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.

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.

Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans

2000 ◽  
Vol 113 (22) ◽  
pp. 4001-4012 ◽  
Author(s):  
F. Liu ◽  
I. Ortiz ◽  
A. Hutagalung ◽  
C.C. Bauer ◽  
R.G. Cook ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Immunoelectron Microscopy ◽  
Body Wall ◽  
Developmental Stages ◽  
Body Wall Muscle ◽  
Developmentally Regulated ◽  
C Elegans ◽  
Novel Proteins ◽  
Thick Filaments ◽  
Associated Proteins

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

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 Heat Shock in C. elegans Induces Downstream of Gene Transcription andAccumulation of Double-Stranded RNA

2018 ◽  
Author(s):  
Marko Melnick ◽  
Patrick Gonzales ◽  
Joseph Cabral ◽  
Mary A. Allen ◽  
Robin D. Dowell ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Gene Transcription ◽  
Specific Monoclonal Antibody ◽  
Rna Seq ◽  
Antisense Transcripts ◽  
Double Stranded Rna ◽  
C Elegans

AbstractWe observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find antisense transcripts are generally increased after heat shock, and a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.

scholarly journals Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

2021 ◽  
Author(s):  
Moriya Slavin ◽  
Joanna Zamel ◽  
Keren Zohar ◽  
Siona Eliyahu ◽  
Merav Braitbard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Full Length ◽  
Integrative Model ◽  
Cross Linking ◽  
Cross Link ◽  
Integrative Modeling ◽  
Cellular Context ◽  
Cross Links ◽  
The Cross

AbstractAtomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

scholarly journals LET-711, the Caenorhabditis elegans NOT1 Ortholog, Is Required for Spindle Positioning and Regulation of Microtubule Length in Embryos

2006 ◽  
Vol 17 (11) ◽  
pp. 4911-4924 ◽  
Author(s):  
Leah R. DeBella ◽  
Adam Hayashi ◽  
Lesilee S. Rose
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Regulation Of Gene Expression ◽  
Early Embryo ◽  
Cell Cortex ◽  
Loss Of Function ◽  
Wild Type ◽  
Spindle Positioning ◽  
C Elegans ◽  
Associated Proteins

Spindle positioning is essential for the segregation of cell fate determinants during asymmetric division, as well as for proper cellular arrangements during development. In Caenorhabditis elegans embryos, spindle positioning depends on interactions between the astral microtubules and the cell cortex. Here we show that let-711 is required for spindle positioning in the early embryo. Strong loss of let-711 function leads to sterility, whereas partial loss of function results in embryos with defects in the centration and rotation movements that position the first mitotic spindle. let-711 mutant embryos have longer microtubules that are more cold-stable than in wild type, a phenotype opposite to the short microtubule phenotype caused by mutations in the C. elegans XMAP215 homolog ZYG-9. Simultaneous reduction of both ZYG-9 and LET-711 can rescue the centration and rotation defects of both single mutants. let-711 mutant embryos also have larger than wild-type centrosomes at which higher levels of ZYG-9 accumulate compared with wild type. Molecular identification of LET-711 shows it to be an ortholog of NOT1, the core component of the CCR4/NOT complex, which plays roles in the negative regulation of gene expression at transcriptional and post-transcriptional levels in yeast, flies, and mammals. We therefore propose that LET-711 inhibits the expression of ZYG-9 and potentially other centrosome-associated proteins, in order to maintain normal centrosome size and microtubule dynamics during early embryonic divisions.

scholarly journals Regulators of the Actin Cytoskeleton Mediate Lethality in a Caenorhabditis elegans dhc-1 Mutant

2010 ◽  
Vol 21 (15) ◽  
pp. 2707-2720 ◽  
Author(s):  
Aleksandra J. Gil-Krzewska ◽  
Erica Farber ◽  
Edgar A. Buttner ◽  
Craig P. Hunter
Keyword(s):  
Caenorhabditis Elegans ◽  
Actin Cytoskeleton ◽  
Cytoplasmic Dynein ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Capping Protein ◽  
C Elegans ◽  
Wide Range ◽  
Actin Capping Protein ◽  
Associated Proteins

