bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular relationship between actin and alpha-actinin in a whole corneal epithelial tissue
Authors: Khoory Wissam , Wu Elisa , Svoboda Kathy Kay Hartford 
Published: April 2021

ABSTRACT
Alpha-actinin is an actin crosslinking protein that may be one of the proteins involved in the attachment of the actin cytoskeletal framework to the plasma membrane. We investigated the distribution of-actinin in whole-mount embryonic chick corneal epithelia using confocal laser scanning analysis. The intracellular-actinin dis-tribution was compared with F-actin using phalloidin, or total actin using an anti-actin antibody. Corneal epithelial tissues were isolated with or without the basal lamina (+ or BL), and fixed immediately. In addition, epithelia isolated BL were cultured for 2 hours with either control medium, laminin-supplemented medium or laminin and cytochalasin D (CD)-containing medium. The single- and double-labeled epithelia showed that-actinin delineated the cell borders and microvilli of the periderm cells in the most apical optical sections of control and laminin-treated epithelia. At the optical plane through the basal cell nuclei, the-actinin was distributed diffusely throughout the cytoplasm, whereas the actin was sparse, only associated with the lateral cell membranes. Epithelia (BL) cultured in control medium had cytoplasmic protrusions or blebs on the basal cell surface. The blebs contained both actin and α-actinin. In epithelia cultured with laminin, the basal cell surface was flat. The actin cortical mat became reorganized within two hours. Actin and-actinin were colocalized in the re-formed basal cytoskeletal network. In cells cultured with cytochalasin D (CD) and laminin the actin cortical mat was not reorganized. Actin net-works from both cell layers were eliminated and replaced by aggregates scattered throughout the cyto-plasm. The-actinin remained diffusely distributed in the cytoplasm and failed to colocalize with the actin aggregates. The-actinin appeared closer to the basal cell membrane than the actin in cross-sectional views of the tissue. Results from these double-labeling experiments confirmed the intimate association of alpha-actinin and actin in the laminin-stimulated actin cortical mat reorganization. This study is the first to demonstrate that CD-aggregated F-actin does not capture the-actinin. The-actinin appeared to remain diffuse throughout the cytoplasm and separate from F-actin; however, there was some overlap with G-actin.
The analysis of 40 kDa nuclear protein, p40, in interphase cells and mitotic cells
Authors: Kaneda Yoshinao , Kinoshita Katsunari , Sato Manabu , Tanaka Kiyoji , Kaneda Yasufumi 
Published: April 2021

ABSTRACT
We previously reported that the monoclonal antibody M108 recognized a 40 kDa protein both in the nucleus and the cytoplasm. This nuclear 40 kDa antigen was located in the nuclear envelope in interphase cells and in the perichromosomal region during mitosis. Now, we have analyzed this nuclear 40 kDa protein (p40) further, through morphological and biochemical approaches. At the beginning of mitosis, the perinuclear p40 detached from the nuclear envelope and moved to surround the condensing chromatin, while in the late stage of mitosis, the perichromosomal p40 moved back to the reassembled nuclear envelope. Most of the perichromo-somal p40 on the metaphase chromosome was solubilized only by DNase I treatment, not by either high salt or detergent treatment. On the other hand, the perinuclear p40 was not solubilized by DNase1 alone, or high salt detergent alone. Sequential treatments with DNase I and high salt detergent were required to extract p40 in interphase nuclei. These results suggest that p40 was associated both with the nuclear envelope and chro-matin DNA in interphase nuclei, while it bound only to chromatin DNA in mitosis.
Involvement of Ypt7p, a small GTPase, in traffic from late endosome to the vacuole in yeast
Authors: Schimmöller Frauke , Riezman Howard 
Published: April 2021

