Interaction of substance P with tubulin

Binding of the peptide neurotransmitter substance P to brain tubulin in vitro inhibits self-assembly of the protein into microtubules and disrupts preassembled microtubules. This cooperative inhibition of the maximum extent of self-assembly by substance P is explicable in terms of preferential binding to the protomer state as compared to the polymer state of tubulin. The inhibition is relieved by the microtubule-associated protein MAP2, which evidently acts in a mixed competitive-noncompetitive fashion. Substance P interacts directly with the isolated C-terminal 4-kDa peptide fragment of tubulin, which appears to contain the specific binding area for MAP2, but is without effect on the self-assembly of the larger (48-kDa) part of the tubulin molecule called S-tubulin. The results are consistent with the C-terminal fragment having a binding site for the cationic substance P as well as for MAP2. However, factors other than electrostatic interaction must be operative, since the sulfoxide of substance P, a derivative with oxidized methionine but similar electrostatic characteristics, is inactive in inhibiting the extent of microtubule assembly.     

Interaction of tyrosine hydroxylase with tubulin

Bovine adrenal medulla tyrosine hydroxylase associates with microtubules during tubulin assembly. Limited proteolytic digestion of tyrosine hydroxylase does not affect the enzymatic activity but prevents its association with tubulin. A possible interpretation is that an ionic interaction occurs between microtubules and a negatively charged region of the enzyme which is removed by the protease treatment. Tyrosine hydroxylase is able to induce purified tubulin assembly as do the microtubule associated proteins; however, the association induced by tyrosine hydroxylase corresponds to the formation of aggregates or organized structures different from microtubules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electron microscopy of proteins obtained from bovine adrenal medulla show the presence of tubulin in this tissue.     

Interaction of thiabendazole with fungal tubulin

Thiabendazole, 2-(4′-thiazolyl)benzimidazole, at 80 μM completely inhibits mitosis in hyphae of Aspergillus nidulans, growing in liquid culture. DNA and RNA synthesis and mycelial growth are only partially inhibited at this concentration.Binding studies with cell-free mycelial extracts from Penicillium expansum showed that thiabendazole competitively inhibits [¹⁴C]carbendazim binding to tubulin, which suggests that the antimitotic activity of thiabendazole is based on interference with microtubule assembly.Tubulin from a thiabendazole-resistant and carbendazim-highly sensitive mutant of P. expansum has a lower affinity to thiabendazole and a higher affinity to carbendazim than tubulin from a wild-type strain. This indicates that in this mutant the structure of the binding site is affected.The data presented suggest that several sites of both the tubulin and ligand molecule are involved in the binding of benzimidazole compounds to fungal tubulin.     

High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits

A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis. ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed gamma-subunit and the two-headed alpha/beta-heteropolymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.     

Interaction of receptor-activity-modifying protein1 with tubulin

Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors.     

Interaction of tubulin with non-denaturing amphiphiles.

Soluble purified calf brain tubulin contains extensive and easily accessible regions capable of hydrophobic interactions. The binding of non-ionic and mild anionic detergents to this protein has been characterized by difference absorption spectroscopy and equilibrium gel chromatography with labelled ligands. Tubulin bound reversibly and co-operatively 0.42 +/- 0.05 g deoxycholate per g protein and bound octyl glucoside at a minimal stoichiometry of 0.26 g per g protein. Binding of deoxycholate and octyl glucoside perturbed the protein absorption, quenched the fluorescence, and produced a moderate change in the far u.v. circular dichroism of tubulin. These changes have been interpreted as the result of detergent binding near aromatic amino acids and the production of a structural change different from detergent-induced denaturation. Deoxycholate and octyl glucoside inhibited colchicine binding. Octyl glucoside and Triton X-100 inhibited the in vitro self-assembly of tubulin into microtubules, whereas small concentrations of deoxycholate were found to enhance microtubule formation.     

Interaction of thiabendazole with fungal tubulin.

