bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast
Authors: Huang Kristen M. , Gullberg Lisa , Nelson Karen K. , Stefan Christopher J. , Blumer Kendall , Lemmon Sandra K. 
Published: April 2021

ABSTRACT
Clathrin is a major coat protein involved in sorting and retention of proteins at the late Golgi and in endocytosis from the cell surface. The clathrin triskelion contains three heavy chains, which provide the structural backbone of the clathrin lattice and three light chains, which are thought to regulate the formation or disassembly of clathrin coats. To better understand the function of the clathrin light chain, we characterized yeast strains carrying a disruption of the clathrin light chain gene (CLC1). Light chain-deficient cells showed phenotypes similar to those displayed by yeast that have a disruption in the clathrin heavy chain gene (CHC1). In clc1-Δ cells, the steady state level of the clathrin heavy chain was reduced to 20%-25% of wild-type levels and most of the heavy chain was not trimerized. If CHC1 was overexpressed in clc1-Δ cells, heavy chain trimers were detected and several clc1-Δ phenotypes were partially rescued. These results indicate that the light chain is important for heavy chain trimerization and the heavy chain still has some function in the absence of the light chain.
In yeast, deletion of CHC1 is lethal in strains carrying the scd1-i allele, while strains carrying the scd1-v allele can survive without the heavy chain. In previous studies we isolated several multicopy suppressors of inviability of chc1-Δ scd1-i cells. Surprisingly, one of these suppressors, SCD4, is identical to CLC1. Overexpression of CLC1 in viable chc1-Δ scd1-v strains rescued some but not all of the phenotypes displayed by these cells. In the absence of the heavy chain, the light chain was not found in a high molecular mass complex, but still associated with membranes. These results suggest that the light chain can function independently of the clathrin heavy chain in yeast.
Functional analysis of human RPS14 null alleles
Authors: Roufa Donald J. , Martín-Nieto José 
Published: April 2021

ABSTRACT
Previously we described a large collection of cloned human DNAs that encode chemically defined missense mutations within the ribosomal protein S14 sequence. We determined that biologically inactive (i.e. null) alleles resulted primarily from point mutations targeted to two internal segments of the S14-coding sequence and designated these functionally critical regions as domains B and D. Further, we inferred that structural determinants within domains B and D are required for proper incorporation of the S14 protein into nascent 40 S ribosomal particles and/or for the normal function of mature cytoplasmic ribosomes. In this study we have used immunofluorescence to monitor the intracellular trafficking of epitopically labeled human S14 protein isoforms transiently expressed by cultured Chinese hamster cells. Data obtained distinguish null alleles of RPS14 which encode proteins that are not incorporated into pre-ribosomal subunit particles from null alleles whose products are compatible with normal ribosome assembly processes but result in functionally inactive cytoplasmic 40 S ribosomal subunits. Mutations assigned to the first allele class involve amino acid replacements located within S14 domains B and D; whereas mutations assigned to the second class are distributed throughout the S14 proteincoding sequence.
Imaging of Ca2+ transients induced in Paramecium cells by a polyamine secretagogue
Authors: Klauke Norbert , Plattner Helmut 
Published: April 2021

ABSTRACT
In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (&lt;1 second) trichocyst exocytosis has been hampered by various technical problems which we now have overcome. While Fura Red was found appropriate for quantitative double wavelength recordings, Fluo-3 allowed to follow, semi-quantitatively but with high time resolution, [Ca2+]i changes by rapid confocal laser scanning microscopy (CLSM). Resting values are between 50 and 70 nM in the strains analysed (7S wild type, as well as a non-discharge and a trichocyst-free mutant, nd9-28°C and tl). In all strains [Ca2+]i first increases at the site of AED application, up to 10-fold above basal values, followed by a spillover into deeper cell regions. This might: (i) allow a vigorous Ca2+ flush during activation, and subsequently (ii) facilitate re-establishment of Ca2+ homeostasis within ≥20 seconds. Because of cell dislocation during vigorous trichocyst exocytosis, 7S cells could be reasonably analysed only by CLSM after Fluo-3 injection. In 7S cells cortical [Ca2+]i transients are strictly parallelled by trichocyst exo-cytosis, i.e. in the subsecond time range and precisely at the site of AED application. Injection of Ca2+ is a much less efficient trigger for exocytosis. Ca2+-buffer injections suggest a requirement of [Ca2+]i &gt;1 to 10 μM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent.
Sec12p requires Rer1p for sorting to coatomer (COPI)-coated vesicles and retrieval to the ER
Authors: Boehm Johannes , Letourneur François , Ballensiefen Wolfgang , Ossipov Dmitri , Démollière Corinne , Schmitt Hans Dieter 
Published: April 2021

