bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transmembrane domain of CD44 is required for its detergent insolubility in fibroblasts
Authors: Perschl Astrid , Lesley Jayne , English Nicole , Hyman Robert , Trowbridge Ian S. 
Published: April 2021

ABSTRACT
The hyaluronan receptor CD44 is an abundant glycopro-tein expressed on a variety of different cell types. In fibrob-lasts a significant portion of receptor molecules remain in the detergent-insoluble fraction after Triton X-100 extrac-tion. Detergent insolubility of these CD44 molecules has been interpreted to reflect their association with the cytoskeleton. In this study we examined the structural features of CD44 required for its Triton X-100 insolubility in murine fibroblasts. We expressed in L cells the wild-type hematopoietic form of CD44, a mutant CD44 lacking the cytoplasmic domain, and two mutant CD44 molecules with substituted transmembrane domains. Immunofluorescence and cell surface iodination were performed and the detergent extraction profile of the transfected CD44 molecules was determined. No difference in detergent sol-ubility was observed between wild-type and tailless mutant-transfected molecules. However, both CD44 mutants with a heterologous transmembrane domain, derived from either the CD3 ζ chain or CD45, were completely soluble in Triton X-100. These results demonstrate that the transmembrane region but not the cytoplasmic domain of CD44 is required for the detergent-insolubility in these cells. No obvious colocalization of CD44 and actin stress fibers was observed before or after treatment with cytochalasin D, and no change in the detergent extraction profile of wild-type and mutant CD44 molecules was effected by cytochalasin D. In equilibrium density sucrose gradients the Triton-insoluble CD44 component was found in the low density fractions, indicating an association with Triton X-100-insoluble lipids. Together, these results suggest that Triton X-100 insolubility of a fraction of the CD44 molecules in fibroblasts is not the result of interac-tion with the actin-based cytoskeleton but rather due to an association with Triton-insoluble lipids, which is, in turn, dependent on the transmembrane domain of CD44.
Molecular genetic analysis of myoc, a Dictyostelium myosin I
Authors: Peterson Michelle D. , Novak Kristine D. , Reedy Mary C. , Ruman Jane I. , Titus Margaret A. 
Published: April 2021

ABSTRACT
The protozoan myosin Is are widely expressed actin-based motors, yet their in vivo roles remain poorly understood. Molecular genetic studies have been carried out to determine their in vivo function in the simple eukaryote Dictyostelium, an organsim that contains a family of four myosin Is. Here we report the characterization of myoC, a gene that encodes a fifth member of this family. Analysis of the deduced amino acid sequence reveals that the myoC gene encodes a myosin that is homologous to the well-described Acanthamoeba myosin Is as well as to Dic-tyostelium myoB and-D. The expression pattern of the myoC mRNA is similar to that of myoB and myoD, with a peak of expression at times of maximal cell migration, around 6 hours development. Deletion of the myoB gene has been previously shown to result in mutant cells that are defective in pseudopod extension and phagocytosis. However, no obvious differences in cell growth, develop-ment, phagocytosis or motility were detected in cells in which the myoC gene had been disrupted by homologous recombination. F-actin localization and ultrastructural organization also appeared unperturbed in myoC− cells. This apparent ‘lack’ of phenotype in a myosin I single knockout cannot be simply explained by redundancy of function. Our results rather suggest that the present means of assessing myosin I function in vivo are insufficient to identify the unique roles of these actin-based motors.
Change in intracellular K+ concentration caused by external osmolality change regulates sperm motility of marine and freshwater teleosts
Authors: Takai Hiroyuki , Morisawa Masaaki 
Published: April 2021

