bluuebunny/crossref_metadata_2025_split huggingface dataset:
A key function of non-planar membranes and their associated microtubular ribbons in contractile vacuole membrane dynamics is revealed by electrophysiologically controlled fixation of Paramecium
Authors: Tominaga Takashi , Naitoh Yutaka , Allen Richard D. 
Published: April 2021

ABSTRACT
The contractile vacuole complex of the fresh water protozoan Paramecium multimicronucleatum exhibits periodic exocytotic activity. This keeps cytosolic osmolarity at a constant value. The contractile vacuole, the central exocytotic vesicle of the complex, becomes disconnected from its surrounding radial arms and rounds before its fluid content is expelled. We previously proposed a hypothesis that the rounding of the contractile vacuole corresponds to an increase in its membrane tension and that a periodic increase in membrane tension governs the exocytotic cycle. We also proposed a hypothesis that transformation of excess planar membrane of the contractile vacuole into 40 nm diameter tubules, that remain continuous with the contractile vacuole membrane, is a primary cause for the tension development in the planar membrane. In order to investigate tension development further, we have examined electron microscopically the contractile vacuole membrane at the rounding phase. To do this, we developed a computer-aided system to fix the cell precisely at the time that the contractile vacuole exhibited rounding. In this system a decrease in the electrical potential across the contractile vacuole membrane that accompanied the vacuole’s rounding was monitored through a fine-tipped microelectrode inserted directly into the in vivo contractile vacuole. A decrease in membrane potential was used to generate an electric signal that activated an injector for injecting a fixative through a microcapillary against the cell at the precise time of rounding. Subsequent electron micrographs of the contractile vacuole membrane clearly demonstrated that numerous ∼40 nm membrane-bound tubules formed in the vicinity of the vacuole’s microtubule ribbons when the vacuole showed rounding. This finding suggested that membrane tubulation was the cause for topographical isolation of excess membrane from the planar membrane during the periodic rounding of the contractile vacuole. This together with stereo-pair images of the contractile vacuole complex membranes suggested that the microtubule ribbons were intimately involved in enhancing this membrane tubulation activity. Electron micrographs of the contractile vacuole complexes also showed that decorated tubules came to lie abnormally close to the contractile vacuole in these impaled cells. This suggested that the contractile vacuole was capable of utilizing the smooth spongiome membrane that lies around the ampullae and the collecting canals to increase its size.
Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1
Authors: Bastin Philippe , Pullen Timothy J. , Sherwin Trevor , Gull Keith 
Published: April 2021

ABSTRACT
The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intraflagellar structure that represents an excellent system in which to study flagellum assembly. Molecular ablation of one of its major constituents, the PFRA protein, in the snl-1 mutant causes considerable alteration of the PFR structure, leading to cell paralysis. Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells. This phenotype was complemented by transformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene. In the snl-1 mutant, other PFR proteins such as the second major constituent, PFRC, accumulated at the distal tip of the growing flagellum, forming a large dilation or ‘blob’. This was not assembled as filaments and was removed by detergent-extraction. Axonemal growth and structure was unmodified in the snl-1 mutant and the blob was present only at the tip of the new flagellum. Strikingly, the blob of unassembled material was shifted towards the base of the flagellum after cell division and was not detectable when the daughter cell started to produce a new flagellum in the next cell cycle. The dynamics of blob formation and regression are likely indicators of anterograde and retrograde transport systems operating in the flagellum. In this respect, the accumulation of unassembled PFR precursors in the flagellum shows interesting similarities with axonemal mutants in other systems, illustrating transport of components of a flagellar structure during both flagellum assembly and maintenance. Observation of PFR components indicate that these are likely to be regulated and modulated throughout the cell cycle.
Expression of human β3-adrenergic receptor induces adipocyte-like features in CHO/K1 fibroblasts
Authors: Gros Jérôme , Gerhardt Cindy C. , Strosberg A. Donny 
Published: April 2021

