bluuebunny/crossref_metadata_2025 huggingface dataset:
Adhesion of cho cells to fibronectin is mediated by functionally and structurally distinct adhesion plaques
Authors: Tranqui Léone , Usson Yves , Marie Christiane , Block Marc R.
Published: April 2021

We have investigated the dynamics between free fibronectin receptors and clusters of them organized into adhesion plaques on CHO cells using the ability of these free integrins to be endocytosed and recycled to the plasma membrane. Indirect inhibition of the endo- cytic cycle by monensin resulted in the subsequent internalization of free receptors, which we followed by indirect immunostaining and confocal microscopy. Consequently, all the adhesive structures that were in equilibrium with free integrins became progessively disorganized. The cellular morphological changes were analyzed and correlated with the distribution of cellsubstratum contacts viewed by confocal images obtained after immunostaining with antibodies raised against the fibronectin receptor, talin, vinculin and actin. After cell adhesion to fibronectin, blockage of the endocytic cycle induced disruption of the adhesion plaques that were mainly localized at the cell periphery, and disappearance of the stress fibers. However, the cells remained firmly attached to the substratum through focal contacts localized in the central part of the cell. These central focal contacts, but not the peripheral adhesion plaques, could form when the vesicular traffic was blocked prior to adhesion and they allowed the cells to attach and flatten onto the substratum. Whereas both adhesive structures contained the same receptors linked to talin and vinculin, the central adhesive structures were attached to a short stretch of actin but never permitted the organization of stress fibers.
Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts: implications for their roles in cell-substratum adhesion
Authors: Yamagata Masahito , Saga Shinsuke , Kato Masato , Bernfield Merton , Kimata Koji
Published: April 2021

We showed previously that a large chondroitin sulfate proteoglycan, PG-M (also known as versican), inhibits cell-substratum adhesion, while basement membrane heparan sulfate proteoglycan (recently named perlecan) does not (Yamagata et al. (1989) J. Biol. Chem. 264, 8012-8018). To extend our understanding of the adhesive function of these proteoglycans, we examined the pericellular localization of the proteoglycans and their ligands and also that of some matrix receptors and cytoskeletal molecules in various fibroblast culture systems. PG-M was abundant in the subcellular space of fibroblasts, but was excluded selectively from focal contacts where vinculin, integrins and fibronectin were localized. Hyaluronan, CD44 and tenascin were distributed similarly as PG-M. In contrast, perlecan was associated with fibronectin and was included in focal contacts. Syndecan-1, a membrane heparan sulfate/chon-droitin sulfate proteoglycan, was associated with fibronectin at the cell surface, partly at focal contacts and in association with stress fibers. Thus, complexes of PG-M with hyaluronan, tenascin and CD44, are not involved in focal contacts. On the other hand, perlecan and syndecan-1 together with fibronectin may participate in focal contacts. The difference in localization between these proteoglycans may be related to their glycosaminoglycan content and to their distinctive roles in cell-substratum adhesion.
Mutations in the Drosophila melanogaster gene three rows permit aspects of mitosis to continue in the absence of chromatid segregation
Authors: Philp Alastair Valentine , Axton J. Myles , Saunders Robert D. C. , Glover David M.
Published: April 2021

We have cloned the three rows (thr) gene, by a combination of chromosome microdissection and P element tagging. We describe phenotypes of embryos homozygous for mutations at the thr locus. Maternal mRNA and protein appear to be sufficient to allow 14 rounds of mitosis in embryos homozygous for thr mutations. However, a small percentage of cells in syncytial blastoderm stage thr embryos sink into the interior of the embryo as if they have failed to divide properly. Following cellularisation all cells complete mitosis 14 normally. All cells become delayed at mitosis 15 with their chromosomes remaining aligned on the spindle in a metaphase-like configuration, even though both cyclins A and B have both been degraded. As cyclin B degradation occurs at the metaphase-anaphase transition, subsequent to the microtubule integrity checkpoint, the delay induced by mutations at the thr locus defines a later point in mitotic progression. Chromosomes in the cells of thr embryos do not undertake anaphase separation, but remain at the metaphase plate. Subsequently they decondense. A subset of nuclei go on to replicate their DNA but there is no further mitotic division.
Chediak-higashi syndrome is not due to a defect in microtubule-based lysosomal mobility
Authors: Perou Charles M. , Kaplan Jerry
Published: April 2021

