bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reorganization of the centrosome and associated microtubules during the morphogenesis of a mouse cochlear epithelial cell
Authors: Henderson Craig G. , Tucker John B. , Chaplin Mark A. , Mackie John B. , Maidment Steven N. , Mogensen Mette M. , Paton Calum C. 
Published: April 2021

ABSTRACT
Reorganization of centrosomal microtubule-organizing centres and the minus ends of microtubules occurs as the centrosomal ends of large microtubule bundles are reposi-tioned and anchored to cell junctions in certain epithelial cells called inner pillar cells in the mouse organ of Corti.
The microtubule bundle that assembles in each cell consists of two distinct microtubule arrays that run closely alongside each other. Both arrays are attached to the cell surface at their upper and lower ends. One of the arrays spans the entire length of a cell but the other is confined to its lower portion.
Initially, about 3,000 microtubules elongate downwards from an apically situated centrosome in each cell. Subse-quently, the minus ends of these microtubules, and the cen-trosome and its two centrioles, migrate for about 12 μm to the tip of a laterally directed projection. Then, a meshwork of dense material accumulates to link microtubule minus ends and the centrosome to cell junctions at the tip of the projection. Pericentriolar satellite bodies, which form after the initial burst of microtubule nucleation, may represent a condensed and inactive concentration of microtubule-nucleating elements.
Suprisingly, as a cell matures, about 2,000 microtubules are eliminated from the centrosomal end of the micro-tubule bundle. However, about 2,000 microtubules are added to the basal portion of each bundle at levels that are remote with respect to the location of the centrosome. Possibly, these microtubules have escaped from the cen-trosome. If this is the case, then both the plus and minus ends of most of the errant microtubules are captured by sites at the cell surface where the ends are finally anchored. Alternatively, each cell possesses at least one other major microtubule-nucleating site (which does not possess centri-oles) in addition to its centrosome.
Cytochemical features common to nucleoli and cytoplasmic nucleoloids of Olea europaea meiocytes: detection of rRNA by in situ hybridization
Authors: Alché J. D. , Fernández M. C. , Rodríguez-García M. I. 
Published: April 2021

ABSTRACT
We used light and electron microscopic techniques to study the composition of cytoplasmic nucleoloids during meiotic division in Olea europaea. Nucleoloids were found in two clearly distinguishable morphological varieties: one similar in morphology to the nucleolus, and composed mainly of dense fibrillar component, and another surrounded by many ribosome-like particles. Cytochemical and immuno- cytochemical techniques showed similar reactivities in nucleoloids and the nucleolus: both are ribonucleoproteic in nature, and possess argyrophillic, argentaffinic and highly phosphorylated proteins. Immunohistochemical techniques failed to detect DNA in either structure. In situ hybridization to a 18 S rRNA probe demonstrated the presence of ribosomal transcripts in both the nucleolus and nucleoloids. These similarities in morphology and compo- sition may reflect similar functionalities.
Site of transcription of ribosomal RNA and intranucleolar structure in HeLa cells
Authors: Hozák Pavel , Cook Peter R. , Schöfer Christian , Mosgöller Wilhelm , Wachtler Franz 
Published: April 2021

ABSTRACT
Sites of transcription of ribosomal RNA in HeLa cells were visualized by electron microscopy. Cells were either incubated with Br-uridine, or permeabilized and then incubated with BrUTP, before sites containing Br-RNA were immunolabelled with gold particles. Short incuba-tions ensured that most incorporated analogue remained at synthetic sites. Fibrillar centres were unlabelled except at their periphery; label was concentrated over certain regions of the surrounding dense fibrillar component. These results suggest that the dense fibrillar component is the site of rRNA transcription. After dispersing the granular component and the dense fibrillar component by a hypotonic treatment, removal of most chromatin and preparation of resinless sections, fibrillar centres remained fixed to a nucleoskeleton. These structural and functional features are incorporated into a model for rRNA tran-scription.
Tissue-specific expression of the fibril-associated collagens XII and XIV
Authors: Wälchli Chantal , Koch Manuel , Chiquet Matthias , Odermatt Bernhard F. , Trueb Beat 
Published: April 2021

