bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.
Microtubule assembly and kinetochore directional instability in vertebrate monopolar spindles: Implications for the mechanism of chromosome congression
Authors: Cassimeris Lynne , Rieder Conly L. , Salmon E. D. 
Published: April 2021

ABSTRACT
We have proposed previously a kinetochore motor-polar ejection model for chromosome congression to the metaphase plate where forces generated at the kinetochore are antagonized by away-from-the pole forces generated within each half-spindle on the chromosome arms. This model was based in large part on observations of the behavior of chromosomes on monopolar spindles. In these cells chromosomes typically become attached to the pole by only one kinetochore fiber. These mono-oriented chromo-somes move to positions away from the pole even though they are pulled poleward at their kinetochores. Their arms are also ejected away from the pole when severed from the centromere. Here we have characterized further the prop-erties of monopolar spindles in newt lung epithelial cells to determine the similarities between monopolar and bipolar spindles. We found no significant differences between monopolar and bipolar spindles over the parameters examined, which included: microtubule dynamics as measured by fluorescence redistribution after photo-bleaching; the ability of polar microtubule arrays to push chromosome arms away from the pole; the dependence of chromosome position relative to the pole on microtubule assembly; the number of kinetochore microtubules per kinetochore; and the directional instability of kinetochore motion during chromosome oscillations poleward and away-from-the-pole. As in bipolar spindles, kinetochore directional instability is characterized by abrupt switching between constant velocity phases of poleward and away-from-the-pole motion. From these data we conclude that the mechanism(s) responsible for chromosome positioning in monopolar spindles are fundamentally the same as those in bipolar spindles; only the geometry of the two spindle forms and the interplay between sister kinetochore direc-tional instabilities are different. We also found no correla-tion in the kinetochore-to-pole distance with kinetochore microtubule number in monopolar spindles, but a strong qualitative correlation with microtubule density. This finding indicates that oscillations of mono-oriented chro-mosomes in both monopolar and bipolar spindles occur because chromosomes persist in poleward motion until they reach a density of polar microtubules sufficiently high to promote switching to away-from-the-pole motion. As the kinetochore and chromosome arms move away-from-the-pole, microtubule density decreases and the kinetochore switches to poleward motion, pulling the chromosome arms back into regions of higher microtubule density. The mechanism regulating kinetochore switching between poleward and away-from-the-pole motion is poorly under-stood, but may depend on tension at the kinetochore generated by pushing forces on the chromosome arms produced by the polar microtubule arrays.
The cell adhesion molecule, connectin, and the development of the Drosophila neuromuscular system
Authors: Meadows Lisa A. , Gell David , Broadie Kendal , Gould Alex P. , White Robert A. H. 
Published: April 2021

ABSTRACT
The connectin gene of Drosophila has been identified as a candidate direct target of homeotic gene control and has also been implicated in the formation of specific neuro-muscular connections. The gene product, connectin, is a member of the leucine-rich repeat protein family and we show that it is attached to the cell surface via a glycosyl-phosphatidylinositol linkage and that it can mediate homotypic cell-cell adhesion in vitro. The expression of connectin protein during Drosophila embryogenesis provides support for a role in adhesion in vivo. In the central nervous system, it is initially expressed on longitu-dinal glia and on a few identified neurons. These cells extend processes and connect up to form a continuous scaffold of connectin-expressing cells, presaging the devel-opment of axonal pathways. Later, connectin is expressed on specific axons as they track along the connectin scaffold. Glial expression then declines and connectin appears on axons that fasciculate with pre-existing connectin-positive bundles. Thus scaffold formation, axon pathfinding and fasciculation involve specific contacts between connectin-positive cells. The timing and pattern of connectin expression suggest that it may play an important role in mediating specific interactions through homotypic cell adhesion.
Cloning of a cdna encoding human centrin, an ef-hand protein of centrosomes and mitotic spindle poles
Authors: Errabolu Ramesh , Sanders Mark A. , Salisbury Jeffrey L. 
Published: April 2021

ABSTRACT
A human cDNA expression library was screened using anti-centrin antibodies to obtain a cDNA clone encoding human centrin. The cDNA clone contains an open reading frame of 516 base pairs and predicts a product of 172 amino acids with a calculated molecular mass of 19,528 and a pI of 4.61. Sequence analysis demonstrates that human centrin and centrins from higher plants, protozoa, algae, Xenopus and the yeast CDC31 gene product are closely related members of a subfamily of the EF-hand superfamily of calcium-binding proteins. The human centrin sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation and western blot studies from HeLa cells confirm that human centrin is a protein of ∼20,000 Mr as predicted from the cDNA clone. Indirect immunofluorescence analysis of HeLa cells demonstrates that centrin is localized at the cen-trosome of interphase cells and that it redistributes to the region of the spindle poles during mitosis. When taken together with earlier genetic studies, these results demon-strate that centrin is a ubiquitous component of centro-somes and mitotic spindle poles of diverse organisms and suggest that centrin plays a role in centrosome separation at the time of mitosis.