bluuebunny/crossref_metadata_2025_split huggingface dataset:
An in vitro model for the analysis of intestinal brush border assembly I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells
Authors: Peterson Michelle D. , Mooseker Mark S. 
Published: April 2021

ABSTRACT
Intestinal epithelial cells assemble and maintain a polarized, highly organized membrane-cytoskeleton array, the brush border. We describe an in vitro, cell contact-induced brush border assembly model using the Caco-2BBe clones. Subconfluent cells were ‘depolarized’ by brief passage through suspension culture in the presence of cytochalasin D and re-plated on filters at high density in low-Ca2+ medium. Upon return to regular medium, these small, rounded cells with bleb-like protrusions formed, over the course of 19 days, a polarized monolayer of tall, columnar cells with a well-defined brush border. Ultrastructural changes were documented by both transmission and scanning electron microscopy. The earliest events of microvillar assembly coincided with a short period of cell aggregation. Inter-cellular cysts were occasionally observed within these aggregates, and junction formation between cells which had no contact with the filter was also observed. Monolayer formation was completed within 48 hours, and cell height steadily increased approximately 3.5-fold over 19 days. Concurrent with monolayer formation and the increase in cell height, sparse microvilli with a few actin core filaments gradually became more dense and better organized. By the third day, the actin core bundles had begun to extend into the subjacent cytoplasm, while terminal web assembly was underway at five days. The mature morphology of the brush border was first observed at nine days, although cell height and microvillar density continued to increase during the subsequent ten days. Microvillar density rose approximately nine-fold throughout brush border assembly in the Caco-2BBe cells. With the exception of the formation of cellular aggregates at the onset of the time course, this sequence of morphological changes is comparable to that observed during brush border assembly in embryonic intestinal epithelial cells. The Caco-2BBe assembly model provides a useful system in which to investigate various molecular aspects of brush border assembly.
Inhibition of protein synthesis separates autophagic sequestration from the delivery of lysosomal enzymes
Authors: Lawrence B. Paige , Brown William J. 
Published: April 2021

ABSTRACT
To investigate the role of newly synthesized proteins during autophagic sequestration and degradation, the effects of protein synthesis inhibition on autophagic vacuole (AV) formation and degradation were analyzed. The inhibition of protein synthesis was found to separate autophagic sequestration from the delivery of lysosomal enzymes to (AVs). Pretreatment with cycloheximide for ≥ 3 h caused a drastic inhibition of autophagy-induced degradation. Surprisingly, morphological analyses showed that the inhibition of protein synthesis for up to 12 h did not block the formation of nascent AVs; however, it did prevent their conversion into degradative AVs. Using immunoperoxidase cytochemistry with an antibody against cathepsin D and labeling of lysosomes with endocytosed colloidal gold, we found that the nascent AVs that formed during prolonged cycloheximide pretreatment had not received lysosomal markers. The inhibition of autophagic degradation and lysosomal enzyme delivery were rapidly reversed following the removal of cycloheximide. These results suggest that there is a fairly rapid turnover of protein(s) that are necessary for lysosomal fusion, but that the initial formation of AVs is independent of new protein synthesis for a long period of time.
The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain
Authors: Boschelli Frank , Uptain Susan M. , Lightbody James J. 
Published: April 2021

ABSTRACT
The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.
Transitions between in situ and isolated chromatin
Authors: Giannasca P. J. , Horowitz R. A. , Woodcock C. L. 
Published: April 2021

ABSTRACT
We show that the mechanism by which chromatin displaying higher-order structure is usually isolated from nuclei involves a transition to an extended nucleosomal arrangement. After being released from nuclei, chromatin must refold in order to produce the typical chromatin fibers observed in solution. For starfish sperm chromatin with a long nucleosome repeat (222 bp), isolated fibers are significantly wider than those in the nucleus, indicating that the refolding process does not regenerate the native higher-order structure. We also propose that for typical eukaryotic nuclei, the concept that the native state of the (inactive) bulk of the genome is a chromatin fiber with defined architecture be reconsidered.
Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic map located at the spindle pole and characterized as a homologous protein to NuMA
Authors: Maekawa Takami , Kuriyama Ryoko 
Published: April 2021

