Abstract. The subcellular distribution of microtubules containing acetylated a-tubulin in mammalian cells in culture was analyzed with 6-11B-l, a monoclonal antibody specific for acetylated a-tubulin. Cultures of 3T3, HeLa, and PtK2 cells were grown on coverslips and observed by immunofluorescence microscopy after double-staining by 6-11B-1 and B-5-1-2, a monoclonal antibody specific for all ¢t-tubulins. The antibody 6-11B-1 binds to primary cilia, centrioles, mitotic spindles, midbodies, and to subsets of cytoplasmic microtubules in 3T3 and HeLa cells, but not in PtK2 cells. These observations confirm that the acetylation of a-tubulin is a modification occurring in different microtubule structures and in a variety of eukaryotic cells. Some features of the acetylation of cytoplasmic microtubules of mammalian cells are also described here. First, acetylated a-tubulin is present in microtubules that, under depolymerizing conditions, are more stable than the majority of cytoplasmic microtubules. In addition to the specific microtubule frameworks already mentioned, cytoplasmic microtubules resistant to nocodazole or colchicine, but not cold-resistant microtubules, contain more acetylated a-tubulin than the rest of cellular microtubules. Second, the ¢t-tubulin in all cytoplasmic microtubules of 3T3 and HeLa cells becomes acetylated in the presence of taxol, a drug that stabilizes microtubules. Third, acetylation and deacetylation of cytoplasmic microtubules are reversible in cells released from exposure to 0°C or antimitotic drugs. Fourth, the epitope recognized by the antibody 6-11B-1 is not absolutely necessary for cell growth and division. This epitope is absent in PtK2 cells. The acetylation of et-tubulin could regulate the presence of microtubules in specific intracellular spaces by selective stabilization.

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of a-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic o~-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only a-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal a-tubulin isoform acetylated in vivo on the ~-amino group of lysine(s) (L'Hernault, S. W., and J. L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal atubulin, unassembled o~-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic a-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated ~-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of a-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated a-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.Microtubules are implicated in a variety of cellular functions including mitosis, cytokinesis, intracellular transport, the maintenance of cell shape, and the formation of motile systems such as eukaryotic cilia and flagella (5). In many cases, microtubules involved in different functions are organized into morphologically different arrays. Although the structure of a variety of microtubule frameworks has been described in great detail, the molecular mechanisms that specify the assembly of morphologically.and functionally different microtubule arrays have yet to be determined. The diversity among microtubular structures may be generated by the association of microtubule components with accessory proteins localized in different parts of the cell (37), by the co-polymerization of different a-and #-tubulin subunits, or by a c...

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.

Proteins necessary for maintenance and function of eukaryotic f lagella are synthesized in the cell body. Transport of the inner dynein arm subunit p28 IDA4 in Chlamydomonas f lagella requires the activity of the kinesin KHP1 FLA10 , a protein inactive at restrictive temperature in fla10, a temperature-dependent mutant of f lagellar assembly. To identify other molecules involved in active transport of inner dynein arms within f lagella we searched for polypeptides of the cytoplasmic matrix of f lagella that fulfill two conditions: they bind to p28 and require the activity of KHP1 to be present in f lagella. We found that the cytoplasmic matrix of f lagella contains a previously unidentified ''17S'' complex of at least 13 polypeptides that in part is associated with p28. The 17S complex is present at permissive but not at restrictive temperature in fla10 f lagella. It also turns over in the cytoplasmic matrix more frequently than dynein arms within the axoneme. This evidence suggests that the 17S complex transports precursors of inner dynein arms within f lagella.Intracellular transport of protein complexes, or membranebound vesicles, often requires the participation of microtubules and the activity of molecular motors, such as dyneins and kinesins (1). Molecular motors are recruited in specific cellular compartments and display their activity by moving a cargo along the microtubules. The questions of whether the motors themselves are transported or diffuse toward their final site of activity are relevant for understanding the dynamics of intracellular and microtubule-based transport. We addressed these questions by the analysis of Chlamydomonas flagella, a model system for microtubule-based movement (2, 3) that can be dissected by genetics (4, 5).Our previous work was focused on the inner dynein arms, a group of six dyneins (6) binding to doublet microtubules in the internal part of the axonemal shaft. The inner dynein arms, but not the outer dynein arms, are required for the formation of both ciliary and flagellar type of waveforms of flagella (7). They are located in asymmetric positions along (8) and around (9) the axoneme. Some of them bind to microtubules at different times during flagellar assembly (8).Knowledge of both subunit composition and axonemal location of inner dynein arms allowed us to address the question of whether the dyneins themselves are transported toward their final site of activity (10). To identify the mode of transport of a subset of inner dynein arms within flagella we recently analyzed the movement of p28 IDA4 (6), a light chain of two types of inner dynein arms. p28, but not the outer dynein arm subunit IC69 ODA6 (11), requires the activity of the kinesin homologous protein 1, KHP1 FLA10 (12), to reach the distal part of flagella (10).KHP1 carries an ATP binding site (12, 13) and presumably moves over outer doublet microtubules and underneath the membrane during the transport of a cargo within the cytoplasmic matrix of flagella (14). The cargo may consist of large protein co...

