bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell cycle-associated accumulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) in the nuclei of human gingival fibroblasts
Authors: Zhao Wan-Qian , Li Hang , Yamashita Kyoko , Guo Xiao-Kui , Hoshino Takeshi , Yoshida Shonen , Shinya Takashi , Hayakawa Taro 
Published: April 2021

ABSTRACT
We first confirmed an earlier immunohistochemical study showing that immunoreactive TIMP-1-like protein accumulated in the nuclei of human gingival fibroblasts (Gin-1 cells), reaching a maximum in the S phase of the cell cycle (Li, H., Nishio, K., Yamashita, K., Hayakawa, T. and Hoshino, T. (1995). Nagoya J. Med. Sci. 58, 133-142). Then we isolated this protein from a nuclear extract of Gin-1 cells and demonstrated it to be identical to human recombinant TIMP-1 by western blotting, by a sandwich enzyme immunoassay for TIMP-1 and by an assay for matrix metalloproteinase inhibition. The amount of TIMP-1 in the cytosolic fraction of quiescent Gin-1 cells after stimulation by fetal calf serum increased continuously for 48 hours, whereas that in the nuclear extract showed a maximum at 24 hours (S phase) and significantly decreased thereafter. Gin-1 cells expressed mRNAs for both TIMP-2 and TIMP-3 together with mRNA for TIMP-1. However, neither TIMP-2 nor TIMP-3 proteins seemed to accumulate in the nuclei of Gin-1 cells. These facts strongly suggest that TIMP-1 accumulates specifically in the nuclei of Gin-1 cells in a cell cycle-dependent manner.
Vitronectin regulates smooth muscle contractility via αv and β1 integrin(s)
Authors: Dahm Lisa M. , Bowers Chauncey W. 
Published: April 2021

ABSTRACT
Previous work from this laboratory has established a method for maintaining physiological contractility of dissociated avian smooth muscle in a defined medium at low density. The present report emphasizes the dramatic potency of serum to alter smooth muscle phenotype and induce a loss of contractility. Vitronectin, a molecule purified from plasma, mimicked these effects of serum via an integrin that is RGD-sensitive. Studies utilizing blocking antibodies against vitronectin demonstrated that the presence of this specific adhesion molecule was necessary for the serum-induced loss of contractility. Based on the actions of function-blocking antibodies and RGD-containing peptides, the integrin αvβ1 appears to be the primary receptor involved in vitronectin’s ability to induce phenotypic transformation in amniotic smooth muscle. The influence of vitronectin on smooth muscle contractility is particularly relevant, because this molecule is abundant in whole blood and plasma (approx. 400 μg/ml). The results suggest that smooth muscle needs to be continually protected from normal blood constituents in vivo. The implications of these results for smooth muscle-related diseases like atherosclerosis, restenosis and Kaposi’s sarcoma are discussed.
Opposite sorting and transcytosis of the polymeric immunoglobulin receptor in transfected endothelial and epithelial cells
Authors: Su Tao , Stanley Keith K. 
Published: April 2021

ABSTRACT
We have transfected a polarised endothelial cell line, ECV 304, and an epithelial cell line, MDCK, with a well characterised epithelial protein, the rat polymeric immunoglobulin receptor (pIgR), in order to study the protein sorting and transcytosis in endothelial cells. The expressed protein was normally processed and the steady state distribution between apical and basolateral surfaces was similar in both cell types. MDCK cells, however, showed a marked polarity in the delivery of newly synthesised pIgR to the cell surface, and in the release of secretory component. 88% of newly synthesised pIgR in MDCK cells was first delivered to the basolateral surface and 99% of secretory component was released from the apical surface. In contrast the basolateral targeting signal of pIgR was only partially recognised in endothelial cells, with 63% of the newly synthesised pIgR being first delivered to the basolateral surface. At steady state only 43% of the pIgR was found on the basolateral membrane. The direction of dimeric IgA transcytosis in endothelial cells was from apical to basolateral surfaces, opposite to that in MDCK cells. These data suggest that endothelial cells poorly recognise the targeting signals of proteins from epithelial cells, and that the direction of transcytosis is linked to the biological role of the cells.
Identification of an interchromosomal compartment by polymerization of nuclear-targeted vimentin
Authors: Bridger Joanna M. , Herrmann Harald , Münkel Christian , Lichter Peter 
Published: April 2021

