bluuebunny/crossref_metadata_2025_split huggingface dataset:
A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome
Authors: Domínguez J. E. , Buendia B. , López-Otín C. , Antony C. , Karsenti E. , Avila J. 
Published: April 2021

ABSTRACT
The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubule nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immuno-fluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps.
These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericen-triolar material.
Role of different domains in the self-association of rat nucleoporin p62
Authors: Buss Folma , Kent Helen , Stewart Murray , Bailer Susanne M. , Hanover John A. 
Published: April 2021

ABSTRACT
We have expressed rat nucleoporin p62 cDNA in Escherichia coli to obtain material for structural and self-association studies. Electron microscopy and circular dichroism spectroscopy are consistent with a rod-shaped molecule with an α-helical coiled-coil domain at its C terminus and a cross-β structure at its N terminus, separated by a threonine-rich linker, which has a less-defined secondary structure. Electron microscopy and the solubility properties of fragments produced using thrombin and CNBr digestion indicate that p62 molecules associate to form linear chains and that a small region near the C terminus is an important determinant of assembly. This association may have important consequences for pore structure and function; for example, one way p62 could associate would be to form rings in nuclear pores that could function like barrel hoops.
Tubulin and tektin in sea urchin embryonic cilia: pathways of protein incorporation during turnover and regeneration
Authors: Stephens R. E. 
Published: April 2021

ABSTRACT
Axonemal precursor tubulin is the major protein component of the detergent-soluble membrane/matrix fraction of sea urchin embryonic cilia. Its unusual abundance may reflect the rapid turnover of these cilia, a process that is further documented here. However, whether during induced regeneration or normal turnover and growth, most other newly synthesized axonemal proteins are not detectable in the membrane/matrix fraction, raising the question of how non-tubulin precursors transit the growing cilium to the distal tip where assembly is generally thought to occur. Three potential explanations were con-sidered: (1) the assembly of these components is proximal; 2 their relative concentration is too low to detect; or (3) tubulin alone is conveyed via a membrane/matrix pathway while most other axonemal proteins are transported in association with the axoneme. Light microscope autoradi-ography of axonemes pulse-chase labeled with [3H]leucine showed relatively uniform labeling, with no evidence for proximal incorporation. Fully grown cilia and cilia at early stages of regeneration were isolated from labeled embryos, fractionated into membrane/matrix, axonemal tubulin and architectural remnant components, and their labeled protein compositions were compared. Heavily labeled axonemal proteins, most notably the integral microtubule doublet component tektin-A, were not detected in the membrane/matrix fraction of emerging cilia, even though nearly half of the total ciliary tubulin appeared in that fraction, arguing against membrane-associated or soluble matrix transit for the architectural proteins at low con-centrations. However, after thermal fractionation of axonemes from growing cilia, labeled proteins characteris-tic of the architectural remnant dominated the solubilized microtubule fraction, supporting axoneme-associated transport of the non-tubulin proteins during growth, in contrast to a membrane/matrix pathway for tubulin.
Ammonia hypersensitivity of slugger mutants of D. discoideum
Authors: Gee Kathryn , Russell Frances , Gross Julian D. 
Published: April 2021

ABSTRACT
The weak base ammonia inhibits aggregation and culmi-nation of wild-type amoebae of Dictyostelium discoideum. Here we have examined its effect on a series of ‘slugger’ mutants previously assigned to 10 complementation groups, and so-called because they remain as slugs for extended periods. We show that the mutants accumulate normal levels of ammonia and hence may be abnormally susceptible to the ammonia they produce. In agreement with this we find that representatives of the slugger com-plementation groups are hypersensitive to ammonia inhi-bition at three clearly recognisable morphological stages of development: aggregation, tip formation and culmination. This finding suggests that a common ammonia-sensitive process underlies each of these developmental events.
Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells
Authors: Stevenson Bruce R. , Begg David A. 
Published: April 2021

