bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-muscle myosin isoforms and cell heterogeneity in developing rabbit vascular smooth muscle
Authors: Giuriato Luca , Scatena Marta , Chiavegato Angela , Tonello Marta , Scannapieco Gianluigi , Pauletto Paolo , Sartore Saverio
Published: April 2021

A panel of monoclonal antibodies specific for cytoskeletal and cytocontractile protein markers has been used to study the expression of vimentin, desmin and α-smooth muscle (SM) actin, as well as non-muscle (NM) and SM myosin isoforms, in developing rabbit aorta. Immunofluorescence experiments show that in the vascular smooth muscle cells (SMC): (1) vimentin and α-actin of SM-type are homogeneously expressed among SMC, since the early stage (day 19, in uterus) of development; (2) desmin is heterogeneously distributed throughout all the developmental stages examined (from day 19, foetal, to day 90, post-natal); and (3) myosin isoform content in pre- and post-natal vascular SM is different when analyzed by anti-SM myosin (SM-E7) and anti-NM myosin (NM-F6, NM-A9 and NM-G2) antibodies. SM myosin in vascular SM is present as early as day 19 in uterus, being especially evident in the region facing the lumen of aortic wall, but not in the outermost layer in which NM myosin is present exclusively. Western blotting and immunofluorescence assays indicate that the foetal aortic SM is specifically labeled by all the three anti-NM myosin antibodies. However, immunoreactivity of aortic SM with NM-F6 and NM-A9 disappears completely around birth. Conversely, NM-G2 binding is maintained during post-natal development up to day 45; between day 45 and day 90 immunoreactivity of aortic SMC with this antibody diminished progressively, without disappearing, in a small number of cells. In aortic SMC cultures from foetal and adult rabbits, NM myosin immunoreactivities appear to be differently distributed, i.e. according to the stress fiber system (NM-F6 and NM-G2), in a diffuse manner (NM-A9) or mainly localized at the level of the cortical cytoplasm (NM-G2).

The fact that a different pattern of NM myosin antigenicity can also be shown in other cell types, such as in the endothelium and the cardiac pericytes as well as in the renal parenchyma, is consistent with the existence of multiple NM myosin in vascular SM isoforms whose expression is developmentally regulated.
DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function
Authors: Cox Lynne S.
Published: April 2021

The lectin, wheat germ agglutinin (WGA), has previously been shown to prevent transport into the cell nucleus. This paper shows that WGA also inhibits nuclear DNA replication, under the same conditions that prevent transport. Although WGA eliminates sperm nuclear DNA replication in a cell-free extract of Xenopus eggs, DNA synthesis on a single-stranded template proceeds normally. Inhibition of nuclear DNA replication is partially reversed by addition of A’-acetylgluco-samine, and completely reversed by triacetylchitotriose. Sensitivity to inhibition by WGA is greatest during the nuclear assembly phase, and nuclear formation on sperm chromatin is blocked. DNA replication in preformed nuclear templates is also sensitive to WGA inhibition. I propose that WGA blocks DNA replication by preventing nuclear transport. The data presented here also indicate that, under certain circumstances, the elongation stage of DNA replication does not proceed in the absence of an intact nuclear envelope. The roles of the nuclear envelope and active nuclear transport in DNA replication are discussed.
DNA-binding domain of RCC1 protein is not essential for coupling mitosis with DNA replication
Authors: Seino Hiroaki , Hisamoto Naoki , Uzawa Satoru , Sekiguchi Takeshi , Nishimoto Takeharu
Published: April 2021

The RCC1 protein that is required for coupling mitosis with the S phase has a DNA-binding domain in the N- terminal region outside the repeat. We found that RCC1 protein without any DNA-binding activity complemented the tsBN2 mutation with the same efficiency as that of intact RCC1 protein. In ts+ transformants of tsBN2 cells transfected with the RCC1 cDNA lacking the DNA-binding domain, an endogenous RCC1 disappeared at 39.5°C, and the deleted RCC1 protein encoded by the transfected cDNA was found in the cytoplasm, but a significant amount of it was also found in the nuclei. This deleted RCC1 protein was eluted from the nuclei with the same concentration of NaCl and DNase I as was used for the intact RCC1 protein in BHK21 cells. Furthermore, the deleted RCC1 protein co-migrated with the nucleosome fraction on sucrose density gradient analysis. These results indicate that the RCC1 protein binds chromatin with the aid of other unknown protein(s). Thus, the DNA-binding domain of RCC1 protein is not essential for coupling between the S and M phases, but was shown instead to function as a nuclear translocation signal.
Antagonistic effects of TGF-β1 and MSF on fibroblast migration and hyaluronic acid synthesis: Possible implications for dermal wound healing
Authors: Ellis Ian , Grey Ann Marie , Schor Ana M. , Schor Seth L.
Published: April 2021

