bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plectin is a linker of intermediate filaments to Z-discs in skeletal muscle fibers
Authors: Hijikata Takao , Murakami Tohru , Imamura Michihiro , Fujimaki Noboru , Ishikawa Harunori 
Published: April 2021

ABSTRACT
Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments, and its deficiency causes the disruption of myofibrils, or muscular dystrophy. To better understand the functional role of plectin in skeletal muscle fibers, we have examined the topological and structural relationships of plectin to intermediate filaments and Z-discs in rat diaphragm muscles by confocal and immunoelectron microscopy. Immunofluorescence analysis revealed that plectin was colocalized with desmin at the periphery of Z-discs. This plectin localization around Z-discs was constantly maintained irrespective of the contracted or extended state of the muscle fibers, suggesting either direct or indirect association of plectin with Z-discs. Immunogold labeling in skinned muscle fibers clearly demonstrated that plectin-labeled fine threads linked desmin intermediate filaments to Z-discs and connected intermediate filaments to each other. These results indicate that through plectin threads desmin intermediate filaments form lateral linkages among adjacent Z-discs, preventing individual myofibrils from disruptive contraction and ensuring effective force generation.
Myosin II-independent F-actin flow contributes to cell locomotion in Dictyostelium
Authors: Fukui Yoshio , Kitanishi-Yumura Toshiko , Yumura Shigehiko 
Published: April 2021

ABSTRACT
While the treadmilling and retrograde flow of F-actin are believed to be responsible for the protrusion of leading edges, little is known about the mechanism that brings the posterior cell body forward. To elucidate the mechanism for global cell locomotion, we examined the organizational changes of filamentous (F-) actin in live Dictyostelium discoideum. We labeled F-actin with a trace amount of fluorescent phalloidin and analyzed its dynamics in nearly two-dimensional cells by using a sensitive, high-resolution charge-coupled device. We optically resolved a cyclic mode of tightening and loosening of fibrous cortical F-actin and quantitated its flow by measuring temporal and spatial intensity changes. The rate of F-actin flow was evaluated with respect to migration velocity and morphometric changes. In migrating monopodial cells, the cortical F-actin encircling the posterior cell body gradually accumulated into the tail end at a speed of 0.35 μm/minute. We show qualitatively and quantitatively that the F-actin flow is closely associated with cell migration. Similarly, in dividing cells, the cortical F-actin accumulated into the cleavage furrow. Although five times slower than the wild type, the F-actin also flows rearward in migrating mhcA− cells demonstrating that myosin II (‘conventional’ myosin) is not absolutely required for the observed dynamics of F-actin. Yet consistent with the reported transportation of ConA-beads, the direction of observed F-actin flow in Dictyostelium is conceptually opposite from a barbed-end binding to the plasma membrane. This study suggests that the posterior end of the cell has a unique motif that tugs the cortical actin layer rearward by means of a mechanism independent from myosin II; this mechanism may be also involved in cleavage furrow formation.
A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish
Authors: Johansson Mats W. , Holmblad Torbjörn , Thörnqvist Per-Ove , Cammarata Matteo , Parrinello Nicolò , Söderhäll Kenneth 
Published: April 2021

ABSTRACT
Peroxinectin, a cell-adhesive peroxidase (homologous to human myeloperoxidase), from the crayfish Pacifastacus leniusculus, was shown by immuno-fluorescence to bind to the surface of crayfish blood cells (haemocytes). In order to identify a cell surface receptor for peroxinectin, labelled peroxinectin was incubated with a blot of haemocyte membrane proteins. It was found to specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin. Purified 230/90 kDa complex also bound peroxinectin in the same assay. In addition, the 230 kDa band binds the crayfish β-1,3-glucan-binding protein. The 230 kDa band could be reduced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa units.
The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immuno-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain homologous to CuZn-containing superoxide dismutases, and a basic, proline-rich, C-terminal tail, but no membrane-spanning segment. In accordance, the 90 and 230 kDa bands had superoxide dismutase activity. Immuno-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surface; it could be released from the membrane with high salt. It was thus concluded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD) and a peripheral membrane protein, presumably kept at the cell surface via ionic interaction with its C-terminal region.
This interaction with a peroxidase seems to be a novel function for an SOD. The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinectin may mediate, or regulate, cell adhesion and phagocytosis; it may also be important for efficient localized production of microbicidal substances.
RhoA activity is required for fibronectin assembly and counteracts β1B integrin inhibitory effect in FRT epithelial cells
Authors: Calì Gaetano , Mazzarella Claudia , Chiacchio Marianna , Negri Rosa , Retta Saverio Francesco , Zannini Mariastella , Gentile Flaviana , Tarone Guido , Nitsch Lucio , Garbi Corrado 
Published: April 2021