ABSTRACT
The effects of different concentrations of the actin-disrupting drug cytochalasin D on tight junction permeability and distribution of actin filaments in MDCK epithelial cells were examined. Consistent with previous studies, 2 μg/ml cytochalasin D caused a significant decrease in transepithelial resistance, indicative of an increase in tight junction permeability. Surprisingly, increasing concentrations of cytochalasin D caused progressively smaller decreases in transepithelial resistance. The effects of cytochalasin D were reversible. Light microscopic analysis utilizing rhodamine-conjugated phalloidin demonstrated two distinct populations of actin filaments in MDCK cells: an apical peripheral ring of actin, presumably associated with the zonula adherens, and larger actin bundles more basally situated. When treated with 2 μg/ml cytochalasin D, both actin populations were severely disrupted and cells became flattened. Actin in the apical ring aggregated along cell boundaries, and these aggregates co-localized with similarly disrupted focal accumulations of the tight junction-associated protein ZO-1. The basal actin filament bundles also reorganized into focal aggregates. Increasing concentrations of cytochalasin D caused gradually less perturbation of the apical actin ring, consistent with the transepithelial resistance observations. However, the basal actin bundles were disrupted at all concentrations of cytochalasin D tested, demonstrating that the two actin populations are differentially sensitive to cytochalasin D and that apical actin filaments are more important in the regulation of tight junction permeability. Finally, treatment of cells with cytochalasin D inhibited the decrease in transepithelial resistance induced by the chelation of extracellular Ca2+. This indicates that the opening of tight junctions caused by removal of Ca2+ requires a functional actin cytoskeleton.
Cytostellin distributes to nuclear regions enriched with splicing factors
Authors: Bregman David B. , Du Lei , Li Yi , Ribisi Stephen , Warren Stephen L. 
Published: April 2021

ABSTRACT
Cytostellin, a ∼240 kDa phosphoprotein found in all cells examined from human to yeast, is predominantly intranuclear in interphase mammalian cells and undergoes continuous redistribution during the cell cycle. Here, mammalian cytostellin is shown to localize to intranuclear regions enriched with multiple splicing proteins, including spliceosome assembly factor, SC-35. Cytostellin and the splicing proteins also co-localize to discrete foci (called ‘dots’), which are distributed throughout the cell during mitosis and part of G1. The cytostellin that is localized to these dots resists extraction by Triton X-100, indicating that it is tightly associated with insoluble cell structures. All immunostainable cytostellin reappears in the nucleus before S-phase. Although cytostellin and the splicing proteins co-localize in interphase and dividing cells, cytostellin is not detected in purified spliceosomes, and it associates with six unidentified proteins, forming a macro-molecular complex that is biochemically distinct from the proteins that comprise spliceosomes. This macromolecular complex is detected at constant levels throughout the cell cycle, and the level of cytostellin protein remains constant during the cell cycle. Nevertheless, intranuclear cytostellin immunostaining fluctuates markedly during the cell cycle. The monoclonal antibody (mAb) H5 epitope of cytostellin is ‘masked’ in serum-starved cells, but 60 minutes after serum stimulation intense cytostellin immunoreactivity appears in the nuclear speckles. This rapid induction of cytostellin immunoreactivity in subnuclear regions enriched with many splicing factors, as well as accumulations of RNA polymerase II (Pol II) transcripts, suggests that cytostellin may have a function related to mRNA biogenesis.
Actin isoform compartments in chicken gizzard smooth muscle cells
Authors: North Alison J. , Gimona Mario , Lando Zeev , Small J. Victor 
Published: April 2021