Chediak-Higashi Syndrome is an autosomal recessive disorder that affects intracellular vesicle formation. The diagnostic feature of Chediak-Higashi Syndrome is the presence of ‘giant’ lysosomes clustered near the nucleus. Lysosome morphology in macrophages is maintained by microtubules and microtubule-based motors, such as kinesin. Dramatic changes in lysosome morphology can be induced by lowering cytoplasmic pH or by adding phorbol esters. When macrophages from beige mice (a murine homolog of Chediak-Higashi Syndrome) were subjected to these protocols they were able to alter their lysosomal distribution and morphology to the same degree as macrophages from control mice. These results indicate that lysosomes in Chediak cells are capable of interacting with the microtubule-based motor system, suggesting that the defective gene product is not an altered microtubular element involved in lysosomal movement.
Calcium homeostasis in plants
Authors: Gilroy Simon , Bethke Paul C. , Jones Russell L.
Published: April 2021

Many aspects of Ca2+ homeostasis in plants are similar to those in animals and fungi (Poovaiah and Reddy, 1989), but an understanding of how Ca2+ transport and function are integrated from the level of the whole plant to the sub-cellular level remains elusive. At the whole-plant level, a constant supply of Ca2+ in the range 1-10 mM is required to maintain normal growth and development (Epstein, 1972; Clarkson and Hanson, 1980). Calcium uptake by roots leads to millimolar concentrations of Ca2+ in plant tissues, and in most plants Ca is the second most abundant metal and the fifth most abundant element, after C, H, O and K (Epstein, 1972). At the subcellular level, certain organelles, such as the large central vacuole, may have similarly high Ca concentrations, but cytoplasmic Ca2+ levels are three to four orders of magnitude lower. Despite the abundance of Ca in plant tissues and the small amounts required for most cellular processes, the supply of Ca to the plant must be uninterrupted. Removal of Ca from the nutrient supply results in rapid death of cells in the apical meristem and a cessation of growth (Epstein, 1972). Just why this calcium starvation occurs when Ca levels in the plant are so high is not fully understood, but the low mobility of Ca within the plant body must be at least partially responsible.
Characterization of a PC12 cell sub-clone (PC12-c41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis
Authors: Teng Kenneth K. , Georgieff Irene S. , Aletta John M. , Nunez Jacques , Shelanski Michael L. , Greene Lloyd A.
Published: April 2021

To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, -tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (± NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non- permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (± NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.
Incorporation of tubulin subunits into dimers requires gtp hydrolysis
Authors: Fontalba Ana , Paciucci Rosanna , Avila Jesus , Zabala Juan C.
Published: April 2021

A toroid multisubunit complex of 800-900 kDa has been implicated in assisting protein folding of at least two cytoplasmic proteins, actin and tubulin. This process is dependent on the presence of magnesium ions and ATP hydrolysis. In vitro translation of cDNAs encoding different α- and β-tubulin isotypes also gives rise to the formation of complexes of about 300 kDa. These complexes have been functionally implicated in the incorporation of tubulin monomers within the tubulin heterodimer. This work shows that, in addition to ATP hydrolysis, the incorporation of newly synthesized tubulin subunits into functional heterodimers requires GTP hydrolysis in the presence of magnesium ions. A two-step process is suggested, a first ATP-dependent step in which the 900 kDa complexes are implicated in a similar way to the step taking place in actin folding, and a second GTP-dependent step in which the 300 kDa complexes are involved in the assembly of the heterodimer.
Inhibition of constitutive protein secretion from lactating mouse mammary epithelial cells by FIL (feedback inhibitor of lactation), a secreted milk protein
Authors: Rennison Michelle E. , Kerr Marian , Addey Caroline V. P. , Handel Susan E. , Turner Mark D. , Wilde Colin J. , Burgoyne Robert D.
Published: April 2021

The effect of a protein feedback inhibitor of lactation (FIL) on casein synthesis and secretion was examined using isolated acini from lactating mouse mammary gland. As previously found, FIL partially inhibited protein synthesis but produced an additional inhibition of constitutive casein secretion. The inhibition of synthesis and secretion showed similar dose-dependency and the inhibition was fully reversible. Constitutive secretion of pre-formed protein was inhibited by FIL in a pulsechase protocol, indicating that the inhibitor regulated protein secretion by reducing protein movement through the secretory pathway independently of any initial inhibition of synthesis. Regulated exocytosis was not inhibited since casein release due to elevation of cytosolic Ca2+ concentration by the ionophore ionomycin was unaffected. Brefeldin A, which is known to block ER-to-Golgi transport, also inhibited both protein synthesis and secretion in mammary cells. The action of FIL on synthesis and secretion and previously described actions on casein degradation would be consistent with a block at an early stage in the secretory pathway. In support of this idea FIL treatment was found to result in vesiculation and swelling of the endoplasmic reticulum. These data provide evidence for a novel control of a constitutive secretory pathway by a physiological extracellular regulatory protein.
Involvement of a phosphoprotein on the zymogen granule membrane in the control of regulated exocytosis in the exocrine pancreas
Authors: MacLean Carol M. , Marciniak Stefan J. , Hall Diane V. , Edwardson J. Michael
Published: April 2021

