bluuebunny/crossref_metadata_2025 huggingface dataset:
Biogenesis of the apical endosome–lysosome complex during differentiation of absorptive epithelial cells in rat ileum
Authors: Wilson Jean M. , Whitney J. Andrew , Neutra Marian R.
Published: April 2021

Absorptive cells of the neonatal rat ileum have an elaborate apical endocytic complex consisting of tubular and vesicular endosomes, multivesicular bodies (MVB), and a giant lysosomal vacuole. This system develops rapidly over the last 3 days (20–22) of gestation. We followed the assembly of this complex by ultrastructural analysis and immunocytochemistry using antigenic markers for microvilli, endosomal tubules and lysosomal membranes. At 19 days gestation, low levels of lactase appeared on microvilli but specialized apical endosomal tubules and lysosomes were absent. At 20 days, expression of microvillar lactase increased and the endosomal marker entubin appeared, in parallel with the appearance of specialized apical endosomal tubules. The compartments of the apical endosomelysosome system were assembled sequentially after differentiation of the apical plasma membrane domains; first endosomal tubules and vesicles, followed by MVB, and ending with the assembly of the giant lysosome shortly after birth. During early stages of the assembly process, membrane components. of the tubular endosomes and lysosomes appeared in the apical plasma membrane before being restricted to their respective intracellular compartments
Comparison of biological and immunochemical properties indicates that scatter factor and hepatocyte growth factor are indistinguishable
Authors: Furlong Robert A. , Takehara Toyohiro , Taylor William G. , Nakamura Toshikazu , Rubin Jeffrey S.
Published: April 2021

Scatter factor, a stimulant of epithelial cell motility, and Hepatocyte Growth Factor (HGF) were compared by cross-biological studies using naturally occurring and recombinant proteins in four bioassays. Both scatter factor and HGF produced similar effects in cell motility and DNA synthesis assays. Antibodies to scatter factor or HGF neutralized the biological activities of each cytokine, and in immunoblotting reacted with species of the same Mr. These results, together with the available sequence data, suggest that scatter factor and HGF are the same protein.
Quantitation of actin polymerization in two human fibroblast sub-types responding to mechanical stretching
Authors: Pender Neil , Mcculloch Christopher A. G.
Published: April 2021

To study early reorganization of the cytoskeleton in response to physical forces, human gingival and periodontal ligament fibroblasts were cultured on flexible plastic substrata and stretched by mechanical deformation of the substratum. F-actin was measured by quantitative spectrofluorimetry of FITC-phalloidin-stained cells. Fluorescence due to FITC-phalloidin was reduced stoichiometrically by co-incubation with unlabeled phalloidn. Increases in stretch-induced fluorescence were blocked by preincubation of cells with cytochalasin D. Compared to baseline values, F-actin in gingival fibroblasts was reduced by 50% at 10 s after stretching but was increased more than 100% at 50 s after stretching. Increases of F-actin were also dependent on the amount of deformation of membrane: 3.3% stretching produced the largest increase and 9.2% stretching produced the least. Stretch-activated actin polymerization was blocked by pre-incubation of cells with 500 ng ml−1 pertussis toxin or 10 mM EGTA. Compared to gingival cells, periodontal ligament fibroblasts exhibited twice the total actin per cell and three times the baseline F-actin per mm2, however, there was no increase in fluorescence after stretching. In both cell types, stretching caused increased ruffling activity and no detectable shape changes at 60 s, but at 180 s the cells shortened and pseudopods retracted. The microfilament system of fibroblasts exhibits rapid dynamic responses to mechanical deformation prior to cell shape changes and appears to be dependent on the cell type.
Evidence for an inequality in the forces that generate principal and reverse bends in sperm flagella
Authors: Eshel Dan , Shingyoji Chikako , Yoshimura Kenjiro , Gibbons Ian R. , Takahashi Keiichi
Published: April 2021