ABSTRACT
FRT thyroid epithelial cells synthesize fibronectin and organize a network of fibronectin fibrils at the basal surface of the cells. Fibronectin fibril formation is enhanced by the overexpression of the ubiquitous β1A integrin and is inhibited by the expression of the dominant-negative β1B subunit. We tested the hypotheses that RhoA activity might mediate the integrin-dependent fibronectin fibrillogenesis and might counteract β1B integrin inhibitory effect. FRT-β1A cells were transfected with a vector carrying a dominant negative form of RhoA (RhoAN19) or treated with the C3 transferase exoenzyme. Both treatments inhibited fibronectin assembly and caused loss of actin microfilaments and adhesion plaques. On the other hand, FRT-β1B cells were transfected with the constitutively activated form of RhoA (RhoAV14) or treated with the E. coli cytotoxic necrotizing factor 1, which directly activates RhoA. Either treatment restored microfilament and adhesion plaque assembly and promoted fibronectin fibril organization. A great increase in fibronectin fibril assembly was also obtained by treatment of FRT-β1B cells with TGF-β.
Our data indicate that RhoA is required to promote fibronectin matrix assembly in FRT cells and that the activation of the signal transduction pathway downstream of RhoA can overcome the inhibitory effect of β1B integrin.
Pre-mRNA and mRNA binding of human nuclear DNA helicase II (RNA helicase A)
Authors: Zhang Suisheng , Herrmann Christine , Grosse Frank 
Published: April 2021

ABSTRACT
Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A, seems to function as a pre-mRNA and mRNA binding protein in human cells. Immunofluorescence studies of NDH II gave a highly diffused nucleoplasmic staining that was similar to that of hnRNP A1 but differed from the localization of the RNA splicing factor Sc-35. Upon transcriptional inhibition, NDH II migrated from the nucleus into the cytoplasm. During mitosis, NDH II was released into the cytoplasm during pro-to metaphase, and was gradually recruited back into telophase nuclei. The timing of nuclear import of NDH II at telophase was found to be later than that of hnRNP A1 but paralleled that of Sc-35. At the ultrastructural level, both NDH II and hnRNP A1 were identified within perichromatin ribonucleoparticle fibrils. However, the subnuclear distributions of NDH II and hnRNP A1 were not overlapping. NDH II could be extracted together with poly(A)-containing mRNA from HeLa cell nuclei and, to a much lesser extent, from the cytoplasm. Following transcriptional inhibition, NDH II was preferentially associated with mRNA from the cytosol, which biochemically confirmed the microscopic observations. Although NDH II is mainly a nuclear enzyme, it is apparently not associated with the nuclear matrix, since it could be extracted with 2 M NaCl from DNase I-treated nuclei. Our cellular and biochemical observations strongly suggest that NDH II is a pre-mRNA and mRNA binding protein. Its significant affinity for ssDNA, but not for dsDNA, points to a transient role in DNA binding during the process of transcript formation. According to our model, single-stranded DNA might be necessary to retain NDH II in the nuclear compartment.
Carboxy-terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin-like activity required for incorporation in the matrix
Authors: Raghunath Michael , Putnam Elizabeth A. , Ritty Timothy , Hamstra Daniel , Park Eun-Sook , Tschödrich-Rotter Mathias , Peters Reiner , Rehemtulla Alnawaz , Milewicz Dianna M. 
Published: April 2021