The migration of adult skin fibroblasts into threedimensional collagen gel matrices is differentially affected by cell density, with subconfluent cells displaying a significantly elevated level of migration compared to confluent ones. Fetal fibroblasts differ from adult cells in that they display an elevated level of migration at both subconfluent and confluent cell densities. We have previously reported that this difference in behaviour results from the secretion by fetal fibroblasts of a ‘migration stimulating factor’ (MSF) which is not made by their normal adult counterparts, and that MSF appears to act by stimulating the synthesis of hyaluronic acid (HA). Data presented in this communication indicate that (a) MSF specifically stimulates the synthesis of high molecular weight species of HA, (b) TGF-β inhibits the elevated migration of adult fibroblasts plated at subconfluent cell density, (c) under these conditions, TGF-β1induces a parallel decrease in the synthesis of high molecular weight HA and increase in the synthesis of low molecular weight HA, (d) TGF-β1 is a potent antagonist of MSF, effectively blocking its stimulation of cell migration and synthesis of high molecular weight HA, and (e) the inhibition of fibroblast migration by TGF-β1 does not appear to be a chemotactic response dependent upon the existence of a concentration gradient of the cytokine. Our observations regarding the inhibitory effects of TGF-β1 on fibroblast migration into 3D collagen gels stand in marked contrast to various published reports indicating that this cytokine stimulates the migration of human skin fibroblasts through the pores of polycarbonate filters as used in modified Boyden chamber assays; this discrepancy underscores the importance of the substratum in modulating cellular response to cytokines. Our results are discussed in terms of the possible combined contribution of MSF and TGF-β1 to wound healing.
Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes
Authors: Kubiak J. Z. , Weber M. , Géraud G. , Maro B.
Published: April 2021

When metaphase Il-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M Il/M III transition resembles the natural transition between the first and second meiotic metaphases (M 1/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosoma! proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone Hl kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M 1/M II and M Il/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.
Constitutive secretion of a bacterial enzyme by polarized epithelial cells
Authors: Soole Kathleen L. , Hall Judith , Jepson Mark A. , Hazlewood Geoffrey P. , Gilbert Harry J. , Hirst Barry H.
Published: April 2021

The constitutive (or default) pathway for protein secretion was investigated in two epithelial cells, Madin-Darby canine kidney (MDCK) and human colonic adenocarcinoma (Caco-2), using a bacterial enzyme. The choice of a bacterial protein was based on the requirement to identify a protein devoid of sorting signals. The sorting of a bacterial endoglucanase derived from Clostridium thermocellum, endoglucanase E, from stably transfected MDCK and Caco-2 cells was examined. The choice of a bacterial endoglucanase for these studies has advantages of simple, sensitive and quantitative detection, while higher eukaryotic cells do not express endoglucanase activity. Both cell lines secreted a 50 kDa form of the bacterial protein, while smaller intracellular forms were also observed. In polarized layers of MDCK cells the endoglucanase was secreted into both membrane domains in the ratio 62% apical and 38% basolateral. In Caco-2 cells secretion was predominantly, 70%, through the basolateral membrane. These results define the constitutive pathway for protein secretion in these two model epithelial cells.
Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells
Authors: Dluzewski A. R. , Mitchell G. H. , Fryer P. R. , Griffiths S. , Wilson R. J. M. , Gratzer W. B.
Published: April 2021

We have attempted to determine whether the parasitophorous vacuole membrane, in which the malaria parasite (merozoite) encapsulates itself when it enters a red blood cell, is derived from the host cell plasma membrane, as the appearance of the invasion process in the electron microscope has been taken to suggest, or from lipid material stored in the merozoite. We have incorporated into the red cell membrane a haptenic phospholipid, phosphatidylethanolamine, containing an NBD (N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)) group, substituted in the acyl chain, and allowed it to translocate into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, Plasmodium falciparum, immuno-gold electron microscopy was used to follow the distribution of the labelled lipid; this was found to be overwhelmingly in favour of the host cell membrane relative to the parasitophorous vacuole. Merozoites of P. knowlesi were allowed to attach irreversibly to red cells without invasion, using the method of pretreatment with cytochalasin. The region of contact between the merozoite and the host cell membrane was in all cases devoid of the labelled phosphatidylethanolamine. These results lead us to infer that the parasitophorous vacuole membrane is derived wholly or partly from lipid preexisting in the merozoite.
Syringe loading introduces macromolecules into living mammalian cell cytosol
Authors: Clarke Mark S. F. , McNeil Paul L.
Published: April 2021