The response of the mechanism initiating flagellar bends to imposed mechanical transients has been studied by holding the head of a sea urchin sperm in the tip of a sinusoidally vibrating micropipet and then displacing the micropipet laterally at a speed of up to 1.15 μm ms−1 for 1.5 beat cycle, without vibration, before resuming sinusoidal vibration with the initial phase, frequency and amplitude at the new location of the pipet This transient displacement of the micropipet delays the initiation of the bend that was due to initiate 0.5 beat cycle after onset of the displacement. The amount of this delay increases with the speed of the displacement, for speeds up to 1 μm ms−1. Analysis of the flagellar waveforms during the transient showed that with imposed displacements at speeds of equal magnitude, the initiation of a principal bend was delayed to a longer extent thanthat of a reverse bend. At a micropipet speed of 0.75 μm ms−1, there was an average delay of 0.21 beat cycle in the initiation time of a principal bend as compared to a delay of only about 0.04 beat cycle in the initiation time of a reverse bend during displacements in the opposite direction. For both principal and reverse bends, the second bend due to initiate during the transient displacement initiated in most of the cases with no delay, regardless of the micropipet speed. Our results suggest that the force generated by microtubule sliding to initiate a new reverse bend is greater than that generated to initiate a principal bend.
Synchronised secretory organelle docking in Paramecium saltatory movement along microtubules transiently formed from ciliary basal bodies and selective exclusion of microinjected heterologous organelles
Authors: Glas-Albrecht René , Kaesberg Birgit , Knoll Gerd , Allmann Karl , Pape Regina , Plattner Helmut
Published: April 2021

Ln certain strains (nd) of Paramecium tetraurelia all secretory organelles (trichocysts) can be detached from the cell surface and then reattached synchronously. To account for the lability of microtubules involved in trichocyst docking during the preparation procedures we analysed this process by combining video microscopy, analogue contrast enhancement microscopy, laser scanning fluorescence microscopy (both after fast freezing, freeze-substitution and anti-tubulin antibody fluorescence labelling), and electron microscopy after cryoflxation. We found that synchronous trichocyst docking is saltatory, occurring along transiently formed microtubules (not normally recognised in these cells), which emanate from ciliary basal bodies, acting as organising centres. Hence, trichocyst transport proceeds from the plus to the minus end of microtubules (in contrast to gland cells), with the ‘correct’ polarity (trichocyst tips pointing to the cell surface). We then injected chromaffin granules (isolated from bovine adrenal medullae) during the trichocyst detachment phase and analysed cells by electron microscopy during and after synchronised redocking of trichocysts. We used a chromate reaction for chromaffin granule identification on semithin sections by X-ray microanalysis (scanning transmission EM). While chromaffin granules remained intact (as judged by morphology and Cr signals) and although cell function was unimpaired (as judged by complete trichocyst attachment), we determined that heterologous organelle transport was not detectable, probably because of inverse microtubule polarity.
Multiple families of proteins in the secretory granules ofParamecium tetraurelia:Immunological characterization and immunocytochemical localization of trichocyst proteins
Authors: Shih Shian-Jiun , Nelson David L.
Published: April 2021

Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14–25×103), acidic (pI4.4–5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx.

The antibodies defined four distinct protein groups. Group I proteins (defined by mAb Al-3 and B5–5) showed a relatively wide range of pl values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condensed and extended trichocysts, as judged by electron-microscopic immunocytochemistry. Group n proteins (defined by mAb B4–4 and B3–5) were more acidic, also present as heterodimers and specifically localized in a 150 nm wide cortex in ctmx and in a much thinner cortex in xtmx. In xtmx, antibodies against group II proteins stained the outer surface on the regions between the electrondense striations with 55 nm intervals. However, these regions were not accessible to antibody B4–4 in ctmx. Group IH proteins (defined by mAb B7–4) are monomeric proteins; group IV are two subunits of heterodimers. Proteins of groups IH and IV were localized in the core of ctmx, but were distributed uniformly in xtmx.