ABSTRACT
In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER. The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins. In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi. In contrast, rer3 mutants accumulate Sec12-derived hybrid proteins carrying early Golgi modifications. We found that rer3 mutants harbour unique alleles of the α-COP-encoding RET1 gene. ret1 mutants, along with other coatomer mutants, fail to retrieve KKXX-tagged type I transmembrane proteins from the Golgi back to the ER. Surprisingly rer3-11(=ret1-12) mutants do not affect this kind of ER recycling. Pulse-chase experiments using these mutants show that α-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins. Rer1p protein may be directly involved in the retrieval process since it also recycles between the early Golgi and ER in a coatomer (COPI)-dependent manner. Rer1p may act as an adapter coupling the recycling of non-KKXX transmembrane proteins like Sec12p to the coatomer (COPI)-mediated backward traffic.
The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi
Authors: Nothwehr Steven F. , Hindes Anna E. 
Published: April 2021

ABSTRACT
Genetic analysis of late Golgi membrane protein localization in Saccharomyces cerevisiae has uncovered a large number of genes (called GRD) that are required for retention of A-ALP, a model late Golgi membrane protein. Here we describe one of the GRD genes, VPS5/GRD2, that encodes a hydrophilic protein similar to human sorting nexin-1, a protein involved in trafficking of the epidermal growth factor receptor. In yeast cells containing a vps5 null mutation the late Golgi membrane proteins A-ALP and Kex2p were rapidly mislocalized to the vacuolar membrane. A-ALP was delivered to the vacuole in vps5 mutants in a manner independent of a block in the early endocytic pathway. vps5 null mutants also exhibited defects in both vacuolar morphology and in sorting of a soluble vacuolar protein, carboxypeptidase Y. The latter defect is apparently due to an inability to localize the carboxypeptidase Y sorting receptor, Vps10p, to the Golgi since it is rapidly degraded in the vacuole in vps5 mutants. Fractionation studies indicate that Vps5p is distributed between a free cytosolic pool and a particulate fraction containing Golgi, transport vesicles, and possibly endosomes, but lacking vacuolar membranes. Immunofluorescence microscopy experiments show that the membrane-associated pool of Vps5p localizes to an endosome-like organelle that accumulates in the class E vps27 mutant. These results support a model in which Vps5p is required for retrieval of membrane proteins from a prevacuolar/late endosomal compartment back to the late Golgi apparatus.
A nematode gene required for sperm vesicle fusion
Authors: Achanzar William E. , Ward Samuel 
Published: April 2021

ABSTRACT
During maturation of spermatids to motile spermatozoa in Caenorhabditis elegans, large vesicles called membranous organelles (MOs) fuse with the spermatid plasma membrane. Mutations in the gene fer-1 cause abnormal spermatozoa in which the MOs do not fuse, although they abut the plasma membrane normally. Here we describe the fer-1 gene, which we found to be approximately 8.6 kb in length and to encode a 6.2 kb transcript whose expression is limited to the primary spermatocytes, the cells in which the MOs form. fer-1 is predicted to encode a 235 kDa protein which is highly charged except for a putative transmembrane domain near the C terminus. We identified the mutations associated with five fer-1 alleles, all of which are missense mutations causing single amino acid changes. FER-1 is not similar to any characterized proteins in sequence databases, nor does it contain known functional motifs other than the predicted transmembrane domain. The C-terminal transmembrane domain makes FER-1 resemble some viral fusion proteins, suggesting it may play a direct role in MO-plasma membrane fusion. FER-1 does show significant sequence similarity to several predicted human proteins of unknown function. Two of the identified fer-1 mutations are located in regions of similarity between FER-1 and two of these predicted proteins. This strengthens the biological significance of these similarities and suggests these regions of similarity represent functionally important domains of FER-1 and the human proteins.
Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion
Authors: Bonavaud Sylvie , Charrière-Bertrand Cécile , Rey Christiane , Leibovitch Marie-Pierre , Pedersen Nina , Frisdal Eric , Planus Emmanuelle , Blasi Francesco , Gherardi Romain , Barlovatz-Meimon Georgia 
Published: April 2021

