Basal body-associated DNA: in situ studies in Chlamydomonas reinhardtii.

We have explored the localization of the uni chromosome (LG XIX) of Chlamydomonas reinhardtii using the technique of in situ hybridization. Using standardized methods of cell fixation together with large chromosome-specific probes we have studied the position of uni DNA sequences in metaphase and interphase cells. We find that in dividing cells uni probes identify a condensed metaphase chromosome that shows no specialized orientation. In interphase cells uni hybridization signals occur on the anterior edge of the nucleus at a position where basal bodies are normally associated with the nuclear envelope. These data reveal an underlying spatial organization of uni chromosomal DNA within the interphase nucleus that may be significant in terms of the fact that this chromosome encodes numerous functions affecting basal body and flagellar assembly.

Basal body/centriolar DNA: molecular genetic studies in Chlamydomonas.

In Chlamydomonas reinhardtii, mutations on an unusual linkage group, the uni linkage group (ULG), affect structure and function of basal bodies. The ULG shows Mendelian segregation, but its genetic map is circular. Molecular cloning of fragments of the ULG was accomplished by taking advantage of restriction fragment length polymorphisms generated by crosses to Chlamydomonas smithii. These clones were used as probes to determine the size and form of the ULG chromosome; it is a 6-9 megabase linear molecule. Use of the probes for in situ DNA hybridization in cells localized the ULG chromosome to basal bodies.

Loci affecting flagellar assembly and function map to an unusual linkage group in Chlamydomonas reinhardtii.

The uni1 mutant of Chlamydomonas reinhardtii lacks one of the paired flagella seen in wild-type cells. The missing flagellum is cis to the eye spot and at this site the basal body is incomplete, lacking a transition zone. Together with nine other loci affecting flagellar assembly and two loci affecting flagellar function uni1 defines an approximately equal to 100-centimorgan linkage group with a circular map. Measurements of gene-centromere distances on the uni linkage group are consistent with the ordering of loci determined by recombination analysis and place the centromere near pf29, a motility-defective mutant. In well-synchronized meiotic cycles, recombination frequencies between loci on the uni linkage group but not loci on other linkage groups show striking temperature sensitivity during a 1- to 2-hr period 5 days before meiosis.

Phosphorylation in isolated Chlamydomonas axonemes: a phosphoprotein may mediate the Ca2+-dependent photophobic response.

An in vitro system was devised for studying phosphorylation of Chlamydomonas reinhardtii axonemal proteins. Many of the polypeptides phosphorylated in this system could be identified as previously described axonemal components that are phosphorylated in vivo. The in vitro system apparently preserved the activities of diverse axonemal kinases without greatly altering the substrate specificity of the enzymes. The in vitro system was used to study the effect of calcium concentration on axonemal protein phosphorylation. Calcium has previously been demonstrated to initiate the axonemal reversal reaction of the photophobic response; the in vitro system made it possible to investigate the possibility that this calcium effect is mediated by protein phosphorylation. Calcium specifically altered the phosphorylation of only two axonemal proteins; the phosphorylation of an otherwise unidentified 85,000 Mr protein was repressed by calcium concentrations greater than or equal to 10(-6) M, while the phosphorylation of the previously identified 95,000 Mr protein b4 was stimulated by calcium at concentrations greater than 10(-6) M. Protein b4 is one of six polypeptides that are deficient in the mbo mutants, strains that do not exhibit a photophobic reversal reaction. Therefore, this calcium-stimulated phosphorylation may be involved in initiating the photophobic response. Neither calmodulin nor the C-kinase could be implicated in b4 phosphorylation. The calcium-dependent activation of the b4 kinase was not affected by several drugs that bind to and inhibit calmodulin, or by the addition of exogenous calmodulin. Activators and inhibitors of the calcium-phospholipid-dependent C kinase also had no effect on b4 phosphorylation.

Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant.

Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.

Mutant strains of Chlamydomonas reinhardtii that move backwards only.

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 of the 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 mbo1 , 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 32P-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 mbo1 flagella and the mbo1 , 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.

Genetic and biochemical dissection of the eucaryotic flagellum.

The axoneme is the basic functional unit of the eucaryotic flagellum. Periodic structures appended to its 9+2 microtubule core are responsible for generation of flagellar bending. An account of biochemical and genetic studies of flagellar-defective mutants of Chlamydomonas reinhardtii is presented. These studies provide insights into the complex molecular composition of the appended structures, their mode of assembly, and the way in which they interact to modulate flagellar function.

Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.

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 pf16 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.

Bending patterns of chlamydomonas flagella I. Wild-type bending patterns.

Using a uniflagellate mutant of Chlamydomonas and flash photomicrography at 300 Hz, we have obtained detailed information on the forward and reverse beating modes of Chlamydomonas flagella and on the relationship between rotation of the uniflagellate cell and the bending cycle of the forward mode. Flagella ranging in length from 5 to 15.5 micron were photographed. There is a decrease in wavelength and an increase in curvature in the principal bends when the length of the flagellum is less than the normal length of 12-13 micron, but these changes are not sufficient to maintain similarity of the bending pattern. In the reverse mode, the flagellum propagates symmetrical, planar, undulatory waves with a shear amplitude which is the same as in the forward mode; there is a 19% increase in beat frequency and a similar decrease in wave length. The reorientation of the flagellar beat direction towards the axis of the cell in the reverse mode is caused both by the decrease in asymmetry of beat and by activation of sliding in the principal bends at an earlier time in the beat cycle, relative to the time of activation of sliding in reverse bends. There are additional rare modes of beating which may be related to intermediate stages in the transition between forward and reverse beating modes.