Our results show that these very similar tmx proteins are not structurally equivalent. Within the highly regular structures of condensed and extended tmx, immunologically distinct families of tmx proteins occupy specific and different positions in the paracrystalline array. One family of tmx proteins (group II) is buried in the condensed tmx and only becomes accessible to antibodies upon trichocyst extension. Our results suggest that the 150 nm cortex of condensed tmx expands lengthwise, while decreasing in the thickness, to form the outer shell of extended tmx, and the core expands in length without decreasing in diameter to form the inside structure of the extended tmx.
A Drosophila anti-rna polymerase ii antibody recognizes a plant nucleolar antigen, rna polymerase i, which is mostly localized in fibrillar centres
Authors: Martin Marta , Medina Francisco Javier
Published: April 2021

The distribution of nucleolar RNA polymerase in the nucleolus of onion root meristematic cells has been studied by means of an antibody originally raised against Drosophila RNA polymerase II. This antibody recognizes the homologous domains of the large subunit of the enzyme, which are highly conserved throughout evolution in the three classes of eucaryotic RNA polymerases. Given that RNA polymerase I is confined to the nucleolus, and that the onion cell nucleolus lacks digitations of extranuc-leolar chromatin, we conclude that the nucleolar enzyme localized is RNA polymerase I. A quantitative approach, independent of the existence of borderlines between nucleolar fibrillar centres and the dense fibrillar component, allowed us to show that the enzyme is localized in fibrillar centres and in the transition area between them and the dense fibrillar component, in parallel with the nucleolar DNA.

These results, together with previous autoradiographic, cytochemical and immunocytochemical results, in this and other species, lead us to conclude that the activation of rDNA for transcription occurs in the fibrillar centres and pre-rRNA synthesis is expressed at the transition area between fibrillar centres and the dense fibrillar component. Fibrillar centres are connected to each other by extended RNA polymerase-bound DNA fibres, presumably active in transcription. This work provides evidence of the high evolutionary conservation of some domains of the large subunit of RNA polymerases and of the existence of fibrillar centres in the nucleolus of plant cells, totally homologous to those described in mammalian cells.
Visualization of centromeric and nucleolar DNA in fission yeast by fluorescence in situ hybridization
Authors: Uzawa Satoru , Yanagida Mitsuhiro
Published: April 2021

The nucleolar and centromeric DNAs of the fission yeast Schizosaccharomyces pombe were visualized in the nucleus by fluorescence in situ hybridization using repetitive ribosomal and centromeric DNAs as the probes. The rDNAs were seen in the nuclear domain previously assigned as nucleolar, that is, the region into which the rod-like chromatin protrudes from the hemispherical chromosomal domain. Using mitotically-arrested cells containing condensed chromosomes, it was demonstrated that the rDNAs were present on the smallest chromosome HI, consistent with genetic data. Using a centromeric repetitive element as the hybridization probe, the centromere of chromosome HI, cen3, which contains the largest number of the repetitive elements, was visualized. The centromere in interphase cells is located near the periphery of the nucleus as a single dot. Early in mitosis, however, it divides into two and is situated in the middle of the short mitotic spindle. After spindle extension in anaphase, the centromeric DNA is present at both ends of the spindle, that is, near the spindle pole bodies. The movement of cen3 during mitosis (anaphase A and B) is discussed in relation to spindle dynamics and chromosome separation.
Protein kinase C involvement in focal adhesion formation
Authors: Woods Anne , Couchman John R.
Published: April 2021

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form.

Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concen trations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin β1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cellbinding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.
Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes
Authors: Terasaki Mark , Reese Thomas S.
Published: April 2021

The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the significance of the structures labeled by DiOC6(3) in CV1 cells, a monkey epithelial cell line. First, we show that the network stained in living CV-1 cells is preserved by glutaraldehyde fixation and then we co-label it with an antibody against BiP (immunoglobulin binding protein), a protein commonly accepted to be present in the endoplasmic reticulum. Anti-BiP labeled the same network as that labeled by DiOC6(3), so this network now is identified as being part of the endoplasmic reticulum. DiOC6(3) labels many other membrane compartments in addition to the endoplasmic reticulum. This, along with its lipophilic properties, suggests that DiOC6(3) stains all intracellular membranes. However, the extensive reticular network in the thin peripheral regions of cultured cells is easily distinguished from these other membranes. Thus, staining by DiOC6(3) is a useful method for localizing the endoplasmic reticulum, particularly in thin peripheral regions of cultured cells.
Diacylglycerol activation of protein kinase C results in a dual effect on Na+,K+-ATPase activity from intact renal proximal tubule cells
Authors: Bertorello Alejandro M.
Published: April 2021