ABSTRACT
It is reported here that CHO/K1 cells stably transfected with the human β3 AR gene (CHO/K1-β3), grown in the presence of differentiation-stimulating agents accumulate triglycerides. This lipid formation is mediated through the β3 AR, since non-transfected CHO/K1 cells, or cells expressing the human β2 AR, accumulate no significant amount of lipids when grown in supplemented medium. Moreover, lipid production can be inhibited significantly by the β AR antagonist bupranolol. CHO/K1 cells expressing the W64R polymorphism (Trp to Arg polymorphism at position 64 of the human β3 AR), which has been associated with morbid obesity, show increased lipid accumulation as compared to CHO/K1 cells expressing the wild-type β3 AR. Semi-quantitative RT-PCR experiments reveal that a major gene regulating adipocyte differentiation, peroxisome-proliferator-activated-receptor γ (PPARγ), is expressed in CHO/K1 cells. Concomitantly with the formation of lipid droplets, the expression of PPARγ mRNA is increased in CHO/K1-β3 cells, but not in non-transfected CHO/K1 cells. We furthermore detected constitutive expression of another adipocyte-associated protein: hormone sensitive lipase, while leptin or uncoupling protein-1 transcripts were not expressed. These data suggest that the frequently used CHO/K1 fibroblasts display several preadipocyte-like features, and that the sole expression of the β3 AR modifies the expression of PPARγ mRNA in these cells, and induces lipid formation under certain culture conditions.
A secreted Frizzled related protein, FrzA, selectively associates with Wnt-1 protein and regulates Wnt-1 signaling
Authors: Dennis Steve , Aikawa Mina , Szeto Wayne , d’Amore Patricia A. , Papkoff Jackie 
Published: April 2021

ABSTRACT
The Wnt gene family encodes proteins that serve key roles in differentiation and development. Wnt proteins interact with seven transmembrane receptors of the Frizzled family and activate a signaling pathway leading to the nucleus. A primary biochemical effect of Wnt-1 signaling is the stabilization of cytoplasmic β-catenin which, in association with transcription factors of the Lef/tcf family, regulates gene expression. The recent identification of a new class of secreted proteins with similarity to the extracellular, ligand-binding domain of Frizzled proteins, soluble Frizzled related proteins (sFRP), suggested that additional mechanisms could regulate Wnt signaling. Here we demonstrate that FrzA, a sFRP that is highly expressed in vascular endothelium and a variety of epithelium, specifically binds to Wnt-1 protein, but not Wnt-5a protein, and modulates Wnt-1 signaling. FrzA associated with Wnt- 1 either when expressed in the same cell or when soluble FrzA was incubated with Wnt-1-expressing cells. FrzA efficiently inhibited the Wnt-1 mediated increase in cytoplasmic β-catenin levels as well as the Wnt-1 induction of transcription from a Lef/tcf reporter gene. The effects of FrzA on β-catenin levels could be demonstrated when co- expressed with Wnt-1 or when individual cells expressing FrzA and Wnt-1 were co-cultured. These data demonstrate the existence of a negative regulatory mechanism mediated by the selective binding of FrzA to Wnt-1 protein.
Rac GTPases localize at sites of actin reorganization during dynamic remodeling of the cytoskeleton of normal embryonic fibroblasts
Authors: Albertinazzi Chiara , Cattelino Anna , de Curtis Ivan 
Published: April 2021

ABSTRACT
Rac GTP-binding proteins are implicated in the dynamic organization of the actin cytoskeleton, and the mechanisms utilized for this purpose are not understood yet. In this paper we have analysed the effects of the expression of Rac proteins on the organization of the cytoskeleton, and their subcellular distribution in chicken embryo fibroblasts. In these cells, overexpression of wild-type Rac GTPases induces disassembly of stress fibers, and production of long, highly branched actin-rich protrusions, with consequent dramatic changes in cell morphology. The formation of these protrusions is mediated by adhesion to the substrate, and is prevented by incubation with anti-β1 function- blocking antibodies. Rac-mediated cell shape changes require a wild-type GTPase, since expression of constitutively active V12-Rac proteins affects actin organization differently in these cells, without causing alterations in their morphology. Localization studies performed on ventral plasma membranes from fibroblasts transfected with wild-type or mutant GTPases show codistribution of Rac along stress fibers, before their disassembly and the formation of the actin-rich protrusions. These data show a link between Rac protein distribution, and their effects on the actin cytoskeleton. Altogether, our results are indicative of an active role of Rac proteins in stress fiber disassembly, and show that Rac, which can cycle its bound nucleotide, produces unique dynamic effects on actin organization.
Cell adhesion molecules and extracellular-matrix constituents in kidney development and disease
Authors: Müller Ulrich , Brändli André W. 
Published: April 2021

ABSTRACT
Functional analyses of cell-matrix interactions during kidney organogenesis have provided compelling evidence that extracellular-matrix glycoproteins and their receptors play instructive roles during kidney development. Two concepts are worthy of emphasis. First, matrix molecules appear to regulate signal transduction pathways, either by activating cell-surface receptors such as integrins directly or by modulating the activity of signaling molecules such as WNTs. Second, basement membranes are highly organized structures and have distinct molecular compositions, which are optimized for their diverse functions. The importance of these findings is highlighted by the fact that mutations affecting basement-membrane components lead to inherited forms of kidney disease.
Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor
Authors: Skretting Grethe , Torgersen Maria Lyngaas , van Deurs Bo , Sandvig Kirsten 
Published: April 2021

ABSTRACT
We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glyco-sylphosphatidylinositol-linked molecule, a glycosyl-phosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-β-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.
Cell-sorting in aggregates of Dictyostelium discoideum
Authors: Nicol Alastair , Rappel Wouter-Jan , Levine Herbert , Loomis William F. 
Published: April 2021

ABSTRACT
When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.
HIV-induced T-cell syncytia release a two component T-helper cell chemoattractant composed of Nef and Tat
Authors: Shutt Damon C. , Soll David R. 
Published: April 2021

ABSTRACT
Using a newly developed gradient chamber to provide independent measurements of chemokinesis (stimulated motility) and chemotaxis (stimulated motility up a concentration gradient) of individual T-helper cells, it was recently demonstrated that HIV-induced T-cell syncytia release two distinct chemotactic activities that are separable by their rates of diffusion. The molecular masses of the two chemoattractant activities were estimated to be 30 and 120 kDa. The higher molecular mass activity was demonstrated to be the viral glycoprotein gp120. In an attempt to identify the lower molecular mass activity, chemotaxis and chemokinesis of T-helper cells were analyzed in individual concentration gradients of the virally encoded proteins Rev, p24, Tat and Nef. None functioned alone as a chemoattractant, but both Tat and Nef alone functioned as chemokinetic stimulants. When Tat and Nef were used together to generate parallel gradients, they stimulated chemotaxis. Antibody to either Tat or Nef neutralized the lower molecular mass chemotactic activity released by syncytia. The addition of antibody to the CD4 receptor or the addition of soluble CD4 inhibited high molecular mass chemotactic activity but not the low molecular mass chemotactic activity in HIV-induced syncytium-conditioned medium, demonstrating that the former but not the latter activity is mediated through the CD4 receptor. These results identify the combination of Nef and Tat as the lower molecular mass T cell chemoattractant released by HIV-induced syncytia, and provide the first evidence suggesting that parallel concentration gradients of two proteins are necessary for chemotaxis.
The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments
Authors: Assmann Volker , Jenkinson David , Marshall John F. , Hart Ian R. 
Published: April 2021

ABSTRACT
We reported recently on the intracellular localisation of the hyaluronan receptor RHAMM/IHABP in human cancer cells. Here we describe the colocalisation of RHAMM/IHABP proteins with microtubules, both in interphase and dividing cells, suggesting that RHAMM/IHABP represents a novel member of the family of microtubule-associated proteins (MAPs). We have identified four different splice variants of RHAMM/IHABP, all of which colocalise, at least transiently, with microtubules when expressed as GFP fusion proteins in HeLa cells. Using microtubule-binding assays and transient transfection experiments of deletion-bearing RHAMM/IHABP mutants, we localised the microtubule-binding region to the extreme N terminus of RHAMM/IHABP. This interaction domain is composed of two distinct subdomains, one of which is sufficient to mediate binding to the mitotic spindle while both domains are required for binding of RHAMM/IHABP proteins to interphase microtubules. Sequence analysis revealed that the projection domain of RHAMM/IHABP is predicted to form coiled-coils, implying that RHAMM/IHABP represents a filamentous protein capable of interacting with other proteins and we found that RHAMM/IHABP interacts with actin filaments in vivo and in vitro. Moreover, in vitro translated RHAMM/IHABP isoforms efficiently bind to immobilised calmodulin in a Ca2+- dependent manner via a calmodulin-binding site within the projection domain of RHAMM/IHABP (residues 574-602). Taken together, our results strongly suggest that RHAMM/IHABP is a ubiquitiously expressed, filamentous protein capable of interacting with microtubules and microfilaments and not, as numerous previous reports suggest, a cell surface receptor for the extracellular matrix component hyaluronan.
Pattern of expression of the Jun family of transcription factors during the early development of the inner ear: implications in apoptosis
Authors: Sanz Carmen , León Yolanda , Cañón Susana , Alvarez Luis , Giraldez Fernando , Varela-Nieto Isabel 
Published: April 2021

ABSTRACT
Jun transcription factors have been implicated in the regulation of cell proliferation, differentiation and apoptosis. We have investigated the relationship between Jun expression and cell death in the developing chicken inner ear. c-jun and junD transcripts were expressed in the epithelium of the otic placode and otic vesicle. c-jun expression was restricted to the dorsal area of the otic pit (stages 14-17), dorsal otic vesicle and cochleo-vestibular ganglion (stages 18-20). junD expression was transient and occurred in the dorsal and upper medial aspects of the otic pit and otic cup, but it was down-regulated in the otic vesicle. A parallel TUNEL analysis revealed that expression of c-jun co-located within areas of intense apoptosis. Furthermore, phosphorylation of c-Jun at serine-63 by Jun amino-terminal-kinases was detected in the dorsal otic pit, otic vesicle and cochleo-vestibular ganglion. c-Jun protein exhibited DNA binding activity, as assessed by gel mobility shift assays. The association between c-Jun and apoptosis was further demonstrated by studying nerve growth factor-induced apoptosis in cultured otic vesicles. Nerve growth factor-induced cell death and c-Jun phosphorylation that were suppressed by insulin-like growth factor-I and by viral-mediated overexpression of Raf, which had survival effects. In conclusion, the precise regulation of the expression and activity of Jun proteins in the otic primordium suggests that it may operate as a fundamental mechanism during organogenesis.
Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes
Authors: Ojima K. , Lin Z. X. , Zhang Z. Q. , Hijikata T. , Holtzer S. , Labeit S. , Sweeney H. L. , Holtzer H. 
Published: April 2021

ABSTRACT
While over a dozen I-Z-I proteins are expressed in postmitotic myoblasts and myotubes it is unclear how, when, or where these first assemble into transitory I-Z-I bodies (thin filament/Z-band precursors) and, a short time later, into definitive I-Z-I bands. By double-staining the growth tips of transfected myotubes expressing (a) MYC-tagged s-α-actinins (MYC/s-α-actinins) or (b) green fluorescent protein-tagged titin cap (GFP/T-cap) with antibodies against MYC and I-Z-I band proteins, we found that the de novo assembly of I-Z-I bodies and their maturation into I-Z-I bands involved relatively concurrent, cooperative binding and reconfiguration of, at a minimum, 5 integral Z-band molecules. These included s-α-actinin, nebulin, titin, T-cap and α-actin. Resolution of the ∼1.0 µm polarized α-actin/nebulin/tropomyosin/troponin thin filament complexes occurred subsequent to the maturation of Z-bands into a dense tetragonal configuration. Of particular interest is finding that mutant MYC/s-α-actinin peptides (a) lacking spectrin-like repeats 1-4, or consisting of spectrin-like repeats 1-4 only, as well as (b) mutants/fragments lacking titin or α-actin binding sites, were promptly and exclusively incorporated into de novo assembling I-Z-I bodies and definitive I-Z-I bands as was exogenous full length MYC/s-α-actinin or GFP/T-cap.
Factors involved in the cell density-dependent regulation of nuclear/cytoplasmic distribution of the 11.5-kDa Zn2+-binding protein (parathymosin-α) in rat hepatocytes
Authors: Trompeter Hans-Ingo , Schiermeyer Andreas , Blankenburg Gabriele , Hennig Elisabeth , Söling Hans-Dieter 
Published: April 2021

ABSTRACT
Although the 11.5 kDa Zn2+-binding protein (ZnBP, parathymosin-α) possesses a functional bipartite nuclear localization signal it was found in most tissues in the cytoplasm. The cultivation of freshly isolated rat hepatocytes for 24 hours under standard conditions was associated with an almost complete translocation of ZnBP from the cytoplasm to the nuclei. Here we demonstrate, that this translocation is negatively correlated with cell density. The translocation of ZnBP to the nucleus can be inhibited or abolished by inhibitors of protein synthesis (cycloheximide) or transcription (actinomycin D). Moreover, cycloheximide can induce a relocation of ZnBP to the cytoplasm when applied after the appearance of ZnBP in the nuclei. DMSO, an inhibitor of dedifferentiation of cultured hepatocytes, abolishes also the translocation of ZnBP into the nucleus. Thinly seeded cells keep their ZnBP in the cytoplasm if they are co-cultured with plasma membranes from Morris MH7777 hepatoma cells or antibodies against E-cadherin indicating the involvement of cell adhesion proteins. We have enriched a protein from the cytosol of fresh hepatocytes which inhibits the translocation of ZnBP, but not that of albumin-NLS into the nucleus in a permeabilized cell system. Such an activity could not be found in the cytoplasm of permanent cell lines which harbor ZnBP only in the nucleus. A model for the regulation of the nuclear import of ZnBP is proposed.