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L A Amos

Publications and source records attributed to L A Amos.

At least 37 records · Page 2Linked to original sources
PubMed1996

Microtubule minus ends can be labelled with a phage display antibody specific to alpha-tubulin.

To investigate the orientation of alpha- and beta-tubulin heterodimers within microtubules, we cloned a phage display antibody to alpha-tubulin. The N-terminal 100 residues of alpha-tubulin were bacterially expressed and used to select clones from a large repertoire of antibody-expressing phagemid particles. One clone reacted with the expressed alpha-tubulin N terminus and native tubulin dimer but not with the expressed beta-tubulin N terminus. Electron microscopy showed 30 nm gold beads coated with the antibody binding to one end of brain microtubules. The beads bound to the minus ends of axonemes but not to the brain tubulin extensions from their plus ends. In sliding motility assays with a plus end directed motor, beads were pushed ahead of the microtubules. Our results indicate that an N-terminal epitope of alpha-tubulin is exposed only at the minus ends of microtubules.

Animals↗
PubMed1996

Structural comparison of tektins and evidence for their determination of complex spacings in flagellar microtubules.

Recent structural studies indicate that a tektin heteropolymer forms a unique protofilament of flagellar microtubules. We report here the sequence of tektin C (approximately 47 kDa), predicted from its cDNA (GenBank U38523), compared to tektins A (approximately 53 kDa) and B (approximately 51 kDa) from sea urchin (Strongylocentrotus purpuratus) sperm flagellar microtubules, and compared to partial sequences reported from mouse and human. We are now able to make several observations concerning the tektin family: (1) their common structural features, (2) a comparison of their structure to intermediate filament proteins, and (3) their possible organization in the tektin filament polymer. The predicted amino acid sequence identities/similarities are: for tektins A and C, 42/54%, for tektins A and B, 34/51%; for tektins B and C, 29/42%; for tektin C and a partial cDNA clone from mouse testis, 55/65%; and for tektin B and a partial cDNA clone from the human brain, 45/47%. The three tektins (and the human clone) possess the exact sequence repeat RPNVELCRD. The structural pattern of all three tektin polypeptides is similar to intermediate filament proteins. Tektins are predicted to form extended rods composed of two alpha-helical segments (approximately 180 residues long) capable of forming coiled coils, which are interrupted by short non-helical linkers. The two segments are homologous in sequence and secondary structure, indicating a gene duplication event prior to the divergence of the three tektins. Along each tektin rod cysteine residues occur with a periodicity of approximately 8 nm, coincident with the axial repeat of tubulin dimers in microtubules. From EM data and calculations of secondary structure, the segment length of tektin AB heterodimers is likely to be 16 nm. Both segments of tektin C may be 24 nm long, but one may be 16 nm. On the basis of the available evidence, we propose that coassembly of tektin AB heterodimers with tektin C dimers produces filaments with overall repeats of 8, 16, 24, 32, 40, 48 and 96 nm, generating the basis for the complex spatial arrangements of axonemal components.

Amino Acid Sequence↗
PubMed1995

Re-examination of the polarity of microtubules and sheets decorated with kinesin motor domain.

Electron microscope images of microtubules and tubulin sheets decorated with kinesin head domains have shown the main mass of the kinesin head domain to be superimposed on one subunit of each tubulin dimer. We have polymerized brain tubulin extensions on to the ends of flagellar axonemes under varied conditions, in order to check the polarity of the tubulin-kinesin head complex. Since the polarity of axonemes incubated with normal brain tubulin may be ambiguous, we also tried 50% N-ethylmaleimide-treated tubulin which specifically blocks minus ends. Our conclusion, which conflicts with recently published results, is that the main mass of the kinesin head is associated with the tubulin subunit closer to the plus end of a microtubule.

Animals↗
PubMed1995

Nucleotide-dependent angular change in kinesin motor domain bound to tubulin.