We describe a simple, efficient, gentle and inexpensive technique for the introduction of normally impermeant macromolecules into the cytosol of living mammalian cells growing in suspension or attached to the culturing substratum. Loading is achieved by the production of transient, survivable plasma membrane disruptions as cells are passed back and forth through a standard syringe needle or similar narrow orifice. The loading volume required, which contains cells and the macromolecule to be loaded, can be as little as 5 μl, thus minimizing the use of valuable reagents. In addition, we report that the surfactant molecule, Pluronic F-68, is capable of altering the physical properties of the plasma membrane in such a way as to increase loading efficiency and the long-term survivability of cells loaded by this and other mechanically based cell-loading techniques.
Conditions for assembly of tubulin-based structures in unfertilized sea urchin eggs Spirals, monasters and cytasters
Authors: Harris Patricia J. , Clason Elise L.
Published: April 2021

Cytasters were induced in the unfertilized eggs of the sea urchin Lytechinus pictus by two different methods: (1) treatment with hexylene glycol or taxol, which are known to lower the tubulin critical concentration in vitro, but do not activate the nuclear cycle, and (2) by raising the cytoplasmic pH from that of unfertilized cytoplasm to that of fertilized, which activated the nuclear cycle and initiated tubulin polymerization. In the unactivated eggs, with increasing concentrations of the inducing agents, temperature and duration of treatment, microtubule structures that formed showed a progression from loose microtubule networks and spiral arrays to tightly organized cytasters. In eggs incubated in sea water containing 2.5 or 10 mM ammonium acetate titrated with HC1 or NaOH in steps from pH 5.0 to pH 9.0, the nuclear cycle and tubulin polymerization were initiated at about pH 7.0. The degree of development attainable after three hours was dependent on the pH, with spirals forming at the threshold level of pH 7.0, monasters at PH 7.5, and at PH 8.5 cells formed cytasters, multipolar spindles and even completed multipolar divisions. In unactivated eggs, tubulin was made available by lowering of the tubulin critical concentration; in activated eggs it was made available from some previously unavailable store. The evidence suggests that the amount of assembly-competent tubulin available, regardless of how it is made available, determines the type of structure that is formed, with microtubule bundles and spirals at the lower concentrations and functional cytasters at the upper. We also describe some details of cytaster formation, including the role of microtubule interactions and the movement of dense granules to the aster centers in hexylene glycol-treated eggs.
Structure of the cortical cytoskeleton in mammalian outer hair cells
Authors: Holley M. C. , Kalinec F. , Kachar B.
Published: April 2021

The cortical cytoskeletal lattice in outer hair cells is a two-dimensional actin-based structure, which can be labelled with rhodamine/phalloidin and disrupted by the enzyme deoxyribonuclease I. Structural information from thin sectioned, freeze-etched and negatively stained preparations shows that it is based upon two types of filament that form a cross-linked lattice of circumferential filaments. The cross-links are 70-80 nm long. Measurements of the spacing between circumferential filaments suggest that the lattice is stiffer circumferen-tially than it is longitudinally. Analysis of the orientation of circumferential filaments shows that it is composed of discrete domains of up to 10 μm2. Relative movements between domains could allow substantial changes of cell shape without disrupting the unit structure of the lattice, thus allowing the cell cortex to retain its elastic responses to high-frequency deformations.
Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells
Authors: Schweiger Anton , Mihalache Oana , Ecke Maria , Gerisch Günther
Published: April 2021

A 45 kDa protein in Dictyostelium discoideum cells that was recognized by a phosphotyrosine-specific antibody was identified by its binding activity to DNase I and its 2D-electrophoretic behavior as actin. The reactivity of actin with the antibody was transiently enhanced for about 30 minutes shortly after starving cells were reintroduced into nutrient medium. This effect indicates a modification of actin that is regulated under physiological conditions. A similar effect was obtained when growing cells were treated with phenylarsine oxide (PAO), an inhibitor of phosphotyrosine phosphatases. This effect was reversed and the cells fully recovered upon addition of the PAO antagonist 2,3-dimercapto-propanol. Starved cells did not show this enhancement of antibody labelling, which indicates that the response to PAO depends on the developmental stage. Phosphorylated amino acid residues were identified after in vivo labelling with [32P]phosphate in the presence of PAO. Part of the radioactivity in the actin band was recovered as phosphotyrosine, another part as phosphoserine.