The pancreatic acinar cell is one of a number of cell types in which phosphoproteins are believed to be involved in the control of regulated exocytosis. We have examined the effects of three agents that affect secretion in the acinar cell on the phosphorylation states of proteins on the zymogen granule membrane. We show that Ca2+ and GTP S, which stimulate secretion, also stimulate the phosphorylation of a protein of Mr 45,000 (p45) on isolated zymogen granules. On the other hand, the protein kinase inhibitor genistein inhibits both secretion and phosphorylation of p45. For all three agents, p45 phosphorylation is affected over concentration ranges identical to those that affect secretion. The stimulatory effect of GTP S and the inhibitory effect of genistein are also seen when the phosphorylation state of p45 on granules within permeabilized cells is examined. Ca2+, however, over the same concentration range, now causes dephosphorylation of p45. Furthermore, the time-course of this effect is similar to that of Ca2+-triggered secretion. Phosphorylation of p45 is exclusively on serine, with no detectable phosphorylation on either threonine or tyrosine. We propose that exocytosis in pancreatic acini is controlled at least in part through the phosphorylation/dephosphorylation of p45, with dephosphorylation acting as a trigger for exocytosis.
Brain-type creatine kinase in photoreceptor cell outer segments: role of a phosphocreatine circuit in outer segment energy metabolism and phototransduction
Authors: Hemmer Wolfram , Riesinger Ingrid , Wallimann Theo , Eppenberger Hans M. , Quest Andrew F. G.
Published: April 2021

Different isoforms of creatine kinase, an important enzyme of vertebrate energy metabolism, were localized in bovine photoreceptor cells, with particular emphasis on the identification and quantification of the brain-type isoform within the outer segment compartment. Using immunofluorescence and immunoelectron microscopy, brain-type creatine kinase was shown to be present in bovine photoreceptor cell outer and inner segments. The presence of this isoenzyme in rod outer segments was additionally confirmed by immunoblotting and immunolabeling of isolated rod outer segments. The content of creatine kinase in rod outer segments was quantified by measuring creatine kinase activity after membrane disruption with detergent. The ATP regeneration potential provided by the creatine kinase in isolated, washed bovine rod outer segments was 1.2 ± (0.4) i.u. mg 1 rhodopsin. This value was calculated to be at least an order of magnitude larger than that necessary to replenish the energy required for cGMP resynthesis in rod outer segments, and high enough to regenerate the entire ATP pool of rod outer segments within the time span of a photic cycle.

A mitochondrial creatine kinase isoenzyme was located within the ellipsoid portions of bovine rod and cone inner segments by immunofluorescence microscopy and, using immunogold staining, was specifically localized in the mitochondria clustered within bovine rod and cone inner segments. These results suggest that vertebrate photoreceptor cells contain a functional phos-phocreatine circuit. Outer segment creatine kinase may play an important role in phototransduction by providing energy for the visual cycle, maintaining high local ATP/ADP ratios and consuming protons produced by enzymes located in the outer segment.
Localisation of granulocyte macrophage colony-stimulating factor in human long-term bone marrow cultures Biological and immunocytochemical characterisation
Authors: Wynter E. de , Allen T. , Coutinho L. , Flavell D. , Flavell S. U. , Dexter T. M.
Published: April 2021

The distribution of granulocyte macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures (HLTBMC) was examined using two monoclonal antibodies raised using purified recombinant GM-CSF and a third commercially available GM-CSF antibody. The antibodies were able to bind to purified recombinant GM-CSF and showed inhibition of GM-CFC colonies in the presence of both recombinant and native protein. All antibodies displayed similar pat-terns of distribution in both permeabilised and non-per-meabilised stromal cell preparations. Fibroblasts were labelled at their periphery in early cultures and both endothelial cells and fibroblasts showed cytoplasmic labelling with anti-GM-CSF. The fact that GM-CSF appears to be sequestered by cells of the bone marrow stroma raises the possibility that it is synthesised by these cells and may regulate activity of the progenitor cells in the haemopoietic foci. In contrast, early progenitor cells within the foci did not stain with any of the anti-GM-CSF antibodies. Adipocytes, which differenti-ate from fibroblasts in these cultures, showed a diffuse staining pattern. Two types of macrophage staining were observed in the non-permeabilised cells; those exhibiting only autofluorescence and those that bound the antibody. Intracellular staining was apparent in a small sub-population. Generally, the staining persisted up to eight weeks of culture and thereafter declined, becoming virtually undetectable after 12 weeks. This correlates with the pattern of GM-CFC production in long-term bone marrow cultures.
Antisense RNA inactivation of gp138 gene expression results in repression of sexual cell fusion in Dictyostelium discoideum
Authors: Fang Hui , Higa Kazuhiro Aiba, Mikihiko , Urushihara Hideko , Yanagisawa Kaichiro
Published: April 2021

