bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunological characterization of lamins in the nuclear matrix of onion cells
Authors: Mínguez A. , Díaz de la Espina S. Moreno
Published: April 2021

We have used polyclonal and monoclonal antibodies against different lamins from vertebrates, and the IFA antibody recognizing all kinds of intermediate filament proteins, to investigate the lamins of the nuclear matrix of Allium cepa meristematic root cells. All the antibodies react in the onion nuclear matrix with bands in the range of 60-65 kDa, which are enriched in the nuclear matrix after urea extraction, and do not crossreact with other antibodies recognizing intermediate filaments in plants (AFB, anti-vimentin and MAC 322), ruling out crossreaction with contaminating intermediate filaments of cytoplasmic bundles. In 2-D blots the chicken antilamin serum reacts with one spot at 65 kDa and pI 6.8 and the anti B-type lamin antibodies with another one at 64 kDa and pI 5.75. Both crossreact with IFA.

The lamin is localized at the nuclear periphery and the lamina by indirect immunofluorescence. Immunogold labelling of nuclear matrix sections reveals that the protein is not only associated with the lamina, but also with the internal matrix. Taken together these results reveal that higher plants, which do not possess an organized network of cytoplasmic intermediate filaments, nevertheless present a well-organized lamina containing lamins in which at least one of them is immunologically related to vertebrate lamin B. Our data confirm that lamins are very old members of the intermediate filament proteins that have been better conserved in plants during evolution than their cytoplasmic counterparts.
The 270 kda splice variant of erythrocyte β-spectrin (βi∑2) segregates in vivo and in vitro to specific domains of cerebellar neurons
Authors: Malchiodi-Albedi Fiorella , Ceccarini Marina , Winkelmann John C. , Morrow Jon S. , Petrucci Tamara C.
Published: April 2021

Spectrin isoforms arise from four distinct genes, three of which generate multiple alternative transcripts. With no βIochemical restrictions on the assembly of αβ het-erodimers, more than 25 distinct heterodimeric spectrin species may exist. Whether (and why) this subtle but substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively spliced regions of α- and β-spectrin have been prepared. Reported here is the localization in rat cerebellar neurons at light and electron microscopic levels of an antibody against a unique sequence (βI∑2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell β-spectrin gene (spectrin I 2). In this version, the 3 sequence of ery-throid β-spectrin (βI∑1) is replaced with an alternative sequence that shares substantial homology with the 3’ sequence of non-erythroid -β-spectrin (βI∑1). The antibody to βI∑2-A stains a single protein band at 270 kDa, determined by western blotting, in both rat cerebellum and in cultured cerebellar granule cells, and does not react with βII∑1 spectrin (β-fodrin). This antibody stains the dendritic spines of Purkinje cells in the molecular layer, and is concentrated at postsynaptic densities (PSDs) adjacent to synapsin I (which is confined to the presynaptic membrane). The soma of Purkinje cells do not stain. In the granular layer, cytoplasmic organelles and the postsynaptic densities of granular cells stain strongly. Astrocytes are also stained. In all cells, plasma membrane staining is confined to postsynaptic densities (PSD). The βI∑2 isoform co-immunoprecipitates with non-erythroid -spectrin (αII∑2*), even though the distribution of II within neurons only partially overlaps that of βI∑2 No hybrid βI∑2 and βII∑1 (β-fodrin) spectrin complexes appear to exist. Spectrin βI∑2 is also polarized in cultured rat cerebellar granule cells, where it is abundant in cell bodies but not neurites. The overall distribution of βI∑2 is as a subset of the distribution of spectrins 240/235E previously detected with a generally reactive erythrocyte αβspectrin antibody. These findings establish the highly precise segregation of a β-spectrin isoform to distinct cytoplasmic and membrane surface domains, indicate that it is complexed (partially) with non-erythroid - spectrin, and demonstrate that cytoskeletal targeting mechanisms are preserved in cultured granular cells. The extreme concentration of βI∑2 spectrin at the PSD and in selected cytoplasmic compartments suggests that unique isoforms of spectrin may play a pivotal role in organizing topographically defined clusters of receptors or cytoplasmic protein complexes.
Interactions between neutrophils and endothelial cells
Authors: Godin Christine , Caprani Adrien , Dufaux Jacques , Flaud Patrice
Published: April 2021

