Search for eukaryotic motility proteins in spirochetes: immunological detection of a tektin-like protein in Spirochaeta halophila

The serial endosymbiotic theory (SET) for the spirochete origin of undulipodia (cilia and eukaryotic flagella) predicts that a greater number of axonemal proteins will show homology to spirochete than to other bacterial proteins. To continue testing, the SET proteins associated with eukaryotic motility (tektin, centrin and calmodulin) were sought in Spirochaeta halophila. Western blot immunological detection techniques (for tektin and centrin) and two-dimensional gel analysis (for calmodulin) were used. Tektins are filamentous proteins extending the length of the axoneme in sperm tails and other undulipodia. Whole cell extracts of S. halophila were probed with antibodies made against three sea urchin (Lytechinus pictus) sperm axonemal tektins (tektins A, B, and C). In the spirochetes, one tektin-like protein was detected as a band on Western blots (a C tektin.) An antibody made against Lytechinus pictus sperm tail axonemes, affinity-purified against the C tektin of another sea urchin, Stronglyocentrotus purpuratus, bound to a 30 kDa protein from Spirochaeta halophila. The C tektin epitope was not detected in Escherichia coli. Both the poly- and monoclonal anti-centrin antibodies cross-reacted with multiple proteins in the control alga (Tetraselmis striata) and in the putatively negative control bacterium E. coli. No cross reaction was seen between any anti-centrin antibody and S. halophila. Neither did a two-dimensional gel analysis reveal the presence of calmodulin in these spirochetes or in the two other prokaryotes tested (Spiroplasma citri, Acholeplasma laidlawii). Although neither centrin nor calmodulin were detected, a 30 kDa tektin-like protein apparently is present in these spirochetes.     

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.     

Relationship between tektins and intermediate filament proteins: an immunological study

Affinity-purified antibodies raised against three flagellar tektins (tektin A, B, and C) from each of two sea urchin species (Lytechinus pictus and Strongylocentrotus purpuratus) were used to study the immunological relationship between tektins and intermediate filament proteins. By immunofluorescence microscopy, several antitektins revealed a staining of intermediate filament-like arrays in three vertebrate cell lines tested. Immunoelectron microscopy substantiated the cross reaction of antitektins with intermediate filaments. When the cells were treated with cytochalasin B, the arrangement of the filaments recognized by anti-(Lp)-tektin B was altered; the alteration observed is typical for keratin filaments. By immunoblot, it was found that anti-(Lp)-tektin B cross reacted with two isoforms or different proteins of approximately 54 kD with pIs of 6.1 and 6.2 in human carcinoma epithelia (HeLa) cells and with two isoforms or different proteins of approximately 55 kD with pIs of 6.1 and 6.3 in pig kidney epithelia (LLC-PK1) cells. Furthermore, when antitektin antibodies were affinity purified with the 54 kD HeLa keratin, these keratin-specific antibodies again restained the original tektins on immunoblots. From these observations, it can be concluded that tektins and keratins are to a certain extent immunologically related. To determine the degree of the immunological relationship, tektin filaments and purified intermediate filaments from HeLa cells were cleaved with alpha-chymotrypsin and examined by quantitative immunoblot analysis. On immunoblots of digested tektins from L. pictus, anti-(Lp)-tektin B recognized several cleavage products in the range of 20 kD to 46 kD. However, when immunoblots of digested intermediate filaments from HeLa cells were probed, the cross reaction of anti-(Lp)-tektin B with HeLa keratins was eliminated by more than 98% within 2 min, suggesting that tektins have epitopes in common with the end domains of certain keratins.     

The Novel Microtubule-Destabilizing Drug BAL27862 Binds to the Colchicine Site of Tubulin with Distinct Effects on Microtubule Organization

Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin–BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37 °C with an IC₅₀ of 1.4 μM and bound to unassembled tubulin with a stoichiometry of 1 mol/mol tubulin and a dissociation constant of 244 ± 30 nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin–BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin–BAL27862 and tubulin–colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization.     

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1/Efhc1 indicated that it is indeed abundantly present in sperm flagella and tracheal cilia but only in a small amount in the brain. It is not present in immotile primary cilia. These observations raise the possibility that malfunction of motile cilia is involved in the development of JME.     

The C‐terminal tails of heterotrimeric kinesin‐2 motor subunits directly bind to α‐tubulin1: Possible implications for cilia‐specific tubulin entry

The assembly of microtubule‐based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin‐2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C‐terminal tail fragments of heterotrimeric kinesin‐2 and α‐tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin‐treated microtubule and LC‐ESI‐MS/MS characterization of the tail‐fragment‐associated tubulin identified an association between the tail domains and α‐tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue‐cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α‐tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non‐particulate form requiring kinesin‐2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia.      

Oligomerization of H-pyrophosphatase and its structural and functional consequences

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.     

Peroxiredoxin 1 is involved in disassembly of flagella and cilia

Cilia/flagella are evolutionarily conserved cellular organelles. In this study, we demonstrated that Dunaliella salina Peroxiredoxin 1 (DsPrdx1) localized to the flagella and basal bodies, and was involved in flagellar disassembly. The link between DsPrdx1 and flagella of Dunaliella salina (D. salina) encouraged us to explore the function of its human homologue, Homo sapiens Peroxiredoxin 1 (HsPrdx1) in development and physiology. Our results showed that HsPrdx1 was overexpressed, and cilia were lost in esophageal squamous cell carcinoma (ESCC) cells compared with the non-cancerous esophageal epithelial cells Het-1A. Furthermore, when HsPrdx1 was knocked down by short hairpin RNA (shRNA) lentivirus in ESCC cells, the phenotype of cilia lost can be reversed, and the expression levels of tumor suppressor genes LKB1 and p-AMPK were increased, and the activity of the oncogene Aurora A was inhibited compared with those in cells transfected with scrambe-shRNA lentivirus. These findings firstly showed that Prdx1 is involved in disassembly of flagella and cilia, and suggested that the abnormal expression of the cilia-related gene including Prdx1 may affect both ciliogenesis and cancernogenesis.     

Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening

Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.     

Characterization of the dinuclear metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants

Prolidases are dipeptidases specific for cleavage of Xaa-Pro dipeptides. Pyrococcus furiosus prolidase is a homodimer having one Co-bound dinuclear metal cluster per monomer with one tightly bound Co(II) site and the other loosely bound (Kd 0.24 mM). To identify which Co site is tight-binding and which is loose-binding, site-directed mutagenesis was used to modify amino acid residues that participate in binding the Co1 (E-313 and H-284), the Co2 site (D-209) or the bidentate ligand (E-327). Metal-content, enzyme activity and CD-spectra analyses of D209A-, H284L-, and E327L-prolidase mutants show that Co1 is the tight-binding and Co2 the loose-binding metal center.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Caenorhabditis elegans UNC-45 is a component of muscle thick filaments and colocalizes with myosin heavy chain B, but not myosin heavy chain A

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.     

Temperature-shock induction of multiple flagella induces additional synthesis of flagellum specific mRNAs and tubulin

Four mRNAs (alpha- and beta-tubulin, flagellar calmodulin and Class-I), specifically expressed when Naegleria amebae differentiate into flagellates, were followed at 5-10 min intervals during the temperature-shock induction of multiple flagella in order to better understand how basal body and flagellum number are regulated. Surprisingly, tubulin synthesis continued during the 37 min temperature shock. An initial rapid decline in alpha- and beta-tubulin and flagellar calmodulin mRNAs was followed by a rapid re-accumulation of mRNAs before the temperature was lowered. mRNA levels continued to increase until they exceeded control levels by 4-21%. Temperature shock delayed flagella formation 37 min, produced twice as much tubulin protein synthesis and three fold more flagella. Labeling with an antibody against Naegleria centrin suggested that basal body formation was also delayed 30-40 min. An extended temperature shock demonstrated that lowering the temperature was not required for return of mRNAs to near control levels suggesting that induction of multiple flagella and the formation of flagella per se are affected in different ways. We suggest that temperature-shock induction of multiple flagella reflects increased mRNA accumulation combined with interference with the regulation of the recently reported microtubule-nucleating complex needed for basal body formation.     

Identification of non-specific lipid transfer protein-1 as a calmodulin-binding protein in Arabidopsis

Although non-specific lipid transfer proteins (nsLTPs) are widely present in plants, their functions and regulations have not been fully understood. In this report, Arabidopsis nsLTP1 was cloned and expressed to investigate its binding to calmodulin (CaM). Gel overlay assays revealed that recombinant nsLTP1 bound to CaM in a calcium-independent manner. The association of nsLTP1 and CaM was corroborated using CaM-Sepharose beads to specifically isolate recombinant nsLTP1 from crude bacterial lysate. The CaM-binding site was mapped in nsLTP1 to the region of 69-80 amino acids. This region is highly conserved among plant nsLTPs, implicating that nsLTPs are a new family of CaM-binding proteins whose functions may be mediated by CaM signaling.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Flagellar filaments of the deep-sea bacteria Idiomarina loihiensis belong to a family different from those of Salmonella typhimurium

The helical filaments of the bacterial flagella so far studied seem to be universal in the bacterial kingdom. Despite the variation in flagellin molecular masses, which range from 24 kDa to 62 kDa in different species, there are only two forms: either the so-called Normal (left-handed) or the Curly (right-handed). The Normal and Curly helical forms are asymmetric; the two characteristic helical parameters, which are the pitch and diameter, of Normal filaments are twice those of Curly filaments. Both the universality of these two helical forms and their asymmetry are biological puzzles. We found that the marine bacteria Idiomarina loihiensis have flagella with left-handed Curly-like filaments. Analysis of the polymorphic forms under different pH conditions showed that the Curly-like filaments are actually Normal filaments having a smaller pitch and diameter than those of Salmonella typhimurium. A minor modification of Calladine's model for a filament lattice can explain the variant helical forms. Pseudomonas aeruginosa filaments also belong to the family of I.loihiensis filaments. Thus, there are at least two families of flagella filaments.     

Giant chloroplast development in ethylene response1-1 is caused by a second mutation in ACCUMULATION AND REPLICATION OF CHLOROPLAST3 in Arabidopsis

The higher plants of today array a large number of small chloroplasts in their photosynthetic cells. This array of small chloroplasts results from organelle division via prokaryotic binary fission in a eukaryotic plant cell environment. Functional abnormalities of the tightly coordinated biochemical event of chloroplast division lead to abnormal chloroplast development in plants. Here, we described an abnormal chloroplast phenotype in an ethylene insensitive ethylene response1-1 (etr1-1) of Arabidopsis thaliana. Extensive transgenic and genetic analyses revealed that this organelle abnormality was not linked to etr1-1 or ethylene signaling, but linked to a second mutation in ACCUMULATION AND REPLICATION3 (ARC3), which was further verified by genetic complementation analysis. Despite the normal expression of other plastid division-related genes, the loss of ARC3 caused the enlargement of chloroplasts as well as the diminution of a photosynthetic protein Rubisco in etr1-1. Our study has suggested that the increased size of the abnormal chloroplasts may not be able to fully compensate for the loss of a greater array of small chloroplasts in higher plants.     

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-A resolution. BET3 adopts an alpha/beta-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein-protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.