ABSTRACT
Interstitial collagen fibrils form the supporting scaffold of all connective tissues. The synthesis of this framework is subject to a precise spatial and temporal regulation in order to meet the mechanical needs of every tissue type. A subgroup of non-fibrillar collagens termed FACIT seems to play a role in this regulation by providing specific molecular bridges between fibrils and other matrix com-ponents. Collagens XII and XIV represent such FACIT molecules and occur preferentially in tissues containing banded type I collagen fibrils. We have used the techniques of indirect immunofluorescence and in situ hybridization to investigate the expression patterns of the two molecules during chicken embryonic development. We detected specific differences in these patterns, which may be related to the respective functions of the two proteins within the connective tissues. Collagen XIV was expressed at very few sites in the 6-day-old embryo, but occurred in virtually every collagen I-containing tissue (skeletal muscle, cardiac muscle, gizzard, tendon, periosteum, nerve) by the end of embryonic development. In contrast, collagen XII was fairly abundant in the 6-day-old embryo but was, at later stages, restricted to only a few dense connective tissue structures (bone, tendon, gizzard). Thus, our results suggest that collagen XII and collagen XIV serve different functions during embryonic development although their structures are highly similar.
Protein composition of cornified cell envelopes of epidermal keratinocytes
Authors: Steven Alasdair C. , Steinert Peter M. 
Published: April 2021

ABSTRACT
Terminally differentiated mammalian epidermal cells are lined with a 15 nm thick layer of proteins cross-linked by isodipeptide and disulfide bonds, called the cornified cell envelope (CE). A number of proteins, including involucrin, loricrin, cystatin A, filaggrin, a cysteine-rich protein (CRP) and the ‘small proline-rich’ proteins (SPRRs) have been reported to be components of this complex, but little infor-mation has been obtained as to their relative abundances because the acute insolubility of the CEs has precluded direct methods of analysis. To address this question, we have determined the amino acid compositions of isolated CEs, and then modelled them in terms of linear combina-tions of the candidate proteins. The results show that stratum corneum CEs have a loricrin content of 65-70% (w/w) in human, and 80-85% in mouse. In human epidermal CEs, the secondary contributors are filaggrin and CRP (each ~10%), with smaller amounts of involucrin, SPRR and cystatin A (2-5% each) also present. Mouse epidermal CEs have about the same amount of filaggrin and somewhat more SPRR, but only trace amounts of the other proteins. In marked contrast, the major constituents of the CEs of cultured keratinocytes induced to terminal differentiation in vitro are cystatin A, involucrin and CRP (each ~30%). No significant amount of loricrin was detected except in sloughed mouse cells, which represent a more advanced state of terminal differentiation than attached cells. These results demonstrate that the CEs of cultured cells are quite different from mature CEs from native epidermis, and further suggest that generation of the CE in vivo is a multi-stage process in which involucrin, SPRR, CRP and cystatin A are initially attached to the cell membrane, and are subsequently overlaid with a heavy deposition of loricrin together with some filaggrin. Our data suggest that cultured CEs approximate only the first stage(s) of this process.
Cytolocation of prosome antigens on intermediate filament subnetworks of cytokeratin, vimentin and desmin type
Authors: Olink-Coux Marguerite , Arcangeletti Cristina , Pinardi Federica , Minisini Rosalba , Huesca Michèle , Chezzi Carlo , Scherrer Klaus 
Published: April 2021

ABSTRACT
Analysis by double-label indirect immunofluorescence of PtK1 and HeLa cells had previously demonstrated that prosome* antigens form networks that superimpose on those of the intermediate filaments of the cytokeratin type. We show here that in PtK1 cells various prosomal antigens also reside to a variable extent on intermediate filaments subnetworks of the vimentin type. In proliferating human fibroblasts the prosome and vimentin networks were found to coincide, while in proliferating myoblasts of the C2.7 mouse myogenic cell line the prosomal antigens seem to superimpose on the intermediate filaments of the desmin type. Thus, the prosomes, which are RNP particles of variable composition and subcomplexes of untranslated mRNP, and carry a multicatalytic proteinase activity, seem to co-localize with the specific kind of cytoplasmic intermediate filament in relation to the cell type. These results, which generalize the previous data, are discussed in view of possible role(s) for prosomes in mRNA metabolism and/or intermediate filaments remodelling.
Species specificity and organ, cellular and subcellular localization of the 100 kDa Ras GTPase activating protein
Authors: Mollat Patrick , Fournier Agnès , Yang Chun Zhi , Alsat Eliane , Zhang Yuehe , Evain-Brion Danièle , Grassi Jacques , Thang Ming Nguy 
Published: April 2021