ABSTRACT
Fibrillin-1, the main component of 10-12 nm microfibrils of the extracellular matrix, is synthesized as profibrillin and proteolytically processed to fibrillin. The putative cleavage site has been mapped to the carboxy-terminal domain of profibrillin-1, between amino acids arginine 2731 and serine 2732, by a spontaneous mutation in this recognition site that prevents profibrillin conversion. This site contains a basic amino acid recognition sequence (R-G-R-K-R-R) for proprotein convertases of the furin/PACE family. In this study, we use a mini-profibrillin protein to confirm the cleavage in the carboxy-terminal domain by both fibroblasts and recombinantly expressed furin/PACE, PACE4, PC1/3 and PC2. Site-directed mutagenesis of amino acids in the consensus recognition motif prevented conversion, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Using a PACE/furin inhibitor, we show that wild-type profibrillin is not incorporated into the extracellular matrix until it is converted to fibrillin. Therefore, profibrillin-1 is the first extracellular matrix protein to be shown to be a substrate for subtilisin-like proteases, and the conversion of profibrillin to fibrillin controls microfibrillogenesis through exclusion of uncleaved profibrillin.
Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote
Authors: Hinchcliffe Edward H. , Thompson Elizabeth A. , Miller Frederick J. , Yang Jing , Sluder Greenfield 
Published: April 2021

ABSTRACT
In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for &gt;180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for &gt;120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.
Is arcA3 a possible mediator in the signal transduction pathway during agonist cell cycle arrest by salicylic acid and UV irradiation?
Authors: Perennes Claudette , Glab Nathalie , Guglieni Benjamin , Doutriaux Marie-Pascale , Phan Thi Hai , Planchais Séverine , Bergounioux Catherine 
Published: April 2021

ABSTRACT
Progression of BY-2 tobacco cells through the cell cycle was followed after treatments with ultra violet (UV) and salicylic acid (SA) used as a potent inhibitor of the octadecanoid pathway which can mediate response to UV irradiation. Cells in S phase were more sensitive than G0/G1 or G2 cells to UV irradiation. Although SA efficiently blocked cells in G0/G1 or G2, it did not block S phase synchronized cells. UV and SA applied simultaneously to cells in G0/G1 delayed the cell cycle progression more than each one separately. Therefore UV irradiation and SA act as agonists to arrest BY-2 cells at cell cycle entry.
To further investigate the signalling pathway mediating UV response, we complemented a UV-sensitive Escherichia coli strain with a Nicotiana xanthi cDNA expression library. A cDNA (arcA3) whose coding sequence is identical to the 2,4-D induced arcA cDNA cloned by Ishida et al. (1993) was isolated. We show that arcA3 transcription is induced at cell cycle entry but not directly by the 2,4-D treatment. Moreover, arcA3 transcription is induced prior to the restriction point as shown with the CDK inhibitor roscovitine. The arcA3 transcription level is increased by UV irradiation but prevented by SA. Indeed, addition of SA prior to UV irradiation blocks the induction of arcA3 transcription. This suggests that arcA3 gene is modulated in both UV and SA responses, the SA effect preceding the UV step. Since arcA3 is 67% similar to RACK1 (functional homology), a rat intracellular receptor for protein kinase C, and possesses identical PKC fixation motifs, it is hypothesised that the arcA3 gene is involved in UV and SA cell cycle arrest.
Globular domains of agrin are functional units that collaborate to induce acetylcholine receptor clustering
Authors: Cornish Toby , Chi Jamin , Johnson Steve , Lu Yang , Campanelli James T. 
Published: April 2021

ABSTRACT
Agrin, an extracellular matrix protein involved in neuromuscular junction formation, directs clustering of postsynaptic molecules, including acetylcholine receptors (AChRs). This activity resides entirely in the C-terminal portion of the protein, which consists of three laminin-like globular domains (G-domains: G1, G2 and G3) and four EGF-like repeats. Additionally, alternate mRNA splicing yields G-domain variants G2(0,4) with 0- or 4-amino-acid inserts, and G3(0,8,11,19) with 0-, 8-, 11- or 19-amino-acid inserts. In order to better understand the contributions of individual domains and alternate splicing to agrin activity, single G-domains and covalently linked pairs of G-domains were expressed as soluble proteins and their AChR clustering activity measured on cultured C2 myotubes. These analyses reveal the following: (1) While only G3(8) exhibits detectable activity by itself, all G-domains studied (G1, G2(0), G2(4), G3(0) and G3(8)) enhance G3(8) activity when physically linked to G3(8). This effect is most pronounced when G2(4) is linked to G3(8) and is independent of the order of the G-domains. (2) The deletion of EGF-like repeats enhances activity. (3) Increasing the physical separation between linked G1 and G3(8) domains produces a significant increase in activity; similar alterations to linked G2 and G3(8) domains are without effect. (4) Clusters induced by two concatenated G3(8) domains are significantly smaller than all other agrin forms studied. These data suggest that agrin G-domains are the functional units which interact independently of their specific organization to yield AChR clustering. G-domain synergism resulting in biological output could be due to physical interactions between G-domains or, alternatively, independent interactions of G-domains with cell surface receptors which require spatially localized coactivation for optimal signal transduction.
E-cadherin binding prevents β-catenin nuclear localization and β-catenin/LEF-1-mediated transactivation
Authors: Orsulic Sandra , Huber Otmar , Aberle Hermann , Arnold Sebastian , Kemler Rolf 
Published: April 2021