Functional analysis of cytoplasmic dynein in Caenorhabditis elegans has revealed a wide range of cellular functions for this minus-end–directed motor protein. Dynein transports a variety of cargos to diverse cellular locations, and thus cargo selection and destination are likely regulated by accessory proteins. The microtubule-associated proteins LIS-1 and dynein interact, but the nature of this interaction remains poorly understood. Here we show that both LIS-1 and the dynein heavy-chain DHC-1 are required for integrity of the actin cytoskeleton in C. elegans. Although both dhc-1(or195ts) and lis-1 loss-of-function disrupt the actin cytoskeleton and produce embryonic lethality, a double mutant suppresses these defects. A targeted RNA interference screen revealed that knockdown of other actin regulators, including actin-capping protein genes and prefoldin subunit genes, suppresses dhc-1(or195ts)–induced lethality. We propose that release or relocation of the mutant dynein complex mediates this suppression of dhc-1(or195ts)--induced phenotypes. These results reveal an unexpected direct or indirect interaction between the actin cytoskeleton and dynein activity.

scholarly journals A ZYG-12–dynein interaction at the nuclear envelope defines cytoskeletal architecture in the C. elegans gonad

2009 ◽  
Vol 186 (2) ◽  
pp. 229-241 ◽  
Author(s):  
Kang Zhou ◽  
Melissa M. Rolls ◽  
David H. Hall ◽  
Christian J. Malone ◽  
Wendy Hanna-Rose
Keyword(s):  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Germ Cells ◽  
Nuclear Positioning ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
C Elegans ◽  
Embryo Formation ◽  
Developmental Progression

Changes in cellular microtubule organization often accompany developmental progression. In the Caenorhabditis elegans embryo, the centrosome, which is attached to the nucleus via ZYG-12, organizes the microtubule network. In this study, we investigate ZYG-12 function and microtubule organization before embryo formation in the gonad. Surprisingly, ZYG-12 is dispensable for centrosome attachment in the germline. However, ZYG-12–mediated recruitment of dynein to the nuclear envelope is required to maintain microtubule organization, membrane architecture, and nuclear positioning within the syncytial gonad. We examined γ-tubulin localization and microtubule regrowth after depolymerization to identify sites of nucleation in germ cells. γ-Tubulin localizes to the plasma membrane in addition to the centrosome, and regrowth initiates at both sites. Because we do not observe organized microtubules around zyg-12(ct350) mutant nuclei with attached centrosomes, we propose that gonad architecture, including membrane and nuclear positioning, is determined by microtubule nucleation at the plasma membrane combined with tension on the microtubules by dynein anchored at the nucleus by ZYG-12.

Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans

Development ◽  
1994 ◽  
Vol 120 (10) ◽  
pp. 2823-2834 ◽  
Author(s):  
G. Seydoux ◽  
A. Fire
Keyword(s):  
Caenorhabditis Elegans ◽  
Somatic Cells ◽  
Early Embryogenesis ◽  
Messenger Rnas ◽  
Cell Stage ◽  
P Granules ◽  
C Elegans ◽  
Germline Cells ◽  
Hybridization Protocol

Early embryogenesis in Caenorhabditis elegans is characterized by a series of unequal cleavages that mark the stepwise separation of somatic and germ lineages. We have developed an in situ hybridization protocol to examine the localization of specific maternal and embryonically transcribed messenger RNAs during these early cleavages. We detected three classes of maternal RNAs: RNAs that are maintained in all cells, RNAs that are maintained in germline cells but are lost from somatic cells, and a population of RNAs that are associated with the germline-specific P granules. We observed embryonically transcribed RNAs in somatic cells as early as the 4-cell stage. These transcripts were not detected in germline cells. These observations suggest that mechanisms which distinguish between soma and germline cause asymmetries in mRNA stability and transcription within the first few cleavages of C. elegans embryogenesis.

scholarly journals Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2

2021 ◽  
Vol 118 (34) ◽  
pp. e2103554118
Author(s):  
Moriya Slavin ◽  
Joanna Zamel ◽  
Keren Zohar ◽  
Tsiona Eliyahu ◽  
Merav Braitbard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Full Length ◽  
Integrative Model ◽  
Cross Linking ◽  
Cross Link ◽  
Integrative Modeling ◽  
Cellular Context ◽  
Cross Links ◽  
The Cross

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication–transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

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