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.     

Two attentional deficits in serial target search: the visual attentional blink and an amodal task-switch deficit

Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an Mr 8,000 light chain termed LC8. The function of this chain is unknown, but it is highly conserved between a wide variety of organisms. We have identified deletion alleles of the gene (fla14) encoding this protein in Chlamydomonas reinhardtii. These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules. Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT) (Kozminski, K.G., P.L. Beech, and J.L. Rosenbaum. 1995. J. Cell Biol. 131:1517-1527). IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules. Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown. Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum. This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process. Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes (Cole, D.G., D.R. Deiner, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008), confirming that these complexes are subunits of the rafts. Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT.     

Inner and outer arm axonemal dyneins from the Antarctic rockcod Notothenia coriiceps

Adaptive compensation of enzymatic activities is common among cold-living poikilotherms. Their enzymes often demonstrate higher activities at low temperatures than do homologs from temperate or thermophilic species. To understand the molecular features necessary for cold adaptation of microtubule motor proteins, we have initiated studies of the flagellar dynein ATPases of Antarctic fishes (body temperature range = -1.8 to +2 degrees C). Dyneins were isolated by high-salt extraction of demembranated sperm axonemes from the Antarctic yellowbelly rockcod, Notothenia coriiceps. Although solubilization of inner arms was incomplete, an inner arm dynein was recognized as a discrete complex containing one major dynein heavy chain (DHC) and sedimenting through sucrose gradients at approximately 12 S. Like inner arm dyneins from Chlamydomonas, the fish complex contained an actin-immunoreactive protein of 43 kDa and a 30-kDa protein. One isoform of the inner arm DHC gene family of N. coriiceps was detected by the polymerase chain reaction, and Southern analysis established that this DHC gene is present at one copy per haploid genome. Outer arm dynein was extracted quantitatively by high-salt treatment, contained two DHCs (one major, one minor), and sedimented through sucrose gradients as a polydisperse, aggregating system. Associated with the outer arm DHCs were five presumptive intermediate chains (ICs) of 66-91 kDa, immunologically defined by their cross-reactivity to four monoclonal antibodies specific for ICs from other organisms. The basal (non-microtubule-stimulated) specific ATPase activities of the N. coriiceps inner and outer arm dyneins were approximately 0.07 and approximately 0.04 micromol of P(i) min(-1) mg(-1), respectively, at 0 degrees C, attained their maxima (approximately 0.1 micromol of P(i) min(-1) mg(-1)) at 9 and 19 degrees C, respectively, and at higher temperatures declined substantially. Furthermore, the activities of the fish dyneins at temperatures < or = 15 degrees C were significantly larger than that of outer arm dynein from the mesophile Tetrahymena. These results suggest that the greater catalytic efficiencies of N. coriiceps inner and outer arm dyneins at low temperatures are due to enhanced polypeptide flexibility in the active sites of their protein subunits. We conclude that temperature adaptation of flagellar dyneins from Antarctic fishes is compatible with substantial conservation of primary and quaternary structure.     

Transport of a novel complex in the cytoplasmic matrix of Chlamydomonas flagella

Proteins necessary for maintenance and function of eukaryotic flagella are synthesized in the cell body. Transport of the inner dynein arm subunit p28(IDA4) in Chlamydomonas flagella requires the activity of the kinesin KHP1(FLA10), a protein inactive at restrictive temperature in fla10, a temperature-dependent mutant of flagellar assembly. To identify other molecules involved in active transport of inner dynein arms within flagella we searched for polypeptides of the cytoplasmic matrix of flagella that fulfill two conditions: they bind to p28 and require the activity of KHP1 to be present in flagella. We found that the cytoplasmic matrix of flagella 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 flagella. 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 flagella.     

Is outer arm dynein intermediate chain 1 multifunctional?

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.     

Dynein arms are oscillating force generators

Eukaryotic flagella beat rhythmically. Dynein is a protein that powers flagellar motion, and oscillation may be inherent to this protein. Here we determine whether oscillation is a property of dynein arms themselves or whether oscillation requires an intact axoneme, which is the central core of the flagellum and consists of a regular array of microtubules. Using optical trapping nanometry, we measured the force generated by a few dynein arms on an isolated doublet microtubule. When the dynein arms on the doublet microtubule contact a singlet microtubule and are activated by photolysis of caged ATP8, they generate a peak force of approximately 6pN and move the singlet microtubule over the doublet microtubule in a processive manner. The force and displacement oscillate with a peak-to-peak force and amplitude of approximately 2 pN and approximately 30 nm, respectively. The geometry of the interaction indicates that very few (possibly one) dynein arms are needed to generate the oscillation. The maximum frequency of the oscillation at 0.75 mM ATP is approximately 70 Hz; this frequency decreases as the ATP concentration decreases. A similar oscillatory force is also generated by inner dynein arms alone on doublet microtubules that are depleted of outer dynein arms. The oscillation of the dynein arm may be a basic mechanism underlying flagellar beating.     