Kinesin is a 'motor' molecule, consisting of two head domains, an alpha-helical coiled coil rod, and a tail part that binds to its cargo. When expressed in a bacterial system, the head domain is functional, and can bind to microtubules with the stoichiometry of one head per tubulin dimer. Kinesin moves along microtubules by means of a cyclic process of nucleotide binding, hydrolysis and product release. We have used negative-stain electron microscopy and image analysis to study the structures of microtubules and tubulin sheets decorated with the motor domain (head) of kinesin in three states: in the presence of an unhydrolysable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP); without nucleotides; and with adenosine 5'-diphosphate (ADP). A single kinesin head bound to a microtubule has a pear-shaped structure, with the broader end towards the 'plus' end of the microtubule under all conditions; the reverse motor, ncd, is similarly oriented. Three-dimensional maps reveal that kinesin heads have a spike that is assumed to form the attachment to the tail of a complete kinesin molecule. This spike is perpendicular to the microtubule axis in the presence of ADP, but points towards the plus end (approximately 45 degrees) in the presence of AMP-PNP or absence of nucleotides. Our results provide direct evidence for a conformational change of the kinesin motor domain during the ATPase cycle.

Adenylyl Imidodiphosphate↗
PubMed1995

The microtubule lattice--20 years on.

In 1974, optical diffraction and image analysis indicated that tubulin dimers in the cylindrically complete A-tubule of flagellar doublet microtubules are arranged with helical symmetry, while those in the incomplete B-tubule associate differently. Recently, electron micrographs of reassembled brain microtubules decorated with kinesin heads have shown that the tubulin dimers there are arranged as in the B-tubule. The lack of symmetry of microtubules assembled in vitro prompts Linda Amos to speculate here that the assembly process in vitro may differ from that occurring in the cell.

Journal Article↗
PubMed1994

Kinesin light chain isoforms in Caenorhabditis elegans.

cDNAs for two isoforms of kinesin light chain in Caenorhabditis elegans have been cloned and sequenced; the deduced M(r) values of the polypeptides are 60,338 and 58,938. Surprisingly, two independent cDNA libraries, each derived from worms at mixed stages of development, provided different single isoforms. The two isoforms are apparently derived from a single gene but have differences at both N and C termini of the predicted protein sequences. The sequences are highly homologous to those from other species, except for some minor features that may be related to the shortened form of the kinesin heavy chain, the product of gene unc-116, with which the light chains are assumed to associate.

Amino Acid Sequence↗
PubMed1993

Tektin B1 from ciliary microtubules: primary structure as deduced from the cDNA sequence and comparison with tektin A1.

Tektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules, and they may also be present in centrioles, centrosomes and mitotic spindles. We report here the cloning and sequencing of a cDNA for ciliary tektin B1. Comparison of the predicted amino acid sequence of tektin B1 with the previously published sequence for tektin A1 reveals several features that better define this class of proteins. Like tektin A1, the central region of the tektin B1 polypeptide chain is predicted to form a coiled-coil rod, consisting of four major alpha-helical regions that are separated by non-helical linkers. Between the central rod domains of tektins A and B there is a 34%/20% amino acid sequence identity/similarity, including equivalent 50-residue segments containing 36 identities, and a high probability of long-range structural homology. The tektin polypeptide chains are divided into two major segments that have significant sequence homology to each other, both within a given tektin chain and between tektins A and B, indicative of gene duplication events. The tektins have a secondary structure and molecular design similar to, but a low primary sequence homology with, intermediate filament proteins. Unlike tektin A1, tektin B1 lacks any part of the C-terminal IFP consensus sequence.

Amino Acid Sequence↗
PubMed1992

Primary structure of tektin A1: comparison with intermediate-filament proteins and a model for its association with tubulin.

Tektins are proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules and that have biochemical and immunological properties similar to those of intermediate-filament proteins. We report here the sequence of a cDNA for tektin A1, one of the main tektins from Strongylocentrotus purpuratus sea urchin embryos. By hybridization analysis, tektin A mRNA appears maximally at ciliogenesis. The predicted structure of tektin A1 (M(r) 52,955) is a series of alpha-helical rod segments separated by nonhelical linkers. The two halves of the rod appear homologous and are probably related by gene duplication. Comparison of tektin A1 with intermediate-filament proteins, including nuclear lamins, reveals a low amino acid homology but similar molecular motif, i.e., pattern of helical and nonhelical domains. This study indicates that tektins are unique proteins but may be evolutionarily related to intermediate-filament proteins, and suggests a structural basis for the interaction of tektins and tubulin in microtubules.