ABSTRACT
We previously demonstrated that osmolality isotonic to the seminal plasma suppresses sperm motility in marine and freshwater teleosts, and exposure of sperm to hypertonicity of sea water or hypotonicity of fresh water, respectively, induces the initiation of sperm motility at spawning. The motile sperm became immotile by return of osmolality to the isotonic osmolality both in a marine teleost, the puffer fish, and a freshwater teleost, the zebrafish. The initiation and termination of sperm motility could be repeated several times by changing surrounding osmolality in both species. In demembranated sperm, motility was suppressed by a K+ concentration equivalent to the seminal salt concentration in both puffer and zebrafish. Demembranated puffer sperm were reactivated when K+ concentration of the reactivating solution increased. Conversely, initiation of motility in the demembranated zebrafish sperm was induced by decreasing K+ concentration. The initiation and termination of the demembranated sperm were alternately repeated by changing K+ concentration of the reactivation solution in both species. Furthermore, intracellular K+ concentration rose when sperm motility of the puffer was initiated in hypertonic solutions. These results suggest that change in external osmolality is converted into change in intracellular K+ concentration, and that the change affects the fiagellar axoneme as a signal to initiate or terminate sperm motility. The initiation and termination of motility in the demembranated puffer sperm were caused at a high pH and a low pH of the reactivating solution, respectively, suggesting the contribution of intracellular pH in the regulation of fiagellar motility.
Increased axon regeneration in astrocytes grown in the presence of proteoglycan synthesis inhibitors
Authors: Smith-Thomas Linda C. , Stevens James , Fok-Seang Juin , Faissner Andreas , Rogers John H. , Fawcett James W. 
Published: April 2021

ABSTRACT
We have recently reported that the critical difference between astrocytic cell lines that are good or poor promoters of axon growth lies in the extracellular matrix. We demonstrated that much of this difference between matrix produced by permissive and non-permissive cell lines could be ascribed to one or more dermatan/keratan sulphate proteoglycans and that these proteoglycans are able to block the neurite-promoting effect of laminin. These proteoglycans are also produced by cultures of primary astrocytes. In the present study, we have demonstrated that treatment of both astrocytic cell lines and primary astro-cytes with inhibitors of proteoglycan synthesis, β-D-xylosides and sodium chlorate, can strongly influence the axon growth promoting properties of both matrix and whole cells. Dorsal root ganglia grown on matrix or in con-ditioned medium from cultures treated with β-D-xylosides or sodium chlorate had twice as many axons and the axons grew to twice the length as in control cultures. Following treatment of Neu7 cells with proteoglycan synthesis inhibitors there was also a significant reduction in the ability of Neu7 conditioned medium to block the neurite-promoting effect of laminin. Dorsal root ganglia grown on Neu7 cells treated with sodium chlorate extended 2 to 3 times the number of axons for approximately 300 mm longer distance than on control cultures. Treatment of Neu7 cells with β-D-xylosides, however, did not make the cells less inhibitory to axon growth. We have also examined the effects of proteoglycan synthesis inhibitors on three-dimensional primary astrocyte cultures, which closely mimic the in vivo effects of astrocytes on axon growth. Here again, dorsal root ganglia extended many more axons for a longer distance than in control cultures. We conclude from these results that interfering with astrocyte proteo-glycan synthesis makes them better able to support axon regeneration.
The topogenic fate of the polytopic transmembrane proteins, synaptophysin and connexin, is determined by their membrane-spanning domains
Authors: Leube Rudolf E. 
Published: April 2021

ABSTRACT
The synaptophysins and connexins are polytopic trans-membrane proteins of similar secondary structure that accumulate as multiple homo-oligomers in specialized membrane regions, the presynaptic transmitter vesicles or gap junctions. Transfection and expression of the respective genes in cultured epithelial cells results in the de novo formation of either small cytoplasmic, synaptophysin-rich vesicles, or functional gap junctions consisting of clustered connexin molecules. To examine the molecular requirements for the specific enrichment and topogenesis of both types of molecule, chimeric cDNAs were constructed composed of different parts of the rat synaptophysin and rat liver connexin32 genes. Expression of the encoded chimeric polypeptides in hepatocellular carcinoma-derived cells showed that only chimeras with all four transmem-brane domains from either parent molecule were delivered to their specific destination. In contrast, chimeras with transmembrane domains from both connexin32 and synap-tophysin were always retained in the endoplasmic reticulum. The topogenic nature of the transmembrane domains was further demonstrated by deletion mutagen-esis, indicating that removal of cytoplasmic end domains or intravesicular loops does not abolish targeting. On the other hand, excision of individual transmembrane domains or introduction of point mutations in transmembrane segments resulted in retention in the endoplasmic reticulum.
Role of the carboxyl-terminal Fib2 domain in fibronectin matrix assembly
Authors: Ichihara-Tanaka Keiko , Titani Koiti , Sekiguchi Kiyotoshi 
Published: April 2021