This study evaluated the effect of L-1-oleoyl-2-acetoyl sn-3-glycerol (OAG) on ouabain-sensitive Na,K-depen dent oxygen consumption (Na,K-QO2) in intact renal proximal tubule cells (RPTC). Basal Na,K-QO2 (nmol O2/mg protein per min) was 20.0±1.0. Incubation with 10 nM of OAG induced a dual effect on Na,K-QO2, with an initial stimulation (maximal at 10 min, 37.1±5.0), followed by an inhibition (significant at 20 min, 16.3±1.0). No changes in ouabain-insensitive QO2 were observed in any of the protocols. The effects were abolished by sphingosine, a protein kinase C inhibitor. Stimulation was abolished by amHorlde 0.1 mM. Amiloride had no effect on Na,K-QO2 at the concen tration used. Stimulation was not potentiated by the sodium ionophore, amphotericine B, and the later inhibition was still observed in the presence of ampho tericine B. The initial stimulation was attributed to an increase in sodium permeability, while the later inhi bition was attributed to a direct effect on the Na,K pump. Regulation of Na+,K+-ATPase activity by pro tein kinase C in intact RPTC can be accomplished by a direct effect on the protein or as a secondary effect consequent upon changes in intracellular sodium.
Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells
Authors: Almahbobi Ghanim , Williams Lindy J. , Hall Peter F.
Published: April 2021

Light microscopy of living and extracted adrenal cells (Y-l mouse adrenal tumour cells and cultured bovine fasciculata cells), using Nomarski optics and fluorescence with nile red to stain lipid, revealed in both cell types that lipid droplets remain attached to intermediate filaments when the cells are extracted to prepare these structures.

Electron microscopy of thin sections shows the presence of lipid droplets in both cell types. The droplets differ in appearance but are, in both cases, surrounded by a complete capsule 5nm wide. The droplets in Y-l cells include those associated with lysosomes and crystalline structures in addition to typical rounded forms. Only the latter type is seen in bovine fasciculata. Intermediate filaments apparently ending in droplets can also be seen. Immunoelectron microscopy with anti-vimentin and Protein A conjugated to gold particles together with measurement of the diameter of these structures identifies them as intermediate filaments. When adrenal cells are permeabilised and extracted under mild or severe conditions using Triton X-100, thin sections showed that lipid droplets remain associated with the cytoskeleton and in particular intermediate filaments. Extraction under mild and severe conditions cleared the cell contents, revealing attachment of intermediate filaments to lipid droplets with greater clarity than in unextracted cells, i.e. homogenised cells or cells subjected to lysis. Such attachment was unequivocally demonstrated in stereo pairs. These observations support our earlier studies showing attachment of droplets to intermediate filaments, which suggests a role for these filaments in intracellular transport of cholesterol.
Endosomal system of Paramecium: coated pits to early endosomes
Authors: Allen Richard D. , Schroeder Christopher C. , Fok Agnes K.
Published: April 2021

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188±41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 μm diameter, each potentially bearing one or more ∼80-nm-wide coated évaginations. These coated évaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated évaginations pinch off to form a second group of coated vesicles (90±17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.
Cloning and expression of Drosophila HP1 homologs from a mealybug, Pianococcus citri
Authors: Epstein Helen , James Tharappel C. , Singh Prim B.
Published: April 2021