ABSTRACT
The YPT7 gene encodes the Saccharomyces cerevisiae homolog of mammalian rab7 protein. Data obtained from studies on a ypt7 mutant suggested that Ypt7p is involved in the endocytic pathway in yeast (Wichmann et al., Cell 71, 1131-1142, 1992). We report here that endocytosed pheromonefactor accumulates in late endosomes in ypt7 cells, indicating that Ypt7p is involved in the regulation of transport steps from late endosomes to the vacuole. We also show that-factor can be degraded in a PEP4-dependent manner in a pre-vacuolar/endosomal compartment in ypt7 cells, pro-viding independent evidence that the pathways of vac-uole biogenesis and endocytosis in yeast may intersect in the endosomal membrane system.
Cultured microvascular endothelial cells (MVEC) differ in cytoskeleton, expression of cadherins and fibronectin matrix A study under the influence of interferon-γ
Authors: Fenyves A. M. , Behrens J. , Spanel-Borowski K. 
Published: April 2021

ABSTRACT
Endothelial cells are known to undergo transitions in cell shape during long-term culture. Thus, the assumption that the separate phenotypes of microvascular endothelial cells (MVEC) recently isolated from bovine corpus luteum represent constitutively different cell strains cannot automatically be made. For this reason, particular morphological qualities from four of five reported MVEC types were studied. Confluent cultures of MVEC types 1, 3, 4 and 5 were either left untreated or exposed to recombinant bovine interferon-(IFN-; 200 units/0.5 ml culture medium) for 3 days. Paraformaldehyde-fixed monolayers were permeabilized with TritonR X-100 prior to the detection of filamentous actin, using phalloidin-FITC. Vimentin filaments, cytokeratin filaments, microtubules, E- and N-cadherins as molecules of cell adhesion plaques, and fibronectin filaments were localized by the application of specific antibodies in combination with epifluorescence microscopy. Cells from untreated single cultures uniformly and reproducibly showed an actin cytoskele-ton that distinguished the particular MVEC type. MVEC type 1 presented a circular band of fine actin fil-aments. MVEC type 3 preferentially had developed a starburst-like actin pattern. MVEC type 4 mainly exhibited a polygonal network. MVEC type 5 showed a prominent circular band of thick microfilament bundles from which short filaments radiated. Cytokeratin filaments were noted in MVEC type 1 only. Vimentin filaments occurred as a dense network constricted to the central area in MVEC type 1, while they were spread out in MVEC types 3 and 4. A wavy path comparable to the course of microtubules was apparent in MVEC type 5. Fibronectin assembled into two differently shaped layers at the basal cell side of each MVEC type. Under IFN-treatment, cytoskeletal diversities were maintained between the MVEC types, yet each MVEC type showed specific modulations to its cytoskeleton and to its fibronectin matrix. Upregulation of anti-E-cad-herin labelling was detected in MVEC type 1, showing a fluorescent cell border of linear contour. The upregulation of E-cadherin by IFN-treatment could also be demonstrated by western blotting, which revealed a 135 kDa full-sized molecule and a 95 kDa tryptic fragment characteristic of cadherins. Anti-N-cadherin labelling was evident for MVEC type 5, giving rise to a fluorescent punctate cell margin. Our investigations support the existence of truly separate MVEC types.
Tektin B1 from ciliary microtubules: primary structure as deduced from the cDNA sequence and comparison with tektin A1
Authors: Chen Runtian , Perrone Catherine A. , Amos Linda A. , Linck Richard W. 
Published: April 2021

ABSTRACT
Tektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar micro-tubules, and they may also be present in centrioles, cen-trosomes and mitotic spindles. We report here the cloning and sequencing of a cDNA for ciliary tektin B1. Comparison of the predicted amino acid sequence of tektin B1 with the previously published sequence for tektin A1 reveals several features that better define this class of proteins. Like tektin A1, the central region of the tektin B1 polypeptide chain is predicted to form a coiled-coil rod, consisting of four majorhelical regions that are separated by non-helical linkers. Between the central rod domains of tektins A and B there is a 34%/20% amino acid sequence identity/similarity, including equivalent 50-residue segments containing 36 identities, and a high probability of long-range structural homology. The tektin polypeptide chains are divided into two major segments that have significant sequence homology to each other, both within a given tektin chain and between tektins A and B, indicative of gene duplication events. The tektins have a secondary structure and molecular design similar to, but a low primary sequence homology with, intermedi-ate filament proteins. Unlike tektin A1, tektin B1 lacks any part of the C-terminal IFP consensus sequence.
HIV-induced syncytia of a T cell line form single giant pseudopods and are motile
Authors: Sylwester Andrew , Wessels Deborah , Anderson Stephanie A. , Warren Ronald Q. , Shutt Damon C. , Kennedy Ronald C. , Soll David R. 
Published: April 2021