A glycoprotein, gp138, is implicated in the sexual cell fusion of Dictyostelium discoideum. We previously cloned and sequenced the two genes encoding the gp138 protein, GP138A and GP138B (Fang et al. (1993)Dev. Biol. 156, 201-208). Here, we have constructed a vector producing antisense RNA for the gp138 genes and have transformed the vector into Dictyostelium cells. The transformed cells showed a reduction in the amounts of gp138 mRNA and protein and their sexual cell fusion activity was considerably repressed.
The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A
Authors: Roa Michèle , Cornet Véronique , Yang Chun-Zhi , Goud Bruno
Published: April 2021

Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 g/ml, induced redistribution of rab6p according to a biphasic process: during the first 10-15 minutes, tubulo-vesicular structures – colabelled with a bona fide medial Golgi marker called CTR 433-were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein β-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center.

The kinetics of reversion of BFA action on these anti-gens was also followed by immunofluorescence. Consis-tent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.
Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment
Authors: Bryant Nia J. , Boyd Alan
Published: April 2021

One of the Golgi compartments of Saccharomyces cere - visiae is defined by the presence of a specific endopro-teinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi com-partment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.
The phosphotyrosine phosphatase inhibitor vanadyl hydroperoxide induces morphological alterations, cytoskeletal rearrangements and increased adhesiveness in rat neutrophil leucocytes
Authors: Bennett Paul A. , Dixon Richard J. , Kellie Stuart
Published: April 2021

The functional consequences of treating rat neutrophils with the potent tyrosine phosphatase inhibitor vanadyl hydroperoxide (pervanadate) has been investigated. Pervanadate induced rapid increases in cellular protein phosphotyrosine content in a dose-dependent manner. This treatment also resulted in a change in morphology of the cells from a rounded to a polarised morphology, with many cells exhibiting uropods, pseudopodia and increased membrane activity. Pervanadate induced a transient actin polymerisation and reorganisation similar to that in agonist-stimulated cells. The pervanadate-induced increases in tyrosine phosphorylation, shape change and actin polymerisation were inhibited by the tyrosine kinase inhibitors tyrphostin and erbstatin, indicating that these phenomena were mediated by the constitutive activity of cellular tyrosine kinases. Double flu-orescence experiments demonstrated that there was a co-localisation of tyrosine phosphorylated proteins with F-actin in both pervanadate- and agonist-stimulated neutrophils. Pervanadate also induced spreading of neutrophils on tissue culture substrata with concurrent changes in F-actin localisation including unusual F-actin-containing structures. These results demonstrate that morphological changes and cytoskeletal reorganisation in neutrophils are regulated by tyrosine phosphorylation, and that inhibition of tyrosine phosphatase activity in neutrophils is sufficient to activate motile machinery of these cells. These results suggest that an alternative pathway involved in neutrophil stimulation might be via inhibition of endogenous tyrosine phosphatases rather than activation of tyrosine kinases.
Co-localization of class II antigen and exogenous antigen in the rat enterocyte
Authors: Gonnella Patricia A. , Wilmore Douglas W.
Published: April 2021

The role of class II major histocompatibility antigens in classical antigen-presenting cells has been described (Unanue (1984)Annu. Rev. Immunol. 2, 395-428; Watts and McConnell (1987)Rev. Immunol. 5, 461-475). Whether enterocytes, which also express class II antigens, can act as antigen-presenting cells in vivo is not known. One pre-requisite for a role for enterocytes in antigen presentation is an interaction between exogenous antigen and class II antigens. Our results demonstrate that class II antigen and exogenous antigen absorbed from the gastrointestinal tract are co-localized within endocytic compartments and along the basolateral membranes of enterocytes.
The locomotion, shape and pseudopodial dynamics of unstimulated Dictyostelium cells are not random
Authors: Killich Thomas , Plath Peter J. , Wei Xiang , Bultmann Heino , Rensing Ludger , Vicker Michael G.
Published: April 2021

