Geometry drives the "deviated-bending" of the bi-tubular structures of the 9 + 2 axoneme in the flagellum

The axoneme "9 + 2" is basically a system constituted of a cylinder of 9 microtubule doublets surrounding a central pair of microtubules. These bi-tubular structures are considered as the support system of the active molecular complexes that generate and regulate the axonemal movement. Schoutens has calculated their moments of inertia [Schoutens, 1994: Journal of Theoretical Biology 171:163-177]. The results obtained allowed us to assume that these bi-tubular systems are endowed with dynamic properties that could be involved in the regulation of the axonemal machinery. For the first time, using the finite elements methods and the resistance of material principles, we have now calculated that the curvature of the axoneme induces the deviated-bending of the bi-tubular structures of the axoneme, because of their geometry only; they behave as beams in a framework. This approach is similar to the one used to measure the deflection of a single microtubule [Kasas et al., 2004: Chem Phys Chem 5:252-257]. These behaviors induce internal movement or constraints of either couples or triplets of doublets within the axonemal cylinder that could be directly involved in a constrained or a spontaneous "convergence/divergence" equilibrium of the cylindrical generatrices that they draw along the axonemal cylinder, which could apparently regulate the activity of the axonemal motors (the dynein arms). These results are discussed here, taking into consideration the dynamic propagation of the wave train along the flagellar axoneme, and the regulated balance between the activities of the two opposite sides of the axoneme during the beat. This study raises a few questions about the architecture-activity duo of the axonemal doublets.     

Ca2+ spikes in the flagellum control chemotactic behavior of sperm

The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-and-run'). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.     

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.     

Kinesin 9 family members perform separate functions in the trypanosome flagellum

Numerous eukaryote genome projects have uncovered a variety of kinesins of unknown function. The kinesin 9 family is limited to flagellated species. Our phylogenetic experiments revealed two subfamilies: KIF9A (including Chlamydomonas reinhardtii KLP1) and KIF9B (including human KIF6). The function of KIF9A and KIF9B was investigated in the protist Trypanosoma brucei that possesses a single motile flagellum. KIF9A and KIF9B are strongly associated with the cytoskeleton and are required for motility. KIF9A is localized exclusively in the axoneme, and its depletion leads to altered motility without visible structural modifications. KIF9B is found in both the axoneme and the basal body, and is essential for the assembly of the paraflagellar rod (PFR), a large extra-axonemal structure. In the absence of KIF9B, cells grow abnormal flagella with excessively large blocks of PFR-like material that alternate with regions where only the axoneme is present. The functional diversity of the kinesin 9 family illustrates the capacity for adaptation of organisms to suit specific cytoskeletal requirements.     

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.     

Ultrastructure of E1 + 2 + 9 + 12 inversion breakpoints in Drosophila subobscura

The ultrastructure of the Drosophila subobscura chromosome regions around the breakpoints of the complex E1 + 2 + 9 + 12 gene arrangement was analyzed. This overlapping inversion is formed by the association of the E1, E2, E9, and E12 simple inversions. Ultrastructure of sections involving 58D/59A, 61C/D, 62D/63A, 64B/C, 67A/B, and 68B/C breakpoints on Est chromosomes were compared with the ultrastructure of sections involving chromosomes were compared with the ultrastructure of sections involving 58D/68B, 62D/64C, 59A/63A, 64B/68C, 67B/61C, and 67A/61B breakpoints on E1 + 2 + 9 + 12 chromosomes. No detectable changes of structural organization on banding patterns induced by the E1 + 2 + 9 + 12 inversion were found. Ultrastructural analysis of the two E12 breakpoints has, however, facilitated the analysis of the left boundary of E12 inversion. Accordingly, we propose 61B/C as a new breakpoint instead of 61C/D.     

Ab initio modeling of glycosyl torsions and anomeric effects in a model carbohydrate: 2-ethoxy tetrahydropyran

A range of ab initio calculations were carried out on the axial and equatorial anomers of the model carbohydrate 2-ethoxy tetrahydropyran to evaluate the level of theory required to accurately evaluate the glycosyl dihedral angle and the anomeric ratio. Vacuum CCSD(T)/CBS extrapolations at the global minimum yield DeltaE = E(equatorial) - E(axial) = 1.42 kcal/mol. When corrected for solvent (by the IEFPCM model), zero-point vibrations and entropy, DeltaG(298) = 0.49 kcal/mol, in excellent agreement with the experimental value of 0.47 +/- 0.3 kcal/mol. A new additivity scheme, the layered composite method (LCM), yields DeltaE to within 0.1 kcal/mol of the CCSD(T)/CBS result at a fraction of the computer requirements. Anomeric ratios and one-dimensional torsional surfaces generated by LCM and the even more efficient MP2/cc-pVTZ level of theory are in excellent agreement, indicating that the latter is suitable for force-field parameterization of carbohydrates. Hartree-Fock and density functional theory differ from CCSD(T)/CBS for DeltaE by approximately 1 kcal/mol; they show similar deviations in torsional surfaces evaluated from LCM. A comparison of vacuum and solvent-corrected one- and two-dimensional torsional surfaces indicates the equatorial form of 2-ethoxy tetrahydropyran is more sensitive to solvent than the axial.     