Uniflagellar mutants of Chlamydomonas: evidence for the role of basal bodies in transmission of positional information.

A series of uniflagellar mutants isolated following mutagenesis of Chlamydomonas reinhardtii (strain 137c) with ICR-191 show a remarkable positional phenotype. The flagellum that fails to develop is cis to the eyespot in more than 95% of the cells examined. Both the positional and the uniflagellar phenotypes are transmitted stably through mitotic and meiotic divisions, and in backcrosses the meiotic segregation is two mutant to two wild-type progeny. Four of the mutants, uni1, uni2, uni3 and uni4, have been studied extensively. They appear to be alleles of a single gene locus or to be closely linked (less than or equal to 0.06 map units). The characteristic expression of the uniflagellar defect in cells under different growth conditions or in stable diploids indicates that the mutations alter the rate of development of the flagellum in the cis-eyespot flagellum. Electron microscopic studies suggest that the developmental defect resides in the basal body. Extensive recombination analysis to 33 nuclear markers representing the 16 linkage groups failed to establish linkage. The uni mutants, however, showed linkage to four unmapped mutant loci. Mutations for each of these loci also affect flagellar assembly.

Temperature-Sensitive, Assembly-Defective Flagella Mutants of CHLAMYDOMONAS REINHARDTII.

We describe an efficient selection procedure for the isolation of mutants of Chlamydomonas reinhardtii with temperature sensitive flagella defects, with final yields of up to 11% of the population being mutant. Several mutants, all showing an inability to maintain flagellar integrity at the restrictive temperature, are described. We have examined flagellar stability and reassembly at various temperatures in the mutants. Mapping data are provided for these, as well as for some previously described mutants.

Analysis of the movement of Chlamydomonas flagella:" the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella.

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 suppf-3, show the typical asymmetric bending pattern associated with forward swimming in Chlamydomonas, although suppf-1 flagella have about one-half the normal beta 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-2 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 suppfpf-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.

Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for Flagellar function.

Reversion analysis of flagellar-motility mutants of Chlamydomonas reinhardtii yields an unusual class of intergenic suppressor mutations that restore flagellar activity to paralyzed radial-spoke or central-pair mutants without altering the structural or molecular defects associated with the original mutations. Four suppressors representing independent genetic loci were studied in detail. Two of the mutations, suppf1 and suppf2, restore flagellar motility to either radial-spoke or central-pair mutants of different genes. The mutants suppf3 and suppf 4 suppress flagellar paralysis associated only with mutants defective for the radial spokes. Analyses of the axonemal polypeptides of suppf1, suppf3 and suppf4 mutants indicate that the mutations restore flagellar activity to paralyzed radial-spoke or central-pair mutants by altering other components of the flagellar axoneme. suppf1 shows an altered electrophoretic migration for a 325,000 molecular weight polypeptide known to be a subunit of an outer-arm dynein. suppf3 and suppf4 are missing different axonemal polypeptides with molecular weights of 60,000 (in the case of suppf3), and 40,000 and 29,000 (in the case of suppf4). Genetic evidence has been obtained indicating that the polypeptides affected in suppf3 and suppf4 are components of a newly identified functional and/or structural compartment of the flagellar axoneme. The suppressor mutations described here reveal the operation of a control mechanism that inhibits the operations of flagellar movements in the presence of radial-spoke or central-pair defects. Suppressor mutations release the inhibition. The molecular defects of suppf1, suppf3 and suppf4 provide evidence that the inhibitory mechanism can be interrupted at two different levels of axonemal function.

Inner arm dyneins from flagella of Chlamydomonas reinhardtii.

Two dyneins have been isolated from axonemes of Chlamydomonas flagella by a three step procedure consisting of extraction in a high salt containing buffer, hydroxyapatite chromatography and sedimentation in sucrose gradient. A dynein with Mg+2- dependent ATPase activity 6.0 mumole Pi/min/mg, sedimenting at 12.5S was found associated with a polypeptide of molecular weight 310,000. A second dynein with specific activity of 3.7, sedimenting at 10-11S was found associated with a polypeptide of molecular weight 315,000. In their most purified forms, the two dyneins are complexed with nonstoichiometric amounts of four polypeptides ranging in molecular weight between 42,000 and 19,000. The 42,000 component has been identified previously as an actin-like protein. The high molecular weight subunits of both dyneins and two polypeptides of 28,000 and 19,000 molecular weight were found to be phosphorylated by in vivo pulse-labeling with 32P-phosphoric acid. All components of the 12.5S and 10-11S dynein complexes, with the exception of the 19,000 polypeptide, form a subset of polypeptides found to be deficient in pf-23, a chlamydomonas mutant, which is defective for inner arms.

Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants.

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 35S-labeled axonemal 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 32P-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-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta 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.

Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components.

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S-containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 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 greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.

Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function.

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.