Independently of their protective influence, leukocytes can have harmful effects in pathological situations such as diabetes (Benhamou et al., 1990), ischemia-reperfusion injury (Hernandez et al., 1987; Schmid-Schönbein and Engler, 1990; Thornton et al., 1989), inflammatory disorders (Fiebig et al., 1991; Grant, 1973; Jutila, 1992) and vascular permeability disorders (Kubes et al., 1991a; Schoenberg et al., 1991). Their action is mainly due to a rolling phenomenon along venular walls, adhesion to vascular endothelial cells and migration across the endothelium. A number of factors govern the interactions between leukocytes and endothelial cells, including the expression of molecules on the surface of activated leukocytes and/or endothelial cells, certain products of neutrophil activation, and shear forces within blood vessels that tend to sweep leukocytes along the endothelial cell surface (Harlan, 1985; Jutila, 1992; Ley, 1989; Tonnesen, 1989; Zimmerman et al., 1992).
Is the gap junction channel - The Connexon - made of connexin or ductin?
Authors: Finbow Malcolm E. , Pitts John D.
Published: April 2021

“For a‘ that, an a’ that

Our toils obscure an a’ that”

Robert Burns

The liver gap junction and Halobacterium purple membrane were among the first organised membrane complexes to be isolated. The retention of their paracrystalline form during isolation provided opportunities for physical analysis and allowed early glimpses of the structural organisation of integral membrane proteins. The seminal study of Henderson and Unwin (1975) on the purple membrane, using electron diffraction analysis and image reconstruction, revealed a detailed topology of the subunits that could be related to the structure of the component protein, bacteriorhodopsin. The work provided a framework for understanding the helical nature of the protein within the membrane bilayer and this model now serves as a structural archetype for a super-family of proteins with seven transmembrane α-helices. Other integral membrane proteins have now been crystallised that confirm the suggestion from Henderson and Unwin’s study that the secondary structure and packing fea-tures of such proteins are very similar to those of their water-soluble counterparts.
The differentiation and lineage development of goblet cells in the murine small intestinal crypt: experimental and modelling studies
Authors: Paulus Ursula , Loeffler Markus , Zeidler Joachim , Owen Gary , Potten Christopher S.
Published: April 2021

The objective of this study was to provide a new insight into the origin and lineage development of mucus-producing cells in the small intestinal crypt. For this, new experimental data were obtained from both crypt sections and whole mounts. Model simulation studies were undertaken to investigate which rules are most likely to govern the dynamic cellular development and goblet cell pedigree.

We have measured the frequency of mucus-secreting goblet cells (using alcian blue and periodic acid Schiff’s stains) at each cell position in the ileal murine crypt. These measurements, made on sections, overestimate the number of goblet cells because of the size and centripetal position of the stained cytoplasm. The correction factor for this overscoring has been measured to be 0.25 by two independent methods. The data suggest that there are about 12 functional goblet cells per crypt many of which retain an ability to divide. We have also determined the labelling index of the crypt goblet cells at each cell position. Spatially, goblet cells exhibit a small degree of clustering in the crypt and show a good mixture with columnar cells.

We have adapted our earlier dynamic matrix-based computer simulation model to take into account goblet cell differentiation. The modelling suggested the following conclusions: firstly, goblet cells do not have their own stem cells but share a common stem cell with the columnar cells; secondly, the goblet lineage differentiates from the transit population two to three generations before the end of the lineage; and thirdly, the decision to switch on goblet properties is stochastic at a specific step in the development of columnar cells.
Lipid and membrane protein transfer from human neutrophils to schistosomes is mediated by ligand binding
Authors: Rogers Rick A. , Jack Richard M. , Furlong Stephen T.
Published: April 2021