The dynamic periphery of unstimulated, preaggregation, hunger-stage Dictyostelium discoideum amoebae was investigated by time-lapse videomicroscopy and digital image processing. Circular maps (i.e. of each of 360 radii around the cell transformed upon Cartesian coordinates) were constructed around the centroid of individual cell images and analysed in time series. This novel technique generated spatiotemporal structures of various degrees of order in the maps, which resemble classical wave interference patterns. The patterns thus demonstrate that cell movement is not random and that cells are intrinsically vibrating bodies, transited by self-organized, superpositioned, harmonic modes of rotating oscillatory waves (ROWS). These waves appear to depend upon spatiotemporal oscillations in the physico-chemical reactions associated with actin polymerization, and they govern pseudopodial movements, cell shape and locomotion generally. ROWS in this case are unrelated to the cyclic-AMP-regulated oscillations, which characterize later, aggregative populations of Dictyostelium. However, the exposure of aggregation-stage cells to a pulse of the chemoattractant cyclic-AMP induces a characteristic sequence of changes in the global cellular concentration and spatiotemporal distri-bution of fibrillar (F-)actin. This reaction begins with what appears to be a phase resetting of ROWS and it may, therefore, underlie the cellular perception of and response to chemotactic signals. We also develop here an analytical mathematical description of ROWS, and use it to simulate cell movements accurately.
Formation of hemidesmosomes in cells of a transformed murine mammary tumor cell line and mechanisms involved in adherence of these cells to laminin and kalinin
Authors: Sonnenberg A. , de Melker A. A. , de Velasco A. M. Martinez , Janssen H. , Calafat J. , Niessen C. M.
Published: April 2021

Keratinocytes attach to an underlying basement mem-brane by adhesion junctions called hemidesmosomes. We have characterized a cell line, RAC-11P/SD, estab-lished from a murine mammary tumor, which differen-tiates into squamous epithelium and forms well defined hemidesmosomes. These hemidesmosomes contain the integrin 6 4 as well as the hemidesmosomal plaque proteins BP230 and HD1 and are associated with a matrix containing kalinin and laminin. We examined how these cells adhere to laminin and to kalinin present in matrices as well as immunopurified kalinin. We show that adhesion to laminin is energy dependent but does not require an intact actin-containing cytoskeleton. The affinity for kalinin proved to be greater and binding to kalinin was still observed when cells had been treated with deoxyglucose and azide to inhibit metabolic energy. Binding to laminin (or fragment E8), but not to kalinin was partially blocked by a monoclonal antibody specific for the integrin 6 subunit, and only in the initial phase of adhesion. The antibody efficiently blocked adhesion to laminin of cells treated with the microfilament dis-rupting drug cytochalasin B, but only partially blocked the adhesion of cytochalasin B-treated cells to kalinin, while adherence of cells treated with deoxyglucose and azide to kalinin was blocked completely. The integrin α6β4 is redistributed to the basal surface during adhe-sion and then is organized into ring-like structures when cells are bound to laminin and localized into hemidesmosomes in cells adhered to kalinin. We suggest that anti-α6 hinders the binding of theα6β4 integrins to its ligands laminin and kalinin, but cannot prevent adhe-sion when clustering of the integrin has become com-plete. In addition, there is evidence that adhesion to kalinin is mediated by a second receptor, which associ-ates with the actin-containing cytoskeleton. The pres-ence of such a second receptor is suggested because the cells can spread on kalinin, but not when they have been treated with cytochalasin B. On laminin spreading does not occur, irrespective of whether cells have been treated with cytochalasin B or not. The integrin α3β1, which has been identified as a receptor for kalinin but not for laminin, is strongly expressed in RAC-11P/SD cells and it seems likely that this integrin is responsible for spreading of cells on kalinin.
Vacuolar H+-ATPase of Dictyostelium discoideum A monoclonal antibody study
Authors: Fok Agnes K. , Clarke Margaret , Ma Le , Allen Richard D.
Published: April 2021