The mealybug chromosome cycle is one of the most dramatic examples of genomic imprinting known. In embryos that are to become male the entire paternal chromosome set becomes heterochromatic and inactive at the blastoderm stage, while the maternal set remains active and euchromatic. HP1 is a protein from Drosophila melanogaster, which binds preferentially to heterochromatin on polytene chromosomes and is likely to be a modifier of position effect variegation. This paper describes the isolation and sequencing of two cDNA clones encoding HP1 homologs from the mealybug, Pianococcus citri. The protein product of the cDNA clone that was closer to HP1 in sequence was expressed as a fusion protein in Eschericia coli, and polyclonal rat antibodies were raised against it. Immunohistochemistry to mealybug squash preparations showed that this protein was a male-specific nuclear protein, but that it was not specifically associated with the heterochromatic set of chromosomes.
Genetic interactions in the control of septation in Schizosaccharomyces pombe
Authors: Marks John , Fankhauser Christian , Simanis Viesturs
Published: April 2021

We have used genetic and molecular techniques to investigate the interactions among genes required for the initiation and regulation of septum formation in Schizosaccharomyces pombe. Our data suggest that the products of the cdc7, cdc11, cdcl4 and cdcl6 genes interact. These activities may regulate the function of the cdcl5 gene product. A model for the control of septation in fission yeast is presented.
Lymphocyte adhesion to high endothelium is mediated by two β1 integrin receptors for fibronectin, α4β1 and α5β1
Authors: Szekanecz Zoltan , Humphries Martin J. , Ager Ann
Published: April 2021

Using a rat model we have previously proposed a role for fibronectin as an adhesive ligand on high endothelial cells (HEC) for recirculating lymphocytes. Lymphocyte adhesion to high endothelial cells was blocked by CS1 peptide (from the type IH connecting segment of fibronectin) and RGD-containing peptides using two different in vitro assays of lymphocyte-HEC recognition, the frozen section assay and cultured HEC. In order to study the receptors utilised by lymphocytes to bind to HEC we have developed a xenogeneic model in which the adhesion of human lymphocytes to HEC cultured from rat lymph nodes is measured. The basic properties of lymphocyte-HEC interaction were retained using human lymphocytes. CS1 peptide and RGD-containing peptides gave similar profiles of inhibition of lymphocyte adhesion as found previously using rat cells.

FACS analysis showed that the majority of peripheral blood lymphocytes expressed two β1 integrin receptors, α4β1 and α5β1, which are known to recognise distinct adhesion domains in fibronectin. A subpopulation of lymphocytes also expressed α3β1, which, like α5β1, has been reported to be an RGD-dependent adhesion receptor for the central cell binding domain of fibronectin. Antiα4 and anti-β5 subunit monoclonal antibodies maximally inhibited adhesion to HEC by 60% and 65%, respectively. Monoclonal antibodies to the common & subunit gave slightly higher inhibition at 70%. These results suggest that lymphocytes employ one or both of two different β1 integrin fibronectin receptors to bind to HEC. The simultaneous or alternate engagement of two fibronectin receptors on the lymphocyte surface by immobilised fibronectin in the endothelial layer may contribute to the stabilisation of adhesive contacts or to the subsequent transendothelial migration of lymphocytes. In contrast to lymphocytes, peripheral blood neutrophils did not express any members of the β1 integrin family. The selective expression of β1 integrins by lymphocytes and not neutrophils contrasted with the widespread distribution of the other homing-associated adhesion molecules, LECAM-1, CD44 and LFA-1, on these two cell types. It is thus possible that the selective expression of β1 integrins regulates the constitutive migration of lymphocytes but not neutrophils into organised lymphoid tissues.
Commentary structure and dynamics of transcriptionally active chromatin
Authors: Ausio Juan
Published: April 2021

Chromatin is a highly dynamic macromolecular complex that undergoes continuous structural modification during the various stages of genetic activity. Such dynamic behavior is the result of a complex and perfectly balanced network of interactions involving histones, DNA and ions in an aqueous environment. In the past eighteen years since the discovery of the nucleosome particle, a great deal of information has been obtained on interactions responsible for maintaining the static three-dimensional structure of inactive chromatin (see Van Holde, 1988, for a recent review on chromatin). More recently, structural studies of chromatin have focused on the analysis of the molecular events involved in chromatin activation. A variety of models have been proposed, primarily to explain the structural transitions undergone by chromatin during transcription. In the following sections, I will briefly review what is known about the structure of transcriptionally active chromatin and propose a model for “activation” that would account for the chemical and ionic interactions that are possibly involved. I hope that this model will stimulate design of new experimental approaches with which to understand better the structure-function relationship in chromatin.
Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles
Authors: Leibovici Michel , Monod Guillaume , GÉraudie Jacqueline , Bravo Rodrigo , Méchali Marcel
Published: April 2021