ABSTRACT
Differentiated smooth muscle cells typically contain a mixture of muscle (α and γ) and cytoplasmic (β and γ) actin isoforms. Of the cytoplasmic actins the β-isoform is the more dominant, making up from 10% to 30% of the total actin complement. Employing an antibody raised against the N-terminal peptide specific to β-actin, which labels only the β-isoform on two-dimensional gel immunoblots, we have shown that this isoform has a restricted localisation in smooth muscle. Using double-label immunofluorescence and immunoelectron microscopy of ultrathin sections of chicken gizzard, β-actin was localised in the dense bodies and in longitudinal channels linking consecutive dense bodies that were also occupied by desmin. It was additionally found in the membrane-associated dense plaques, but was excluded from the actomyosin-containing regions of the contractile apparatus. Taken together with earlier results these findings identify a cytoskeletal compartment containing intermediate filaments, cytoplasmic actin and the actin cross-linking protein filamin.
Using an antibody specific only for muscle actin, labelling was found generally around the myosin filaments of the contractile apparatus, but was absent from the core of the dense bodies that contained β-actin. Thus, if dense bodies act as dual-purpose anchorage sites, for the cytoskeletal actin and the contractile actin, the thin filaments of the contractile apparatus must be anchored at the periphery of the dense bodies. A model of the structural organisation of the cell is presented and the possible roles of the cytoskeleton are discussed.
Unusual chromosome structure of fission yeast DNA in mouse cells
Authors: McManus John , Perry Paul , Sumner Adrian T. , Wright Diana M. , Thomson Eric J. , Allshire Robin C. , Hastie Nicholas D. , Bickmore Wendy A. 
Published: April 2021

ABSTRACT
Chromosomes from the fission yeast Schizosaccharomyces pombe have been introduced into mouse cells by protoplast fusion. In most cell lines the yeast DNA integrates into a single site within a mouse chromosome and results in striking chromosome morphology at metaphase. Both light and electron microscopy show that the yeast chromosome region is narrower than the flanking mouse DNA. Regions of the yeast insert stain less intensely with propidium iodide than surrounding DNA and bear a morphological resemblance to fragile sites. We investigate the composition of the yeast transgenomes and the modification and chromatin structure of this yeast DNA in mouse cells. We suggest that the underlying basis for the structure we see lies above the level of DNA modification and nucleosome assembly, and may reflect the attachment of the yeast DNA to the rodent cell nucleoskeleton. The yeast integrant replicates late in S phase at a time when G bands of the mouse chromosomes are being replicated, and participates in sister chromatid exchanges at a high frequency. We discuss the implications of these studies to the understanding of how chromatin folding relates to metaphase chromosome morphology and how large stretches of foreign DNA behave when introduced into mammalian cells.
A hypothesis on the traffic of MG160, a medial Golgi sialoglycoprotein, from the trans-Golgi network to the Golgi cisternae
Authors: Johnston Patricia A. , Stieber Anna , Gonatas Nicholas K. 
Published: April 2021

ABSTRACT
We have reported that MG160, an intrinsic membrane sialoglycoprotein of the Golgi apparatus (GA), resides in the medial cisternae of the organelle (Gonatas et al. (1989)J. Biol. Chem. 264, 646-653). In order to resolve the question whether MG160 acquires sialic acid residues in the trans cisternae or trans-Golgi network (TGN) prior to its retrograde transport, we have examined the effects of brefeldin A (BFA) on the post-translational processing of MG160, and the distribution of internalized wheat germ agglutinin covalently linked with HRP (WGA-HRP), which labels the TGN (Gonatas et al. (1977)J. Cell Biol. 73, 1-13).
In BFA-treated PC12 cells, MG160 acquires resistance to endo H, but fails to be sialylated. This effect occurs in parallel with the redistribution of MG160 into an ER compartment dispersed throughout the cytoplasm including the nuclear envelope, and the collapse of the WGA-HRP-labelled TGN into vesicles and tubules surrounding the centriole. These results suggest that MG160 is not sialylated in BFA-treated cells because it is sequestered from the sialyltransferase enzyme(s), presumably located in the TGN, and provide evidence supporting the hypothesis for a retrograde transport pathway that recycles resident GA proteins, including MG160, between the Golgi cisternae and the TGN.
To examine further the above hypothesis we studied cells treated with BFA and then allowed to recover from the effect of the drug for various lengths of time. After 15 minutes of recovery, cisternae of the Golgi apparatus, typically found in the pericentriolar region, are labeled by both MG160 and WGA-HRP. Thirty minutes after removal of BFA, the sialylation of MG160 has begun and by one hour of recovery the protein has matured to its final apparent molecular mass. These results are consistent with the hypotheses that under physiologic conditions, either MG160 is sialylated in a distal Golgi compartment and then returns to the medial Golgi, or that the sialic acid transferase(s) undergo retrograde transport. Additional morphological evidence of a retrograde pathway is provided by the retrograde flow of internalized WGA-HRP into all of the Golgi cisternae during prolonged exposure to the lectin. Taken together, these results provide evidence for the existence, under physiologic conditions, of a retrograde transit pathway active in the distal Golgi apparatus. This hypothesis may be tested when cloned cDNA for MG160, and antibodies specific for rat neural cell sialyltransferases become available.
Pertussis toxin inhibition of T-cell hybridoma invasion is reversed by manganese-induced activation of LFA-1
Authors: Rivière Geertje La , Klein Gebbinck Jacqueline W. T. M. , Driessens Mariëtte H. E. , Roos Ed 
Published: April 2021