ABSTRACT
A p100-GAP isoform, generated by an alternative splicing mechanism that eliminates the 180 hydrophobic amino acids at the amino terminus of p120-GAP, has been described in human placenta, in addition to the known p120GAP and neurofibromin. This p100-GAP possesses full Ras-GTPase stimulating activity. p120-GAP is ubiquitously localized in the cytosol while the localization of p100-GAP is unknown. Here we have explored the precise localization of p100-GAP and show that p100-GAP is present only in extracts of primate placenta. It is abundant in both human and Maccaca Rhesus placentae, where it is present in far larger amounts than p120-GAP. The p100-GAP is species-specific since it was not detected in the placenta of pig, sheep, mouse or rat. p100-GAP was also found to be organ-specific, since it was not detectable in organs other than the placenta. In this connection, we substantiated our previous finding that p100-GAP is mainly localized in the trophoblasts. Both subcellular trophoblast fractionation and immunofluorescence analyses showed that this protein was distributed between the cytosol, plasma membrane and a fraction bound to the nucleus, but not inside it. This highly restrictive specificity of p100-GAP localization in relation to species, organ and cell type, confirms the extreme singularity of this protein, and strongly suggests a particular specific function in the trophoblast.
Calponin is localised in both the contractile apparatus and the cytoskeleton of smooth muscle cells
Authors: North Alison J. , Gimona Mario , Cross Robert A. , Small J. Victor 
Published: April 2021

ABSTRACT
Calponin and caldesmon are two thin filament-binding proteins found in smooth muscle that have both been attributed a role in modulating the interaction of actin and myosin. Using high-resolution dual-label immunocyto-chemistry we have determined the distribution of calponin relative to the contractile and cytoskeletal compartments of the smooth muscle cell. We show, using chicken gizzard smooth muscle, that calponin occurs in the cytoskeleton, with β-cytoplasmic actin, filamin and desmin, as well as in the contractile apparatus, with myosin and caldesmon. According to the observed labelling intensities, calponin was more concentrated in the cytoskeleton and it was additionally localised in the cytoplasmic dense bodies as well as in the adhesion plaques at the cell surface, which both harbour the β-cytoplasmic isoform of actin.
It is probable that these results explain earlier conflicting reports on the composition of smooth muscle thin filaments and suggest that calponin, together with a Ca2+-receptor protein, could just as likely serve a role in the cytoskeleton of smooth muscle as in the contractile apparatus.
Molecular analysis of the γ heavy chain of Chlamydomonas flagellar outerarm dynein
Authors: Wilkerson Curtis G. , King Stephen M. , Witman George B. 
Published: April 2021

ABSTRACT
We report here the complete sequence of the γ dynein heavy chain of the outer arm of the Chlamydomonas flagellum, and partial sequences for six other dynein heavy chains. The γ dynein heavy chain sequence contains four P-loop motifs, one of which is the likely hydrolytic site based on its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein heavy chain sequences reveals regions that are highly conserved in all dynein heavy chains sequenced to date, regions that are conserved only among axonemal dynein heavy chains, and regions that are unique to individual dynein heavy chains. The presumed hydrolytic site is absolutely conserved among dyneins, two other P loops are highly conserved among cytoplasmic dynein heavy chains but not in axonemal dynein heavy chains, and the fourth P loop is invariant in axonemal dynein heavy chains but not in cytoplasmic dynein. One region that is very highly conserved in all dynein heavy chains is similar to a portion of the ATP-sensitive microtubule-binding domain of kinesin. Two other regions present in all dynein heavy chains are predicted to have high α-helical content and have a high probability of forming coiled-coil structures. Overall, the central one-third of the γ dynein heavy chain is most conserved whereas the N-terminal one-third is least conserved; the fact that the latter region is divergent between the cytoplasmic dynein heavy chain and two different axonemal dynein heavy chains suggests that it is involved in chain-specific functions.
Association of cytoplasmic dynein with manchette microtubules and spermatid nuclear envelope during spermiogenesis in rats
Authors: Yoshida Toshimichi , Ioshii Sergio O. , Imanaka-Yoshida Kyoko , Izutsu Kosaku 
Published: April 2021