ABSTRACT
The human immunodeficiency virus, HIV, induces syncytium formation in cultures of many T cell lines. These syncytia have previously been viewed as disorganized fusion products in the throes of death. Evidence is presented that in HIV1-infected SupT1 cultures, syncytia five times to over one hundred times larger than single cells organize their many nuclei into blastula-like balls, reorganize their cytoskeleton to mimic that of a single cell, and extend single, giant pseudopods in a polar fashion. Medium-sized syncytia are capable of translocation through extension of these giant pseudopods. The rate of translocation of syncytia is comparable to that of single cells. Single cell motility, syncytium motility and pseudopod extension also appear to play roles in the recruitment of cells into syncytia. Finally, condensation of F-actin at cell-syncytium and syncytium-syncytium adhesion sites suggests the involvement of the cytoskeleton in the adhesion and/or subsequent fusion event. These results suggest that the fusion events involved in HIV-induced syncytia formation involve both cell motility and reorganization of the cytoskeleton, and demonstrate that syncytia are highly organized, motile entities.
Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle
Authors: Fang Fang , Newport John W. 
Published: April 2021

ABSTRACT
RP-A is a single-stranded DNA-binding protein, which has been shown to be required for DNA replication using an SV40 model system. The protein has also been shown to be phosphorylated at the G1-S phase transition. Using Xenopus cell-free extracts we have investigated the role of RP-A in nuclear replication and characterized the kinases and conditions that lead to phosphorylation of RP-A during the cell cycle. By immunodepleting RP-A from Xenopus extracts we have shown that RP-A is essential for replication of chromosomal DNA. Our results show that, during S phase, only that RP-A which is associated with nuclei is phosphorylated. Furthermore our results indicate that during S phase RP-A is only phosphorylated when associated with single-stranded DNA. By immunodepleting cdk2 kinase we show that cdk2 kinase is required for the observed phosphorylation of RP-A in nuclei during S phase. However, using purified cdk2 kinase and RP-A we are unable to detect a direct phosphorylation of RPA by cdk2 kinase. This observation suggests that phos-phorylation of DNA-bound RP-A at S phase is carried out by a kinase distinct from cdk2. Consistent with this we find that when single-stranded DNA is added to S phase extracts depleted of cdk2 kinase, RP-A is phos-phorylated. Together these results suggest that cdk2 kinase participates in the activation of DNA replication at a stage prior to the binding of RP-A to the initiation complex. In addition to RP-A phosphorylation in S phase, we have also found that at the onset of mitosis RP-A is quantitatively phosphorylated and that phos-phorylation is directly mediated by cdc2 kinase. How-ever, at this time during the cell cycle, cdc2-dependent phosphorylation of RP-A is independant of DNA bind-ing. These observations further demonstrate the dis-tinctions between cdk2 and cdc2 kinases.
A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules
Authors: Dráberová Eduarda , Dráber Pavel 
Published: April 2021

