scholarly journals Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

scholarly journals The isolation and in situ location of adligin: the microtubule cross-linking protein from Caenorhabditis elegans.

1989 ◽  
Vol 108 (3) ◽  
pp. 955-963 ◽  
Author(s):  
E Aamodt ◽  
R Holmgren ◽  
J Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Structural Protein ◽  
Microtubule Network ◽  
C Elegans ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross ◽  
Labeling Pattern

Microtubules isolated from the nematode Caenorhabditis elegans contain long stretches of periodic cross-links formed by microtubule-associated proteins (MAPs). These cross-links are 5.7 nm long, 3 nm wide, and occur at one tubulin dimer (8-nm) intervals along the walls of microtubules (Aamodt, E., and J. Culotti, 1986. J. Cell Biol. 103:23-31). The structural protein of the cross-links was isolated from the MAPs by centrifugation and exclusion chromatography. The cross-links were formed exclusively from the most prevalent MAP, a 32,000 mol wt protein. We suggest the name adligin for this MAP. Adligin eluted from the exclusion column at 33,000 mol wt indicating that it was a monomer in solution. Antibodies were made against the purified adligin and affinity purified. The affinity-purified antibodies were used to locate adligin in situ and to determine its distribution relative to that of tubulin by the use of double label immunofluorescence. The anti-adligin antibodies labeled a fibrous network in the cytoplasm of most cells of C. elegans. Neurons were labeled especially well. This labeling pattern was similar to the labeling pattern obtained with antitubulin, but anti-adligin labeled some granules in the gut that were not labeled with antitubulin. These results suggest that adligin may be part of the interphase microtubule network in C. elegans.

PTL-1, a microtubule-associated protein with tau-like repeats from the nematode Caenorhabditis elegans

1996 ◽  
Vol 109 (11) ◽  
pp. 2661-2672 ◽  
Author(s):  
M. Goedert ◽  
C.P. Baur ◽  
J. Ahringer ◽  
R. Jakes ◽  
M. Hasegawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Tandem Repeats ◽  
Microtubule Assembly ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Functional Characterisation ◽  
Associated Proteins ◽  
Mammalian Origin

Tau, MAP2 and MAP4 are structural microtubule-associated proteins (MAPs) that promote the assembly and stability of microtubules. They share three or four imperfect tandem repeats of an amino acid motif, which is involved in the binding to microtubules. All sequences to data containing this motif are of mammalian origin. We report here the cloning and functional characterisation of a new member of this family of proteins from the nematode Caenorhabditis elegans. This protein exists as two isoforms of 413 and 453 amino acids with four or five tandem repeats that are 50% identical to the tau/MAP2/MAP4 repeats. Both isoforms bind to microtubules and promote microtubule assembly, with the five-repeat isoform being more effective at promoting assembly than the four-repeat isoform. When expressed in COS cells, the five-repeat isoform co-localises with microtubules and induces the formation of microtubule bundles, whereas its expression in Sf9 cells leads to the extension of long unipolar processes. In view of its length, amino acid sequence and functional characteristics, we have named this invertebrate structural MAP ‘Protein with Tau-Like Repeats’ (PTL-1). In C. elegans PTL-1 is expressed in two places known to require microtubule function. It is first seen in the embryonic epidermis, when circumferentially oriented microtubules help to distribute forces generated during elongation. Later, it is found in mechanosensory neurons which contain unusual 15 protofilament microtubules required for the response to touch. These findings indicate that MAPs of the tau/MAP2/MAP4 family are found throughout much of the animal kingdom, where they may play a role in specialised processes requiring microtubules.

scholarly journals Processing of a Psoralen DNA Interstrand Cross-link by XPF-ERCC1 Complex in Vitro

2007 ◽  
Vol 283 (3) ◽  
pp. 1275-1281 ◽  
Author(s):  
Laura A. Fisher ◽  
Mika Bessho ◽  
Tadayoshi Bessho
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Dependent Manner ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
Stalled Fork

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5′ to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3′-side of an ICL. The ICL-specific 3′-incision, along with the 5′-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.

scholarly journals Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures.

1988 ◽  
Vol 106 (6) ◽  
pp. 1985-1995 ◽  
Author(s):  
H F Epstein ◽  
G C Berliner ◽  
D L Casey ◽  
I Ortiz
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Gradient Centrifugation ◽  
Blue Staining ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Thick Filaments ◽  
Cell Biol ◽  
Associated Proteins ◽  
Body Wall Muscles

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.

scholarly journals 270K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon.