ABSTRACT
Pertussis toxin (PT) inhibits invasiveness of T-cell hybridomas in vitro and metastasis formation in vivo. We present evidence for the hypothesis that PT interferes with functional activation of LFA-1. Invasion by TAM2D2 T-cell hybridoma cells of fibroblast monolayers was completely blocked by PT pretreatment, but the cells regained invasiveness in the presence of Mn2+, which activates LFA-1. This invasion was blocked by anti-LFA-1 mAb, and Mn2+ did not stimulate invasiveness of LFA-1-deficient TAM2D2 mutants. TAM2D2 cells did not adhere to surfaces coated with the LFA-1 counterstructure ICAM-1, but Mn2+ induced adhesion. Hence, LFA-1 on TAM2D2 cells requires activation before it can participate in the invasion process.
The hypothesis is further supported by the slightly different results obtained with the TAM8C4 T-cell hybridoma. PT inhibited invasion strongly but not completely. This reduced invasion was increased by Mn2+. TAM8C4 cells did adhere to ICAM-1, but Mn2+ enhanced adhesion. Thus, part of LFA-1 on TAM8C4 cells is constitutively active, allowing for some PT-insensitive invasion, but further activation is required for optimal adhesion and invasion.
PT blocks G-protein-mediated signals, suggesting that an extracellular factor is involved. This is not a serum component or an autocrine motility factor, since the PT effect was serum-independent, and PT did not inhibit motility. Therefore, it is probably produced by the fibroblasts, and either secreted or associated with the cell surface. These results are in line with the hypothesis that a fibroblast constituent activates LFA-1 via a PT-sensitive G-protein and thus stimulates invasion of T-cell hybridomas into the fibroblast monolayer.
Regulation of epithelial cell surface polarity reversal by β1 integrins
Authors: Ojakian George K. , Schwimmer Randi 
Published: April 2021