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.
Extrajunctional distribution of N-cadherin in cultured human endothelial cells
Authors: Salomon Daniela , Ayalon Oran , Patel-King Ramila , Hynes Richard O. , Geiger Benjamin
Published: April 2021

Human endothelial cells contain prominent Ca2+-dependent intercellular adherens-type junctions (AJ), which are associated, at their cytoplasmic surfaces, with actin, vinculin and plakoglobin. The transmembrane adhesion molecules present in these sites are members of the cadherin family, which are recognized by a pan-cadherin serum, directed against the conserved C terminus of these molecules. Immunoblotting analysis of cultured human endothelial cells using these antibodies revealed three immunoreactive bands with apparent molecular masses of 135,130 and 120 kDa. Cloning and sequencing of the 135 kDa cadherin from an endothelial cDNA expression library indicated that this molecule is a typical cadherin, essentially identical to N-cadherin. Transfection of cDNA encoding this molecule into CHO cells resulted in the induction of AJ formation and an apparent epithelialization of the cells. Immunofluor-escent labeling with antibodies to chicken N-cadherin indicated that the molecule is associated with intercellular junctions in the transfectants. In contrast, cultured human umbilical cord endothelial cells exhibited a largely diffuse N-cadherin labeling over the entire cell surface with only occasional enrichment in cell-cell junctions. Comparison of this pattern with the discrete junctional labeling obtained with the pan-cadherin antibody suggests that different cadherins, co-expressed in the same endothelial cells, may undergo differential surface distribution.
Modulation of anaphase spindle microtubule structure in stamen hair cells of Tradescantia by calcium and related agents
Authors: Zhang D. H. , Wadsworth P. , Hepler P. K.
Published: April 2021

We have previously shown that changes in the intracellular free calcium concentration ([Ca2+]i) modulate the rate of anaphase chromosome motion in stamen hair cells of Tradescantia. Elevations between 0.8–1.0 μM accelerate motion, while increases above 2.0 μM or decreases below resting level inhibit motion. The related signaling agent GTPγS also accelerates motion, however, by a mechanism that does not appear to involve changes in [Ca2+)i. To explore further the mechanism by which Ca2+ and GTPγS regulate chromosome motion we have analyzed the direct effect of these agents on the structure of the spindle microtubules (MTs). First, we injected carboxyfluorescein-derivatized brain tubulin and allowed it to incorporate into spindle MTs. Then, during appropriate times of anaphase, we injected Ca2+ or related agents and monitored their effect on spindle MT fluorescence using a confocal laser scanning microscope. A high level of Ca2+ (10 μM), known to inhibit motion, causes extensive degradation of spindle MT structure. An intermediate level (2 μM), which slows but does not stop movement, produces a distinct decay of fluorescence. A level of Ca2+ (0.8–1 μM) known to accelerate motion, however, generates only a small change in which the kinetochore fibers appear less distinct, and the overall spindle fluorescence is more diffuse. The Ca2+ buffer EGTA, which transiently blocks motion, has no detectable effect on spindle structure. GTPγS, which enhances motion also has no discernible effect on spindle structure. The results with Ca2+ support the idea that the ion facilitates anaphase motion through depolymerization of kinetochore MTs. However, if the depolymerization is extensive then inhibition of motion ensues. GTPγS, on the other hand, accelerates motion without inducing a detectable effect on spindle MT structure.
Expression of tau protein in non-neuronal cells: microtubule binding and stabilization
Authors: Lee Gloria , Rook Susan L.
Published: April 2021