ABSTRACT
Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA7PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA7PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.
Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading
Authors: Planus Emmanuelle , Barlovatz-Meimon Georgia , Rogers Rick A. , Bonavaud Sylvie , Ingber Donald E. , Wang Ning 
Published: April 2021

ABSTRACT
Urokinase plasminogen activator and its receptor are both found at the surface of the cell membrane in many cell types. The plasminogen activator inhibitor type-1 (PAI-1) is often associated with the extracellular matrix. The spatial localization of these three molecules could account for their involvement in cell adhesion and/or migration. We have shown previously that the urokinase receptor mediates mechanical force transmission across the cell surface to the cytoskeleton. Here we investigated whether immobilized plasminogen activator inhibitor type 1 (PAI-1) could regulate cell spreading and cytoskeleton reorganization. Serum deprived human myogenic cells were plated in serum free medium onto bacteriologic dishes precoated with different extracellular matrix ligands (fibronectin, vitronectin, or type 1 collagen) or PAI-1 at increasing concentrations. The number of adherent cells and their projected area were quantitated after 3 hours of plating. PAI-1 promoted cell adhesion and spreading in a dose dependent manner. Addition of antibodies to PAI-1 inhibited the adhesion on PAI-1 coated dishes in a dose dependent way. The PAI-1 mediated cell adhesion required the presence of urokinase at the cell surface. Removal of the glycosylphos-phatidylinositol (GPI)-linked proteins abolished cell adhesion on PAI-1 dish, suggesting its dependence on the presence of the urokinase receptor, a GPI-linked receptor. Furthermore, addition of antibodies against αvβ3 integrin completely inhibited cell adhesion on PAI-1, suggesting that αvβ3 might be the transmembrane molecule that physically connects the complex of PAI-1, urokinase, and urokinase receptor to the cytoskeleton. Visualization of spread cells stained for filamentous actin with confocal microscopy showed a dose-dependent increase of filopodia on PAI-1 coated dishes and cytoskeletal reorganization, suggesting a migratory profile. These data indicate that PAI-1 plays a direct role in dynamic cell adhesion particularly at the leading edge, where increased levels of urokinase plasminogen activator (uPA) and its receptor (uPAR) are localized in migrating cells. Immobilized PAI-1 could therefore serve to bridge the cell surface with the extracellular matrix via the formation of a multimolecular complex that includes αvβ3 integrins in myogenic cells.
Occludin confers adhesiveness when expressed in fibroblasts
Authors: Itallie Christina M. Van , Anderson James Melvin 
Published: April 2021

ABSTRACT
Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cellcell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.
Myeloperoxidase mediates cell adhesion via the αMβ2 integrin (Mac-1, CD11b/CD18)
Authors: Johansson Mats W. , Patarroyo Manuel , Öberg Fredrik , Siegbahn Agneta , Nilsson Kenneth 
Published: April 2021