ABSTRACT
A protein of Mr 210 000 was identified in 3T3 cells by immunoblotting and by immunoprecipitation with a monoclonal antibody MA-01. The protein was thermo-labile and was located on 3T3 microtubules prepared by taxol-driven polymerization in vitro. On fixed cells the MA-01 antigen was located on interphase and mitotic microtubular structures, vinblastine paracrystals, taxol bundles and colcemid-resistant microtubules. Microin-jection experiments with purified MA-01 antibody fol-lowed by double immunofluorescence have shown that the injection of antibody led to disruption of vimentin filaments, whereas the distribution of cytoplasmic microtubules was unchanged. The collapse of vimentin filaments started 30 minutes after injecting the antibody at immunoglobulin concentrations 2 mg ml 1 or higher and reached its maximum 3-6 hours after the injection. Within 20 hours after the injection vimentin filaments became reconstituted. Microinjection of the antibody into cells pre-treated with vinblastine resulted in local-ization of the MA-01 antigen on vinblastine paracrys-tals as well as on coiled vimentin filaments. The data presented suggest that the MA-01 antigen is a new microtubule-interacting protein that mediates, directly or indirectly, an interaction between microtubules and vimentin intermediate filaments.
Molecular mechanisms of neural crest cell attachment and migration on types I and IV collagen
Authors: Perris Roberto , Syfrig Josef , Paulsson Mats , Bronner-Fraser Marianne 
Published: April 2021

ABSTRACT
We have examined the mechanisms involved in the interaction of avian neural crest cells with collagen types I and IV (Col I and IV) during their adhesion and migration in vitro. For this purpose native Col IV was purified from chicken tissues, characterized biochemically and ultrastructurally. Purified chicken Col I and Col IV, and various proteolytic fragments of the collagens, were used in quantitative cell attachment and migration assays in conjunction with domain-specific collagen antibodies and antibodies to avian integrin sub-units. Neural crest cells do not distinguish between different macromolecular arrangements of Col I during their initial attachment, but do so during their migration, showing a clear preference for polymeric Col I. Interaction with Col I is mediated by the α1 β1 integrin, through binding to a segment of the β1(I) chain composed of fragment CNBr3. Neural crest cell attachment and migration on Col IV involves recognition of con-formation-dependent sites within the triple-helical region and the noncollagenous, carboxyl-terminal NC1 domain. This recognition requires integrity of inter- and intrachain disulfide linkages and correct folding of the molecule. Moreover, there also is evidence that interaction sites within the NC1 domain may be cryptic, being exposed during migration of the cells in the intact collagen as a result of the prolonged cell-substratum con-tact. In contrast to Col I, neural crest cell interaction with Col IV is mediated by β1-class integrins other than α1 β1.
In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium
Authors: Walczak Claire E. , Nelson David L. 
Published: April 2021

ABSTRACT
Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca2+-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity.
In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca2+-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.
The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling
Authors: Dauvois Sophie , White Roger , Parker Malcolm G. 
Published: April 2021

ABSTRACT
The mouse estrogen receptor was shown to be constantly shuttling between the nucleus and cytoplasm although under steady-state conditions it is detected predomi-nantly in the cell nucleus in both the absence and pres-ence of estradiol. Shuttling was demonstrated by moni-toring the transfer of protein between nuclei in heterokaryons and by examining the subcellular distri-bution of mutant receptors. In the presence of the par-tial antiestrogen 4-hydroxytamoxifen the receptor was retained in the nucleus whereas it accumulated in the cytoplasm when cells were treated with the pure antie-strogen ICI 182780. The effect of the pure antiestrogen was to inhibit nucleocytoplasmic shuttling of the recep-tor by blocking its nuclear uptake. Thus although ligand binding is not required by the estrogen receptor to undergo nucleocytoplasmic shuttling, this process can be disrupted by the binding of a pure antiestrogen.
Intercellular calcium signaling induced by extracellular adenosine 5′-triphosphate and mechanical stimulation in airway epithelial cells
Authors: Hansen Michael , Boitano Scott , Dirksen Ellen R. , Sanderson Michael J. 
Published: April 2021