Attachment of human neutrophils to schistosomula of Schistosoma mansoni involves leukocyte receptors recognizing carbohydrate, complement and/or IgG ligands on the parasite surface. Here, we examined the transfer of a fluorescent fatty acid analog (BOFA) from human neutrophils to schistosomula coated with concanavalin A (Con A), immune serum or nonimmune serum under co-culture conditions by fluorescence confocal laser scanning microscopy (CLSM). Coating schistosomes with Con A or immune serum and co-culturing them for 24 hours with BOFA-labeled neutrophils resulted in a specific lipid transfer to the surface tegument of the schistosomes. Tegumental labeling was absent when nonimmune serum was used. No significant difference (P<0.001) was found in the number of neutrophils bound to the worm surface between Con A-coated schis-tosomes (4.1 ± 0.345 cells/worm) and worms incubated in immune serum (4.261 ± 0.362). The number of neutrophils bound to the schistosomula (2.7 ± 0.223) was significantly reduced in the presence of nonimmune serum (P<0.0001). The viability of the schistosomula was 98% in nonimmune treated co-cultures, and 91% in co-cultures treated with immune serum. HPLC analysis of labeled neutrophils demonstrated that BOFA was incorporated into both phospholipids and neutral lipids, which were almost exclusively triglycerides and, after 18 hours of culture, all of the fatty acid analog was incorporated into complex lipids. Double-label experiments in which schistosomula bearing Con A were first incubated with BOFA-labeled neutrophils and subsequently immunolabeled revealed that the neutrophil membrane proteins, MHC class I, CR1 and CR3 were co-transferred with neutrophil lipids to the parasite tegument. Thus, the parasite acquires necessary lipids as well as host membrane proteins in a native conformation that may play a role in parasite protection via mimicry (class I), regulation of complement activation (CR1) and binding of host integrin ligands (CR3).
Regulation of cd18 expression in human neutrophils as related to shape changes
Authors: Volz Alexandra
Published: April 2021

The study analyses the distribution and quantitative expression of surface CD18 of neutrophils exposed to distinct stimuli that produce different types of continuous shape changes, including types that are associated with locomotion and others that are not. The chemotactic peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanine, colchicine and nocodazole were used to induce a polarized locomotor morphology, phorbol myristate acetate, 1,2-dioctanoylglycerol and 1-oleoyl-2-acetylglycerol to induce non-polar motile cells ruffling all over the surface and 2H2O to induce non-polar cells performing circus movements as have been previously described. Except for colchicine and nocodazole, these stimuli increased surface expression of CD18. Thus, stimulated shape changes are frequently, though not always, associated with increased surface expression of CD18. High concentrations (10 7 to 10 5 M) of phorbol myristate acetate but not of chemotactic peptide induced down-regulation of surface CD18. Cytochalasin D (10 4 M) stimulated CD18 expression even though it inhibited shape changes. The surface distribution of CD18 determined by light microscopy was uniform in unstimulated cells or in various forms of stimulation except for cells treated with 10 5 M cytochalasin D. Cytochalasin D (10 5 M) produced CD18 accumulation at the pole opposite the F-actin cap. Experiments with colchicine, nocodazole, 2H2O and cytochalasin D suggest that microtubules as well as microfilaments modulate surface expression of CD18.

The results suggest that protein kinase C and phosphatases play a role in regulating surface expression of CD18 in neutrophils. Increased CD18 expression in response to phorbol myristate acetate, but not in response to chemotactic peptide was inhibited by the protein kinase C inhibitor Ro 31-8220 (10 6 to 10 5 M). Ro 31-8220 (10 5 M) alone increased CD18 expression of initially resting cells. Furthermore, the phosphatase inhibitor okadaic acid (5 M) and calyculin A (0.04 μM) suppressed CD18 expression in chemotactic peptide-treated cells, but there was no detectable effect in phorbol myristate acetate-stimulated cells. This indicates that at least two different pathways mediating CD18 expression are operative in human neutrophils, one being protein kinase C-dependent, the other(s) probably being protein kinase C-independent.
Calcium buffer injections inhibit cytokinesis in Xenopus eggs
Authors: Miller Andrew L. , Fluck Richard A. , McLaughlin Jane A. , Jaffe Lionel F.
Published: April 2021