ABSTRACT
Using a human autoantibody, SP-H, we identified a 200-230 kDa mitotic MAP in a variety of mammalian cell lines which shows affinity for the minus end of microtubules and also becomes associated with the spindle pole during mitosis. To examine the detailed structure and functional organization of the protein, the gene coding for the end-specific MAP was isolated and characterized by screening a human placenta gt11 expression library using SP-H as a probe. Overlapping cDNA clones, which covered the entire length of the coding region of the SP-H antigen, were obtained. Polyclonal antibodies raised against fusion proteins generated from non-overlapping cDNA fragments stained the HeLa SP-H antigen in interphase and mitotic cells, and recognized a single 215 kDa band on immunoblots, as did the original SP-H antibody. Analysis of the nucleotide sequence revealed a 7,091 nucleotide sequence with an open reading frame of 6,345 nucleotides encoding a 2,115 amino acid polypeptide with a calculated molecular mass of 238,376 Da. The predicted amino acid sequence showed the protein to be composed of an -helical domain, flanked by globular domains located at the amino and carboxy termini. The sequence contained five repeats of the hypothetical leucine zipper motif: one is in the N-terminal globular domain, and four are in the central -helical stalk. Comparison with other sequences in the database shows that the SP-H antigen is identical to the NuMA protein reported by Yang et al. (1992)J. Cell Biol. 116, 1303-1317, but there are differences between the SP-H antigen and NuMA sequence reported by Compton et al. (1992)J. Cell Biol. 116, 1395-1408. cDNA inserts of the truncated SP-H antigen were expressed in both insect Sf9 cells and in cultured mammalian cells. The recombinant protein corresponding to the C-terminal half of the protein was restricted to the nucleus, whereas the N-terminal half of the protein was localized in the cytoplasm, suggesting the presence of a nuclear translocation signal(s) in the C-terminal domain. The C-terminal polypeptide expressed in mitotic COS cells was shown to specifically localize at the spindle pole. Microtubule-binding assays using in vitro transcribed/translated polypeptide products from different domains of the SP-H antigen further suggested that the SP-H antigen interacts with microtubules through the globular domain at the C-terminus.
Avian pluripotent haemopoietic progenitor cells: detection and enrichment from the para-aortic region of the early embryo
Authors: Cormier Françoise 
Published: April 2021

ABSTRACT
In the avian embryo, the wall of the aorta is a site where haemopoiesis occurs in large diffuse foci from day 3 to day 10. In contrast to haemopoiesis in other organs of the embryo, para-aortic haemopoiesis is sustained by stem cells, which emerge in situ. Previous studies have demonstrated that the para-aortic region from the day-4 chick embryo harbours committed myeloid progenitors and committed erythroid progenitors. The present paper reports the in vitro development of para-aortic progenitors with both myelomonocytic and erythroid potentialities. Three types of myelo-erythroid progenitors were observed, giving rise to erythroblasts and monocytes, to erythroblasts and granulocytes, or to erythroblasts, monocytes and granulocytes. Their frequency in the para-aortic cell suspension was 1 per 10,000 cells. In cell sorting experiments, they co-sorted with committed progenitors in the cell population that immunolabeled with the VI-A2 monoclonal antibody, which is specific for chicken haemopoietic cells. Cell sorting also demonstrated that these multipotential progenitors did not express the BEN cell surface molecule, in contrast to late myeloid progenitors. The BEN molecule belongs to the immunoglobulin superfamily and is expressed by haemopoietic progenitors from bone marrow, selective sets of neurons and epithelial cells from the bursa of Fabricius. The myelo-erythroid progenitors were enriched 4 times in the VI-A2-positive cell population, and 2 to 5 times in the BEN-negative population. These results represent the first in vitro demonstration of avian normal myelo-erythroid progenitors.
α3β1 Integrin is moved into focal contacts in kidney mesangial cells
Authors: Grenz Harald , Carbonetto Salvatore , Goodman Simon L. 
Published: April 2021

ABSTRACT
The movement of integrins into focal adhesive structures accompanies cell attachment to extracellular matrix. The kinetics of incorporation of integrins into focal contacts was studied during attachment to matrix of mesangial cells of the kidney glomerulus. On collagen, fibronectin, laminin and vitronectin, the number and intensity of talin-focal contacts increased with time. Talin-containing focal contacts were present in mesangial cells within 2 h of plating and in control cells (HT1080 and Rugli) within 1 h. Integrin α-chains colocalized with talin, dependent on the matrix substrate. The attachment, spreading and organization of integrin into focal contacts was not affected when endogenous protein synthesis was suppressed with cycloheximide.
In Rugli, α1 β1 organized into focal contacts on collagen and laminin, while in HT1080 α2 β1 organized on collagen type I, α5 β1 on fibronectin, α6 β1 on laminin, and α3 β1 and α4 β1 were diffusely distributed on all substrates. These distributions mirrored the usage and expression patterns previously established for integrins in these cells and was as predicted from the literature. In mesangial cells, however, α3 β1 was also organized into prominent focal contact arrays on collagen, fibronectin, EHS and human placental laminins, but not on vitronectin, while α6 β1 was not organized.
Initial attachment and spreading of mesangial cells was absolutely dependent on divalent cations. Mg2+ and Mn2+ supported attachment on all substrates, while Ca2+ stimulated attachment on laminin (E8), fibronectin and vitronectin. The data suggest that the functional integrins on mesangial cells include α1 β1 (on collagen and laminin) α2 β1 (on collagen), α5 β1 (on fibronectin) and αV β3 (on vitronectin). However, mesangial cells do not use α6 β1 on laminin, and the data support a role for α3 β1 as putative receptor for fibronectin, collagen and laminin.
Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D
Authors: Friederich E. , Kreis T. E. , Louvard D. 
Published: April 2021

