profilin's ability to bind G-actin in vivo. However, when compared with other profilin isoforms, CrPRF has a relatively low affinity for poly-L-proline and for phosphatidylinositol (4,5) bisphosphate micelles. Furthermore, and surprisingly, CrPRF inhibits exchange of adenine nucleotide on G-actin in a manner similar to human ADF or DNase I. Thus, we postulate that a primary role for CrPRF is to sequester actin in Chlamydomonas. The unusual biochemical properties of CrPRF offer a new opportunity to distinguish specific functions for profilin isoforms.

Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes.

Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.

Flagellar radial spoke protein 3 is an A-kinase anchoring protein (AKAP).

Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144-180. Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

Peter Satir -- investigating the structural basis for cell function.

Peter Satir has devoted his research career to elucidating the structural basis for ciliary motility. His ingenious use of structural analysis, combined with identification of powerful model systems, provided a model for the sliding microtubule hypothesis of ciliary bending and led to the discovery that dynein is a 'minus-end'-directed motor whose regulated activity underpins the bending motion of cilia. Here, we focus on ciliary motility to illustrate Satir's pioneering contributions to cell biology.

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity.

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.

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.

Characterization of a Chlamydomonas insertional mutant that disrupts flagellar central pair microtubule-associated structures.

Two alleles at a new locus, central pair-associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke-defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

The Mr 140,000 intermediate chain of Chlamydomonas flagellar inner arm dynein is a WD-repeat protein implicated in dynein arm anchoring.

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on beta-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/beta-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.

The Chlamydomonas IDA7 locus encodes a 140-kDa dynein intermediate chain required to assemble the I1 inner arm complex.

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm that ida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that the ida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

Regulation of flagellar dynein by phosphorylation of a 138-kD inner arm dynein intermediate chain.

One of the challenges in understanding ciliary and flagellar motility is determining the mechanisms that locally regulate dynein-driven microtubule sliding. Our recent studies demonstrated that microtubule sliding, in Chlamydomonas flagella, is regulated by phosphorylation. However, the regulatory proteins remain unknown. Here we identify the 138-kD intermediate chain of inner arm dynein I1 as the critical phosphoprotein required for regulation of motility. This conclusion is founded on the results of three different experimental approaches. First, genetic analysis and functional assays revealed that regulation of microtubule sliding, by phosphorylation, requires inner arm dynein I1. Second, in vitro phosphorylation indicated the 138-kD intermediate chain of I1 is the only phosphorylated subunit. Third, in vitro reconstitution demonstrated that phosphorylation and dephosphorylation of the 138-kD intermediate chain inhibits and restores wild-type microtubule sliding, respectively. We conclude that change in phosphorylation of the 138-kD intermediate chain of I1 regulates dynein-driven microtubule sliding. Moreover, based on these and other data, we predict that regulation of I1 activity is involved in modulation of flagellar waveform.

Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas.

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATP gamma S or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein. We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from approximately 2 microns/second to approximately 7 microns/second when inner arm dynein from pf14pf28 axonemes ws reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATP gamma S, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.

Regulation of dynein-driven microtubule sliding by an axonemal kinase and phosphatase in Chlamydomonas flagella.

The following is a summary of physiological and pharmacological studies of the regulation of dynein-driven microtubule sliding in Chlamydomonas flagella. The experimental basis for the study is described, and data indicating that an axonemal cAMP-dependent protein kinase can regulate inner arm dynein activity are reviewed. In addition, preliminary data are summarized indicating that an axonemal type 1 phosphatase can also regulate dynein-drive microtubule sliding velocity. It is predicted that the protein kinase, phosphatase, and an inner dynein arm component form a regulatory complex in the axoneme.

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.

Kinesin-related proteins in eukaryotic flagella.

To identify kinesin-related proteins that are important for ciliary and eukaryotic flagellar functions, we used affinity-purified, polyclonal antibodies to synthetic peptides corresponding to conserved sequences in the motor domain of kinesin (Sawin et al. (1992) J. Cell Sci. 101, 303-313). Using immunoblot analysis, two antibodies to distinct sequences (LNLVDLAGSE, 'LAGSE' and, HIPYRESKLT, 'HIPYR') reveal a family of proteins in flagella and axonemes isolated from Chlamydomonas. Similar analysis of axonemes from mutant Chlamydomonas strains or fractionated axonemes indicates that none of the immunoreactive proteins are associated with dynein arm or spoke structures. In contrast, one protein, approximately 110 kDa, is reduced in axonemes from mutant strains defective in the central pair apparatus. Immunoreactive proteins with masses of 96 and 97 kDa (the '97 kDa' proteins) are selectively solubilized from isolated axonemes in 10 mM ATP. The 97 kDa proteins co-sediment in sucrose gradients at about 9 S and bind to axonemes or purified microtubules in a nucleotide-dependent fashion characteristic of kinesin. These results reveal that flagella contain kinesin-related proteins, which may be involved in axonemal central pair function and flagellar motility, or directed transport involved in morphogenesis or mating responses in Chlamydomonas.

The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules.

Glass-adsorbed intact sea urchin outer arm dynein and its beta/IC1 subunit supports movement of microtubules, yet does not form a rigor complex upon depletion of ATP (16). We show here that rigor is a feature of the isolated intact outer arm, and that this property subfractionates with its alpha heavy chain. Intact dynein mediates the formation of ATP-sensitive microtubule bundles, as does the purified alpha heavy chain, indicating that both particles are capable of binding to microtubules in an ATP-sensitive manner. In contrast, the beta/IC1 subunit does not bundle microtubules. Bundles formed with intact dynein are composed of ribbon-like sheets of parallel microtubules that are separated by 54 nm (center-to-center) and display the same longitudinal repeat (24 nm) and cross-sectional geometry of dynein arms as do outer doublets in situ. Bundles formed by the alpha heavy chain are composed of microtubules with a center-to-center spacing of 43 nm and display infrequent, fine crossbridges. In contrast to the bridges formed by the intact arm, the links formed by the alpha subunit are irregularly spaced, suggesting that binding of the alpha heavy chain to the microtubules is not cooperative. Cosedimentation studies showed that: (a) some of the intact dynein binds in an ATP-dependent manner and some binds in an ATP-independent manner; (b) the beta/IC1 subunit does not cosediment with microtubules under any conditions; and (c) the alpha heavy chain cosediments with microtubules in the absence or presence of MgATP2-. These results suggest that the structural binding observed in the intact arm also is a property of its alpha heavy chain. We conclude that whereas force-generation is a function of the beta/IC1 subunit, both structural and ATP-sensitive (rigor) binding of the arm to the microtubule are mediated by the alpha subunit.

Regulation of dynein-driven microtubule sliding by the radial spokes in flagella.

The regulation of microtubule sliding in flagellar axonemes was studied with the use of Chlamydomonas mutants and in vitro assays. Microtubule sliding velocities were diminished in axonemes from mutant cells missing radial spoke structures but could be restored upon reconstitution with dynein from axonemes with wild-type radial spokes. These experiments demonstrate that the radial spokes activate dynein's microtubule sliding activity.