ABSTRACT
A truncated form of fibronectin consisting of the N-terminal 70 kDa and C-terminal 37 kDa regions, desig-nated r70F2, retained the ability to assemble into the extra-cellular matrix when expressed in cultured fibroblasts (Ichihara-Tanaka et al. (1992)FEBS Lett. 299, 155-158). To elucidate the role of the C-terminal 37 kDa region in fibronectin matrix assembly, we expressed a panel of mutant forms of r70F2 with various deletions and amino acid substitutions in mouse L cells. Although substitution of Ser for two Cys residues in the C-terminal dimer-forming segment led to a marked reduction in the matrix assembly activity of r70F2, the resulting monomeric r70F2 still retained a low, but significant activity to assemble into the matrix. Neither the N-terminal 70 kDa nor the C-terminal 37 kDa regions, when expressed as monomeric forms, exhibited any residual activity, suggesting that the core domain of the 37 kDa region consisting of III15 and I10 through I12 modules, termed Fib2 domain, is actively involved in the matrix assembly of r70F2. In support of the role of Fib2 domain, the proteolytic fragment derived from the 37 kDa region inhibited the assembly of r70F2. Fur-thermore, en bloc deletion of the Fib2 domain or deletion of the I10 through I12 modules from r70F2 resulted in a marked decrease of the matrix assembly activity. Since deletion of any one of the three type I modules led to a much lesser decrease in activity, it seems likely that a cluster of the three type I modules in the Fib2 domain, but not any one in particular, serves as a functional unit for the matrix assembly of r70F2. Further supporting the active role of the Fib2 domain, a recombinant homodimer of the 37 kDa region was found to be incorporated into the deoxy-cholate-insoluble matrix. These results, taken together, indicate that the Fib2 domain per se has an intrinsic ability to assemble into the matrix and is actively involved in the matrix assembly of fibronectin.
Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction Evidence for a primary capacitation event in boar spermatozoa
Authors: Gadella B. M. , Lopes-Cardozo M. , Golde L. M. G. van , Colenbrander B. , Gadella T. W. J. 
Published: April 2021

ABSTRACT
In order to extend the static information of immunola-belling sulphogalactolipids in fixed boar spermatozoa, a fluorescent sulphogalactolipid analogue, galactose(3-sulphate)-β1-1′[(N-lissamine rhodaminyl)-12-aminodode-canoyl]-sphingosine, was incorporated into plasma membranes of living spermatozoa and its lateral distribu-tion over the sperm head was studied. The fluorescent lipid was enriched in the apical ridge subdomain of freshly ejac-ulated sperm cells. After sperm binding to the zona pellucida the lipid redistributed to the equatorial segment of the sperm surface. A similar shift occurred during capacitation in vitro with 2 mM CaCl2 or with 4% (w/v) bovine serum albumin. The desulphated derivative galactose-β1-1′[(N-lissamine rhodaminyl)-12-aminodo-decanoyl]-sphingosine was also incorporated into the plasma membrane of freshly ejaculated sperm cells and clearly stained the apical ridge subdomain and the (pre)-equatorial subdomains of the sperm heads. The desulphogalactolipid analogue showed a slightly faster migration to the equatori-al segment of the sperm plasma membrane than did its sulphated counterpart. The measured fluorescence intensity distributions correlated linearly with the spatial probe dis-tribution, which was checked by fluorescence lifetime imaging microscopy. The observed migration of the incor-porated glycolipids precedes the acrosome reaction and is one of the underlying molecular events likely to be important in the process of sperm capacitation. The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.
Cerebral microenvironment influences expression of the vitronectin gene in astrocytic tumors
Authors: Gladson Candece L. , Wilcox Josiah N. , Sanders Luraynne , Gillespie G. Yancey , Cheresh David A. 
Published: April 2021