Amino Acid Sequence↗
PubMed1991

The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy.

Individual microtubules can be visualised by confocal microscopy in reflection mode; when associated with a glass surface, they show up as black lines against the bright reflection from the surface. The high contrast imaging allows details of the behaviour of sliding microtubules to be studied easily. Taxol-stabilised microtubules sliding over kinesin-coated surfaces are normally straight, but can bend into tight loops if the leading end sticks to the surface. Some remain curved after release and move in circles. In such cases, the microtubule lattice must have become stably deformed. Electron microscopy of microtubules fixed during sliding shows no gross rearrangement of the subunit lattice and indicates that microtubule bending is mainly achieved by increased twisting of the longitudinal protofilaments around the microtubule.

Adenosine Triphosphatases↗
PubMed1989

Brain dynein crossbridges microtubules into bundles.

Cytoplasmic dynein was purified from pig brain, using a modified version of published procedures, in order to study its interaction with microtubules. Since the preparation produces ATP-dependent sliding of taxol-stabilized purified microtubules over glass and runs on SDS-containing gels as a major band exceeding 300,000 Mr plus a medium chain band at about 75,000 Mr, it is assumed to be identical to the mammalian brain dynein (MAP 1C) purified by Vallee and colleagues. When viewed by electron microscopy in negative stain, individual particles show two distinct configurations. Some are clearly similar to the two-headed bouquet structure already shown for MAP 1C. A larger number of molecules in the present preparation appear to have two heads fused together, forming a dimeric globular particle with two separate tails. They are referred to as phiparticles, because of their resemblance to the greek letter phi. A model for the structural relationship between the two molecular forms is presented. The stems of two associated dynein subunits may separate beyond the base, to form a bouquet, or they may remain fused to form the larger tail of a phi-particle. The smaller tail probably represents a combined pair of features equivalent to the 'stalks' shown to emanate from axonemal dynein heads by Goodenough and colleagues. Both tails of a phi-particle can bind to microtubules, even in the presence of ATP, and cause microtubule bundling. These results suggest a complete structural homology between axonemal and cytoplasmic dynein.

Adenosine Triphosphatases↗
PubMed1987

Kinesin from pig brain studied by electron microscopy.

Kinesin, a protein that transports particles along microtubules, has been purified from pig brain, following published methods. By electron microscopy of both shadowed and negatively stained specimens, it appears to be a rod with a large branched structure at one end and a small fork at the other. The total length of the structure is about 100 nm. The rod has a diameter of 2-4 nm. Close to the middle of the rod there may be a flexible joint. The molecules appear to be attached to microtubules most often by their forked ends in the presence of AMP.PNP. However, the large branched end can also attach to microtubules and individual molecules occasionally have been seen cross-linking two microtubules.

Animals↗
PubMed1986

Studies of tektin filaments from flagellar microtubules by immunoelectron microscopy.

Chemically resistant 2-3 nm filaments with a high alpha-helical content, isolated from sea-urchin sperm flagellar doublet microtubules, consist of proteins that have been named tektins. Polyclonal affinity-purified antibodies to tektins labelled sperm tails all along their lengths, as shown by indirect immunofluorescence microscopy, provided the specimens were not too well fixed. Results obtained for unfixed specimens studied by immunoelectron microscopy suggested the tektins are normally masked by tubulin. A monoclonal anti-tektin antibody labelled bare tektin filaments at longitudinal intervals of approximately 48 nm, which fits in well with the 96 nm longitudinal repeat of axonemes. We discuss a possible scheme for the regular interaction of tubulin monomers with an alpha-helical coiled coil.