ABSTRACT
Myeloperoxidase is a leukocyte component able to generate potent microbicidal substances. A homologous invertebrate blood cell protein, peroxinectin, is not only a peroxidase but also a cell adhesion ligand. We demonstrate in this study that human myeloperoxidase also mediates cell adhesion. Both the human myeloid cell line HL-60, when differentiated by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid, and human blood leukocytes, adhered to myeloperoxidase; however, undifferentiated HL-60 cells showed only minimal adhesion. No cells adhered to horseradish peroxidase, and cell adhesion to myeloperoxidase was not decreased by catalase, thus showing that peroxidase activity, per se, was neither sufficient nor necessary for the adhesion activity. Mannan, which has been reported to inhibit the binding of peroxidases to cells, did not affect adhesion to myeloperoxidase. However, adhesion to myeloperoxidase was inhibited by monoclonal antibodies to αM (CD11b) or to β2 (CD18) integrin subunits, but not by antibodies to αb (CD11a), αx (CD11c), or to other integrins. Native myeloperoxidase mediated dosedependent cell adhesion down to relatively low concentrations, and denaturation abolished the adhesion activity. It is evident that myeloperoxidase supports cell adhesion, a function which may be of considerable importance for leukocyte migration and infiltration in inflammatory reactions, that αMβ2 integrin (Mac-1 or CD11b/CD18) mediates this adhesion, and that the αMβ2 integrin-mediated adhesion to myeloperoxidase is distinct from the previously reported ability of this integrin to bind to certain denatured proteins at high concentrations.
Mutational analysis of regulated exocytosis in Tetrahymena
Authors: Melia Sharon M. , Cole Eric S. , Turkewitz Aaron P. 
Published: April 2021

ABSTRACT
Genetic analysis of regulated exocytosis can be accomplished in ciliates, since mutants defective in stimulus-dependent secretion of dense-core vesicles can be identified. In Tetrahymena thermophila, secretion in wild-type cells can result in their encapsulation by the proteins released from vesicle cores. Cells with defects in secretion were isolated from mutagenized homozygous cells that were generated using a highly efficient method. Screening was based both on a visual assay for encapsulation, and on a novel panning step using differential centrifugation to take advantage of the selective mobility of mutants that fail to encapsulate upon stimulation. 18 mutants with defects in several ordered steps have been identified. Defects in a set of these could be localized to three stages: granule formation, transport to cell surface docking sites, and exocytosis itself. Mutants with defects in this last stage can be ordered into successive steps based on several criteria, including their responsiveness to multiple secretagogues and Ca2+ ionophores. The results of both somatic and genetic complementation on selected pairs also help to characterize the defective factors.
Degradation of phagosomal components in late endocytic organelles
Authors: Tjelle Torunn Elisabeth , Saigal Babita , Frøystad Marianne , Berg Trond 
Published: April 2021

ABSTRACT
Phagosomes are formed when phagocytic cells ingest particles such as bacteria, viruses or synthetic beads of different kinds. The environment within the phagosome gradually changes to generate degradative conditions. These changes require multiple interactions between the maturing phagosomes and the endocytic and the biosynthetic pathway. The phagosomes probably communicate with endocytic organelles by a transient fusion event, often referred to as the ‘kiss- and-run’ hypothesis.
We have studied the role of endocytic organelles in the phagocytic pathway of J774 cells, a mouse macrophage cell line. We have used magnetic Dynabeads coated with 125ITC-IgG and 125ITC-OVA as phagocytic probes and were able to isolate the phagosomal fraction by means of a magnet. To separate lysosomes from other organelles in the endocytic pathway we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three endocytic fractions corresponding to early endosomes (the light Percoll fraction), late endosomes (the dense Percoll fraction) and lysosomes (the gold fraction).
We observed that the proteins linked to the ingested beads are initially cleaved in the phagosomes. This cleavage is inhibited by leupeptin, a thiol-protease inhibitor, and requires an acidic environment. However, efficient communication between the phagosomes and the endocytic pathway leads to the transfer of dissociated phagocytosed peptides of different sizes to late endosomes and lysosomes for further processing. Consequently, the late endosomes and the lysosomes may be involved in the degradation of phagocytosed compounds.
Targeted gene disruption reveals a role for vacuolin B in the late endocytic pathway and exocytosis
Authors: Jenne Nicole , Rauchenberger Robert , Hacker Ulrike , Kast Thomas , Maniak Markus 
Published: April 2021

ABSTRACT
Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, an endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B− mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.