ABSTRACT
Airway epithelial cells in culture respond to extracellular adenosine 5′-triphosphate (ATP) by increasing their intracellular Ca2+ concentration ([Ca2+]i). The effective concentration of ATP that elicited a Ca2+ response equal to 50% of the maximal response (EC50) was 0.5 μM. Release of ATP from a pipette to form a local gradient of ATP increased [Ca2+]i of individual cells in a sequential manner. Cells closest to the pipette showed an immediate increase in [Ca2+]i while more distal cells displayed a delayed increase in [Ca2+]i. This response to the local release of ATP appeared as a wave of increasing [Ca2+]i that spread to several cells and, in this respect, was similar to the intercellularly communicated Ca2+ waves initiated by mechanical stimulation in airway epithelial cells (Sanderson et al., Cell Regul. 1, 585-596, 1990). In the presence of a unidirectional fluid flow, the Ca2+ response to a local release of ATP was biased such that virtually all the cells responding with an increase in [Ca2+]i were downstream of the release site. By contrast, an identical fluid flow did not bias the radial propagation of intercellular Ca2+ waves induced by mechanical stimulation. Suramin, a P2-purinergic receptor antagonist, did attenuate the Ca2+ response induced by ATP but did not block the propagation of mechanically induced Ca2+ waves. Cells from young cultures (3-5 days) or those at the leading edge of an outgrowth elevated their [Ca2+]i in response to ATP. However, these cells do not respond to mechanical stimulation by the propagation of a Ca2+ wave. From these results we conclude that the intercellular Ca2+ waves elicited by mechanical stimulation are not the result of ATP or another compound released from the stimulated cell, diffusing through the extracellular fluid. This conclusion is consistent with previous experimental evidence suggesting that intercellular Ca2+ signaling in epithelial cells is mediated by the movement of inositol trisphosphate through gap junctions (Boitano et al., Science 258, 292-295, 1992).
Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: Correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells
Authors: Jungbluth Andreas , Arnim Veronika von , Biegelmann Elke , Humbel Bruno , Schweiger Anton , Gerisch Günther 
Published: April 2021

ABSTRACT
When oxidative phosphorylation is inhibited in cells of Dictyostelium discoideum, the phosphorylation of tyrosine residues on actin is strongly increased. This increase is fully reversible. Under the same conditions the amoeboid cells undergo a series of shape changes. Within three minutes the pseudopods are withdrawn and replaced by cell surface blebs. Subsequently, the cells are rounding up to become immobile. In parallel with the changes in cell shape, the distribution of actin filaments is grossly altered within the cells. The cortical network of actin filaments of normal cells is broken down, and the F-actin forms large, irregular clusters deep within the cytoplasm. In these clusters the actin is associated with myosin II and with the het-erodimeric F-actin capping protein cap32/34. After restoration of oxidative phosphorylation the actin returns within less than four minutes to its normal cortical position. A causal relationship between tyrosine phosphorylation and changes in the distribution of actin remains to be established. The rearrangements in the actin system that result from the inhibition of oxidative phosphorylation indicate that the organisation of this system and its maintenance in a functional state depend on the continuous supply of energy by ATP.
tau confers drug stability but not cold stability to microtubules in living cells
Authors: Baas Peter W. , Pienkowski Thomas P. , Cimbalnik Kelly A. , Toyama Kimberly , Bakalis Shelley , Ahmad Fridoon J. , Kosik Kenneth S. 
Published: April 2021

ABSTRACT
We previously defined two classes of microtubule polymer in the axons of cultured sympathetic neurons that differ in their sensitivity to nocodazole by roughly 35-fold (Baas and Black (1990)J. Cell Biol. 111, 495-509). Here we demonstrate that virtually all of the microtubule polymer in these axons, including the drug-labile polymer, is stable to cold. What factors account for the unique stability properties of axonal microtubules? In the present study, we have focused on the role of tau, a microtubule-associated protein that is highly enriched in the axon, in determining the stability of microtubules to nocodazole and/or cold in living cells. We used a baculovirus vector to express very high levels of tau in insect ovarian Sf9 cells. The cells respond by extending processes that contain dense bundles of microtubules (Knops et al. (1991)J. Cell Biol. 114, 725-734). Cells induced to express tau were treated with either cold or 2 μg/ml nocodazole for times ranging from 5 minutes to 6 hours. The results with each treatment were very different from one another. Virtually all of the polymer was depolymerized within the first 30 minutes in cold, while little or no microtubule depolymerization was detected even after 6 hours in nocodazole. Based on these results, we conclude that tau is almost certainly a factor in conferring drug stability to axonal microtubules, but that factors other than or in addition to tau are required to confer cold stability.