PAO caused the cells to form elongated blebs, to round up and finally to become immobilized. Fluorescence labelling with phalloidin of cells that were fixed at different times of PAO treatment revealed a progressive decrease in the staining for actin filaments and showed that these alterations in cytoskeleton organization were readily reversible, in accordance with the reversal of tyrosine phosphorylation at actin.
Formation of highly organized skeletal muscle fibers in vitro Comparison with muscle development in vivo
Authors: Swasdison Somporn , Mayne Richard
Published: April 2021

Two methods were developed in which long-term cultures of quail skeletal muscle were established so that all of the muscle fibers develop in a highly oriented manner. The muscle fibers became spontaneously and vigorously contractile and established strong connections with the extracellular matrix at their ends that closely duplicate the structure of the myotendinous junction. A continuous basal lamina was formed around each muscle fiber that contained type IV collagen, laminin and heparan sulfate proteoglycan. With one of the methods, an extensive extracellular matrix developed around each muscle fiber that was highly organized with the formation of a distinctive épimysium, perimysium and endomysium. Analysis of the cultures by both methods for different isoforms of myosin showed expression of an adult form of myosin by some of the muscle cells. The results therefore demonstrate that muscle development in the present culture systems proceeds extensively for several weeks. It will now be possible to investigate directly the structure of the connections between muscle fibers and the extracellular matrix.
Formation of skeletal muscle in vivo from the mouse C2 cell line
Authors: Morgan Jennifer E. , Moore Stephen E. , Walsh Frank S. , Partridge Terence A.
Published: April 2021

The C2 muscle cell line is myogenic in vitro and has been extensively used in studies of muscle cell differentiation. Here, we have investigated the myogenicity in vivo of C2 cells implanted into suitable sites in the mouse.

Large amounts of new muscle were formed when C2 cells were implanted into sites in nude mice which were undergoing regeneration following whole muscle grafting and in scaffolding of freeze-killed muscle or vicryl suture in the anterior tibial compartment. When implanted into regenerating muscle, C2 cells fused with the host muscle to form mosaic fibres; when implanted into inert sites, they formed muscle of largely donor origin. C2-derived muscle fibres appeared to become innervated, but the progression of N-CAM (neural cell adhesion molecule) isoform changes in such regenerates indicated that they did not become fully mature. Proliferating, undifferentiated cells of C2 origin form tumours in older grafts; however, this was more pronounced in the absence of competition from host muscle cells.

In the short term, C2 cells can form large amounts of muscle in vivo for biochemical analysis. In addition, C2 cells are easily manipulable in vitro; genes of interest may be transfected into them prior to implantation of the cells into skeletal muscle and the effects of these genes in vivo may thus be examined.
Total inhibition of involucrin synthesis by a novel two-step antisense procedure Further examination of the relationship between differentiation and malignancy in hybrid cells
Authors: Griffin Elaine F. , Harris Henry
Published: April 2021

A novel procedure involving the sequential use of two different antisense constructs has been used to inhibit the synthesis of involucrin in a hybrid cell line formed by the fusion of a human cervical carcinoma cell with a normal human keratinocyte (ESH100P6). In this cell line, and other similar hybrids, malignancy, as measured by progressive growth in vivo, is suppressed; and it has been shown that the keratinocyte imposes its own programme of terminal differentiation on the non-malignant hybrid cell. In particular, involucrin, a precursor of one of the major components of the cornified envelope of mature kératinocytes, continues to be produced. When, however, malignant segregants arise in the hybrid cell population, the terminal differentiation programme of the keratinocyte is not expressed and involucrin ceases to be made. It seemed possible that if the synthesis of involucrin, a critical marker of keratinocyte terminal differentiation, could be completely inhibited, this differentiation programme might be disrupted, and the malignant phenotype might then reappear in the non-malignant hybrids. This question was investigated further in the present paper. Total, and specific, inhibition of involucrin synthesis was indeed achieved by a sequential two-step antisense procedure, which might provide a systematic general method for the complete inactivation of other selected target genes.
Aortic endothelial cell heterogeneity in vitro Lack of association between morphological phenotype and collagen biosynthesis
Authors: Canfield A. E. , Wren F. E. , Schor S. L. , Grant M. E. , Schor A. M.
Published: April 2021