A Dictyostelium membrane fraction rich in vacuolar proton pumps, previously described by Nolta et al. (J. Biol. Chem. 266, 18,318-18,323, 1991), was used as the immunogen for production of monoclonal antibodies. We obtained antibodies that recognized polypeptides of 100 kDa and 68 kDa, corresponding to the two largest subunits of the vacuolar proton pump. In indirect immunofluorescence experiments, these two subunits were located on an interconnected collection of tubules and vacuoles. On frozen thin sections they were found principally on membranes of vacuoles and collections of small vesicles typically located just internal to the plasma membrane. These vesicles and vacuoles had elec-tron-translucent lumens. No other structures in axeni-cally grown Dictyostelium cells were labeled to a signif-icant extent by these two antibodies. Using an affinity-purified antibody to calmodulin and a mono-specific antibody to the B subunit of the chromaffin granule vacuolar ATPase, markers known to label the membranes of the contractile vacuole complex in Dictyostelium (Zhu and Clarke, J. Cell Biol. 118, 347-358, 1992; Heuser et al., J. Cell Biol. 121, 1311-1327, 1993), we showed that the 100 kDa and 68 kDa subunits had the same distribution as these two markers. Co-local-ization was seen in both interphase and mitotic cells. Thus, our results support the conclusion that vacuolar proton pumps are located principally on the membranes of the contractile vacuole complex in Dictyostelium. In addition, in indirect immunofluorescence experiments, these monoclonal antibodies provided improved images of the organization of the contractile vacuole system.
Internalisation of desmosomes and their entry into the endocytic pathway via late endosomes in MDCK cells: Possible mechanisms for the modulation of cell adhesion by desmosomes during development
Authors: Burdett I. D. J.
Published: April 2021

MDCK cells grown in media with normal levels of Ca2+ (∼2 mM) contain internalised desmosomes, referred to as desmosome-associated vacuoles (DAVs). The DAVs consist of one to three plaques retained in the plane of a surrounding vacuolar membrane, and their entry into the endocytic pathway has been investigated using HRP, cationised ferritin and BSA/gold in combination with electron microscopy and immunogold labelling of frozen sections.

Endocytic tracers supplied from the apical and baso-lateral surfaces to filter-grown MDCK cells met in a common perinuclear compartment but DAVs were not labelled during short (5-30 minutes) pulses of marker, whether applied apically or basolaterally. Only when the tracers were taken up from the basolateral surface and then chased for periods of 2-18 hours, were DAVs labelled. It is proposed that entry of an endocytic tracer to DAVs occurs by the association of the desmosomal vacuole with late endosomes. Immunolabelling studies with antibodies to desmosomal components (to Dsg, DPI/II), to HRP and to the cation-independent mannose 6-phosphate receptor (MPR), confirmed that Dsg and DPI/II are located within DAVs and late endosomes, but not in early endosomes. Passage of Dsg, but to a lesser extent DPI/II, was detected in MPR structures (lyso-somes). DAV-like structures have also been observed in developing tissues such as mouse kidney. Such engulf-ment may provide a general mechanism for handling insoluble junctional proteins, particularly where rapid morphogenetic changes are occurring in the pattern of cell-cell adhesion.
Talin distribution and phosphorylation in thrombin-activated platelets
Authors: Bertagnolli Maria E. , Locke Sarah J. , Hensler Mary E. , Bray Paul F. , Beckerle Mary C.
Published: April 2021

We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333-3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phospho-rylation and proteolytic cleavage as possible mechanisms by which talin’s subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with throm-bin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are defi-cient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.
Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating
Authors: Salathe Matthias , Pratt Melanie M. , Wanner Adam
Published: April 2021

The present study examined whether protein kinase C phosphorylated a ciliary protein and whether this phos-phorylation event was temporally correlated with a decrease in ciliary beat frequency. Activation of protein kinase C decreased ciliary beat frequency of sheep tracheal epithelium, an effect fully blockable by pretreat-ment of the tissue pieces with H-7, a protein kinase inhibitor. Using cilia removed from these epithelial sur-faces and incubated in solutions containing stimulators of protein kinase C along with [-32P]ATP or [-35S]ATP, a single protein target of ciliary protein kinase C activity was identified. The protein is a polypeptide of molecular mass 37 kDa (p37) as estimated by SDS-polyacrylamide gel electrophoresis. Protein kinase C dependency of p37 phosphorylation was proven by showing that Calphostin C, a specific protein kinase C inhibitor, blocked label incorporation into p37 com-pletely, and by demonstrating that purified protein kinase C phosphorylated p37. Inhibitors of cAMP-dependent kinase and calcium/calmodulin-dependent

kinase did not change the phosphorylation of p37 in the presence of protein kinase C activators. p37 was recovered in a Triton X-100-extractable fraction of this cil-iary preparation, suggesting that p37 is membrane associated. This hypothesis was further supported by the fact that p37 was present in a pellet representing reconstituted membranes. Thin-layer electrophoresis revealed that p37 was phosphorylated on serine and tyrosine residues, suggesting that the activation of protein kinase C also stimulated tyrosine kinase activity. p37 did not precipitate with annexin I or II antibodies. These results show that sheep tracheal cilia contain protein kinase C activity and that activated protein kinase C phosphory-lates a membrane-associated ovine ciliary target, an effect temporally related to a protein kinase C-mediated decrease in ciliary beat frequency.
Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells
Authors: Turner Mark D. , Handel Susan E. , Wilde Colin J. , Burgoyne Robert D.
Published: April 2021