莱茵衣藻鞭毛内运输蛋白IFT81调控鞭毛组装

利用插入突变的方式获得了一株衣藻不运动突变体ift81,该突变体表现出鞭毛缺失或者短鞭毛的性状.基因序列分析表明,外源基因aphⅧ插入了突变体中IFT81(intraflagellar transport,IFT)基因的第五个外显子内,并导致该外显子原有的52个碱基对被替换.把含有完整IFT81基因的重组质粒导入突变体ift81后,其鞭毛恢复为野生型且可以检测到IFT81-HA融合蛋白的表达,这证明突变体的鞭毛缺陷确实是由于IFT81基因突变所导致.电镜观察显示突变体中鞭毛的显微结构发生改变,免疫荧光实验证实IFT81蛋白主要定位于基体和鞭毛部位.上述结果表明:IFT81蛋白缺失会导致衣藻鞭毛组装缺陷,在鞭毛组装所需蛋白的运输过程中,IFT81蛋白是必不可少的.      

Purealin blocks the sliding movement of sea urchin flagellar axonemes by selective inhibition of half the ATPase activity of axonemal dyneins

Ciliary and flagellar movements are explained by active sliding between the outer doublet microtubules of an axoneme via their inner and outer dynein arms. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, blocked the motility of Triton-demembranated sea urchin sperm flagella within 5 min at concentrations above 20 microM. In a similar concentration range, purealin blocked the sliding movement of the flagellar axonemes in vitro within a few minutes judging from the turbidity measurements. The ATPase activity of axonemes was partially inhibited by purealin in a concentration-dependent manner. The maximum inhibition reached approximately 50% at concentrations above 20 microM, indicating that half the axonemal ATPase activity is sensitive to purealin. Similar results were observed on the ATPase activity of outer-arm-depleted axonemes and that of a mixture of 21S dynein and salt-extracted axonemes. On the other hand, ATPase activity of isolated 21S dynein was not inhibited by purealin. The inhibitory action of purealin on the axonemal ATPases was reversed by dilution of purealin. The effect of purealin on the double-reciprocal plot of the ATPase activity as a function of ATP concentrations showed that the inhibition was not a competitive type. In accord with this finding, purealin did not affect the vanadate-mediated UV photocleavage of axonemal dyneins. These results suggest that purealin binds reversibly to a site other than the catalytic ATP-binding site and inhibits half the ATPase activity of axonemes. Taken together, our results suggest that purealin-sensitive ATPase activity of the dynein arms plays an essential role in generating the sliding movement of flagellar axonemes.     

Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery

The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete. The complex PF are composed of multiple class A and class B polypeptides. Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro. To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1. To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed. The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively. The inactivated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis. Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant. These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins. The resulting flaA1 flaB1 mutant exhibited altered motility in vitro. Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S. hyodysenteriae in the gut. Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism. The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract. This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus.     

Genetic interactions among cytoplasmic dynein, dynactin, and nuclear distribution mutants of Neurospora crassa

Cytoplasmic dynein is a multisubunit, microtubule-associated, mechanochemical enzyme that has been identified as a retrograde transporter of various membranous organelles. Dynactin, an additional multisubunit complex, is required for efficient dynein-mediated transport of vesicles in vitro. Recently, we showed that three genes defined by a group of phenotypically identical mutants of the filamentous fungus Neurospora crassa encode proteins that are apparent subunits of either cytoplasmic dynein or dynactin. These mutants, designated ropy (ro), display abnormal hyphal growth and are defective in nuclear distribution. We propose that mutations in other genes encoding dynein/dynactin subunits are likely to result in a ropy phenotype and have devised a genetic screen for the isolation of additional ro mutants. Cytoplasmic dynein/dynactin is the largest and most complex of the cytoplasmic motor proteins, and the genetic system described here is unique in its potentiality for identifying mutations in undefined genes encoding dynein/dynactin subunits or regulators. We used this screen to isolate > 1000 ro mutants, which were found to define 23 complementation groups. Unexpectedly, interallelic complementation was observed with some allele pairs of ro-1 and ro-3, which are predicted to encode the largest subunits of cytoplasmic dynein and dynactin, respectively. The results suggest that the Ro1 and Ro3 polypeptides may consist of multiple, functionally independent domains. In addition, approximately 10% of all newly isolated ro mutantsdisplay unlinked noncomplementation with two or more of the mutants that define the 23 complementation groups. The frequent appearance of ro mutants showing noncomplementation with multiple ro mutants having unlinked mutations suggests that nuclear distribution in filamentous fungi is a process that is easily disrupted by affecting either dosage or activity of cytoplasmic dynein, dynactin, and perhaps other cytoskeletal proteins or regulators.     