Previous reports dealing with the characterisation of endothelial cells derived from the same tissue have produced apparently conflicting results in fundamental cellular attributes such as matrix biosynthesis and the ability to form sprouts in vitro. One potential explanation for this discrepancy is that endothelial cells actually comprise a heterogeneous population of cells displaying a significant degree of intra-site variation in phenotype. In order to address this question, we have characterised both cloned and uncloned lines of bovine aortic endothelial cells with respect to (a) their ability to adopt both the cobblestone and sprouting cell phenotypes and (b) matrix biosynthesis by cells displaying these two phenotypes. Data are presented indicating that all of the 18 cloned and 20 uncloned cell lines examined were capable of undergoing a reversible transition between the cobblestone and sprouting cell phenotypes in response to culture conditions. In all cases, sprouting occurred spontaneously in the presence of either serum or platelet-poor plasma and did not require the addition of exogenous factors to the medium. Twelve lines of cells were examined with respect to protein biosynthesis; these lines produced different types of collagens in differing proportions. The pattern of collagen synthesis displayed by every cell line was stable and did not vary with either passage number or batch of serum. The presence of a 3-D gel of native type I collagen increased specifically the synthesis of type IV collagen by one cell line. However, in four other cell lines, even though total synthesis was increased, the type of proteins secreted by these cells was not altered. Transition from a cobblestone to a sprouting cell phenotype by all five cell lines examined was not accompanied by any changes in the specific types of collagens synthesised by these cells. A decrease in thrombospondin and an increase in type IV collagen secretion was observed in one out of five cell lines. These data are discussed in terms of the possible significance of endothelial cell heterogeneity to the angiogenic response.
Cytostellin: A novel, highly conserved protein that undergoes continuous redistribution during the cell cycle
Authors: Warren Stephen L. , Landolfi Amalia S. , Curtis Carmel , Morrow Jon S.
Published: April 2021

Cytostellin, a 240 kDa protein, has been purified from mammalian cells by immunoaffinity chromatography using monoclonal antibody H5. Immunofluorescence microscopy shows diffuse and punctate cytostellin immunoreactivity in interphase nuclei. Nuclease digestion and salt extraction are not required to expose the epitope. The onset of prophase is marked by the appearance of multiple intensely immunofluorescent cytostellin-containing ‘bodies’ within the nucleus. Nuclear disassembly is heralded by the movement of cytostellin bodies from the nucleus to multiple positions throughout the cell. Cytostellin bodies in metaphase, anaphase and telophase cells are widely dispersed, including some in cell processes far removed from the mitotic spindle apparatus. However, a distinct subset of larger, more intensely staining bodies surrounds the mitotic spindle apparatus. Cytostellin bodies remain in the cytoplasm of the daughter cells and disappear after the appearance of nascent nuclei. Cytostellin is immunologically distinct from other nuclear and cytoplasmic proteins, and it has been detected by immunoblot analysis in all species tested from yeast to humans. Based upon these findings, we postulate that cytostellin has a cell cycle-dependent function which is conserved in higher and lower eukaryotic cells.
Purification and immunological detection of pea nuclear intermediate filaments: Evidence for plant nuclear lamins
Authors: McNULTY A. K. , Saunders M. J.
Published: April 2021

A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1-3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.
Indiscriminate synapsis in achiasmate Allium fistulosum L. (Liliaceae)
Authors: Jenkins G. , Okumus A.
Published: April 2021

Seedlings of Allium fistulosum (2n=2x=16) were treated with aqueous colchicine with the intention of inducing tetraploidy. One treated, but undoubled, diploid mutant is described which consistently fails to form any chiasmata at diakinesis and metaphase I of meiosis. Electron microscopy of whole-mount surface-spread synaptonemal complex complements of pollen mother cell nuclei revealed that the achiasmate condition is probably due not only to the failure to complete synapsis, but also to the indiscriminate way in which the chromosomes form synaptonemal complexes during meiotic prophase. Synapsis begins and progresses with complete disregard to homology, with frequent exchanges of pairing partners resulting in the formation of multiple associations comprising heterologous chromosomes. Intrachromosomal synapsis is also evident as fold-back loops. Up to 78% of lateral element length is incorporated into synaptonemal complex, the morphology of which is not unlike that of normal A. fistulosum and other Allium species described previously. However, all the synaptonemal complexes are ineffective in terms of supporting chiasmata, since 16 univalents enter metaphase I and disjoin irregularly at anaphase I. The mutant is as a consequence completely male sterile. The synaptic behaviour observed confirms that the recognition of homology is an independent process and not a prerequisite for synaptonemal complex formation. It is hoped this mutant will be a valuable tool for probing the molecular basis of homology.
Attachment of human vascular smooth muscle cells to intact microfibrillar assemblies of collagen VI and fibrillin
Authors: Kielty Cay M. , Whittaker Stephen P. , Grant Michael E. , Shuttleworth C. Adrian
Published: April 2021

