No abstract

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex . Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes . This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule . The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pfl6 axonemes . No structural gene product has been identified for the PF6 locus . The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments . Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule .The central pair microtubules offlagella more closely resemble the cytoplasmc microtubules than the outer microtubules of the flagella by virtue of their structure and their lability . They are singlet microtubules with 13 protofilaments unlike the nearby outer doublet microtubules that are composed of acomposite set ofmicrotubules with 13 and 11 protofilaments (1). The outer doublet microtubules, in contrast to the central pair microtubules, arise as a continuation ofthe microtubules of the basal bodies. The central pair complex originates distal to the transition zone of the basal body and terminates in specialized structures associated with the flagellar membrane at its tip (2) . In Chlamydomonas reinhardtii the central pair microtubules are sensitive to depolymerization by low ionic strength dialysis and by exposure to detergents (3, 4), whereas the outer doublet microtubules retain their integrity under these conditions .The two microtubules of the central pair complex also differ from one another (5-7). In squid sperm, Linck and coworkers (8) have shown that the morphology of the Cl microtubule differs from that of the C2 microtubule . In Chlamydomonas the C 1 microtubule has two projections that are 18 nm long as measured by thin-section electron micros-THE JOURNAL OF CELL BIOLOGY " VOLUME 98 JANUARY 1984 229-236 0 The Rockefeller University Press -0021-9525/84/01/0229/08 $1 .00 copy. The C2 microtubule has two shorter projections of -8 nm each. Furthermore, the C2 microtubule is more labile than the C 1 microtubule (4) .We have analyzed mutations at two unlinked loci in Chlamydomonas that affect the C1 microtubule specifically with the aim of unders...

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 .

Polypeptides from flagella or axonemes of Chlamydomonas reinhardth were analyzed by labeling cellular proteins by prolonged growth on 3r S-containing media and using one-and two-dimensional electrophoretic techniques which can resolve >170 axonemal components . By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8-7 .1 . Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads . The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild-type axonemes . Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question . Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1 . No Ca"-or Mg"-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads . In vivo pulse 32p incorporation experiments provide evidence that >80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.Eukaryotic cilia and flagella are complex organelles that retain their intrinsic motility when separated from cells . In most cases, their core structure, the axoneme, consists of nine doublet microtubules forming a cylinder that surrounds a pair of central microtubules . Attached to these continuous structures are appendages that occur at precise longitudinal intervals. On the A subfiber of the outer doublet are attached inner and outer dynein arms, interdoublet links, and radial spokes ; the latter extend toward projections attached to the central pair (for review, see reference 1).For several years our laboratory has been engaged in genetical, morphological, and biochemical studies of flagellar motility mutants of Chlamydomonas reinhartdii as a means of dissecting the complex structure and function of the flagellar axoneme (2-7) . In this communication we report the analysis of the polypeptides that compose the radial spokes .Although radial spokes in different organisms differ somewhat in ultrastructure and in longitudinal periodicity, they are

The mutation uni-1 gives rise to uniflagellate Chlamydomonas cells which rotate around a fixed point in the microscope field, so that the flagellar bending pattern can be photographed easily . This has allowed us to make a detailed analysis of the wild-type flagellar bending pattern and the bending patterns of flagella on several mutant strains. Cells containing uni-1, and recombinants of uni-1 with the suppressor mutations, suppf-1 and sup,,-3, show the typical asymmetric bending pattern associated with forward swimming in Chlamydomonas, although suppf-1 flagella have about one-half the normal beat frequency, apparently as the result of defective function of the outer dynein arms . The pf-17 mutation has been shown to produce nonmotile flagella in which radial spoke heads and five characteristic axonemal polypeptides are missing. Recombinants containing pf-17 and either suppf -1 or suppf -3 have motile flagella, but still lack radial-spoke heads and the associated polypeptides . The flagellar bending pattern of these recombinants lacking radial-spoke heads is a nearly symmetric, large amplitude pattern which is quite unlike the wild-type pattern . However, the presence of an intact radial-spoke system is not required to convert active sliding into bending and is not required for bend initiation and bend propagation, since all of these processes are active in the suppf pf-17 recombinants . The function of the radial-spoke system appears to be to convert the symmetric bending pattern displayed by these recombinants into the asymmetric bending pattern required for efficient swimming, by inhibiting the development of reverse bends during the recovery phase of the bending cycle.Chlamydomonas has proven to be a valuable organism for genetic and biochemical investigations of flagellar structure .Flagellar function in Chlamydomonas has received more limited attention, in part because of the difficulty of observing or photographically recording the activity of flagella on swimmin g cells. We describe here an analysis of the movement of Chlamydomonas flagella, which has been facilitated by making use of a mutant uni-1 (B . Huang, Z . Ramanis, S . Dutcher, and D . J . L. Luck, manuscript in preparation) . In uni-1 a high proportion of cells are uniflagellate. When flagella beat in the asymmetric or "breast-stroke" mode which propels biflagellate cells forward, uni-1 cells rotate, and in most cases the cells show little precession. The flagellar beat is executed in a plane perpendicular to the axis of rotation, and the bending cycle of cells in which the bending plane is stabilized by proximity to the surface of a microscope slide can be recorded easily by stroboscopic dark-field microscopy. We have used this method to analyze the normal, wild-type, "breast-stroke" mode of flagellar beating, and one type of variant beating pattern associated with a flagellar motility mutation.Among the "paralyzed" flagellar mutants isolated fromChlamydomonas reinhardtii, which show little or no flagellar motility, are several muta...

(14). Flagella were removed from cells suspended at a concentration of approximately 2 X 107/ml in 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) (pH 7.2), 0.5 mM CaCl2, and 1 mM dithiothreitol by pH shock (3) or by addition of Nonidet P40 (14). Flagella or axonemes were isolated by differential centrifugation, solubilized in sodium dodecyl sulfate (NaDodSO4) and analyzed by two-dimensional gel electrophoresis. Isoelectric focusing was started within 3-5 hr of the time flagella were detached from cell bodies in order to limit artifactual modification of flagellar proteins. The method of analysis described by Ferro-Luzzi Ames and Nikaido (13) was modified as follows: to obtain a linear pH gradient in the range of 3.5-9.5, ampholines were mixed according to the LKB (Bromma, Sweden) application note on the model 2117 Multiphor; the amount of riboflavin added for polymerization of the slab gel used for the isoelectric focusing was 10% of the amount indicated; the polyacrylamide gel slab chosen for the second electrophoresis was prepared by a modification (14) of the method of Neville (16); and, protein samples of approximately 3 ,ug (1 X 105 cpm/,ug) were mixed with one-half volume of undiluted Nonidet P40 and 2 volumes of the sample buffer before being applied to the gels. The proteins used as standards were: spectrin heavy subunit, W-galactosidase, bovine-serum albumin, ovalbumin, chymotrypsinogen, and hemoglobin. Exposure time for autoradiograms was approximately 1 week. RESULTS Two-dimensional separation of flagellar polypeptidesTo detect flagellar components present in low quantities, we labeled cells to high specific activity by including [a5S]sulfate in the medium. We observed that cell growth was not altered by use of the isotope at a specific activity of 500 Ci/mol of sulfate. Consequently, our analyses will detect only sulfurcontaining proteins and the number of components observed will also be a function of the sensitivity of the film and the exposure time used for autoradiography.The two-dimensional method we used (13) included isoelectric focusing in one dimension and gradient gel electrophoresis with NaDodSO4 in the other. In analyzing the maps we obtained, including the gel strip from the first dimension after electrophoresis in the second dimension, we recognized that there were other limits to the types of polypeptides we could resolve. While the majority of components fell into a molecular weight range of 300,000-15,000 with isoelectric points between 7 and 4 and were well resolved (Fig. 1A), molecules with isoelectric points between 8.5 and 7 formed long streaks. We also observed that polypeptides wth molecular weight higher than 300,000 did not migrate to their isoelectric points; when loaded at the anode for the first dimension instead 1600 Abbreviation: NaDodSO4, sodium dodecyl sulfate. * We did the mapping studies after finding that two of these cultures maintained in our collection were incorrectly identified.

The motility mutant of Chlamydomonas reinhardtii pfl4 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its 35S-labeled flagellar proteins, Six of these same proteins are missing in fi, which lacks spokeheads. To determine whether any of the missing roteins represent the mutant gene product two experimenta approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with 35S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pfl4 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pfl only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pfl gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pfI4, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.Previously we have analyzed flagellar proteins in paralyzed mutants of Chlamydomonas reinhardtii using a two-dimensional system of isoelectric focusing followed by electrophoresis in the presence of sodium dodecyl sulfate (1). In the case of the mutant pfl4, a two-dimensional map of flagellar proteins was shown to lack 12 characteristic polypeptides that were regularly present in wild-type flagella. This deficiency could be correlated with the total absence of radial spokes and associated spokeheads in the axonemal structures of pfl4. Another mutant, pfl was shown to lack 6 of these 12 flagellar polypeptides, and analysis of axonemal structures revealed that the radial spokes were present but that spokeheads were absent.Since pfl4 and pfl have been shown to be independent single-site mutations (2), the deficiency of all spoke structures in one case and of spokeheads in the other indicates that the mutant gene products play critical roles in the assembly of as many as 12 different proteins in the case of pfl4 and 6 in the case of pfl. Two complementary approaches have been undertaken to determine if the mutant gene products of pfl4 and/or pfl are structural components of the radial spoke.The first type of experiments makes use of quadriflagellate dikaryon cells which are produced after gametic fusion in the Chlamydomonas mating cycle. It has been observed that in dikaryons of some paralyzed mutants of C. moewusii (3) or C. reinhardtni (4) with the counterpart wild-type stra...

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally ( pf-18, pf-19, and pf-15) or nearly totally ( pf-20) the central microtubular pair complex in isolated axonemal preparations . Analysis of "S-labeled axonernal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes . Analyses of axonemal proteins labeled by cellular growth on 32 P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated .Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-a or anti-/3 tubulin sera prepared from Chlamydomonas tubulins . The immunoprecipitates identify two major a tubulins, a major # tubulin, and a minor component which is also precipitated by the anti-f3 serum . None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella .The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules . Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.The analysis of axonemal polypeptides in mutants of Chlamydomonas reinhardtii with defects in flagellar structures has proved to be a valuable approach to the study of assembly and function of eukaryotic flagella. Using electrophoretic methods which identify nearly two hundred axonemal polypeptides, wild-type axonemes have been compared with those from mutants lacking radial spokes (1, 2, 3) and inner or outer dynein arms (4) . In each case, the comparison revealed that the mutant axonemes were deficient in a set of polypeptides, each set being unique for the missing structure . The missing polypeptides identify possible structural components of the radial spokes or dynein arms; they also serve as a signature by THE JOURNAL OF CELL BIOLOGY " VOLUME 91 OCTOBER 1981 69-76 © The Rockefeller University Press -0021-9525/81/10/0069/08 $1 .00 which additional mutants affecting these structures can be identified or by which polypeptides present in the course of enzyme purification can be identified with specific axonemal structures . The usefulness of such signatures is exemplified by the identification of several mutants affecting radial spokes (2, 3) and by progress in purification of dyneins (4,5) .This paper is concerned with the flagellar and axonemal polypeptides of a series of mutants defective in the central microtubules and their appended struct...