ABSTRACT
During spermiogenesis, the shape of the spermatid nucleus, which is spherical, changes and it becomes the sperm head. A microtubular structure called a manchette is thought to be involved in this morphogenetic process. In this report, we demonstrate the localization of cytoplasmic dynein and manchette development by a double immunofluorescence technique using anti-bovine brain MAP 1C and antitubulin. Before step 6 of the Leblond and Clermont staging, the microtubules showed a fine reticular network, and the dynein staining was homogeneous. In step 6, the microtubular network was concentrated around the nucleus. The manchette developed in step 7 spermatids, and was fully formed, with a skirt-like appearance, covering the nuclear surface in step 8. Dynein fluorescence was associated with the microtubular manchette in steps 7-10. During these steps, the nucleus was protruded from the cytoplasm. In steps 11-13, the most active stages in nuclear shaping, the dynein was densely localized at the nuclear surface covered by the manchette. As the nucleus acquired a shape similar to the mature spermatozoon at step 14, the dynein fluorescence was localized only at the concave side of the nuclear caudal edge. The manchette became narrower and elongated. In step 15, the manchette extended into the elongated cytoplasm, diminishing during steps 16-18. The localization of the dynein was limited to the ventral aspect of the caudal head in these steps. There was little dynein fluorescence in mature spermatozoa. Immunoelectron microscopy showed positive reactions in the nuclear envelope and the inner region of the microtubular manchette. These observations suggest that cytoplasmic dynein, possibly bound to the nuclear envelope, and manchette microtubules are involved in the protrusion of the spermatid nucleus from the cytoplasm.
Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes
Authors: Mitchell David R. , Brown Kimberly S. 
Published: April 2021

ABSTRACT
We have sequenced genomic clones spanning the complete coding region of one heavy chain (beta) and the catalytic domain of a second (alpha) of the Chlamydomonas reinhardtii flagellar outer arm dynein ATPase. The beta heavy chain gene (ODA-4 locus) spans 20 kb, is divided into at least 30 exons, and encodes a predicted 520 kDa protein. Comparison with sea urchin beta dynein sequences reveals homology that extends throughout both proteins. Over the most conserved central catalytic region, the Chlamydomonas alpha and beta chains are equally divergent from the sea urchin beta chain (64% and 65% similarity, respectively), whereas the Chlamydomonas gamma chain is more divergent from urchin beta (54% similarity). The four glycine-rich loops identified as potential nucleotide-binding sites in other dynein heavy chains are also present in Chlamydomonas alpha and beta dyneins. Two of these four nucleotide-binding motifs are highly conserved among flagellar dyneins, but only the motif previously identified as the catalytic site in sea urchin dynein is highly conserved between flagellar and cytoplasmic dynein heavy chains. Predictions of secondary structure suggest that all dynein heavy chains possess three large domains, with the four nucleotide-binding consensus sequences located in a central 185 kDa domain that is bounded on both sides by regions that form multiple, short alpha-helical coiled-coils.
The distribution of cytoplasmic bacteria in the early Drosophila embryo is mediated by astral microtubules
Authors: Callaini Giuliano , Riparbelli Maria Giovanna , Dallai Romano 
Published: April 2021