Animals↗
PubMed1985

Proteins closely similar to flagellar tektins are detected in cilia but not in cytoplasmic microtubules.

Affinity-purified antibodies against Strongylocentrotus purpuratus sperm flagellar tektin polypeptides have been tested for cross-reactivity with microtubules isolated from various sources, using indirect immunofluorescent staining and antibody binding to nitrocellulose replicas of SDS polyacrylamide gels. The antitektins reacted with sperm tail axonemes from four genera of sea urchins and with cilia from sea urchin embryos. Antibody binding was observed only if the specimens were prefixed by methods that would not preserve them well at an ultrastructural level. However, even after such fixation regimes, no antibody binding was detected to cytoplasmic microtubule arrays in the same embryos, to mitotic spindles isolated from sea urchin eggs or to gill cilia from a mollusc. We conclude that, if tektins are present in sea urchin egg cytoplasmic microtubules, they are sufficiently different from the sperm tektins to have no common strongly antigenic determinants.

Animals↗
PubMed1985

Localization of tektin filaments in microtubules of sea urchin sperm flagella by immunoelectron microscopy.

Extraction of doublet microtubules from the sperm flagella of the sea urchin Strongylocentrotus purpuratus with sarkosyl (0.5%)-urea (2.5 M) yields a highly pure preparation of "tektin" filaments that we have previously shown to resemble intermediate filament proteins. They form filaments 2-3 nm in diameter as seen by negative stain electron microscopy and are composed of approximately equal amounts of three polypeptide bands with apparent molecular weights of 47,000, 51,000, and 55,000, as determined by SDS PAGE. We prepared antibodies to this set of proteins to localize them in the doublet microtubules of S. purpuratus and other species. Tektins and tubulin were antigenically distinct when tested by immunoblotting with affinity-purified antitektin and antitubulin antibodies. Fixed sperm or axonemes from several different species of sea urchin showed immunofluorescent staining with antitektin antibodies. We also used antibodies coupled to gold spheres to localize the proteins by electron microscopy. Whereas a monoclonal antitubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol. 93:576-582) decorates intact microtubules along their lengths, antitektins labeled only the ends of intact microtubules and sarkosyl-insoluble ribbons. However, if microtubules and ribbons attached to electron microscope grids were first extracted with sarkosyl-urea, the tektin filaments that remain were decorated by antitektin antibodies throughout their length. These results suggest that tektins form integral filaments of flagellar microtubule walls, whose antigenic sites are normally masked, perhaps by the presence of tubulin around them.

Animals↗
PubMed1984

Arrangement of protofilaments in two forms of tubulin crystal induced by vinblastine.

Tubulin crystals produced by incubation with vinblastine sulphate have been investigated by X-ray diffraction, electron microscopy and density measurement. The results show that crystals produced in vitro from bovine brain tubulin are made of packed helices, each consisting of a single continuous protofilament, whereas crystals produced in vivo in sea urchin eggs are built from pairs of such helices. The symmetry, cell dimensions, percentage protein and composition of the crystals are consistent with either 24 or 30 tubulin monomers per turn of the helix. The sea urchin egg crystals are more highly ordered and thus more suitable for further analysis.

Animals↗
PubMed1983

Three-dimensional arrangement of the cell wall protein of Sulfolobus acidocaldarius.

The three-dimensional structure of the S-layer that surrounds the bacterium Sulfolobus acidocaldarius is described in detail. Pieces of the S-layer, which are two-dimensional crystals with p6 symmetry, have been studied by crystallographic analysis of electron micrographs of tilted specimens. In the density map, each asymmetric unit appears to consist of several domains connected by strong hinges. On the basis of the ragged appearance of the structure at torn edges, we now suggest that the single species of polypeptide is in a highly extended conformation, with much overlap between different molecules, and show how such molecules might be weaved together to produce the morphological domains. We show that the closed surface lattice of the intact cell wall contains 5-fold and 7-fold vertices and show how the subunit structure appears to be well suited to form 5-fold and 7-fold symmetric rings at these points in place of the 6-fold rings of the hexagonal lattice.

Bacterial Proteins↗