Human vascular smooth muscle cells have been used to assess the implied role of connective tissue microfibrils as cellular ligands. Preparations of intact high-Mr microfibrillar assemblies of collagen VI and of fibrillin, respectively, were isolated from foetal bovine skin and used as ligands in cell attachment and spreading assays. Intact collagen VI microfibrils were capable of mediating cell attachment and partial spreading. Cell attachment assays using ligands composed of defined collagen VI fragments generated by pepsin or bacterial collagenase digestions demonstrated that both the triplehelical and non-collagenous domains of collagen VI had cell adhesion activity, although at reduced levels relative to intact microfibrils. Fibronectin was identified as a modulator of intact collagen VI microfibril-mediated cell attachment. These observations are indicative of complex multiple interactions between collagen VI microfibrils and smooth muscle cells. Purified fibrillincontaining microfibrils were also shown to support smooth muscle cell adhesion. Both pepsin-resistant and pepsin-sensitive domains of fibrillin exhibited some cell attachment activity, but at reduced levels relative to the intact fibrillin microfibrils. These data provide the first direct evidence of a physiological role for intact microfibrillar assemblies in cell-matrix interactions, and the involvement of integrin cell surface receptors containing the 1 subunit.
Total internal reflection fluorescence microscopy (TIRFM): II. *Topographical mapping of relative cell/substratum separation distances
Authors: Truskey G.A. , Burmeister J.S. , Grapa E. , Reichert W.M.
Published: April 2021

A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.
Comparative analysis of the effects of dimethyl sulfoxide and retinoic acid on the antigenic pattern of human ovarian adenocarcinoma cells
Authors: Grunt TH.W. , Somay C. , Oeller H. , Dittrich E. , Dittrich CH.
Published: April 2021

HOC-7 malignant ovarian surface epithelial cells have been exposed to differentiation promoters like dimethyl sulfoxide (DMSO) and retinoic acid (RA) and the resulting cell phenotypes were characterized immunologically. Immunocytochemistry revealed that DMSO caused elevation of membrane-associated staining for epidermal growth factor-receptor (EGF-R) and for desmoplakins I and II (DPI+II). DMSO also stimulated cytoplasmic and surface labelling for CA 125 and extracellular deposition of fibronectin (FN). A fixed-cell ELISA system was used for quantification of these differentiation-like responses and revealed that DMSO efficiently induced expression of EGF-R, CA 125, FN and DPI+II in dosedependent manner. Immunocytochemistry, ELISA and Western blotting additionally demonstrated that both DMSO and RA caused down-regulation of myc oncoproteins. Densitometer evaluation of electrophoresed proteins revealed a 50% DMSO- and a 25% RA-induced myc reduction. Apart from growth reduction, which was seen for both inducers, inhibition of myc gene expression was the only response of HOC-7 cells to RA-treatment. The extent of myc down-regulation seems, therefore, to be crucial for the initiation of maturational processes in the cells. Subsequent phenotypic differentiation of HOC-7 cells causes elevated levels of EGF-R, CA 125, FN and DPI+II. This cell model might be useful for the distinction between induced growth reduction and differentiation of ovarian cancer cells.
Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of madin-darby canine kidney (mdck) cells
Authors: Ojakian George K. , Schwimmer Randi
Published: April 2021

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by ∼33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.
A novel vesicle-associated protein (vap-1) in sea urchin eggs containing multiple rna-binding consensus sequences
Authors: Barton N. R. , Bonder E. M. , Fishkind D. J. , Warren R. H. , Pratt M. M.
Published: April 2021

We have identified a novel high molecular weight, vesicle-associated protein (VAP-1) in the eggs of the sea urchin Strongylocentrotuspurpuratus. Biochemical fractionation and immunofluorescence analysis of unfertilized eggs indicate that VAP-1 is a peripheral membrane protein associated with microsomal membrane fractions. Sequence analysis of partial VAP-1 cDNA clones reveals that the protein contains at least four RNA-bind-ing consensus sequences. The RNA-binding sequences are separated by several glycine rich domains and this organization, RNA-binding domains separated by glycine rich sequences, is common to several RNA-binding proteins including the heterogeneous ribonuclear protein A1 and nucleolin. The characteristics of VAP-1 suggest that the protein may function as a multidomain RNA-binding protein. The possibility that VAP-1 may play a role in nuclear RNA processing is also discussed.
A block in degradation of mhc class ii-associated invariant chain correlates with a reduction in transport from endosome carrier vesicles to the prelysosome compartment
Authors: Zachgo Sabine , Dobberstein Bernhard , Griffiths Gareth
Published: April 2021

Invariant chain (li) associated with MHC class II molecule is processed proteolytically via several distinct intermediates during its intracellular transport through endosomal compartments. Leupeptin added to the culture medium blocks processing of Ii, prevents its dissociation from the class II molecules and leads to an intracellular accumulation of a 22 kDa intermediate form of Ii. We show here that leupeptin has a very general effect on protein transport in the endocytic pathway. When added to Mel Juso cells leupeptin reduces the transport of endocytosed material from multivesicular body-like, endosome carrier vesicles (ECV) to the prelysosomal compartment (late endosome) and leads to a concomitant increase in the number of ECV. Our results argue that one effect of leupeptin, related to antigen processing and presentation, is to block transport of antigen and/or MHC class II molecules to prelysosomal compartments.
The relative positions of condensed chromosomes are maintained between divisions in the epidermis of Calpodes ethlius
Authors: Jeun Gwendolyn , Locke Michael
Published: April 2021

The larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells are twins that remain connected by residual midbodies between mitoses. Twins resemble one another more than their other neighbours in such structural features as the shape and number of nucleolar particles and the number of actin bundles. We have now found that they also resemble one another in the position of the condensed chromosomes that occur in female cells. Female lepidopteran cells contain one or more particles of condensed chromatin, depending on their ploidy. In the epidermis, nuclei with two condensed chromosomes are found in pairs and are separated by the same distances. However, clones of cells with multiple condensed chromosomes are not all alike, suggesting that chromosomes are repositioned at mitosis. Separation distances between chromosomes remain the same between but not through cell divisions, suggesting that determinants for nuclear structure are conserved through interphase and relaxed at mitosis. Although the condensed chromosomes of sibling nuclei resemble one another in their separation, they differ in their orientation, as would be expected if whole nuclei rotate in the plane of the epithelium.
Proteases are not involved in the membrane fusion events of the lysolecithin-mediated guinea pig sperm acrosome reaction
Authors: Flaherty Sean P. , Swann Nicholas J.
Published: April 2021

The guinea pig sperm acrosome reaction is characterized by a complex temporal and structural pattern of membrane fusions. In this study, we have used specific protease inhibitors to determine if proteases regulate this pattern of membrane fusions during the lysolecithin-mediated guinea pig sperm acrosome reaction. Inhibitors were chosen so as to cover a wide range of different types of proteases, and all were used at the highest concentration that did not adversely affect sperm motility. Of the eight inhibitors tested, leupeptin, soya bean trypsin inhibitor (SBTI), p-aminobenzamidine (pAB) and nitrophenyl p′ -guanidino benzoate (NPGB) inhibited completion of the acrosome reaction, while diethylenetriaminepentaacetic acid (DTPA), phosphoramidon, bestatin and pepstatin had no effect. Sperm that had been acrosome-reacted in the presence of each inhibitor were examined by transmission electron microscopy to assess whether the inhibitors altered the pattern of membrane fusions during the acrosome reaction. DTPA, phosphoramidon, bestatin and pepstatin had no effect on membrane fusion or matrix dispersal. Serine protease inhibitors such as leupeptin, SBTI, pAB and NPGB prevented complete dispersal of the acrosomal matrix and completion of the acrosome reaction, but did not alter the temporal sequence or structural pattern of membrane fusions. The undispersed matrix was present along the dorsal and ventral aspects of the apical segment and throughout the principal segment. We conclude that proteases are not involved in regulating the temporal and structural pattern of membrane fusions which occurs during the lysolecithin-mediated acrosome reaction of guinea pig sperm.
Platelet activating factor modulates signal transduction in Dictyostelium
Authors: Sordano Concetta , Cristino Elena , Bussolino Federico , Wurster Bernd , Bozzaro Salvatore
Published: April 2021

During development, Dictyostelium discoideum cells produce platelet activating factor (PAF). When cells are stimulated with external cAMP pulses, PAF is transiently synthesized. To determine whether PAF is involved in signal transduction, we have tested the effect of PAF on some cellular responses which are regulated by cAMP, such as spontaneous light-scattering oscillations of suspended cells, cAMP relay, transient increases of cGMP level, and extracellular calcium uptake.

Our results show that PAF specifically interferes with spontaneous spike-shaped oscillations, without affecting sinusoidal ones. PAF increases the amplitude of a spike, but has no effects on its phase or frequency. When cells fail to oscillate spontaneously, PAF does not induce spikes; however, if administered together with cAMP, it amplifies the light-scattering response to cAMP. Amplification of light-scattering changes is accompanied by a threefold increase in the concentration levels of both cellular cAMP and cGMP. Extracellular Ca2 uptake is also stimulated by PAF. This latter response is independent of endogenous or exogenously added cAMP. All these effects are specific for the naturally occurring Renantiomer of PAF, the S-enantiomer and lyso-PAF being inactive. These results suggest that PAF modulates signal transduction in Dictyostelium, probably by interacting with an intracellular acceptor, which is involved in the pathways regulating membrane Ca2 +channels, adenylate and guanylate cyclase.
Analysis of conserved binding proteins for nuclear localization sequences
Authors: Stochaj Ursula , Bossie Mark A. , Zee Karen van , Whalen Anne M. , Silver Pamela A.
Published: April 2021

Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex. It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore. These NLS-receptors could facilitate an early step of nuclear protein import, i.e. targeting and binding of nuclear proteins at the nuclear pore. We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms. Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells. Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores. One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein. Using isolated yeast nuclei we demonstrate that the antiidiotype antibodies compete for binding of nuclear proteins in vitro.