ABSTRACT
Expression of the vitronectin gene was detected in advanced human astrocytoma by in situ hybridization, whereas vitronectin mRNA was undetectable in low grade tumors or in normal adult brain, indicating that vitronectin is a marker of malignant astrocytoma. We established a model of human astrocytoma by transplanting U-251MG human astrocytoma cells intracerebrally into scid mice (C.B.17 severe combined immunodeficient mice). In this model, tumors progressed rapidly and vitronectin mRNA was preferentially detected at the invading tumor margins, i.e. where tumor cells were adjacent to the normal brain tissue. Surprisingly, when U-251MG cells were injected subcutaneously into scid mice, vitronectin mRNA was undetectable throughout the tumor. Moreover, vitronectin mRNA or protein could not be detected among these cells in culture under a wide variety of growth conditions. These findings demonstrate that the cerebral microenvironment influences the expression of the vitronectin gene in malignant astrocytoma. Importantly, the vitronectin binding integrins αvβ3 and αvβ5 localized to distinct sites within these tumors, with β3 mRNA synthesized among invading cells, and αv and β5 mRNAs detected throughout the tumor. In vitro, both of these receptors were capable of promoting adhesion and invasion of astrocytoma cells on a vitronectin substratum. These findings implicate the expression of the vitronectin gene as a contributing factor to the biological behavior of astrocytomas within the cerebral microenvironment.
Nuclear import of glycoconjugates is distinct from the classical NLS pathway
Authors: Duverger Eric , Pellerin-Mendes Christina , Mayer Roger , Roche Annie-Claude , Monsigny Michel 
Published: April 2021

ABSTRACT
The nuclear import of many proteins depends on a short peptide sequence called the nuclear localization signal. However, glycosylated proteins, which lack such a nuclear localization signal, upon their injection into the cytosol by electroporation, enter the nucleus in a sugar-dependent manner. This paper brings new insights on the mechanism of this process, based on a study of neoglycoprotein nuclear uptake by digitonin-permeabilized cells. The nuclear import of neoglycoproteins is energy dependent: it does not occur when cells are maintained at 4°C or when cells are ATP-depleted by treatment with apyrase. The nuclear import of neoglycoproteins occurs through the nuclear pore: it is inhibited by preincubation of cells with wheat germ agglutinin, a lectin which binds the nuclear pore gly-coproteins and blocks the translocation step of nuclear localization signal bearing proteins through the nuclear pore. Furthermore, the nuclear import of neoglycoproteins does not use the pathway of nuclear localization signal bearing proteins: nuclear import of nuclear localization signal bearing proteins depends on cytosolic factors and is inhibited by treatment of cells with N-ethylmaleimide, while the nuclear import of neoglycoproteins neither requires added cytosolic factors nor is sensitive to alkylation by N-ethylmaleimide. In addition, upon incubation in the presence of a large excess of nuclear localization signal bearing protein, the nuclear import of neoglycoproteins is not inhibited.
Formation of two microtubule-nucleating sites which perform differently during centrosomal reorganization in a mouse cochlear epithelial cell
Authors: Tucker John B. , Mogensen Mette M. , Paton Calum C. , Mackie John B. , Henderson Craig G. , Leckie Lorna M. 
Published: April 2021

ABSTRACT
This report provides evidence for the formation of a cell surface-associated centrosome with two spatially discrete microtubule-nucleating sites that perform differently; the minus ends of microtubules remain anchored to one site but escape from the other. Centrosomal reorganization in the cells in question, outer pillar cells of the organ of Corti, indicates that its pericentriolar material becomes intimately associated with the plasma membrane at the two nucleating sites.
Two large microtubule bundles assemble in each cell. A beam which includes about 1,300 microtubules spans most of the cell apex. It is positioned at right angles to a pillar with about 4,500 microtubules which is oriented parallel to the cell’s longitudinal axis.
The beam’s microtubules elongate from, and remain attached to, a centrosomal region with two centrioles which acts as a microtubule-nucleating site. However, the elon-gating microtubules do not radiate from the immediate vicinity of the centrioles. During beam assembly, the minus ends of the microtubules are concentrated together close to the plasma membrane (less than 0.2 μm away in many cases) at a site which is located to one side of the cell apex. High concentrations of the pillar’s microtubules elongating from one particular site have not been detected. Analyses of pillar assembly indicate that the following sequence of events occurs. Pillar microtubules elongate from an apical cell surface-associated nucleating site, which becomes more distantly separated from the centriolar locality as cell morphogenesis progresses. Microtubules do not accumulate at this apical nucleating site because they escape from it. They migrate down to lower levels in the cell where the mature bundle is finally situated and their plus ends are captured at the cell base.
Cyclical bending movements induced locally by successive iontophoretic application of ATP to an elastase-treated flagellar axoneme
Authors: Shingyoji Chikako , Takahashi Keiichi 
Published: April 2021