The microtubule-associated protein tau is a developmentally regulated family of neuronal phosphoproteins that promotes the assembly and stabilization of microtubules. The carboxy-terminal half of the protein contains three copies of an imperfectly repeated sequence; this region has been found to bind microtubules in vitro. In addition, a fourth copy of the repeat has been found in adult-specific forms of tau protein. To examine the structure and function of tau protein in vivo, we have transiently expressed fetal and adult forms of tau protein and tau protein fragments in tissue culture cells. Biochemical analysis reveals full-length products with heterogeneity in post-translational modification synthesized in the cells. Immunofluorescent staining of transfected cells shows that, under our conditions, sequences on both sides of the repeat region are required for in vivo microtubule co-localization. These additional regions may be required either for enhancing micro tubule contacts or for proper protein folding in the cell. In our expression system, the bundling of cellular microtubules occurs only in transfections using four-repeat tau constructs; any four-repeat construct capable of binding is also able to induce bundling. Our data suggest that the presence of bundles is correlated with enhanced microtubule stability; factors that increase stability such as higher levels of tau protein expression or the presence of the fourth repeat, increase the fraction of transfected cells showing bundles. Finally, the presence of tau protein in the cell allows all interphase microtubules to become acetylated, a post-translational modification usually reserved for a subset of stable cellular microtubules.
Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells
Authors: Rennison Michelle E. , Handel Susan E. , Wilde Colin J. , Burgoyne Robert D.
Published: April 2021

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 μg/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.
Regulation of nuclear envelope precursor functions during cell division
Authors: Vigers G. P. A. , Lohka M. J.
Published: April 2021

Previously, we have shown that nuclear envelope assembly in cell-free extracts of Xenopus eggs requires two distinct vesicle-containing fractions, called Nuclear Envelope Precursor Fractions A and B (NEP-A and NEP-B). These fractions are characterized further in this paper and the manner in which they are regulated during metaphase is examined. Antisera against the NEP-B fraction recognized several proteins common to NEP-B and Xenopus oocyte or liver nuclei, but not to NEP-A or cytosol. A known glycoprotein component of the nuclear pore complex, p62, also co-fractionated with NEP-B, whereas the Xenopus egg lamin LUI did not. Together, these results provide further evidence that the NEP-B fraction contains precursors of the nuclear envelope.

The regulation of NEP-A and -B function during metaphase, when the nuclear envelope is disassembled, was examined by treating each fraction with metaphase cytosol or purified protein kinase preparations isolated from metaphase-arrested eggs. Treatment of NEP-B with metaphase cytosol, under conditions where proteins are irreversibly phosphorylated, inhibited the subsequent assembly of the nuclear envelope by preventing the binding of NEP-B to chromatin. In contrast, similar treatment of NEP-A did not affect its ability to form nuclear envelopes. The changes in NEP-B during metaphase did not appear to be regulated directly by either p34cdc2/cyclin B, S6 kinase II or MAP kinase.
Regulation of fibroblast-mediated collagen gel contraction by platelet-derived growth factor, interleukin-1 α and transforming growth factor-β1
Authors: Tingström Anders , Heldin Carl-Henrik , Rubin Kristofer
Published: April 2021

We have examined the effects of three macrophage-derived cytokines, platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1) and interleukin-la (IL-1 a) on the contraction of collagen type I gels populated by human foreskin fibroblasts. Contraction was quantified as loss in gel weight. Both PDGF-A A and PDGF-BB were found to induce a rapid collagen-gel contraction. TGF-β1 also stimulated gel contraction but with a delayed onset and at a slower rate than the PDGF-stimulated contraction. Rabbit polyclonal IgGs recognizing PDGF-A A and PDGF-BB, respectively, specifically inhibited the effects of the corresponding PDGF isoforms. However, the stimulatory effect of TGF-JÎ1 was not affected by any of the anti-PDGF antibodies. The ability of PDGF to stimulate contraction became less pronounced in collagen gel cultures grown in the absence of growth factors over periods of several days. Under the same conditions, the stimulatory effect of TGF-β1 was not reduced. The reduced response to PDGF may be due to reduced tension on fibroblasts growing in collagen gels, since fibroblasts on free-floating gels showed a marked reduction in PDGF-BB-induced PDGF β-receptor aggregates when compared to fibroblasts on attached collagen gels. IL-1α inhibited initial collagen gel contraction, and at later stages induced a visible degradation of the collagen gels, presumably due to the generation of collagenase activity. The combination of IL-la and PDGF-BB stimulated initial collagen gel contraction, although less effectively than PDGF-BB alone. At later stages, collagen gel degradation was stimulated by this combination of cytokines. In contrast, the combination of IL-la and TGF-β1 did not stimulate collagen gel contraction, or any visible collagen gel degradation. Our data suggest that fibroblast-mediated collagen gel contraction can be modulated by cytokines via different mechanisms. Our data are of importance in the understanding of the modulatory roles of cytokines in connective tissue cell activities in inflammatory processes, such as wound healing.
Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures
Authors: Beresford J. N. , Bennett J. H. , Devlin C. , Leboy P. S. , Owen M. E.
Published: April 2021