ABSTRACT
Maternally inherited cytoplasmic bacteria have occasionally been observed in embryos and adults of different strains of several Drosophila species. While there is a considerable body of data on the relationship between bacteria and embryo viability, little is known about the behavior of these bacteria during the early development of Drosophila. In eggs laid by infected Drosophila melanogaster females we showed that cytoplasmic bacteria were initially concentrated in a thin cortical layer and scattered in the yolk region. During the following syncytial blastoderm mitoses the bacteria mainly accumulated towards the poles of the mitotic spindles, suggesting that astral microtubules play a role in localizing bacteria. This is supported by the observation that treatment of the infected embryos with the microtubule-disrupting drug colchicine led to the partial dissociation of the bacteria from the spindle poles, whereas cytochalasin treatment left almost all the bacterial clusters intact. Moreover, bacteria were not found near the polar bodies and yolk nuclei, which were without astral microtubules. In mitosis-defective embryos, with centrosomes dissociated from the nuclei, the bacteria were concentrated in association with the isolated astral microtubules, and in cold-treated embryos, in which microtubules regrew from isolated centrosomes after recovering, the bacteria clustered around the newly formed asters. These observations, also supported by electron microscope analysis, indicate a close relationship between cytoplasmic bacteria and astral microtubules, and suggest that the latter were able to build discrete cytoplasmic domains ensuring the proper distribution of cytoplasmic components during the blastoderm mitoses, despite the lack of cell membranes.
Cloning of Drosophila β-adaptin and its localization on expression in mammalian cells
Authors: Camidge D. Ross , Pearse Barbara M. F. 
Published: April 2021

ABSTRACT
A Drosophila cDNA (BAD1) encoding a structural and assembly-competent homologue of the mammalian coated pit β-adaptins (β and β’ has been cloned and sequenced. In its amino-terminal region (residues 1-575), the BAD1 sequence appears intermediate between that of the mammalian β-adaptin and a predicted sequence, from cDNA 105a, which appears to code for a version of β’-adaptin. To test its functional characteristics, a ‘myc’-tagged version of BAD1 was expressed in Cos cells. The BAD1 protein was detected most clearly in plasma membrane coated pits, where it colocalized with α-adaptin, although other coated pits were noted which apparently did not contain α-adaptin. However, these are probably γ-adaptin containing pits, as BAD1 was also found colocalized with γ-adaptin in Golgi coated pits in which, typically, α-adaptin is absent. Immunoprecipitation experiments confirmed that the BAD1 protein was present in both types of adaptor complex, unlike β-adaptin which complexes with α-adaptin and β’-adaptin which partners γ-adaptin exclusively. In spite of this, BAD1 expression does not appear to mix α-adaptin and γ-adaptin distribution amongst all the coated pits: thus the location of these adaptor complexes in mammalian cells does not depend on the differences between β subunits but rather on membrane-specific interactions of other adaptor polypeptides. The differential interaction of β with α-adaptin and β’ with γ-adaptin in mammalian cells is likely to depend on the few non-conservative differences between their respective sequences and BAD1. Four of these (one with respect to β and three versus 105a) are clustered in a particular region (residues 155 to 305), which may therefore represent a domain that influences the choice of partner adaptin.
Genetic deficiency in low density lipoprotein receptor-related protein confers cellular resistance to Pseudomonas exotoxin A Evidence that this protein is required for uptake and degradation of multiple ligands
Authors: Willnow Thomas E. , Herz Joachim 
Published: April 2021

ABSTRACT
The low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor implicated in the cellular uptake of functionally diverse ligands. Biochemical evidence suggests that LRP is a clearance receptor for apoE-rich remnant lipoproteins, lipoprotein lipase, α2-macroglobulin/protease complexes, plasminogen activator/inhibitor complexes, the active protease tissue-type plasminogen activator and exotoxin A from Pseudomonas aeruginosa. Mice genetically deficient in LRP die early during gestation, underscoring the essential physiological role of this gene in vivo. To study the effect of LRP deficiency at the cellular level, we have used Pseudomonas exotoxin A (PEA) to select murine embryonic fibroblasts that are genetically deficient in LRP. Our results demonstrate that this single gene defect is sufficient to confer resistance to PEA on cultured cells. In addition, embryonic fibroblasts lacking LRP are unable to bind, internalize and degrade methylamine-activated α2-macroglobulin and complexes of urokinase with plasminogen activator inhibitor-1. Furthermore, cellular uptake and degradation of receptor-associated protein, a 39 kDa accessory protein of LRP, is reduced by 90% in the absence of LRP. These results provide genetic evidence for the multifunctional nature of LRP and its crucial role in protease/inhibitor complex metabolism.