A slow cortical wave of high calcium accompanies the elongation of cleavage furrows in medaka fish eggs as well as in Xenopus eggs. We explored the role of such waves by injecting calcium buffers into Xenopus eggs at various times before and during first and second cleavage. Injection earlier than about 15 minutes before first cleavage normally starts delays it for hours. Injection between about 15 minutes and a few minutes before cleavage normally starts allows a (short) furrow to form on time but usually yields an eccentric one. This forms away from the injection side, often as far off-center as the egg’s equator, and then regresses. Injection soon after it starts quickly arrests elongation of the furrow and eventually induces its regression; while injection a bit later likewise soon arrests elongation but allows delocalized furrow deepening to continue. The dependence of these inhibitory actions upon the dissociation constants and final cytosolic concentrations of the injected buffers indicates that they act as shuttle buffers to suppress needed zones of high calcium in the micromolar range. We conclude that the high calcium that is found within these furrows is needed to induce them, to extend them and even to maintain them.

Moreover, while short, eccentric furrows often form as far off center as the equator, they somehow always form along a meridian through the animal pole. This seems difficult to explain by the orthodox, diastral model. Rather, it suggests that the cleavage furrows in Xenopus- and perhaps in animal cells quite generally- are directly induced by a diastema or telophase disc rather than by the asters.
Nuclear localization of vertebrate cyclin a correlates with its ability to form complexes with cdk catalytic subunits
Authors: Maridor G. , Gallant P. , Golsteyn R. , Nigg E. A.
Published: April 2021

Cyclins control the activities of cyclin-dependent protein kinases (cdks) and hence play a key role in cell cycle regulation. While B-type cyclins associate with p34cdc2 to trigger entry into mitosis, progression through S phase requires cyclin A, presumably in association with p33cdk2. Vertebrate A- and B-type cyclins display strikingly distinct subcellular localizations, but the mechanisms underlying these differential distributions are unknown. Here, we have begun to study the requirements for nuclear localization of cyclin A. We have isolated a cDNA coding for chicken cyclin A and constructed a series of deletion mutants. These were then transfected into HeLa cells, and the subcellular distribution of the mutant cyclin A proteins was determined by indirect immunofluorescence microscopy. In parallel, the cyclin A mutants were assayed for their ability to form complexes with cdk subunits. We found that deletion of more than 100 residues from the N terminus of cyclin A did not impair nuclear localization or cdk subunit binding and kinase activation. In contrast, removal of as few as 15 residues from the C terminus, or deletion of part of the internal cyclin box domain, abolished nuclear localization of cyclin A as well as its ability to bind to and activate cdk subunits. These results suggest that nuclear transport of cyclin A may depend on the formation of multiprotein complexes comprising cdk catalytic subunits.
Cloning of the polyubiquitin cDNA from the marine sponge Geodia Cydonium and its preferential expression during reaggregation of cells
Authors: Pfeifer Karin , Frank Wolfgang , Schröder Heinz C. , Gamulin Vera , Rinkevich Baruch , Batel Renato , Müller Isabel M. , Müller Werner E. G.
Published: April 2021

Ubiquitination of proteins is a critical step in the controlled degradation process of many polypeptides. Here we show that sponges, the simplest multicellular group of eukaryotic organisms, are also equipped with the ubiquitin pathway. The polyubiquitin cDNA was isolated and characterized from the marine sponge Geodia cydonium. The open reading frame contains six ubiquitin moieties, which are lined up head to tail without spacers. A comparison of the predicted amino acid sequence of the six sponge ubiquitin-coding units with those from other organisms revealed a high degree of homology (> 93%). The ubiquitin gene is expressed to almost the same extent in the two main compartments of the sponge, the cortex and the medulla. However, only in the cortex are detectable amounts of the ubiquitin protein synthesized. The ubiquitin protein isolated from the sponge organism was found to initiate protein degradation in the heterologous reticulocyte system in the same manner as bovine ubiquitin. In vitro studies with dissociated sponge cells revealed that the homologous aggregation factor causes (i) a strong increase in the steady-state level of mRNA coding for ubiquitin and (ii) a drastic increase in ubiquitin protein synthesis, while the homologous lectin failed to display that effect in isolated cells. These data suggest that ubiquitin may play a role in sponge morphogenesis.
An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif
Authors: Doll T. , Meichsner M. , Riederer B. M. , Honegger P. , Matus A.
Published: April 2021

Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid ‘core’ sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
A novel phosphoglucomutase-related protein is concentrated in adherens junctions of muscle and nonmuscle cells
Authors: Belkin Alexey M. , Klimanskaya Irina V. , Lukashev Matvey E. , Lilley Kathryn , Critchley David R. , Koteliansky Victor E.
Published: April 2021

Using five monoclonal antibodies raised against a human uterine smooth muscle extract, we have identified a novel antigen which runs as a closely spaced doublet in SDS-gels. The proteins (60/63 kDa) co-purify, are present in a 1:1 ratio as judged by Coomassie Blue staining, and are immunologically closely related, if not identical. No N-terminal sequence could be obtained from a mixture of the 60/63 kDa proteins, but the sequence of four polypeptides liberated by V8 protease or cyanogen bromide cleavage showed that the proteins are closely related to the glycolytic enzyme phosphoglucomutase type 1. Affinity-purified poly-clonal antibodies and three different monoclonal antibod-ies to the 60/63 kDa proteins cross-reacted with rabbit skeletal muscle phosphoglucomutase type 1, whilst two additional monoclonal antibodies were specific for the 60/63 kDa proteins. Peptide maps of the 60/63 kDa proteins and phosphoglucomutase 1 are markedly different, and the purified proteins have no detectable phosphoglucomutase activity.

Staining of cultured smooth muscle cells and fibroblasts with antibodies to 60/63 kDa proteins showed that the antigen is concentrated in focal contacts at the ends of actin bundles and is also associated with actin filaments. About 60% of the cellular 60/63 kDa proteins were found in the detergent-insoluble fraction, suggesting a physical associa-tion with the cytoskeleton. The highest levels of protein immunoreactivity were found in muscles. The antigen is concentrated in muscle adherens junctions, including smooth muscle dense plaques, cardiomyocyte intercalated disks, and striated muscle myotendinous junctions. Among epithelial cells, the 63 kDa isoform of the protein was found only in cultured keratinocytes where immunofluorescent staining was localized in cell-to-cell adherens junctions. Expression of the 60/63 kDa proteins in vascular smooth muscle cells is developmentally regulated and correlates with the differentiated contractile phenotype of these cells.
Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads
Authors: Häuselmann Hans J. , Fernandes Russell J. , Mok Su S. , Schmid Thomas M. , Block Joel A. , Aydelotte Margaret B. , Kuettner Klaus E. , Thonar Eugene J.-M. A.
Published: April 2021

Articular chondrocytes embedded in alginate gel produce de novo a matrix rich in collagens and proteoglycans. A major advantage of this culture system is that the cells can be recovered by chelating the calcium, which otherwise maintains the alginate in its gel state. Chondrocytes thus released are surrounded by tightly bound cell-associated matrix, which seems to correspond to the pericellular and territorial matrices identified in cartilage by electron microscopy. The cells and their associated matrix can be easily separated by mild centrifugation from more soluble matrix components derived principally from the ‘interter-ritorial’ matrix. This new cell culture system thus makes it possible to study the assembly and turnover of molecules present in two distinct matrix pools. Importantly, a signif-icant proportion of the aggrecan molecules in each of these two pools can be extracted using a non-denaturing solvent, thereby making possible studies of the metabolism and turnover of native proteoglycan aggregates. We show in this report that chondrocytes isolated from the full depth of adult bovine articular cartilage and maintained for 8 months in alginate gel are still metabolically active and continue to synthesize cartilage-specific type II collagen and aggrecan. The cells did not synthesize large amounts of type I collagen or of the small nonaggregating proteo-glycans as usually occurs when chondrocytes lose their phe-notypic stability. After this extended period of time in culture, the cells were present as two populations exhibit-ing differences in size, shape and amount of extracellular matrix surrounding them. The first population was found only near the surface of the bead: these cells were flattened and surrounded by a matrix sparse in proteoglycans and collagen fibrils. The second population was found through-out the remaining depth of the bead: the cells were more round and almost always surrounded by a basket-like meshwork consisting of densely packed fibrils running tan-gential to the surface.