The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and HI) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10−8 M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10−8 M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts
Authors: Nuckolls Glen H. , Romer Lewis H. , Burridge Keith
Published: April 2021

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, wellspread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.
An evaluation of cell separation techniques in a model mixed cell population
Authors: Murphy S. J. , Wait D. J. , Jones G. E.
Published: April 2021

Muscle precursor cells may act not only as a means of inserting normal genes into diseased muscle fibres, in order to correct or alleviate a genetically inherited myopathy, but recent demonstrations have shown they may prove an invaluable tool for the expression of, and systemic dissemination of, non-muscle gene products. If muscle precursor cells are proved to act as such widespread vectors in terms of gene therapy, then it is imperative that methods are properly elucidated to produce large populations of pure viable myogenic cells for such purposes. In the past, many methods of cell separation have been investigated but carry with them the problems of either a lack of myogenic purity of the population or poor percentage recovery of the original cell population.

In the present work we have investigated two methods for segregating myogenic from non-myogenic cells and have critically reviewed the efficiency of separation of the two techniques used. To obtain a quantitative measure of separation efficiency, segregation was carried out on a 1:1 mixture of murine C2 myogenic and murine 3T3 fibroblastic cells. To distinguish between C2 and 3T3 cells, the latter were prelabelled with the fluorescent strain carboxyfluoroscein diacetate succinimyl ester (CFSE). Once incorporated into the cell, CFSE remains there, thus preventing transfer of the label to C2 cells. Both methods of separation used depend on the affininty of myogenic cells for the monoclonal antibody Mab H28, which specifically binds to the mouse neuronal cell adhesion molecule N-CAM, but differ in that one method, “panning”, completes segregation by adherence of N-CAM positive cells to a dish precoated with secondary IgG antibody whereas in the other separation proceeds by the use of commercially available IgG-coated magnetic beads. Results indicate magnetic bead separation to be more efficient than panning if the beads are precoated with 0.1% gelatin.
Specific patterns of cortical and endoplasmic microtubules associated with cell growth and tissue differentiation in roots of maize (Zea mays L.)
Authors: BalukŠa F. , Parker J. S. , Barlow P. W.
Published: April 2021

Indirect immunofluorescence using a-tubulin antibodies applied to sections of maize roots prepared using low melting point wax was found to give excellent visualisation of both cortical and endoplasmic microtubule (MT) arrays. This allows, for the first time, these arrays to be investigated in cells of the different tissues at various stages of their growth and development. Many cells in the zone between the meristem and the cell elongation region develop a highly ordered transversal bundling of cortical MTs, which we suggest is related to vacuolation of the cytoplasm and which may also be indispensable for the ensuing rapid cell elongation. On the other hand, there are subtle differences between the cells of the individual tissues regarding the arrangement of their cortical MTs in this zone. The possible physiological significance of these tissue-specific MT arrays is discussed. Endoplasmic MTs were seen to encircle and to connect the nucleus with the cortical MT arrays in both dividing and elongating cells. Even the G1 phase nuclei of the slowly dividing cells of the quiescent centre were encircled by endoplasmic MTs. The continuity of the two MT systems may provide the cell with an important signalling system whereby mechanical and physiological information is relayed from the exterior of the cell to the nucleus.