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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,...

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 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...

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Abstract. A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella <6 #m long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions,causlng complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner ann row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.I NNER and outer dynein arms are motile structures that generate the sliding of adjacent outer doublet micro, tubules within the axoneme of cilia and flagella. Both inner and outer arms participate in complex regulatory mechanisms converting microtubule sliding into axonemal bending. However, they have different functions. The absence of one type of inner arm from axonemes of Chlamydomonas mutants alters mainly the waveforms of flagella whereas the absence of outer arms reduces the beat frequency (3, 13).Inner dynein arms differ from outer arms in their structure (5) and molecular composition (9, 16). At least three types of inner arm compose a unit which is repeated with a periodicity of 96 nm in a row along the axonemes of Chlamydomonas flagella (19). The inner arm referred to as I1 i~ proximal to radial spoke S1, whereas the inner arms 12 an~. 13 are distal to spokes S1 and $2, respectively. Each inner arm is composed of two heavy chains, each having a site for ATP hydrolysis as weU as a variety of intermediate and light chains including actin (17,18). In contrast, only one type of outer dynein arm repeating every 24 nm forms a row parallel to that of the inner arms,. Each outer arm is composed of three heavy chains, two intermediate and several light chains.Immotile and short flagella of several Chlamydomonas mutants appear to be deficient for two heavy chains each forming inner arms 12 and I3, respectively (19). This deficiency may result from the lack of posttranslational processing of inner arms 12 and I3 heavy chains or is a consequence of defective assembly or synthesis of one or more axonemal components.To test these hypotheses we have analyzed inner arm heavy chains from various mutants defective for either motility or length of their flagella. Results of our investigation show that flagella < 6 tzm long are defective for two inner arm heavy chains. Each of these heavy chains forms one type of arm that is located in the proximal portion of the axoneme. Materials and Methods Strains and Culture of Chlamydomonas CellsNomenclature and properties of the strains analyzed in this study are listed in Table I.Cells "~4 × 106 per 100-mm plates were cultured on solid medium at 25°C for 3 d under direct illumination from 34 watt fluorescent tubes that were positioned 3-5 in. from the culture dishes. Culture dishes w...

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility . Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern . The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants . This defect in motility was found to be a property ofthe mutant axonemes themselves : the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition . Axonemes obtained from mbol, mbo2, and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S-or 32 P-labeled axonemes . One of the six polypeptides has previously been identified ; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes . Detailed ultrastructural analysis of the mbol flagella and the mbol, mbo2A, and mbo3 axonemes revealed that the mutants specifically lack the beak-like projections found within the B-tubules of outer doublets 5 and 6.Eucaryotic flagella and cilia constitute a well-studied system of microtubule-based movement . Cilia and flagella have in common a regular ultrastructure composed of microtubules and several types of appended structures (reviewed in reference 1). Although they share a basic ultrastructure, the organelles produce a wide range of beat patterns. The beat patterns vary from a pure ciliary stroke, exemplified by the stroke of the lateral cilia ofthe clam gill (2, 3), to a completely flagellar stroke, which has been well-studied in the vertebrate and invertebrate sperm tail (4, 5). The alga Chlamydomonas reinhardtii has two flagella that display both the ciliary and the flagellar stroke (6). This organism can alternate between the two distinct beat patterns, and is therefore a highly suitable system for studying control ofthe wave form.When Chlamydomonas uses the ciliary-type stroke, the cells swim forwards, in the direction of the flagellated pole of the cell. In response to an increase in light intensity, the cells stop, then move backwards briefly . The cells use a flagellar-type 2026