Distinct mutants of retrograde intraflagellar transport (IFT) share similar morphological and molecular defects

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.     

Human CLAPS2 encoding AP17, a small chain of the clathrin-associated protein complex: cDNA cloning and chromosomal assignment to 19q13.2-->q13.3

We have cloned the cDNA for the human homolog of the rat AP17 gene, a small chain of the clathrin-associated protein complex AP-2. The cDNA is highly conserved between rat and human. Human AP17, gene symbol CLAPS2 (clathrin-associated/assembly/adaptor protein, small 3, 17 kDa), was assigned to chromosome region 19q13.2-->q13.3.     

Mutation in the Arabidopsis PASTICCINO1 gene, which encodes a new FK506-binding protein-like protein, has a dramatic effect on plant development

The pasticcino (pas) mutants of Arabidopsis thaliana are a new class of plant developmental mutants; members of this class show ectopic cell proliferation in cotyledons, extra layers of cells in the hypocotyl, and an abnormal apical meristem. This phenotype is correlated with both cell division and cell elongation defects. There are three complementation groups of pas mutants (pas1, pas2, and pas3, with, respectively 2, 1, and 4 alleles). Here we describe in more detail the pas1-1 allele, which was obtained by insertional mutagenesis. The PAS1 gene has been cloned and characterized; it encodes an immunophilin-like protein similar to the p59 FK506-binding protein (FKBP52). PAS1 is characterized by an FKBP-like domain and three tetratricopeptide repeat units. Although the presence of immunophilins in plants has already been demonstrated, the pas1-1 mutant represents the first inactivation of an FKBP-like gene in plants. PAS1 expression is altered in pas1 mutants and in the pas2 and pas3 mutants. The expression of the PAS1 gene is increased in the presence of cytokinins, a class of phytohormones originally discovered because of their ability to stimulate cell division. These results are of particular relevance as they show for the first time that an FKBP-like protein plays an important role in the control of plant development.     

Photosynthetic deficiency of a pufX deletion mutant of Rhodobacter sphaeroides is suppressed by point mutations in the light-harvesting complex genes pufB or pufA

The pufX gene of the facultative phototroph Rhodobacter sphaeroides encodes a membrane protein that is required for photoheterotrophic growth. Deletion of pufX impairs the photosynthetic generation of a transmembrane potential, suggesting a role for the PufX protein in light-driven cyclic electron transfer [Farchaus, J. W., et al. (1992) EMBO J. 11, 2779-2788]. Here we describe the isolation and characterization of 65 spontaneous suppressor mutants in which photosynthetic competence was restored by secondary mutations. Genetic analysis revealed the occurrence of single point mutations altering highly conserved residues within the light-harvesting complex, B875. One of three tryptophan codons was changed to stop or arginine codons in 89% of these suppressor mutants. Spectral characterization and Western blot analysis were used to examine the B875 assembly and the stable expression of the altered light-harvesting polypeptides. Three different groups of suppressor mutants were found: (1) No stable expression of altered B875 polypeptides was detected for the alpha 43W-->* and beta 44W-->* mutants. (2) There was expression of the mutated B875-beta chain, but no stable B875 assembly in the beta 47W-->R mutants. (3) Intact B875 complexes were found for the alpha 47S-->F or beta 20H-->R mutants. These results provide evidence that the differently altered B875 polypeptides do not substitute directly for the PufX protein but lead to structural rearrangements in the macromolecular membrane organization, thus restoring a sufficiently high capacity for light-driven cyclic electron transfer.     

cheA, cheB, and cheC genes of Escherichia coli and their role in chemotaxis

Motile but generally nonchemotatic (che) mutants of Escherichia coli were isolated by a simple screening method. A total of 172 independent mutants were examined, and four genes were defined on the basis of mapping and complemenvestigated by determining their null phenotypes with nonsense or bacteriophage Mu-induced mutations. The cheA and cheB products were essential in producing changes of swimming direction and flagellar rotation. The checC product appeared to be an essential component of the flagellum; however, specific mutational alterations of this component allowed flagellar assembly but prevented directional changes in swimming. Since some cheB mutants changed directions incessantly, this gene product may also serve to control the direction of flagellar rotation in response to chemoreceptor signals. Thus most or all of the common elements in the signalling process were involved in the generation and regulation of changes in the direction of flagellar rotation.     

The role of the dynein stalk in cytoplasmic and flagellar motility

We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10-12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility.