bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genes that cause aberrant cell morphology by overexpression in fission yeast: a role of a small GTP-binding protein Rho2 in cell morphogenesis
Authors: Hirata Dai , Nakano Kentaro , Fukui Mikiko , Takenaka Hiroshi , Miyakawa Tokichi , Mabuchi Issei 
Published: April 2021

ABSTRACT
To identify the genes involved in cell morphogenesis in Schizosaccharomyces pombe, we screened for the genes that cause aberrant cell morphology by overexpression. The isolated genes were classified on the basis of morphology conferred. One of the genes causing a rounded morphology was identified as the rho2+ gene encoding a small GTP-binding protein. The overexpression of rho2+ resulted in a randomized distribution of cortical F-actin and formation of a thick cell wall. Analyses using cdc mutants suggested that the overexpression of rho2+ prevents the establishment of growth polarity in G1. The rho2+ gene was not essential, but among cells deleted for rho2+, those with an irregular shape were observed. The disruptant also showed a defect in cell wall integrity. An HA-Rho2 expressed in the cell was suggested to be present as a membrane-bound form by a cell fractionation experiment. A GFP-Rho2 was localized at the growing end(s) of the cell and the septation site. The localization of GFP-Rho2 during interphase was partially dependent on sts5+. These results indicate that Rho2 is involved in cell morphogenesis, control of cell wall integrity, control of growth polarity, and maintenance of growth direction. Analysis of functional overlapping between Rho2 and Rho1 revealed that their functions are distinct from each other, with partial overlapping.
Aminopeptidase B: a processing enzyme secreted and associated with the plasma membrane of rat pheochromocytoma (PC12) cells
Authors: Balogh Agnès , Cadel Sandrine , Foulon Thierry , Picart Renée , Garabedian Arsène Der , Rousselet Annie , Tougard Claude , Cohen Paul 
Published: April 2021

ABSTRACT
Aminopeptidase B (Ap-B) is a Zn2+-dependent exopeptidase which selectively removes Arg and/or Lys residues from the N terminus of several peptide substrates. Isolated and characterized from rat testes, this ubiquitous enzyme may participate in the final stages of precursor processing mechanisms. To test this hypothesis, we have investigated the secretion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells). By using a combination of biochemical and immunocytochemical methods, the following observations were made: (i) the level of aminopeptidase B detectable in the cell culture medium increased with time; (ii) 8-bromo-adenosine 3′-5′-cyclic monophosphate and the Ca2+ ionophore A23187 both stimulated enzyme liberation in the culture medium; (iii) brefeldin A, an inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi apparatus, decreased enzyme secretion in a time-dependent manner; (iv) whereas nocodazole, a microtubule depolymerizing agent, inhibited enzyme secretion, cytochalasin D, a microfilament disruption agent, had no effect on released aminopeptidase B level; (v) immunofluorescence demonstrated the presence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescence, electron microscopy and tests of enzyme activity on intact cells showed an association of the peptidase with the external face of the plasma membrane. Together these data strongly argued in favour of the enzyme secretion by PC12 cells. It is concluded that aminopeptidase B may participate in processing events occurring either during its intracellular transport along the secretory pathway or at the plasma membrane level, or both.
Drosophila fascin mutants are rescued by overexpression of the villin-like protein, quail
Authors: Cant Kelly , Knowles Brenda A. , Mahajan-Miklos Shalina , Heintzelman Matthew , Cooley Lynn 
Published: April 2021

ABSTRACT
Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells requires two actin bundling proteins, fascin encoded by the singed gene and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. We used P-element mediated germline transformation to overexpress quail in singed mutants and test whether these proteins have redundant functions in vivo. Overexpression of quail protein in a sterile singed background restores actin bundle formation in egg chambers. The degree of rescue by quail depends on the level of quail protein overexpression, as well as residual levels of fascin function. In nurse cells that contain excess quail but no fascin, the cytoplasmic actin network initially appears wild type but then becomes disorganized in the final stages of nurse cell cytoplasm transport. The ability of quail overexpression to compensate for the absence of fascin demonstrates that fascin is partially redundant with quail in the Drosophila germline. Quail appears to function as a bundle initiator while fascin provides bundle organization.
Intracellular trafficking of the vacuolar H+-ATPase accessory subunit Ac45
Authors: Jansen Eric J. R. , Holthuis Joost C. M. , McGrouther Collette , Burbach J. Peter H. , Martens Gerard J. M. 
Published: April 2021

ABSTRACT
Ac45 is a type I transmembrane protein associated with vacuolar H+-ATPase, a proton pump mediating the acidification of multiple intracellular organelles. In this study, we examined the intracellular routing of Ac45 in transfected CV-1 fibroblasts. Steady state immunolabeling showed that Ac45 is located on the plasma membrane and in a vacuolar compartment in the juxtanuclear region. Antibody internalization experiments revealed that Ac45 is rapidly retrieved from the cell surface and is targeted to the vacuolar structures. The 26-residue cytoplasmic tail of Ac45 was intrinsically capable of mediating endocytosis of the cell surface protein Tac, indicating that the tail contains an autonomous internalization signal. Immunolocalization studies on cells expressing carboxy-terminally truncated Ac45 mutants showed the presence of essential routing information in the membrane-distal region of the cytoplasmic tail. Further mutational analysis of this region, which lacks the recognized tyrosine- or di-leucine-based sorting motifs, suggested that multiple sites rather than a short linear sequence are responsible for the internalization. Collectively, our results indicate that the cytoplasmic tail of Ac45 contains autonomous targeting information distinct from previously described routing determinants.
Ye1013p (Vac8p), an armadillo repeat protein related to plakoglobin and importin α, is associated with the yeast vacuole membrane
Authors: Fleckenstein Diana , Rohde Manfred , Klionsky Daniel J. , Rüdiger Manfred 
Published: April 2021

ABSTRACT
Proteins of the armadillo family are involved in diverse cellular processes in higher eukaryotes. Some of them, like armadillo, β-catenin and plakoglobins have dual functions in intercellular junctions and signalling cascades. Others, belonging to the importin-α-subfamily are involved in NLS recognition and nuclear transport, while some members of the armadillo family have as yet unknown functions.
Here, we introduce the Saccharomyces cerevisiae protein Ye1013p as a novel armadillo (arm) repeat protein. The ORF Ye1013w was identified in the genome project on chromosome V (EMBL: U18530) and codes for an acidic protein of 578 residues with 8 central arm-repeats, which are closely related to the central repeat-domain of Xenopus laevis plakoglobin. We show that Ye1013p (Vac8p) is constitutively expressed in diploid and haploid yeasts and that it is not essential for viability and growth. However, the vacuoles of mutant cells are multilobular or even fragmented into small vesicles and the processing of aminopeptidase I, representing the cytoplasm-to-vacuole transport pathway, is strongly impaired. Consistent with these observations, subcellular fractionation experiments, immunolocalization and expression of green fluorescent protein (GFP) fusion proteins revealed that Ye1013p (Vac8p) is associated with the vacuolar membrane.
Our data provide evidence for the involvement of an arm-family member in vacuolar morphology and protein targeting to the vacuole.
A factor required for nonsense-mediated mRNA decay in yeast is exported from the nucleus to the cytoplasm by a nuclear export signal sequence
Authors: Shirley Renee L. , Lelivelt Michael J. , Schenkman Laura R. , Dahlseid Jeffrey N. , Culbertson Michael R. 
Published: April 2021

ABSTRACT
In Saccharomyces cerevisiae, Upf3p is required for nonsense-mediated mRNA decay (NMD). Although localized primarily in the cytoplasm, Upf3p contains three sequence elements that resemble nuclear localization signals (NLSs) and two sequence elements that resemble nuclear export signals (NESs). We found that a cytoplasmic reporter protein localized to the nucleus when fused to any one of the three NLS-like sequences of Upf3p. A nuclear reporter protein localized to the cytoplasm when fused to one of the NES-like sequences (NES-A). We present evidence that NES-A functions to signal the export of Upf3p from the nucleus. Combined alanine substitutions in the NES-A element caused a re-distribution of Upf3p to a subnuclear location identified as the nucleolus and conferred an Nmd− phenotype. Single mutations in NES-A failed to affect the distribution of Upf3p and were Nmd+. When an NES element from HIV-1 Rev was inserted near the C terminus of a mutant Upf3p containing multiple mutations in NES-A, the cytoplasmic distribution typical of wild-type Upf3p was restored but the cells remained phenotypically Nmd−. These results suggest that NES-A is a functional nuclear export signal. Combined mutations in NES-A may cause multiple defects in protein function leading to an Nmd− phenotype even when export is restored.
The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells
Authors: Lyle Stephen , Christofidou-Solomidou Melpo , Liu Yaping , Elder David E. , Albelda Steven , Cotsarelis George 
Published: April 2021

ABSTRACT
Stem cells are vital for the homeostasis of self-renewing tissues such as the hair follicle. Epithelial stem cells have been implicated in tumorigenesis and wound healing, and their manipulation may have wide ranging applications including gene therapy and tissue transplantation. Rodent hair follicle stem cells have been localized to an area of the follicle called the bulge, however, the identification and characterization of human hair follicle stem cells has been hampered by a lack of cellular markers for this area. We have determined that the C8/144B monoclonal antibody, originally generated against a short intracytoplasmic peptide of CD8, preferentially immunostains hair follicle bulge keratinocytes without staining the remaining hair follicle. Using expression cloning, we identified cytokeratin 15 as the keratinocyte protein recognized by the C8/144B monoclonal antibody. By delineating the bulge using this antibody, we demonstrated that bulge cells possess a stem cell phenotype characterized by their slowly-cycling nature, preferential proliferation at the onset of new hair follicle growth, high level of β1 integrin expression, and expression of cytokeratin 19.
N-terminal domain of Gpa1 (G protein α subunit) is sufficient for plasma membrane targeting in yeast Saccharomyces cerevisiae
Authors: Gillen Kathy M. , Pausch Mark , Dohlman Henrik G. 
Published: April 2021

ABSTRACT
G proteins play a central role in transmitting signals from cell surface receptors to effector proteins inside the cell. Signaling can only occur, however, if all these protein components are properly assembled and localized at the plasma membrane. Past studies have shown that certain segments within the N-terminal region of the G protein α subunit are necessary for membrane attachment. Here we identify a region within the yeast Gα (Gpa1) that is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane. Initially, we constructed chimeric proteins that replace the N terminus of mammalian Gsα with the corresponding sequence from Gpa1. Gsα is inefficiently targeted to the yeast plasma membrane and therefore cannot fully complement the loss of Gpa1. Gpa1-Gsα chimeras were assayed for proper membrane localization by functional complementation of a gpa1Δ mutant, and by sucrose density gradient fractionation of cell membranes. Most of the chimeras tested, including one with only the N-terminal 7 amino acids from Gpa1, exhibited normal membrane targeting and complementing activity. We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expressed in the cytoplasm. The first 67- 36- or 9-amino acids of Gpa1 were all sufficient to direct GST specifically to the plasma membrane in yeast. This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isolating and identifying G protein-targeting proteins within the plasma membrane.
Cell biology of transcription and pre-mRNA splicing: nuclear architecture meets nuclear function
Authors: Misteli Tom 
Published: April 2021

ABSTRACT
Gene expression is a fundamental cellular process. The basic mechanisms involved in expression of genes have been characterized at the molecular level. A major challenge is now to uncover how transcription, RNA processing and RNA export are organized within the cell nucleus, how these processes are coordinated with each other and how nuclear architecture influences gene expression and regulation. A significant contribution has come from cell biological approaches, which combine molecular techniques with microscopy methods. These studies have revealed that the mammalian cell nucleus is a complex but highly organized organelle, which contains numerous subcompartments. I discuss here how two essential nuclear processes – transcription and pre-mRNA splicing – are spatially organized and coordinated in vivo, and how this organization might contribute to the control of gene expression. The dynamic nature of nuclear proteins and compartments indicates a high degree of plasticity in the cellular organization of nuclear functions. The cellular organization of transcription and splicing suggest that the morphology of nuclear compartments is largely determined by the activities of the nucleus.
Patching plasma membrane disruptions with cytoplasmic membrane
Authors: McNeil Paul L. , Vogel Steven S. , Miyake Katsuya , Terasaki Mark 
Published: April 2021