Human DNA damage checkpoint protein hRAD9 is a 3' to 5' exonuclease

Human RAD9 protein (hRAD9) is a homolog of the fission yeast Rad9 protein, one of the six so-called checkpoint Rad proteins involved in the early steps of DNA damage checkpoint response in Schizosaccharomyces pombe. It has been shown previously that, in vivo, a highly modified form of hRAD9 makes a ternary complex with two other checkpoint Rad proteins, hRAD1 and hHUS1 (Volkmer, E., and Karnitz, L. M. (1999) J. Biol. Chem. 274, 567-570; St. Onge, R. P., Udell, C. M., Casselman, R., and Davey, S. (1999) Mol. Biol. Cell. 10, 1985-1995). However, the function of this complex is not known at present. To help define the functions of checkpoint Rad proteins in humans, we expressed hRAD9 in Escherichia coli, purified the recombinant protein and characterized it. We found that hRAD9 is a 3' to 5' exonuclease and located the nuclease active site to the region between residues 51 and 91 of the 391-amino acid-long protein. Our results suggest that exonucleolytic processing of primary DNA lesion by hRAD9 may contribute to DNA damage checkpoint response in humans.     

Ca2+ imaging in single living cells: theoretical and practical issues

The measurement of intracellular calcium ion concentrations [( Ca2+]i) in single living cells using quantitative fluorescence microscopy draws from a diverse set of disciplines, including cellular biology, optical physics, statistics and computer science. Over the last few years, we have devised and built a number of systems for measuring [Ca2+]i with Fura-2, and have applied them in the exploration of a wide range of biological processes controlled by Ca2+. In this report we discuss these systems and their advantages and limitations. We also describe the theoretical and practical problems associated with using Fura-2 to measure [Ca2+]i, and the solutions that we, and others, have developed to overcome them. The approaches described should provide useful guidance for others interested in imaging [Ca2+] distribution in living cells. The factors that limit current methods are discussed, and areas for future development are highlighted.     

A bipartite Ca2+-regulated nucleoside-diphosphate kinase system within the Chlamydomonas flagellum. The regulatory subunit p72

Regulation of flagellar activity in Chlamydomonas involves both Ca(2+) and cAMP-mediated signaling pathways. However, Chlamydomonas and sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular protein (p72) from the Chlamydomonas flagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca(2+). There are close homologues of p72 in both mammalian and insect genomes. The p72 protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that p72 interacts with two or three additional axonemal polypeptides. The sensitivity of p72 to tryptic digestion differed considerably in the presence and the absence of Ca(2+), suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca(2+) regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.     

Interference of heavy metal cations with fluorescent Ca2+ probes does not affect Ca2+ measurements in living cells

In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.     

Direct measurement of Ca2+ concentration in the SR of living cardiac myocytes

Although abnormal sarcoplasmic reticulum (SR) Ca(2+) handling may cause heart failure, there has been no method to directly measure Ca(2+) concentration in SR ([Ca(2+)](SR)) of living cardiomyocytes. We have measured [Ca(2+)](SR) by expressing novel fluorescent Ca(2+) indicators yellow cameleon (YC) 2.1, YC3er, and YC4er in cultured neonatal rat cardiomyocytes. The distribution of YC2.1 was uniform in the cytoplasm, while that of YC3er/YC4er, containing the signal sequence which recruits them to SR, showed reticular pattern and was co-localized with SERCA2a. The treatment with caffeine reversibly decreased the emission ratio (R) in YC3er/YC4er-expressing myocytes, and the treatment with ryanodine and thapsigargin decreased R irreversibly. During the contraction-relaxation cycle, R was changed periodically in the YC2.1- and YC3er-expressing myocytes, but its direction of the change was opposite. These results suggest that YC3er/YC4er were specifically localized and functioned in SR as a [Ca(2+)](SR) indicator. This technique would be useful to understand the function of SR in failing myocardium.     