Abstract. The molecular composition and organization of the row of axonemal inner dynein arms were investigated by biochemical and electron microscopic analyses of Chlamydomonas wild-type and mutant axonemes. Three inner arm structures could be distinguished on the basis of their molecular composition and position in the axoneme as determined by analysis of pf30 and pf23 mutants. The three inner arm structures repeat every 96 nm and are referred to here as inner arms I1, 12, and 13. I1 is proximal to the radial spoke S1, whereas 12 and 13 are distal to spokes S1and $2, respectively. The mutant pf30 lacks I1 whereas the mutant pf23 lacks both I1 and 12 but has a normal inner arm 13. Each of the six heavy chains that was identified as an inner dynein arm subunit has a site for ATP binding and hydrolysis. Two of the heavy chains together with a polypeptide of 140,000 molecular weight form the inner arm I1 and were extracted from the axoneme as a complex that had a sedimentation coefficient close to 21S at high ionic strength. Different subsets of two of the remaining four heavy chains form the inner arms 12 and I3. These arms at high ionic strength are dissociated as l lS particles that include one heavy chain, one intermediate chain, two light chains, and actin. These and other lines of evidence indicate that the inner arm I1 is different in structure and function from the inner arms 12 and 13.T HE inner dynein arms, unlike the outer dynein arms, are both necessary and sufficient to generate ciliary and flagellar axonemal bending (4). However, the molecular organization and composition of the inner dynein arms are not as well understood as those of the outer row of arms (for review see references 8, 16, 17, and 25). Through biochemical and electron microscopic analyses of Chlamydomonas wild-type and mutant axonemes, here we have begun to determine the composition of each inner dynein arm. In addition, we demonstrate that the row of inner arms is formed by three distinct structures, each having a specific location relative to the radial spokes S1 and $2 (see references 7 and 8).Several lines of evidence have suggested that the row of inner dynein arms is formed by more than one type of structure. First, as many as five inner arm heavy chains were identified by combined biochemical and microscopic analysis of Chlamydomonas mutants lacking either outer or inner dynein arms (11). Therefore, the existence of multiple inner arms was implied, as all dynein arms characterized so far are formed by at most three heavy chain subunits (6, 28). Second, in contrast to mutations affecting outer arm assembly, which cause the loss of all outer arm heavy chains along with the outer arms (15), mutations known to affect the assembly of inner arms cause the lack of only a subset of inner arm heavy chains (4,20). Third, EM of quick-frozen and deepetched samples has revealed that there are two types of inner arm structures, referred to as dyads and triads organized in triad-dyad-dyad triplet groups which repeat with a 96-nm interval (7,...

A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

Abstract. 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 12 and 13. Subsets of drc subunits are missing from five second-site suppressors, pf2, p f3, suppI3, suD:4, and suppz 5, 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 13 from axonemes of suppressors pf2, pf3, suppi3, and supp:5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, p f3, and supp:5. Recombinant strains carrying one of the mutations p f2, p f3, or suppy5 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.

In addition to the previously studied pf-14 and pf-1 loci in Chlamydomonas reinhardtii, mutations for another five genes ( pf-17, pf-24, pf-25, pf-26, and pf-27) have been identified and characterized as specifically affecting the assembly and function of the flagellar radial spokes . Mutants for each of the newly identified loci show selective alterations for one or more of the 17 polypeptides in the molecular weight range of 20,000-130,000 which form the radial spoke structure. In specific instances the molecular defect has been correlated with altered radial spoke morphology . Biochemical analysis of in vivo complementation in mutant X wild-type dikaryons has provided indirect evidence that mutations for four of the five new loci ( pf-17, pf-24, pf-25, and pf-26) reside in structural genes for spoke components . In the case of pf-24, the identity of the mutant gene product was supported by analysis of induced intragenic revertants . In contrast to the other radial spoke mutants thus far investigated, evidence suggests that the gene product in pf-27 is extrinsic to the radial spokes and is required for the specific in vivo phosphorylation of spoke polypeptides .