ABSTRACT
A number of structural and functional subnuclear compartments have been described, including regions exclusive of chromosomes previously hypothesized to form a reactive nuclear space. We have now explored this accessible nuclear space and interchromosomal nucleoplasmic domains experimentally using Xenopus vimentin engineered to contain a nuclear localization signal (NLS-vimentin). In stably transfected human cells incubated at 37°C, the NLS-vimentin formed a restricted number of intranuclear speckles. At 28°C, the optimal temperature for assembly of the amphibian protein, NLS-vimentin progressively extended with time out from the speckles into strictly orientated intranuclear filamentous arrays. This enabled us to observe the development of a system of interconnecting channel-like areas. Quantitative analysis based on 3-D imaging microscopy revealed that these arrays were localized almost exclusively outside of chromosome territories. During mitosis the filaments disassembled and dispersed throughout the cytoplasm, while in anaphase-telophase the vimentin was recruited back into the nucleus and reassembled into filaments at the chromosome surfaces, in distributions virtually identical to those observed in the previous interphase. The filaments also colocalized with specific nuclear RNAs, coiled bodies and PML bodies, all situated outside of chromosome territories, thereby interlinking these structures. This strongly implies that these nuclear entities coexist in the same interconnected nuclear compartment. The assembling NLS-vimentin is restricted to and can be used to delineate, at least in part, the formerly proposed reticular interchromosomal domain compartment (ICD). The properties of NLS-vimentin make it an excellent tool for performing structural and functional studies on this compartment.
Microtubules facilitate the stimulated secretion of β-hexosaminidase in lacrimal acinar cells
Authors: Costa Silvia R. da , Yarber Francie A. , Zhang Liping , Sonee Manisha , Hamm-Alvarez Sarah F. 
Published: April 2021

ABSTRACT
Stimulation of lacrimal acini with secretagogues such as carbachol initiates movement and fusion of acinar secretory vesicles with the apical plasma membrane, resulting in release of protein into the nascent tear fluid. Using rabbit lacrimal acini reconstituted in vitro from isolated cells, we have investigated the organization of the apical cytoskeleton and its role in stimulated secretion. Confocal microscopy revealed a microtubule array emanating from the apical region of the acini; the apical region was also enriched in microfilaments and γ-tubulin. Cytokeratin-based intermediate filaments were apically concentrated, and also detected at the cell periphery. Neither confocal microscopy nor biochemical analysis revealed any reorganization of lumenal microfilaments or microtubules which might accompany carbachol-stimulated release of secretory proteins. However, major changes in the acinar microtubule array induced by taxol or nocodazole were correlated with inhibition of carbachol-dependent release of the secreted protein, β-hexosaminidase. Major changes in lumenal microfilaments induced by jasplakinolide or cytochalasin D did not inhibit the carbachol-dependent release of β-hexosaminidase; rather, release of β-hexosaminidase from jasplakinolide-or cytochalasin D-treated carbachol-stimulated acini was markedly increased relative to the release from untreated stimulated acini. Our findings demonstrate that microtubules play a major role in stimulated lacrimal secretion, and suggest a contributory role for microfilaments.
Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells
Authors: Norvell Suzanne M. , Green Kathleen J. 
Published: April 2021

ABSTRACT
The integrity of cell-cell junctions in epithelial cells depends on functional interactions of both extracellular and intracellular domains of cadherins with other junction proteins. To examine the roles of the different domains of E-cadherin and desmoglein in epithelial junctions, we stably expressed full length desmoglein 1 and chimeras of E-cadherin and desmoglein 1 in A431 epithelial cells. Full length desmoglein 1 was able to incorporate into or disrupt endogenous desmosomes depending on expression level. Each of the chimeric cadherin molecules exhibited distinct localization patterns at the cell surface. A chimera of the desmoglein 1 extracellular domain and the E-cadherin intracellular domain was distributed diffusely at the cell surface while the reverse chimera, comprising the E-cadherin extracellular domain and the desmoglein 1 intracellular domain, localized in large, sometimes contiguous patches at cell-cell interfaces. Nevertheless, both constructs disrupted desmosome assembly. Expression of constructs containing the desmoglein 1 cytoplasmic domain resulted in approximately a 3-fold decrease in E-cadherin bound to plakoglobin and a 5-to 10-fold reduction in the steady-state levels of the endogenous desmosomal cadherins, desmoglein 2 and desmocollin 2, possibly contributing to the dominant negative effect of the desmoglein 1 tail. In addition, biochemical analysis of protein complexes in the stable lines revealed novel in vivo protein interactions. Complexes containing β-catenin and desmoglein 1 were identified in cells expressing constructs containing the desmoglein 1 tail. Furthermore, interactions were identified between endogenous E-cadherin and the chimera containing the E-cadherin extracellular domain and the desmoglein 1 intracellular domain providing in vivo evidence for previously predicted lateral interactions of E-cadherin extracellular domains.
Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: In vivo imaging with GFP fusion proteins
Authors: Amieva Manuel R. , Litman Pninit , Ichimaru Laiqiang Huang, Eiji , Furthmayr Heinz 
Published: April 2021