ABSTRACT
Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca2+-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm.
On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the prefered end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxyterminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalain D in living cells can be modulated by actin-binding proteins.
Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein
Authors: Vojtek Anne B. , Cooper Jonathan A. 
Published: April 2021

ABSTRACT
CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cere -visiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxyterminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.
Protein localization to the nucleolus: a search for targeting domains in nucleolin
Authors: Schmidt-Zachmann M. S. , Nigg E. A. 
Published: April 2021

ABSTRACT
Nucleolin, a major nucleolar phosphoprotein, is presumed to function in rDNA transcription, rRNA packaging and ribosome assembly. Its primary sequence was highly conserved during evolution and suggests a multidomain structure. To identify structural elements required for nuclear uptake and nucleolar accumulation of nucleolin, we used site-directed mutagenesis to introduce pointand deletion-mutations into a chicken nucleolin cDNA. Following transient expression in mammalian cells, the intracellular distribution of the corresponding wild-type and mutant proteins was determined by indirect immunofluorescence microscopy. We found that nucleolin contains a functional nuclear localization signal (KRKKEMANKSAPEAKKKK) that conforms exactly to the consensus proposed recently for a bipartite signal (Robbins, J., Dilworth, S. M., Laskey, R. A. and Dingwall, C. (1991) Cell 64, 615-623). Concerning nucleolar localization, we found that the N-terminal 250 amino acids of nucleolin are dispensible, but deletion of either the centrally located RNA-binding motifs (the RNP domain) or the glycine/arginine-rich C terminus (the GR domain) resulted in an exclusively nucleoplasmic distribution. Although both of these latter domains were required for correct subcellular localization of nucleolin, they were not sufficient to target nonnucleolar proteins to the nucleolus. From these results we conclude that nucleolin does not contain a single, linear nucleolar targeting signal. Instead, we propose that the protein uses a bipartite NLS to enter the nucleus and then accumulates within the nucleolus by virtue of binding to other nucleolar components (probably rRNA) via its RNP and GR domains.
ors12, a mammalian autonomously replicating DNA sequence, associates with the nuclear matrix in a cell cycle-dependent manner
Authors: Mah David C. W. , Dijkwel Pieter A. , Todd Andrea , Klein Veronica , Price Gerald B. , Zannis-Hadjopoulos Maria 
Published: April 2021

ABSTRACT
Origin enriched sequences ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.
Carboxyl terminus structural requirements for glycosylphosphatidylinositol anchor addition to cell surface proteins
Authors: Beghdadi-Rais C. , Schreyer M. , Rousseaux M. , Borel P. , Eisenberg R. J. , Cohen G. H. , Bron C. , Fasel N. 
Published: April 2021

ABSTRACT
Glycosyl phosphatidylinositol (GPI)-anchored proteins contain in their COOH-terminal region a peptide segment that is thought to direct glycolipid addition. This signal has been shown to require a pair of small amino acids positioned 10-12 residues upstream of an hydrophobic C-terminal domain. We analysed the contribution of the region separating the anchor acceptor site and the C-terminal hydrophobic segment by introducing amino acid deletions and substitutions in the spacer element of the GPI-anchored Thy-1 glycoprotein. Deletions of 7 amino acids in this region, as well as the introduction of 2 charged residues, prevented the glycolipid addition to Thy-1, suggesting that the length and the primary sequence of the spacer domain are important determinants in the signal directing GPI anchor transfer onto a newly synthesized polypeptide. Furthermore, we tested these rules by creating a truncated form of the normally transmembranous Herpes simplex virus I glycoprotein D (gDI) and demonstrating that when its C-terminal region displays all the features of a GPI-anchored protein, it is able to direct glycolipid addition onto another cell surface molecule.
An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum
Authors: Nolta Kathleen V. , Padh Harish , Steck Theodore L. 
Published: April 2021

ABSTRACT
Antisera were generated in rabbits against the vacuolar proton pump (V-H+-ATPase) purified from Dic -tyostelium discoideum. The antisera inhibited V-H+-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H+-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H+-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium.
Antisera directed against the V-H+-ATPase of Dic -tyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells.
We conclude that the V-H+-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.
Abbreviations used: V-H+-ATPase, vacuolar proton ATPase; buffer T, PBS containing 0.2% Tween-20; PBS, 0.15 M NaCl-50 mM NaPi (pH 7.0); TRITC, Texas Red-isothiocyanate; buffer E, 50 mM sodium cacodylate + 100 mM sucrose (pH 6.8); BSA, bovine serum albumin