Thiabendazole, 2-(4'-thiazolyl)benzimidazole, at 80 micrometer completely inhibits mitosis in hyphae of Aspergillus nidulans, growing in liquid culture. DNA and RNA synthesis and mycelial growth are only partially inhibited at this concentration. Binding studies with cell-free mycelial extracts from Penicillium expansum showed that thiabendazole competitively inhibits [14C]carbendazim binding to tubulin, which suggests that the antimitotic activity of thiabendazole is based on interference with microtubule assembly. Tubulin from a thiabendazole-resistant and carbendazim-highly sensitive mutant of P. expansum has a lower affinity to thiabendazole and a higher affinity to carbendazim than tubulin from a wide-type strain. This indicates that in this mutant the structure of the binding site is affected. The data presented suggest that several sites of both the tubulin and ligand molecule are involved in the binding of benzimidazole compounds to fungal tubulin.     

The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella.

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 I2 and I3. Subsets of drc subunits are missing from five second-site suppressors, pf2, pf3, suppf3, suppf4, and suppf5, 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 I3 from axonemes of suppressors pf2, pf3, suppf3, and suppf5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, pf3, and suppf5. Recombinant strains carrying one of the mutations pf2, pf3, or suppf5 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.     

The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity

In motor movement on microtubules, the anionic C-terminal of tubulin has been implicated as a significant factor. Our digital analyses of movements of cytoplasmic dynein- and kinesin-coated beads on microtubules have revealed dramatic changes when the C-terminal region (2-4-kDa fragment) of tubulin was cleaved by limited subtilisin digestion of assembled microtubules. For both motors, bead binding to microtubules was decreased threefold, bead run length was decreased over fourfold, and there was a dramatic 20-fold decrease in diffusional movements of cytoplasmic dynein beads on microtubules (even with low motor concentrations where the level of bead motile activity was linear with motor concentration). The velocity of active bead movements on microtubules was unchanged for cytoplasmic dynein and slightly decreased for kinesin. There was also a decrease in the frequency of bead movements without a change in velocity when the ionic strength was raised. However, with high ionic strength there was not a decrease in run length or any selective inhibition of the diffusional movement. The C-terminal region of tubulin increased motor run length (processivity) by inhibiting "detachment" but without affecting velocity. Because the major motor binding sites of microtubules are not on the C-terminal tail of tubulin (), we suggest that the changes are the result of the compromise of a weakly attached state that is the lowest affinity step in both motors' ATPase cycles and is not rate limiting.     

A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain

A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.     

Interaction of reduced glutathione with bovine brain tubulin

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.     

Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas

Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme. Similar to the polyglutamylation-deficient mutants tpg1 and tpg2-1, tpg2-3 axonemal tubulin has a greatly reduced level of long polyglutamate side chains. We found that tpg1 and tpg2-1 mutations also promote flagellar elongation in short-flagella mutants, consistent with a polyglutamylation-dependent mechanism of suppression. Double mutants of tpg1 or tpg2-1 and fla10-1, a temperature-sensitive mutant of intraflagellar transport, underwent slower flagellar shortening than fla10-1 at restrictive temperatures, indicating that the rate of tubulin disassembly is decreased in the polyglutamylation-deficient flagella. Moreover, α-tubulin incorporation into the flagellar tips in temporary dikaryons was retarded in polyglutamylation-deficient flagella. These results show that polyglutamylation deficiency stabilizes axonemal microtubules, decelerating axonemal disassembly at the flagellar tip and shifting the axonemal assembly/disassembly balance toward assembly.     

Bull sperm 19S dynein polymerizes brain tubulin into microtubules

Crude dynein extracted from bull sperm flagella polymerized pure phosphocellulose tubulin isolated from brain tissues into microtubules. This effect was predominantly due to the 19S dynein particle in the extract. ATP stimulated up to five fold the polymerization of brain tubulin by bull sperm dynein. Hydrolysis of ATP was not required since vanadate at a concentration sufficient to block dynein ATPase activity did not interfere with ATP stimulation and because the non hydrolyzable ATP analogue adenylyl (beta-gamma-methylene) diphosphate (AMPPCP) had effects similar to those of ATP. These results suggest that, in addition to hydrolyzing ATP to generate the driving force necessary for microtubule sliding within the axoneme, dynein may also interact with ATP to polymerize tubulin into microtubules.     

Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild- type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke- deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP- mediated mechanism.     