ABSTRACT
Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, &gt;90% of N-moesin and &gt;60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.
Localization of intracellular Ca2+ stores in HeLa cells during infection with Chlamydia trachomatis
Authors: Majeed Meytham , Krause Karl-Heinz , Clark Robert A. , Kihlström Erik , Stendahl Olle 
Published: April 2021

ABSTRACT
Chlamydia trachomatis elementary bodies (EBs) enter epithelial cells within membrane-bound endosomes that aggregate with each other in a calcium-regulated process, but avoid fusion with lysosomes. Annexin III but not I translocates to Chlamydia l aggregates and inclusions. In this study, we localize the intracellular Ca2+ stores during the course of infection by analyzing the distribution of three intracellular Ca2+ store proteins: calreticulin, type-1 inositol-1,4,5-trisphosphate receptor (IP3-R), and Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase type 2 (SERCA2) in HeLa cells infected with C. trachomatis serovar L2. In uninfected cells, immunofluorescence staining of the proteins showed a fine granular distributed pattern for all three proteins. After infection with C. trachomatis, calreticulin was found at the periphery of Chlamydia l aggregates and inclusions from 3 to 48 hours post-infection. In infected cells, SERCA2 was intimately associated with Chlamydia l inclusions after 3 and 24 hours, but not after 48 hours. Moreover, IP3-R was translocated to and colocalized with EB aggregates and Chlamydia l inclusions and had a distribution very similar to that of SERCA 2.
After 24 hours incubation with Chlamydia e, there was a local accumulation of [Ca2+]i (105±17 nM) in the proximity of Chlamydia l inclusions, compared to 50±13 nM in other parts of the cell cytoplasm. In the absence of extracellular Ca2+, this local accumulation of Ca2+ increased to 295±50 nM after adding 50 µM ATP, and to a similar extent after adding 100 nM thapsigargin (Tg). These data indicate that during infection of HeLa cells with Chlamydia e, intracellular Ca2+ stores are redistributed, causing local accumulation of Ca2+ in the vicinity of Chlamydia l inclusions. These changes may trigger the association of certain proteins such as annexins with Chlamydia - containing vesicles, and thereby regulation of membrane-membrane interaction during endosome aggregation and inclusion formation.
Key words: Intracellular Ca2+ store protein, Chlamydia, Thapsigargin, Elementary body, HeLa cell
The M-cadherin catenin complex interacts with microtubules in skeletal muscle cells: Implications for the fusion of myoblasts
Authors: Kaufmann Ulrike , Kirsch Joachim , Irintchev Andrej , Wernig Anton , Starzinski-Powitz Anna 
Published: April 2021

ABSTRACT
M-cadherin, a calcium-dependent intercellular adhesion molecule, is expressed in skeletal muscle cells. Its pattern of expression, both in vivo and in cell culture as well as functional studies, have implied that M-cadherin is important for skeletal muscle development, in particular the fusion of myoblasts into myotubes. M-cadherin formed complexes with the catenins in skeletal muscle cells similar to E-cadherin in epithelial cells. This suggested that the muscle-specific function of the M-cadherin catenin complex might be mediated by additional interactions with yet unidentified cellular components, especially cytoskeletal elements. These include the microtubules which also have been implicated in the fusion process of myoblasts. Here we present evidence that the M-cadherin catenin complex interacts with microtubules in myogenic cells by using three independent experimental approaches. (1) Analysis by laser scan microscopy revealed that the destruction of microtubules by nocodazole leads to an altered cell surface distribution of M-cadherin in differentiating myogenic cells. In contrast, disruption of actin filaments had little effect on the surface distribution of M-cadherin. (2) M-cadherin antibodies coimmunoprecipitated tubulin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated epithelial cells ectopically expressing M-cadherin. Vice versa, tubulin antibodies coimmunoprecipitated M-cadherin from extracts of nocodazole-treated myogenic cells but not of nocodazoletreated M-cadherin-expressing epithelial cells. (3) M-cadherin and the catenins, but not a panel of control proteins, were copolymerized with tubulin from myogenic cell extracts even after repeated cycles of assembly and disassemly of tubulin. Moreover, neither M-cadherin nor E-cadherin could be found in a complex with microtubules in epithelial cells ectopically expressing M-cadherin. Our data are consistent with the idea that the interaction of M-cadherin with microtubules might be essential to keep the myoblasts aligned during fusion, a process in which both M-cadherin and microtubules have been implicated.
The subapical actin cytoskeleton regulates secretion and membrane retrieval in pancreatic acinar cells
Authors: Valentijn Karine M. , Gumkowski Francine D. , Jamieson James D. 
Published: April 2021