The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of-, - and -caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of - and -caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mam-mary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of - and -but not -caseins. These results suggest that phospho-rylation of -casein normally occurs in Golgi cisternae whereas that of - and -caseins occurs in the trans-Golgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi com-partments.
Binding of the cytosolic p200 protein to Golgi membranes is regulated by heterotrimeric G proteins
Authors: de Almeida J. Bruno , Doherty Joanne , Ausiello Dennis A. , Stow Jennifer L.
Published: April 2021

The formation of vesicles for protein trafficking requires the dynamic binding of cytosolic coat proteins onto Golgi membranes and this binding is regulated by a variety of GTPases, including heterotrimeric G pro-teins. We have previously shown the presence of the pertussis toxin-sensitive G i-3 protein on Golgi membranes and demonstrated a functional role for G i-3 in the trafficking of secretory proteins through the Golgi complex. We have also described a brefeldin A-sensitive phosphoprotein, p200, which is found in the cytoplasm and on Golgi membranes. The present study investigates the role of heterotrimeric G proteins in the regulation of p200 binding to Golgi membranes. An in vitro binding assay was used to measure the binding of cytosolic p200 to LLC-PK1 cell microsomal membranes and to purified rat liver Golgi membranes in the presence of specific activators of G proteins. The binding of p200 to Golgi membranes was compared to that of the coatomer protein-COP, for which G protein-dependent membrane binding has previously been established. Membrane binding of both p200 and-COP was induced maxi-mally by activation of all G proteins in the presence of GTP S. More selective activation of the heterotrimeric G proteins, with AlFn or mastoparan, also induced membrane binding of p200 and-COP. Pertussis toxin pretreatment of Golgi membranes, to selectively inacti-vate G i-3, reduced the AlFn and mastoparan-induced binding of p200 to Golgi membranes, whereas no sig-nificant effect of pertussis toxin on-COP binding was found in this assay. The effect of pertussis toxin thus implicates G i-3, as one component of a regulatory path-way, in the binding of cytosolic p200 to Golgi mem-branes. The effects of AlFn and pertussis toxin on p200 membrane binding were also shown in intact cells by immunofluorescence staining. AlFn treatment of cells induced translocation of p200 from the cytoplasm onto the Golgi complex, resulting in a conformational change in some Golgi membranes. The translocation of p200 was blocked by pretreatment of intact NRK cells with pertussis toxin. The data presented here support the conclusion that the binding of the p200 protein to Golgi membranes involves regulation by the pertussis toxin-sensitive heterotrimeric G proteins, specifically the G i-3 protein.
Generation of truncated brain AE3 isoforms by alternate mRNA processing
Authors: Morgans Catherine W. , Kopito Ron R.
Published: April 2021

AE3 gene is a member of the AE anion exchanger gene family that is expressed primarily in brain and heart. The principal product of the AE3 gene in rodent brain, FL-AE3p, when expressed in heterologous cell lines, gives rise to chloride-dependent changes in intracellular pH consistent with its operation as an anion exchanger. We have identified two novel isoforms of mouse AE3 that are generated by tissue-specific alternate RNA pro-cessing. One of these isoforms encodes a polypeptide, 14-AE3p, that corresponds to a portion of the NH2-ter-minal cytoplasmic domain of AE3. 14-AE3p lacks the entire transmembrane domain that-in FL-AE3p-forms the anion exchange channel. Immunoblots with antibodies to the NH2- and COOH-termini confirm that FL-AE3 and 14-AE3 are expressed in rat brain as 160 kDa and 74 kDa polypeptides, respectively. Unlike FL-AE3p, however, 14-AE3p is insoluble in non-ionic deter-gent, suggesting a possible association with the cytoskeleton.
Cytoskeletal regulation of ion channel distribution in the tip-growing organism Saprolegnia ferax
Authors: Levina Natalia N. , Lew Roger R. , Heath I. Brent
Published: April 2021