A motile Chlamydomonas flagellar mutant that lacks outer dynein arms

A new Chlamydomonas flagellar mutant, pf-28, which swims more slowly than wild-type cells, was selected. Thin-section electron microscopy revealed the complete absence of outer-row dynein arms in this mutant, whereas inner-row arms and other axonemal structures appeared normal. SDS PAGE analysis also indicated that polypeptides previously identified as outer-arm dynein components are completely absent in pf-28. The two ATPases retained by this mutant sediment at 17.7S and 12.7S on sucrose gradients that contain 0.6 M KCl. Overall swimming patterns of pf-28 differ little from wild-type except that forward swimming speed is reduced to 35% of the wild-type value, and cells show little or no backward movement during photophobic avoidance. Mutant cells will respond to phototactic stimuli, and their flagella will beat in either the forward or reverse mode. This is the first report of a mutant that lacks dynein arms that can swim.     

Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein

The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.     

Interaction of the antitumor compound cryptophycin-52 with tubulin

Cryptophycin-52 (LY355703) is currently undergoing clinical evaluation for cancer chemotherapy. It is a potent suppressor of microtubule dynamics in vitro, and low picomolar concentrations appear to inhibit cancer cell proliferation at mitosis by stabilizing spindle microtubules. In the present study, using [(3)H]cryptophycin-52, we found that the compound bound to tubulin at a single high-affinity site [apparent K(a) (3.6 +/- 1) x 10(6) L/mol, 34 degrees C]. The binding of cryptophycin-52 to tubulin was rapid, not appreciably temperature-dependent, and very poorly reversible. However, we could remove [(3)H]cryptophycin-52 from [(3)H]cryptophycin-52-tubulin complex by denaturing the complex with either urea treatment or boiling. These data suggest that the binding of cryptophycin-52 to tubulin is not covalent. A van't Hoff plot of the binding data indicated that the binding of cryptophycin-52 to tubulin is primarily entropy-driven with a minimum enthalpy contribution. In addition, cryptophycin-52 perturbed the far-ultraviolet circular dichroic spectrum of tubulin and it inhibited the colchicine-induced guanosine triphosphatase activity of tubulin, indicating that its binding to tubulin induces a conformational change in the tubulin. Competition experiments with vinblastine suggest that the binding site for crytophycin-52 may overlap with the vinblastine binding site.     

Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes.

A procedure was developed for isolating Chlamydomonas outer-arm dynein that can functionally combine with the axoneme of an outer-arm-missing mutant, oda1. Previous studies showed that the outer-arm dynein of this organism, containing three heavy chains (alpha, beta, gamma), dissociates upon extraction with a high-salt-concentration buffer solution into an 18-S particle containing the alpha and beta heavy chains and a 12-S particle containing the gamma heavy chain. It was found, however, that the three heavy chains did not dissociate if the high-salt extract was centrifuged in the presence of Mg2+; the three chains constituted a single species (23-S dynein) sedimenting at about 23 S and displayed a three-headed bouquet configuration in electron micrographs. Furthermore, the 23-S dynein had the activity to bind to the axonemes of oda1 and increase the reactivated motility of detergent-extracted cell models; its addition increased the beat frequency from 28 Hz to 53 Hz, a frequency comparable to that of wild-type axoneme. The 18-S and 12-S dyneins, on the other hand, were unable to increase the motility of oda1 axonemes even when added together. The new protocol thus enables purification of outer-arm dynein that retains its functional activity. It will provide a useful experimental system with which to study the mechanism of outer-arm function.     

Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene

We find that two Chlamydomonas outer arm dynein assembly loci, oda6 and oda9, are located on the left arm of linkage group XII, in the vicinity of the previously mapped locus for a 70,000 Mr dynein intermediate chain protein. Restriction fragment length polymorphism mapping indicates that this dynein gene is very closely linked to the oda6 locus. A cDNA clone encoding the 70,000 Mr protein was isolated, sequenced, and used to select genomic clones spanning the corresponding locus from both wild-type and oda6 libraries. When wild-type clones were introduced into cells containing an oda6 allele, the mutant phenotype was rescued, while no rescue was observed after transformation with oda6 clones. Genetic analysis further revealed that newly introduced gene copies were responsible for the rescued phenotype and thus confirms that ODA6 encodes the 70,000 Mr dynein intermediate chain protein. The inability of oda6 mutants to assemble any major outer arm dynein subunits shows that this protein is essential for assembly of stable outer dynein arms. This is the first use of transformation with a wild-type gene to identify the product of a Chlamydomonas mutant.