ABSTRACT
β-catenin is a multifunctional protein found in three cell compartments: the plasma membrane, the cytoplasm and the nucleus. The cell has developed elaborate ways of regulating the level and localization of β-catenin to assure its specific function in each compartment. One aspect of this regulation is inherent in the structural organization of β-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, we found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with β-catenin; the association of β-catenin with LEF-1 was competed out by the E-cadherin cytoplasmic domain. Similarly, LEF-1 and adenomatous polyposis coli (APC) formed separate, mutually exclusive complexes with β-catenin. In Wnt-1-transfected C57MG cells, free β-catenin accumulated and was able to associate with LEF-1. The absence of E-cadherin in E-cadherin−/− embryonic stem (ES) cells also led to an accumulation of free β-catenin and its association with LEF-1, thereby mimicking Wnt signaling. β-catenin/LEF-1-mediated transactivation in these cells was antagonized by transient expression of wild-type E-cadherin, but not of E-cadherin lacking the β-catenin binding site. The potent ability of E-cadherin to recruit β-catenin to the cell membrane and prevent its nuclear localization and transactivation was also demonstrated using SW480 colon carcinoma cells.
Dynein is required for spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes
Authors: Palazzo Robert E. , Vaisberg Eugeni A. , Weiss Dieter G. , Kuznetsov Sergei A. , Steffen Walter 
Published: April 2021

ABSTRACT
Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.
Inhibition of clathrin-coated pit assembly by an Eps15 mutant
Authors: Benmerah Alexandre , Bayrou Marie , Cerf-Bensussan Nadine , Dautry-Varsat Alice 
Published: April 2021

ABSTRACT
Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of α-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.
A switch in the cellular localization of macrophage migration inhibitory factor in the rat testis after ethane dimethane sulfonate treatment
Authors: Meinhardt Andreas , Bacher Michael , O’Bryan Moira K. , McFarlane James R. , Mallidis Con , Lehmann Christof , Metz Christine N. , Kretser David M. de , Bucala Richard , Hedger Mark P. 
Published: April 2021

ABSTRACT
Macrophage migration inhibitory factor (MIF), one of the first cytokines to be discovered, has recently been localized to the Leydig cells in adult rat testes. In the following study, the response of MIF to Leydig cell ablation by the Leydig cell-specific toxin ethane dimethane sulfonate (EDS) was examined in adult male rats. Testicular MIF mRNA and protein in testicular interstitial fluid measured by ELISA and western blot were only marginally reduced by EDS treatment, in spite of the fact that the Leydig cells were completely destroyed within 7 days. Immunohistochemistry using an affinity-purified anti-mouse MIF antibody localized MIF exclusively to the Leydig cells in control testes. At 7 days post-EDS treatment, there were no MIF immunopositive Leydig cells in the interstitium, although distinct MIF immunostaining was observed in the seminiferous tubules, principally in Sertoli cells and residual cytoplasm, and some spermatogonia. A few peritubular and perivascular cells were also labelled at this time, which possibly represented mesenchymal Leydig cell precursors. At 14 and 21 days, Sertoli cell MIF immunoreactivity was observed in only a few tubule cross-sections, while some peritubular and perivascular mesenchymal cells and the re-populating immature Leydig cells were intensely labeled. At 28 days after EDS-treatment, the MIF immunostaining pattern was identical to that of untreated and control testes. The switch in the compartmentalization of MIF protein at 7 days after EDS-treatment was confirmed by western blot analysis of interstitial tissue and seminiferous tubules separated by mechanical dissection. These data establish that Leydig cell-depleted testes continue to produce MIF, and suggest the existence of a mechanism of compensatory cytokine production involving the Sertoli cells. This represents the first demonstration of a hitherto unsuspected pattern of cellular interaction between the Leydig cells and the seminiferous tubules which is consistent with an essential role for MIF in male testicular function.