ABSTRACT
The role of extracellular matrix in the regulation of epithelial cell surface polarity development was studied using MDCK cells. Previous work has demonstrated that MDCK cells cultured in suspension form epithelial cysts having polarized cell surface distributions of several membrane proteins. When MDCK suspension cysts are incubated within collagen gel, a dynamic epithelial membrane remodeling occurs that is accompanied by the reversal of cell surface polarity (Wang et al., 1990b, J. Cell Sci. 95, 153-165), suggesting that extracellular matrix is important in the modulation of epithelial polarity development. To determine if members of the integrin receptor family were involved, MDCK cyst binding studies were done utilizing antifunctional monoclonal antibodies (AIIB2 and AJ2) against the β1 integrin subunit. These antibodies inhibited cyst binding to type I collagen, type IV collagen and laminin, providing evidence that functional β1 integrin heterodimers were present on the cyst outer membrane. Integrin localization on suspension cysts demonstrated that the α2, α3 and α6 integrin subunits had a non-polarized cell surface distribution and were localized to both the apical and basolateral membranes. Interestingly, immunofluorescence microscopy determined that the β1 subunit had a polarized, basolateral membrane distribution although cyst binding studies using inhibitory monoclonal antibodies suggested that functional β1 subunits were present on the cyst outer membrane. After incubation of suspension cysts in collagen gel for 8 hours, the β1 integrin subunit was detected on the outer membrane, suggesting that the formation of additional integrin α/β heterodimers could be involved in epithelial remodeling. To establish the role of β1 integrins in polarity reversal, experiments were done on cysts incubated in collagen gel. After 6 hours in collagen gel, considerable membrane remodeling had occurred as determined by a reduction in outer membrane microvilli. However, the presence of monoclonal antibody AIIB2 inhibited membrane remodeling by preventing both microvillar loss and the endocytosis of the apical membrane glycoprotein gp135. These results provide strong evidence that members of the β1 integrin family are involved in the regulation of epithelial polarity reversal, and demonstrate that MDCK cysts constitute an excellent model system for studying the role of cell-extracellular matrix interactions in the regulation of epithelial plasticity and cell surface polarity development.
Induction of stable microtubules in 3T3 fibroblasts by TGF-β and serum
Authors: Gundersen Gregg G. , Kim Irine , Chapin Cheryl J. 
Published: April 2021

ABSTRACT
Previous studies have shown that fibroblasts induced to migrate into an in vitro wound rapidly generate an array of stable, post-translationally detyrosinated microtubules (Glu MTs) oriented toward the direction of migration. To understand how cells generate a stable array of MTs at a specific location, we have analyzed the contribution of media components to the formation of oriented Glu MTs in wounded monolayers of 3T3 fibroblasts. When confluent monolayers were placed in serum-free medium (SFM) for 2 days before wounding, the cells contained virtually no Glu MTs or nocodazole-resistant MTs and were incapable of generating Glu MTs in response to wounding. Such SFM-treated monolayers were capable of generating oriented Glu MTs within 1 hour of wounding, if calf serum (CS) was added back to the medium. The Glu MTs in the CS refed cells were oriented toward the wound in cells at the wound edge, and were juxtanuclear in cells within the monolayer, demonstrating that CS restored the Glu MT array characteristic of each cell type.
To determine the nature of the ‘Glu MT-inducing’ factor in CS, we subjected CS to different treatments and found that the CS factor was nondialyzable, resistant to heat, mild acid and trypsin, but inactivated by treatment with dithiothreitol. The factor was not absorbed by charcoal and was present in lipoprotein-deficient serum. These properties are consistent with the properties of a number of polypep-tide growth factors, so we screened purified growth factors for their ability to induce Glu MTs in wounded SFM-treated monolayers. Of all the growth factors tested, only TGF-β1 and TGF-β2 induced a significant level (&gt; 70% of the CS response) of oriented Glu MTs. The SFM-treated cells were exquisitely sensitive to TGF-β1, with significant induction of Glu MTs observed at 0.01 ng/ml TGF-β1. Induction of Glu MTs observed by immunofluorescence after CS or TGF-β treatments were paralleled by increases in Glu tubulin detected on western blots. The Glu MTs formed after either CS or TGF-β1 treatment showed enhanced resistance to nocodazole, confirming that both treatments increased the level of stable MTs in cells. The TGF-β1 induction of stable MTs was slower than that of CS (2-4 hours onset versus 1 hour onset), but by 24 hours the level of MT stabilization in TGF-β1 was even greater than that in CS. Unlike CS, TGF-β1 did not stimulate the migration of SFM-treated cells into the wound or the entry of SFM-treated cells into the cell cycle, showing that MT stabilization is independent of these events. These results demonstrate that MT stabilization can be regulated by external factors and that TGF-β is a potent inducing factor for stable MTs.