ABSTRACT
We examined the effects of disruption of the actin cytoskeleton by cytochalasin D (cytoD) on basal and carbamylcholine-stimulated exocytosis and on compensatory membrane retrieval in pancreatic acinar cells. Although the involvement of actin in exocytosis is reasonably well established, its role in these coupled processes is not understood. Our findings suggested that cytoD inhibited stimulated secretion of amylase. However, morphometry revealed that exocytosis had occurred: the number of zymogen granules decreased, the size of the lumen increased, and large vacuolar structures continuous with the lumen formed into which amylase accumulated. Large amounts of amylase were released to the medium on removal of secretagogue and cytoD, suggesting that the subapical actin network provides contractile forces that expel the lumenal contents. Strikingly, we observed that at the apical pole of the cells where exocytosis occurred, cytoD induced an accumulation of membrane invaginations into a vastly enlarged apical membrane. These pits were often surrounded by a clathrin-like coat. Concomitantly, AP-2-, clathrin-, dynamin- and caveolin-like immunoreactivity concentrated around the enlarged lumina, suggesting that incorporation of zymogen granule membrane into the apical plasma membrane triggered the recruitment of these proteins. After wash out of cytoD and carbamylcholine and reformation of the subapical actin cytoskeleton, the coated invaginations largely disappeared in association with a reduction in lumenal size, and relocation of clathrin, AP-2, dynamin and caveolin into the cell. We suggest that the actin terminal web also controls compensatory membrane retrieval following exocytosis.
Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends
Authors: Vaughan Kevin T. , Tynan Sharon H. , Faulkner Nicole E. , Echeverri Christophe J. , Vallee Richard B. 
Published: April 2021

ABSTRACT
Cytoplasmic dynein is a minus end-directed microtubule motor responsible for centripetal organelle movement and several aspects of chromosome segregation. Our search for cytoplasmic dynein-interacting proteins has implicated the dynactin complex as the cytoplasmic dynein ‘receptor’ on organelles and kinetochores. Immunofluorescence microscopy using a total of six antibodies generated against the p150Glued, Arp1 and dynamitin subunits of dynactin revealed a novel fraction of dynactin-positive structures aligned in linear arrays along the distal segments of interphase microtubules. Dynactin staining revealed that these structures colocalized extensively with CLIP-170. Cytoplasmic dynein staining was undetectable, but extensive colocalization with dynactin became evident upon transfer to a lower temperature. Overexpression of the dynamitin subunit of dynactin removed Arp1 from microtubules but did not affect microtubule-associated p150Glued or CLIP-170 staining. Brief acetate treatment, which has been shown to affect lysosomal and endosomal traffic, also dispersed the Golgi apparatus and eliminated the microtubule-associated staining pattern. The effect on dynactin was rapidly reversible and, following acetate washout, punctate dynactin was detected at microtubule ends within 3 minutes. Together, these findings identify a region along the distal segments of microtubules where dynactin and CLIP-170 colocalize. Because CLIP-170 has been reported to mark growing microtubule ends, our results indicate a similar relationship for dynactin. The functional interaction between dynactin and cytoplasmic dynein further suggests that this these regions represent accumulations of cytoplasmic dynein cargo-loading sites involved in the early stages of minus end-directed organelle transport.
Pulsatile influxes of H+, K+ and Ca2+ lag growth pulses of Lilium longiflorum pollen tubes
Authors: Messerli Mark A. , Danuser Gaudenz , Robinson Kenneth R. 
Published: April 2021

ABSTRACT
Fluxes of H+, K+ and Ca2+ were measured with selfreferencing ion-selective probes, near the plasma membrane of growing Lilium longiflorum pollen tubes. Measurements from three regions around short, steadygrowing tubes showed small, steady influx of H+ over the distal 40 μm and a region of the tube within 50-100 μm of the grain with larger magnitude efflux from the grain. K+ fluxes were immeasurable in short tubes. Measurements of longer tubes that were growing in a pulsatile manner revealed a pulsatile influx of both H+ and K+ at the growing tip. The average fluxes at the cell surface during the peaks of the H+ and K+ pulses were 489±81 and 688±144 pmol cm−2 second−1, respectively. Growth was measured by tracking the pollen tips with a computer vision system that achieved a spatial resolution of approximately 1/10 pixel. The high spatial resolution enabled the detection of growth, and thus the changes in growth rates, with a temporal sampling rate of 1 frame/second. These data show that the H+ and K+ pulses have a phase lag of 103±9 and 100±11 degrees, respectively, with respect to the growth pulses.
Calcium fluxes were also measured in growing tubes. During steady growth, the calcium influx was relatively steady. When pulsatile growth began, the basal Ca2+ influx decreased and a pulsatile component appeared, superimposed on the reduced basal Ca2+ flux. The peaks of the Ca2+ pulses at the cell surface averaged 38.4±2.5 pmol cm−2 second−1. Longer tubes had large pulsatile Ca2+ fluxes with smaller baseline fluxes. The Ca2+ influx pulses had a phase lag of 123±9 degrees with respect to the growth pulses.