ABSTRACT
Vesicle-vesicle fusion initiated in cell cytoplasm by high Ca2+ can rapidly erect large membrane boundaries. These might be used as a ‘patch’ for resealing plasma membrane disruptions. Three central predictions of this ‘patch’ hypothesis are here established in sea urchin eggs. First, we show that surface markers for plasma membrane protein and lipid are initially absent over disruption sites after resealing is complete. Second, we demonstrate that resealing capacity is strongly dependent upon local availability of fusion competent cytoplasmic organelles, specifically the reserve or yolk granule. Lastly, we demonstrate that the reserve granule is capable of rapid (t1/2 &lt;1 second), Ca2+-regulated (high threshold) fusion capable of erecting large (&gt;1000 μm2), continuous membrane boundaries. Production of patch vesicles for resealing may proceed by an ‘emergency’ fusion mechanism distinct from that utilized for the much slower, highly regulated, cytosol-requiring organelle-organelle fusion events typical of constitutive membrane trafficking pathways.
Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core
Authors: Shestopalov Valery I. , Bassnett Steven 
Published: April 2021

ABSTRACT
The lens of the eye is composed of concentric layers of tightly packed fiber cells. The oldest fibers, those in the lens core, lose their nuclei and other organelles during terminal differentiation. This is thought to ensure the clarity of the lens. The anucleated core fibers are sustained by gap junction-mediated communication with metabolically active cells near the lens surface. In this study, we expressed autofluorescent proteins and microinjected fluorescent markers to probe cell-to-cell communication in different regions of the developing lens. Our data indicate that a novel cell-cell diffusion pathway becomes patent in the lens core during development. This pathway is remarkable in that it is permeable to proteins and other large molecules and is thus distinct from gap junctions. Diffusion of large molecules probably occurs through regions of membrane fusion observed between neighboring cells in the lens core. Further direct evidence for a continuous plasma membrane system was provided by the observation that exogenous membrane proteins expressed in one core fiber cell were able to diffuse laterally into the membranes of adjacent fibers. Thus, the lens core appears to represent a true syncytium within which both membrane proteins and cytoplasmic proteins freely diffuse. Significantly, the outermost edge of the core syncytium encompasses a shell of nucleated, transcriptionally-competent, fiber cells. This arrangement could facilitate the delivery of newly synthesized protein components to the aged and metabolically quiescent cells in the center of the lens.
HZwint-1, a novel human kinetochore component that interacts with HZW10
Authors: Starr Daniel A. , Saffery Richard , Li Zexiao , Simpson Amanda E. , Choo K. H. Andy , Yen Tim J. , Goldberg Michael L. 
Published: April 2021

ABSTRACT
HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.
Talin controls the exit of the integrin α5β1 from an early compartment of the secretory pathway
Authors: Martel Véronique , Vignoud Lucile , Dupé Sandra , Frachet Philippe , Block Marc R. , Albigès-Rizo Corinne 
Published: April 2021

ABSTRACT
Talin is a major cytosolic protein that links the intracellular domains of β1 and β3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some β1 integrins, including the fibronectin receptor α5β1. To investigate this, we characterized the β1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of β1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin α5β1. In normal cells, talin was found associated with β1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin α chains.
The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells
Authors: Valenti Giovanna , Procino Giuseppe , Carmosino Monica , Frigeri Antonio , Mannucci Roberta , Nicoletti Ildo , Svelto Maria 
Published: April 2021

ABSTRACT
Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient Pf by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 μM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 μM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in Pf.
Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin
Authors: Thompson H. Cao, H. M. , Krueger E. W. , McNiven M. A. 
Published: April 2021

ABSTRACT
The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.