Growing hyphal tips of the oomycete Saprolegnia ferax possess a tip-high gradient of stretch-activated ion channels permeable to calcium. These mechanosensitive channels appear to play a direct role in the polarized tip growth process. Treatment of S. ferax hyphae with cytochalasin E leads to the disruption of plasmalemma-associated, peripheral cytoplasmic actin populations and altered morphology of apical protoplasts, and eliminates the tip-high gradient of stretch-activated channels. Cytochalasin E did not alter the normal aggregation of stretch-activated channels. The density of spontaneous K+ channels was decreased in all regions of the hyphae after treatment with cytochalasin E. These results suggest that the peripheral F-actin network in the growing tip of S. ferax hyphae establishes or maintains the tip-high gradient of SA channels, either by the delivery of channel-bearing vesicles to the apex or by the interactions between the channels and the peripheral actin network.
Identification of serum-inducible genes: Different patterns of gene regulation during g0→s and g1→s progression
Authors: Wick Maresa , Bürger Christiane , Brüsselbach Sabine , Lucibello Frances C. , Müller Rolf
Published: April 2021

We have identified, by differential cDNA library screening, 15 serum inducible genes in the human diploid fibroblast cell line WI-38. The genes fall into two classes that are distinguished by their dependence on protein synthesis for the induction by serum, i.e., primary and secondary genes. While 11 of these genes encode known proteins, 4 other genes have not been described to date. The former genes encode proteins of diverse functions, including the monocyte-derived neutrophil chemotactic factor (MONAP), calmodulin, tropomyosin, tenascin, collagenase, plasminogen activator inhibitor-2a, the ‘sperm-specific’ cleavage signal-1 protein, metallothionein IIa and the mitochondrial chaperonin hsp-60. Interestingly, one of the unknown genes contains a large open reading frame for a polypeptide that is highly homologous to a previously unidentified long open reading frame in the opposite strand of the gene coding for the transcription factor HTF-4. We also studied the regulation of these serum-induced genes during cell cycle progression in normally cycling WI-38 and HL-60 cells separated by counterflow elutriation as well as in serum-stimulated HL-60 cells. Our results clearly show that, in contrast to the prevailing opinion, the expression of most genes induced after mitogen stimulation is not subject to a significant regulation in normally proliferating cells. This supports the hypothesis that the progression into S from either G0 or G1 are distinct processes with specific patterns of gene expression.
Differential induction of ‘metabolic genes’ after mitogen stimulation and during normal cell cycle progression
Authors: Bürger Christiane , Wick Maresa , Brüsselbach Sabine , Müller Rolf
Published: April 2021

Mitogenic stimulation of quiescent cells not only triggers the cell division cycle but also induces an increase in cell volume, associated with an activation of cellular metabolism. It is therefore likely that genes encoding enzymes and other proteins involved in energy metabolism and biosynthetic pathways represent a major class of mitogen-induced genes. In the present study, we investigated in the non-established human fibroblast line WI-38 the induction by mitogens of 17 genes whose products play a role in different metabolic processes. We show that these genes fall into 4 different categories, i.e. non-induced genes, immediate early (IE) primary genes, delayed early (DE) secondary genes and late genes reaching peak levels in S-phase. In addition, we have analysed the regulation of these genes during normal cell cycle progression, using HL-60 cells separated by counterflow elutriation. A clear cell cycle regulation was seen with those genes that are induced in S-phase, i.e. thymidine kinase, thymidylate synthase and dihydrofolate reductase. In addition, two DE genes showed a cell cycle dependent expression. Ornithine decarboxylase mRNA increased around mid-G1, reaching maximum levels in S/G2, while hexokinase mRNA expression was highest in early G1. In contrast, the expression of other DE and IE genes did not fluctuate during the cell cycle, a result that was confirmed with elutriated WI-38 and serum-stimulated HL-60 cells. These observations suggest that G0→S and G1→S transition are distinct processes, exhibiting characteristic programmes of gene regulation, and merging around S-phase entry.
Okadaic acid induces dephosphorylation of histone h1 in metaphase-arrested hela cells
Authors: Paulson James R. , Ciesielski Wayne A. , Schram Brian R. , Mesner Peter W.
Published: April 2021

It is shown here that treatment of metaphase-arrested HeLa cells with okadaic acid (0.15-2.5 μM) leads to dephos-phorylation of histone H1. This effect is presumably due to the specific ability of okadaic acid to inhibit protein phos-phatases 1 and/or 2A, because okadaic acid tetraacetate, which is not a phosphatase inhibitor, has no effect. Dephos-phorylation of H1 does not occur if okadaic acid-treated cells are simultaneously treated with 20 nM calyculin A, or if the okadaic acid concentration is 5.0 μM or greater.

The mechanism behind this phenomenon is not known. However, the results suggest that the chain of events leading to histone dephosphorylation may be negatively controlled by a protein phosphatase 2A, while the phos-phatase which actually dephosphorylates H1 could be a protein phosphatase 1. It remains to be determined whether the phosphatase involved here is the same enzyme as that which dephosphorylates H1 at the end of normal mitosis.