ABSTRACT
To elucidate the mechanism of oscillatory bending in cilia and flagella, we studied the effect of protease digestion on the response of axonemes to localized application of ATP. When the axonemes were treated with elastase and then reactivated locally by ATP iontophoresis, a pair of local bends were formed due to localized unidirectional sliding in the vicinity of the ATP pipette. Upon repeated application of ATP, the direction of bending with respect to the sperm head axis changed cyclically from side to side over several cycles. The bends were planar and similar to those observed in axonemes that had not been treated with elastase. In trypsin-treated axonemes, in contrast, repetitive local reactivation did not induce such cyclical bending; instead, it induced a bend that grew only in one direction upon repeated application of ATP. Moreover, the bends were not planar. Electron microscopy of these protease-digested axonemes showed that both the interdoublet (nexin) links and the radial spokes were disrupted, but the effects of these proteases were different; trypsin disrupted 60-70% of these structures whereas elastase disrupted 20-30% of them. In both cases, spokes no. 3 and no. 8 (and no. 7) were more resistant to digestion than the others, although they tended to be more resistant to elastase than to trypsin. The importance of radial spokes and interdoublet links in the generation of cyclical bending and the determination of the bending plane is discussed.
Single stranded nucleic acid binding structures on chicken lampbrush chromosomes
Authors: Solovei Irina , Macgregor Herbert , Gaginskaya Elena 
Published: April 2021

ABSTRACT
In chicken oocytes, proteins of the K/J family or their analogs, such as are known to be involved in mRNA processing in humans, are closely associated with nascent C-rich RNA transcripts on the loops of lampbrush chromosomes. Using labelled single stranded nucleotide probes and an antibody to protein K, these C-rich transcripts have been mapped to six different pairs of lampbrush loops situated on 3 macrochromosomes, the sex bivalent (ZW) and certain microchromosomes. Each of these loop pairs has a distinctive morphology. The observations represent cytological evidence of the connection between K-proteins and C-rich RNA transcripts. Another structure, the spaghetti marker of macrochromosome II, also preferentially binds C-rich homonucleotides. This spaghetti marker has a highly distinctive fine structural organization that is quite unlike that of lampbrush loops. Its proteins are not recognised by anti-bodies to protein K. Homonucleotide binding loops are recommended as potentially extremely valuable as markers on physical maps of chicken chromosomes.
Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos
Authors: Kellum Rebecca , Alberts Bruce M. 
Published: April 2021

ABSTRACT
Heterochromatin protein 1 is associated with centromeric heterochromatin in Drosophila, mice, and humans. Loss of function mutations in the gene encoding heterochromatin protein 1 in Drosophila, Suppressor of variegation2-5, decrease the mosaic repression observed for euchromatic genes that have been juxtaposed to centromeric heterochromatin. These heterochromatin protein 1 mutations not only suppress this position-effect variegation, but also cause recessive embryonic lethality. In this study, we analyze the latter phenotype in the hope of gaining insight into heterochromatin function.
In our analyses of four alleles of Suppressor of variegation2-5, the lethality was found to be associated with defects in chromosome morphology and segregation. While some of these defects are seen throughout embryonic development, both the frequency and severity of the defects are greatest between cycles 10 and 14 when zygotic transcription of the Suppressor of variegation2-5 gene apparently begins. By this time in development, heterochromatin protein 1 levels are diminished by four-fold in a quarter of the embryos produced by parents that are both heterozygous for a null allele (Suppressor of variegation2-505).
In a live analysis of the phenotype, we find prophase to be lengthened by more than two-fold in Suppressor of variegation2-505 mutant embryos with subsequent defects in chromosome segregation. The elongated prophase suggests that the segregation phenotype is a consequence of defects in events that occur during prophase, either in chromosome condensation or kinetochore assembly or function. Immunostaining with an antibody against a centromere-specific antigen indicates that the kinetochores of most chromosomes are functional. The immunostaining results are more consistent with defects in chromosome condensation being responsible for the segregation phenotype.