Alternating Planarity/Nonplanarity in n-Doped Odd-Membered, All-Trans Polyenes: Molecular Structures of NaCnHn+2 (n = 3, 5, 7, and 9)

The electronic structures of small, odd-membered, all-trans polyenes doped with one Na atom at various positions have been investigated using Hartree-Fock and density functional (B3LYP) theory with a 6-31G(d) basis set. Two distinctly different structural motifs have been identified. In one motif, the dopant atom interacts with an allylic polyene unit in a 4-electron interaction that results in a planar polyene backbone. The other motif has the dopant atom interacting with a pentadienyl polyene unit in a 6-electron interaction, which produces a significantly warped polyene backbone. Independent of structural motif at the doping site, the portion of the polyene structure outside the interaction region remains largely undisturbed in terms of planarity and bond length alternation. For a particular formula unit and potential energy surface, the stationary points corresponding to minima and transition states are remarkably close in energy despite the pronounced changes that occur in the dihedral angles of the polyene backbone at the dopant sites. Whereas internal and external instabilities are found in the Hartree-Fock wavefunctions for NaC₇H₉ and NaC₉H₁₁ structures, the restricted B3LYP wavefunctions are stable for all structures investigated.Electronic Supplementary Material available.     

Alternating access and a pore-loop structure in the Na+-citrate transporter CitS of Klebsiella pneumoniae

CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with 2 Na(+) ions. Reaction of Cys-398 and Cys-414, which are located in a cytoplasmic loop of the protein that is believed to be involved in catalysis, with thiol reagents resulted in significant inhibition of uptake activity. The reactivity of the two residues was determined in single Cys mutants in different catalytic states of the transporter and from both sides of the membrane. The single Cys mutants were shown to have the same transport stoichiometry as wild type CitS, but the C398S mutation was responsible for a 10-fold loss of affinity for Na(+). Both cysteine residues were accessible from the periplasmic as well as from the cytoplasmic side of the membrane by the membrane-impermeable thiol reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) suggesting that the residues are part of the translocation site. Binding of citrate to the outward facing binding site of the transporter resulted in partial protection against inactivation by N-ethylmaleimide, whereas binding to the inward facing binding site resulted in essentially complete protection. A 10-fold higher concentration of citrate was required at the cytoplasmic rather than at the periplasmic side of the membrane to promote protection. Only marginal effects of citrate binding were seen on reactivity with MTSET. Binding of Na(+) at the periplasmic side of the transporter protected both Cys-398 and Cys-414 against reaction with the thiol reagents, whereas binding at the cytoplasmic side was less effective and discriminated between Cys-398 and Cys-414. A model is presented in which part of the cytoplasmic loop containing Cys-398 and Cys-414 folds back into the translocation pore as a pore-loop structure. The loop protrudes into the pore beyond the citrate-binding site that is situated at the membrane-cytoplasm interface.     

Characterization of a molecular chaperone present in the eukaryotic flagellum

Chlamydomonas flagella contain a molecular chaperone now identified as HSP70A, a major cytoplasmic isoform. HSP70A synthesis is upregulated by deflagellation, and its distribution in the flagellum overlaps with the IFT kinesin-II motor FLA10. HSP70A may chaperone flagellar proteins during transport, participating in the assembly and maintenance of the flagellum.     

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.     

Characterization of a cysteine-containing peptide after affinity labelling of Ca2+-ATPase of sarcoplasmic reticulum with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate

3'(2')-O-Biotinyl-thioinosine triphosphate is a substrate of the Ca2+ pump of sarcoplasmic reticulum. Its disulfide inactivates the Ca2+-ATPase with two different velocities. The rapidly inactivated sulfhydryl group cannot be protected by ATP and is therefore considered to be outside the ATP binding site. The slowly reacting sulfhydryl group interacts with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate with a dissociation constant of Kd = 137 microM and an inactivation velocity constant of 1.7 X 10(-3) s-1. It is protected by ATP with two different dissociation constants of the enzyme-ATP complex of Kd = 221 microM and 1130 microM. The slowly reacting sulfhydryl group is therefore considered to be part of the ATP binding site. Since it was impossible to isolate a tryptic peptide by affinity purification on matrix-bound avidin after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate, differential labelling with iodo[2-14C]acetic acid after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate was carried out. Tryptic digestion and FPLC purification led to the isolation of a radioactive carboxymethyl derivative of the cysteine-containing peptide ANACNSVIR. This peptide is equivalent to the cDNA-derived sequence 468-476 of Ca2+-ATPase [Brandl et al. (1986) Cell 44, 597-607] and is located between the phosphorylation site, Asp351, and Lys515, a part of the putative purine binding subsite of ATP. Although the carboxymethylation of Cys471 is hindered by (biotinyl-s6ITP)2, the strong dilution of the specific radioactivity of iodo[2-14C]acetic acid in the isolated peptide 468-476 argues against its direct interaction with the ATP analogue. It is therefore proposed that Cys471 undergoes ATP-dependent conformational changes.