We show that the yeast mutant npl3, which is defective in nuclear protein localization, has an altered distribution of antigens recognized by these anti-idiotype antibodies, at the semi-permissive temperature. Our results suggest that a set of proteins common to various eukaryotes recognizes nuclear localization sequences.
F-actin domains in the syncytial blastoderm of the dipteran Ceratitis capitata
Authors: Callaini Giuliano , Dallai Romano , Riparbelli Maria Giovanna
Published: April 2021

Laser scanning confocal microscopy on rhodamine-phalloidin-treated syncytial embryos of the dipteran Ceratitis capitata allowed us to recognize four different kinds of actin filament distribution in close spatial proximity. One domain is represented by microfilaments localized in the plasma membrane within the microprojections and membrane infoldings. At a slightly lower focal level, rhodamine-phalloidin labelling is concentrated in small irregular aggregates, which are localized around the dividing nuclei. Our results indicate that the organization of the actin aggregates follows that of the microtubules of the mitotic apparatus and suggest that the dynamic reorganization of these structures during mitosis may be microtubule-dependent. A threedimensional network of thin actin filaments fills the whole periplasm and links the spindles together. A fourth actin domain is localized at the poles of the spindles in correspondence with the centrosomal region. The complex network of cortical filament bundles described in the present study may represent the ultrastructural basis of the tension leading to segregation of daughter nuclei at late telophase and to their lateral migration along the embryo surface.
Deregulation of cyclins D1 and E and suppression of cdk2 and cdk4 in senescent human fibroblasts
Authors: Lucibello Frances C. , Sewing Andreas , Brüsselbach Sabine , Bürger Christiane , Müller Rolf
Published: April 2021

The state of cellular senescence is characterised by an irreversible arrest in the G1 phase of the cell cycle. It has previously been shown that three cell cycle genes, cyclin A, cyclin B and cdc2, are not expressed in senes-cent human fibroblasts. All three gene products have functions after S-phase entry, so that their suppression cannot explain the irreversible G1 arrest. Here, we report that the abundance of transcripts from two other cell cycle genes, cdk2 and cdk4, thought to act during G1 S progression, is significantly diminished in senes-cent cells of the diploid human fibroblast line WI-38. Surprisingly, two other cyclins, D1 and E, behave in a completely different way, in that their expression is ele-vated in senescent cells, especially under conditions of serum starvation. Both the synthesis and the steady-state level of cyclin D1 protein were also found to be markedly higher in senescent cells (3-to 6-fold). Cyclins D1 and E are thus the first genes shown to be overex-pressed or deregulated in senescent cells. It is tempting to speculate that this deregulation may be due to the absence, in senescent cells, of a regulatory loop that would normally control their expression. This is sup-ported by our finding that cyclin E-associated kinase activity in senescent cells is reduced approx. 14-fold. Our data also suggest that the deregulated expression of cyclin D1 and E is not sufficient to drive senescent cells into DNA replication.
γ-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells
Authors: Julian M. , Tollon Y. , Lajoie-Mazenc I. , Moisand A. , Mazarguil H. , Puget A. , Wright M.
Published: April 2021

Animal cells undergoing cytokinesis form an inter-cel-lular bridge containing two bundles of microtubules interdigitated at their plus ends, which constitute the midbody. Polyclonal antibodies raised against three specific amino acid sequences of γ-tubulin (EEFATEGGDRKDV, NIIQGEADPTDVHKSL and EYHAATRPDYISWGTQEQ) specifically stained the centrosome in interphase, the spindle poles in all stages of mitosis, and the extremities of the midbody in mam-malian cells (Potorous, human, Chinese hamster, mouse). This staining was prevented by the corre-sponding peptides, by Xenopus γ-tubulin, but was not modified by purified αγ-tubulin heterodimer. An iden-tical staining was obtained with affinity-purified anti-bodies against the carboxyl-terminal amino acid sequence of human γ-tubulin. No γ-tubulin could be detected in the interzone during anaphase and early telophase. Material containing γ-tubulin first appeared in the two daughter cells on each side of the division plane in late telophase, and accumulated transiently at the minus ends of the two microtubule bundles consti-tuting the midbody for one hour after metaphase. Micro-injection of γ-tubulin antibodies into anaphase cells prevented the subsequent formation of the micro-tubule bundles between the two daughter cells. In con-trast with previous views, these observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centres, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells.