bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular characterization of two functional domains of CLIP-170 in vivo
Authors: Pierre Philippe , Pepperkok Rainer , Kreis Thomas E. 
Published: April 2021

ABSTRACT
CLIP-170 is a microtubule-binding protein isolated from HeLa cells that is involved in the interaction of endosomes with microtubules. The basic N-terminal domain of CLIP-170 binds to microtubules in vitro. To characterize further the functional domains of this cytoplasmic linker protein, we have transiently expressed intact and mutant forms of CLIP-170 in mammalian cells (HeLa and Vero cells) and show that the tandem repeat present in the N-terminal domain is essential for its binding to microtubules in vivo as previously found in vitro. With increasing levels of expression of CLIP-170, the sites with which the peripheral ends of microtubules interact enlarge, eventually forming large patches, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the transfected protein. Modification of the microtubule-binding region, particularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact with nor to influence the organization of the intermediate filaments, and collapsing the network of intermediate filaments with microinjected anti-bodies against vimentin has no effect on the distribution of CLIP-170. These data suggest that CLIP-170 has at least two functional domains in vivo, an N-terminal micro-tubule-binding domain, and a C-terminal domain that is involved in the anchoring of microtubules to peripheral cytoplasmic structures.
Lineage-specific regulation of cell cycle gene expression in differentiating myeloid cells
Authors: Bürger Christiane , Wick Maresa , Müller Rolf 
Published: April 2021

ABSTRACT
We have analysed the expression of 7 cyclin and cyclin-associated kinase (cdk) genes in the human myeloid cell line HL-60 at different stages of the cell cycle in non-synchronised cells and during terminal differentiation. A clear cell cycle-dependent expression was found with cyclins A (S+G2), B (G2) and E (late G1 and S), while other cell cycle genes showed only very weak (cdk2) or no periodic expression (cyclin D1, cyclin D2 and cdk4). Induction of macrophage-like differentiation by TPA or granulocytic differentiation by retinoic acid or DMSO was accompanied by a block in G1 and resulted in distinct patterns of gene expression that were lineage- and inducer-specific. These included: (i) a dramatic decrease in the expression of cyclin A, cyclin B and cdk2, and surprisingly an up-regulation of cyclin D1 in TPA-induced macrophage-like cells; (ii) a down-regulation of cyclin E in retinoic acid-induced granulocytic cells; and (iii) a decreased abundance of cyclin D1 and D2, but high levels of cyclin A, B and E RNA in DMSO-induced granulocytic cells. These observations suggest that the mechanisms leading to a differentiation-associated cell cycle arrest are lineage-specific, and that the sustained expression of cyclin and cdk genes does not interfere with the induction of terminal differentiation.
Mutations in the Caenorhabditis elegans p-tubulin gene mec-7: effects on microtubule assembly and stability and on tubulin autoregulation
Authors: Savage Cathy , Xue Yingzi , Mitani Shohei , Hall David , Zakhary Randa , Chalfie Martin 
Published: April 2021

ABSTRACT
We have sequenced 45 mutations in mec-7, a β-tubulin gene required for the production of 15-protofilament microtubules in the nematode Caenorhabditis elegans, and have correlated sequence alterations with mutant phenotypes. The expression patterns of most alleles have also been determined by in situ hybridization and immunocytochemistry. Most (12/16) complete loss-of-function alleles, which are recessive, result from nonsense mutations, insertions, or deletions; three others disrupt a putative GTP-binding domain. Three of the four loss-of-function, missense mutations result in elevated mec-7 message levels, suggesting a defect in tubulin autoregulation that may be attributable to a loss in the ability to form heterodimers.
Most (8/9) mild alleles are caused by missense mutations. Two mild alleles appear to increase microtubule stability and lead to the elaboration of ectopic neuronal processes in mec-7-expressing cells. Most (15/23) mutations that cause severe dominant or semidominant phenotypes are clustered into three discrete domains; four others occur in putative GTP-binding regions. Many of these dominant mutations appear to completely disrupt microtubule assembly.
Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy
Authors: Ghosh Richik N. , Gelman Dana L. , Maxfield Frederick R. 
Published: April 2021

ABSTRACT
Numerous experiments on CHO cells have shown that endosomes are composed of separate vesicular and tubulo-vesicular compartments, such as the sorting endosome, the recycling compartment, and the late endosome. However, Hopkins et al. (Nature 346, 335–339, 1990) have reported that endosomes in HEp2 human carcinoma cells form an extensive tubular reticulum. To resolve their observations with previous results from CHO and other cells, we examined the sorting and intracellular transport of endo-cytosed macromolecules in HEp2 cells, using low density lipoprotein (LDL) and transferrin (Tf) to probe the lyso-somally directed and recycling pathways, respectively. Fluorescent LDL and Tf were observed with laser scanning confocal microscopy to visualize simultaneously both probes’ sorting and subsequent post-sorting behavior in HEp2 cells. Quantifying the 3-dimensional cellular distributions of fluorescent LDL and Tf, after a variety of pulsechase schemes, gave the ligands’ trafficking rates.
Initially, both ligands appear in the same punctate sorting endosomes, and fingers of Tf start extending from these sorting endosomes. Tf rapidly leaves dual-labeled sorting endosomes ( 5 minutes) and enters a postsorting recycling compartment from which it is recycled out of the cell ( minutes). We present both morphological and kinetic data supporting the existence of these two separate compartments along the recycling pathway in HEp2 cells. LDL remains in punctate sorting endosomes that eventually lose the ability to receive newly endocytosed LDL, and mature into late endosomes. The trafficking and sorting of Tf and LDL in HEp2 cells follow the same general scheme as in CHO cells, indicating that the tubular endosomes previously seen may be the tubular parts of the sorting endosomes and recycling compartments in these cells. We propose that the endosomes in the recycling pathway of HEp2 cells, as in CHO cells, are composed of short-lived sorting endosomes, accessible to both Tf and LDL, and long-lived post-sorting recycling compartments, which contain Tf and recycling receptors but not LDL.
Cell kinetic characterization of growth arrest in cultured human keratinocytes
Authors: Ruissen Fred van , Erp Piet E. J. van , Jongh Gijs J. de , Boezeman Jan B. M. , van de Kerkhof Peter C. M. , Schalkwijk Joost 
Published: April 2021

ABSTRACT
In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously. Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-β1. It was found that human keratinocytes grown in KGM plus TGF-β1 were growth-arrested within 52 hours. The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and parallelled the decrease of cells in S-phase. Within 52 hours after addition of TGF-β1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation. However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1. Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest. We found that after termination of the growth arrest in KGM supplemented with TGF-β1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-β1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis.
Tenascin-R (J1 160/180) inhibits fibronectin-mediated cell adhesion-functional relatedness to tenascin-C
Authors: Pesheva Penka , Probstmeier Rainer , Skubitz Amy P. N. , McCarthy James B. , Furcht Leo T. , Schachner Melitta 
Published: April 2021

ABSTRACT
Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different extra- and intra-cellular signals. Fibronectin-mediated cell attachment and spreading can be affected in a negative way by tenascin-C, an extracellular matrix glycoprotein expressed in a tempo-rally and spacially restricted manner during early mor-phogenesis. Tenascin-R (J1-160/180), consisting of two major isoforms of 160 kDa (tenascin-R 160) and 180 kDa (tenascin-R 180) in mammals, is an extracellular matrix glycoprotein of the central nervous system that shares high structural homologies with tenascin-C. Here we show that in relation to fibronectin-mediated adhesion, the two extra-cellular matrix molecules are also functionally closely related. When offered as mixed substrata with other extra-cellular matrix molecules, the two tenascin-R isoforms and tenascin-C derived from mouse brain selectively inhibit fibronectin-dependent cell adhesion and neurite outgrowth, and affect cell morphology of different mesenchymal and neural cells. This effect is partially due to interactions at the substrate level that result in a steric hindrance and/or conformational change of the cell binding sites of the fibronectin molecule. In addition, tenascin-R 180 and tenascin-C interact with cells by an RGD- and β1 integrin-independent mechanism, leading to cell rounding and detachment from such substrata. The expression of tenascin-R and tenascin-C in the nervous system at times and locations where fibronectin-mediated cellular processes take place may be related to the role of inhibitory signals in the extracellular matrix in the regulation of cell migration and differentiation in general.
Fast myosin heavy chains expressed in secondary mammalian muscle fibers at the time of their inception
Authors: Cho Mildred , Hughes Simon M. , Karsch-Mizrachi Ilene , Travis Marilyn , Leinwand Leslie A. , Blau Helen M. 
Published: April 2021

ABSTRACT
Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast myosin heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, mon-oclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development.
Association of the Dictyostelium 30 kDa actin bundling protein with contact regions
Authors: Fechheimer Marcus , Ingalls Hilary M. , Furukawa Ruth , Luna Elizabeth J. 
Published: April 2021

ABSTRACT
‘Contact regions’ are plasma membrane domains derived from areas of intercellular contact between aggregating Dictyostelium amebae (H. M. Ingalls et al. (1986). Proc. Nat. Acad. Sci. USA 83, 4779). Purified contact regions contain a prominent actin-binding protein with an Mr of 34,000. Immunoblotting with monoclonal antibodies identifies this polypeptide as a 34,000 Mr actin-bundling protein (known as 30 kDa protein), previously shown to be enriched in filopodia (M. Fechheimer (1987). J. Cell Biol. 104, 1539). About four times more 30 kDa protein by mass is associated with contact regions than is found in total plasma membranes isolated from aggregating cells. In agreement with these observations, immunostaining of the 30 kDa protein in aggregating cells reveals a prominent localization along the plasma membrane at sites of intercellular contact. By contrast, alpha-actinin does not appear to be significantly enriched at sites of cell to cell contact. Binding experiments using purified plasma membranes, actin and 30 kDa protein indicate that the 30 kDa protein is associated with the plasma membrane primarily through interactions with actin filaments. Calcium ions are known to decrease the interaction of actin with 30 kDa protein in solution. Surprisingly, membrane-associated complexes of actin and the 30 kDa protein are much less sensitive to dissociation by micromolar levels of free calcium ions than are complexes in solutions lacking membranes. These results suggest that the interaction of the 30 kDa protein with F-actin at regions of cell-cell contact may be less sensitive to disruption by free calcium ions than elsewhere in the cell cortex. The positively cooperative assembly of stable complexes of actin and the 30 kDa protein at contact regions may be an important factor in the organization of both the cortex and these membrane domains that are spe-cialized for intercellular adhesion.
Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PG-M/versican
Authors: Yamagata Masahito , Kimata Koji 
Published: April 2021

ABSTRACT
Malignantly transformed cells usually display a rosette-like morphology of substratum adhesions (called podosomes) and disorganized microfilaments, and are often associated with elevated production of chondroitin sulphate. We pre-viously showed that many tissues and cells express alter-natively spliced multiforms of the large chondroitin sulphate proteoglycan termed PG-M (versican is one of the short transcripts). Since PG-M/versican inhibits many types of cell-substratum adhesion and is found to be excluded from focal contacts of cultured fibroblasts, it is likely that this proteoglycan is generally involved in regulating cell-substratum adhesion. We report here that PG-M/versican is selectively excluded from podosomes of human osteosarcoma cells and that specific inhibition of its biosynthesis by an antisense method suppresses such a malignant cell-adhesive phenotype. The results support the idea that PG-M/versican acts as an anti-adhesive molecule and raise the possibility that PG-M/versican controls one type of cancer cell behaviour.
Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2
Authors: Mayer-Jaekel Regina E. , Ohkura Hiroyuki , Ferrigno Paul , Andjelkovic Natasa , Shiomi Kensuke , Uemura Tadashi , Glover David M. , Hemmings Brian A. 
Published: April 2021

ABSTRACT
The 55 kDa regulatory subunit of Drosophila protein phos-phatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. We are unable to detect significant change in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar1 and twinsP, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phospho-rylated by p34cdc2/cyclin B kinase, but not against phos-phorylase a. Thus the mitotic defects of aar1 and twinsP are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A.
Inhibition of 3T3-L1 adipose differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin
Authors: Phillips Marjorie , Enan Essam , Liu Phillip C. C. , Matsumura Fumio 
Published: April 2021

ABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity is particularly striking in adipose tissue, where it causes severe wasting. This phenomenon suggests that TCDD could have effects on adipocyte differentiation, now demonstrated using 3T3-L1 cells as a model system. When cells were treated with 10 nM TCDD before differentiation or during the first two days of induction in the presence of dexamethasone (dex) and isobutylmethylxanthine (IBMX), a reduction occurred in the number of fat cell colonies measured 7-10 days later by Oil Red O staining. Northern blotting showed an accompanying reduction in amounts of mRNA encoding several adipocyte markers. In contrast, when TCDD was added after differentiation, it had no effect on the maintenance of the adipose phenotype. Dose-response and structure-activity relationships were consistent with a process mediated by interaction of TCDD with the Ah receptor. The possibility that TCDD acts by inhibiting the signaling pathways activated by dex and IBMX was investigated. TCDD did not interfere with glucocorticoid-inducible transcription as judged by the unimpaired responsiveness of a transfected reporter construct. Treatment of cells with TCDD augmented the increase in protein kinase A (PKA) activity elicited by either IBMX or forskolin; therefore, if TCDD disrupts the cAMP signaling pathway, it must do so at a step after activation of PKA.
Down regulation of talin alters cell adhesion and the processing of the α5β1 integrin
Authors: Albigès-Rizo Corinne , Frachet Philippe , Block Marc R. 
Published: April 2021

ABSTRACT
The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5′ cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mocktransfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both α5 and β1 subunits. Surprisingly, β1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of β1. We analyzed the processing of β1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature β1, while the talin deficient AT22 clone showed that the β1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of β1 integrins.
Identification and cDNA cloning of a Xenopus nucleolar phosphoprotein, xNopp180, that is the homolog of the rat nucleolar protein Nopp140
Authors: Cairns Carol , McStay Brian 
Published: April 2021

ABSTRACT
The monoclonal antibody G1C7, recognises both Xenopus nucleolin and a protein of 180 kDa present in Xenopus oocyte nucleoli. This antibody was used to obtain a cDNA clone encoding the 180 kDa protein now called xNopp180 (Xenopus nucleolar phosphoprotein of 180 kDa). Analysis of the deduced amino acid sequence from this cDNA shows that xNopp180 is almost entirely composed of alternating acidic and basic domains. We show that xNopp180 is heavily phosphorylated and that it contains multiple consensus sites for phosphorylation by casein kinase II and cdc2 kinase. In addition we show that xNopp180 is the 180 kDa antigen recognised by the monoclonal antibody No-114, thus allowing reinterpretation of previous work with this antibody. xNopp180 appears to be the Xenopus homolog of the rat nucleolar protein Nopp140. Nopp140 is a nuclear localisation signal binding protein that shuttles on curvilinear tracks between the nucleolus and the cytoplasm. Possible roles for xNopp180/Nopp140 in ribosome biogenesis are discussed.
Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth
Authors: Kaminskyj Susan G. W. , Heath I. Brent 
Published: April 2021

ABSTRACT
Saprolegnia ferax contains an integrin homologue, identified by crossreactivity with antiserum to the consensus sequence of human/chick/Xenopus cytoplasmic domain β1-integrin, which is highly conserved. In non-reduced samples, this integrin was larger than the reported size range for β1-integrins, at 178 kDa. In reduced samples, there was a reducing agent-concentration-dependent conversion from 178 kDa to 120 kDa, well within the reported size range for β1-integrins in other organisms. The integrin antiserum stained plasma membrane-associated patches, which had a shallow tip-high gradient. This population was reduced and its distribution perturbed in hyphae whose growth rate was reduced by half with tetrapentyl ammonium chloride. The expected integrin function in cytoplasm-cell wall attachment was shown by differential resistance to plasmolysis-induced separation, which positively correlated with integrin abundance. However, when there was separation, remnants of cytoplasm stayed attached to the wall. These were enriched in actin and integrin. Saprolegnia also has a spectrin homologue identified by crossreactivity with an erythrocyte spectin antibody, which has a size (246 kDa) similar to other organisms. This spectrin had a superficially similar distribution to that of integrin, but it did not participate in cytoplasm-wall anchoring. These data suggest that Saprolegnia hyphae have a plasma membrane which is strengthened by spectrin, and cytoplasm which is attached to the cell wall by integrin.