1986 ◽  
Vol 103 (1) ◽  
pp. 33-39 ◽  
Author(s):  
N Hirokawa
Keyword(s):  
Cross Linking ◽  
Flexible Structure ◽  
High Molecular Weight Protein ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Weight Protein ◽  
The Cross ◽  
Cross Bridges ◽  
Brain Tubulin

MAPs (microtubule-associated proteins) were isolated from crayfish walking leg nerves. A major MAP was identified as a high molecular weight protein (270K). This protein co-migrated with mammalian MAP2, stimulated the polymerization of rat brain tubulin into microtubules, and was heat resistant. Rotary shadowing revealed that the 270K MAP is a long thin flexible structure. It formed cross-bridges of fine strands, linking microtubules with each other in vitro. These strands resemble the cross-bridges between microtubules observed in the crayfish axon permeabilized with saponin and quick-frozen, deep-etched. Antibodies against mammalian MAP2 cross-reacted with this crayfish MAP and stained the axoplasm of the walking leg nerves. Thus MAPs, especially the 270K MAP, appear to be a major component of the cross-linking strands between microtubules observed in the crayfish axon.

scholarly journals Talin requires beta-integrin, but not vinculin, for its assembly into focal adhesion-like structures in the nematode Caenorhabditis elegans.

1996 ◽  
Vol 7 (8) ◽  
pp. 1181-1193 ◽  
Author(s):  
G L Moulder ◽  
M M Huang ◽  
R H Waterston ◽  
R J Barstead
Keyword(s):  
Caenorhabditis Elegans ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
The Body ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Beta Integrin ◽  
Cell Shapes

In cultured cells, the 230-kDa protein talin is found at discrete plasma membrane foci known as focal adhesions, sites that anchor the intracellular actin cytoskeleton to the extracellular matrix. The regulated assembly of focal adhesions influences the direction of cell migrations or the reorientation of cell shapes. Biochemical studies of talin have shown that it binds to the proteins integrin, vinculin, and actin in vitro. To understand the function of talin in vivo and to correlate its in vitro and in vivo biochemical properties, various genetic approaches have been adopted. With the intention of using genetics in the study of talin, we identified a homologue to mouse talin in a genetic model system, the nematode Caenorhabditis elegans. C. elegans talin is 39% identical and 59% similar to mouse talin. In wild-type adult C. elegans, talin colocalizes with integrin, vinculin, and alpha-actinin in the focal adhesion-like structures found in the body-wall muscle. By examining the organization of talin in two different C. elegans mutant strains that do not make either beta-integrin or vinculin, we were able to determine that talin does not require vinculin for its initial organization at the membrane, but that it depends critically on the presence of integrin for its initial assembly at membrane foci.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

scholarly journals Sperm Competition in the Absence of Fertilization in Caenorhabditis elegans

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 201-208 ◽  
Author(s):  
Andrew Singson ◽  
Katherine L Hill ◽  
Steven W L’Hernault
Keyword(s):  
Caenorhabditis Elegans ◽  
Sperm Competition ◽  
Sperm Function ◽  
Sperm Precedence ◽  
Wild Type ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Normal Sperm ◽  
Self Fertilization ◽  
Competition Assays

Abstract Hermaphrodite self-fertilization is the primary mode of reproduction in the nematode Caenorhabditis elegans. However, when a hermaphrodite is crossed with a male, nearly all of the oocytes are fertilized by male-derived sperm. This sperm precedence during reproduction is due to the competitive superiority of male-derived sperm and results in a functional suppression of hermaphrodite self-fertility. In this study, mutant males that inseminate fertilization-defective sperm were used to reveal that sperm competition within a hermaphrodite does not require successful fertilization. However, sperm competition does require normal sperm motility. Additionally, sperm competition is not an absolute process because oocytes not fertilized by male-derived sperm can sometimes be fertilized by hermaphrodite-derived sperm. These results indicate that outcrossed progeny result from a wild-type cross because male-derived sperm are competitively superior and hermaphrodite-derived sperm become unavailable to oocytes. The sperm competition assays described in this study will be useful in further classifying the large number of currently identified mutations that alter sperm function and development in C. elegans.

Sign in / Sign up

Export Citation Format

Share Document