bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acid motifs required for isolated β cytoplasmic domains to regulate ‘in trans’ β1 integrin conformation and function in cell attachment
Authors: Mastrangelo Anthony M. , Homan Suzanne M. , Humphries Martin J. , LaFlamme Susan E.
Published: April 2021

The role of β cytoplasmic domains in regulating β1 integrin conformation and function in cell attachment is not fully understood. In this study, we tested the ability of transiently expressed β cytoplasmic domains connected to an extracellular reporter domain to regulate ‘in trans’ the conformation of endogenous β1 integrins, and compared these effects on cell attachment. We found that chimeric receptors containing either the β1, β3 or β5 cytoplasmic domains inhibited the expression of the conformationally dependent 9EG7 and 12G10 epitopes on endogenous β1 integrins. In contrast, chimeric receptors containing the β4 or α5 cytoplasmic domain, or a control receptor lacking a cytoplasmic domain, had no effect. This inhibition occurred in a dose-dependent manner that required high levels of expression of the chimeric receptor. These results suggest that β1 integrin conformation can be regulated by conserved cytosolic interactions involving β cytoplasmic domains. This is further supported by our findings that mutations within amino acid motifs conserved among these β cytoplasmic domains, specifically the NXXY, NPXY and TST-like motifs, reduced the ability of these chimeric receptors to regulate β 1 integrin conformation. Interestingly, the chimeric receptors inhibited cell attachment in a similar dose-dependent manner and required intact NXXY, NPXY, and TST-like motifs. The β1 chimera also inhibited the binding of soluble fibronectin to endogenous β1 integrins. Thus, the concomitant inhibition in the expression of conformation-dependent integrin epitopes, cell attachment and ligand binding by the chimeras, suggests that the expression of the 9EG7 and 12G10 epitopes correlates with integrin function. However, Mn2+, which is an extracellular activator of integrin function, increased 9EG7 expression to basal levels in the presence of the β1 chimera, but did not rescue cell attachment to the same extent. Thus, although the β1 integrin conformation recognized by mAb 9EG7 may be required for cell attachment, it is not sufficient, suggesting that the β chimeras may be inhibiting both ligand binding and post-ligand binding events required for cell attachment. In addition, the inhibitory effects of the chimeric receptors on cell attachment were not reversed by the addition of the pharmacological agents that inhibit intracellular signals previously shown to inhibit integrin function. This finding, together with the requirement for high levels of the chimeric receptors and the fact that mutations in the same conserved motifs in heterodimeric β1 integrins have been reported to regulate β1 integrin conformation and function in cell attachment, suggest that β cytoplasmic domains regulate these processes by interacting with cytosolic factors and that the regulatory effect of the chimeras may be due to their ability to titrate proteins from endogenous integrins.
Control of morphology, cytoskeleton and migration by syndecan-4
Authors: Longley Robert L. , Woods Anne , Fleetwood Allison , Cowling Graham J. , Gallagher John T. , Couchman John R.
Published: April 2021

Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan which localizes to focal adhesions. Previous studies showed that the syndecan-4 cytoplasmic domain can associate with and potentiate the activity of protein kinase C, which is required for focal adhesion formation. To examine further the role of syndecan-4 in cell adhesion, we expressed syndecan-4 cDNA constructs in CHO-K1 cells. Syndecan-2 transfection was used to confirm effects seen were specific for syndecan-4. Cells overexpressing full length syndecan-4 core protein exhibited a more flattened, fibroblastic morphology, with increased focal adhesion formation and decreased cell motility. Expression of a syndecan-4 core protein with either a partial or complete deletion of the cytoplasmic domain or of an antisense construct led to markedly decreased spreading and focal adhesion formation, a more epithelioid morphology, and decreased motility. Overexpression of syndecan-2 changed the adhesive phenotype, but did not markedly alter focal adhesion and microfilament bundle formation. The data suggest that syndecan-4 is a regulator of focal adhesion and stress fiber formation, and influences both morphology and migration.
Overexpression of normal and mutant Arp1α (centractin) differentially affects microtubule organization during mitosis and interphase
Authors: Clark Imran B. , Meyer David I.
Published: April 2021

Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin’s most abundant component, Arp1α was transiently overexpressed in mammalian cells. Arp1α overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1α in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1α that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1α proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1α, NuMA localization remained unaffected. Although expression of mutant Arp1α proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1α interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1α mutants. We conclude that the amino acids altered in the Arp1α mutant proteins participate in stabilizing interactions between overexpressed Arp1α and components of the endogenous dynactin complex as well as the NuMA protein.
Characterization of bone morphogenetic protein-6 signaling pathways in osteoblast differentiation
Authors: Ebisawa Takanori , Tada Keiichiro , Kitajima Isao , Tojo Katsuyoshi , Sampath T. Kuber , Kawabata Masahiro , Miyazono Kohei , Imamura Takeshi
Published: April 2021

Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor (TGF)-β superfamily, and is most similar to BMP-5, osteogenic protein (OP)-1/BMP-7, and OP-2/BMP-8. In the present study, we characterized the endogenous BMP-6 signaling pathway during osteoblast differentiation. BMP-6 strongly induced alkaline phosphatase (ALP) activity in cells of osteoblast lineage, including C2C12 cells, MC3T3-E1 cells, and ROB-C26 cells. The profile of binding of BMP-6 to type I and type II receptors was similar to that of OP-1/BMP-7 in C2C12 cells and MC3T3-E1 cells; BMP-6 strongly bound to activin receptor-like kinase (ALK)-2 (also termed ActR-I), together with type II receptors, i.e. BMP type II receptor (BMPR-II) and activin type II receptor (ActR-II). In addition, BMP-6 weakly bound to BMPR-IA (ALK-3), to which BMP-2 also bound. In contrast, binding of BMP-6 to BMPR-IB (ALK-6), and less efficiently to ALK-2 and BMPR-IA, together with BMPR-II was detected in ROB-C26 cells. Intracellular signalling was further studied using C2C12 and MC3T3-E1 cells. Among the receptor-regulated Smads activated by BMP receptors, BMP-6 strongly induced phosphorylation and nuclear accumulation of Smad5, and less efficiently those of Smad1. However, Smad8 was constitutively phosphorylated, and no further phosphorylation or nuclear accumulation of Smad8 by BMP-6 was observed. These findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly through Smad1.
A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA
Authors: Pittoggi C. , Renzi L. , Zaccagnini G. , Cimini D. , Degrassi F. , Giordano R. , Magnano A. R. , Lorenzini R. , Lavia P. , Spadafora C.
Published: April 2021

We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of ‘active’ chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.
VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals
Authors: Taubenblatt P. , Dedieu J. C. , Gulik-Krzywicki T. , Morel N.
Published: April 2021

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin.

After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoechiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much ressemble homotypic fusion.
High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion
Authors: Joberty Gérard , Stabila Paul F. , Coppola Thierry , Macara Ian G. , Regazzi Romano
Published: April 2021

Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca2+-triggered exocytosis. Rabphilin is found only in neuroendocrine cells where it co-localises with Rab3A on the secretory vesicle membrane. The Rab3 binding domain (residues 45 to 170), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interaction with the small GTPase. To determine whether binding to Rab3A is necessary for the subcellular localisation of Rabphilin, we synthesised point mutants within the Rab3-binding domain. We found that two unique mutations (V61A and L83A) within an amphipathic α-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of the protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furthermore, both mutants transfected in the HIT-T15 beta cell line stimulate Ca2+-regulated exocytosis to the same extent as wild-type Rabphilin. Surprisingly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulated secretion.

High affinity binding to Rab3 is therefore dispensable for the targetting of Rabphilin to secretory vesicles and for the potentiation of Ca2+-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.
Ca2+/calmodulin and p85 cooperatively regulate an initiation of cytokinesis in Tetrahymena
Authors: Gonda Kohsuke , Katoh Mariko , Hanyu Kazuko , Watanabe Yoshio , Numata Osamu
Published: April 2021

Tetrahymena p85 differs in mobility in two-dimensional SDS-polyacrylamide gel electrophoresis between wild-type and temperature-sensitive cell-division-arrest mutant cdaA1 cell extracts, and is localized to the presumptive division plane before the formation of the division furrow. The p85 contained three identical sequences which show homology to the calmodulin binding site of Ca2+/calmodulin dependent protein kinase Type II in Saccharomyces cerevisiae. We found the p85 directly interacts with Tetrahymena calmodulin in a Ca2+- dependent manner, using a co-sedimentation assay. We next examined the localization of p85 and calmodulin during cytokinesis using indirect immunofluorescence. The results showed that both proteins colocalize in the division furrow. This is the first observation that calmodulin is localized in the division furrow. Moreover, the direct interaction between p85 and Ca2+/calmodulin was inhibited by Ca2+/calmodulin inhibitor N-(6-aminohexyl)- 5-chloro-1-naphthalenesulfonamide HCl. When the cells were treated with the drug just before the beginning of cytokinesis, the drug also inhibited the localization of p85 and calmodulin to the division plane, and the formation of the contractile ring and division furrow. Therefore, we propose that the Ca2+/calmodulin signal and its target protein p85 cooperatively regulate an initiation of cytokinesis and may be also concerned with the progression of cytokinesis in Tetrahymena.
Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes
Authors: Smith Susan , de Lange Titia
Published: April 2021

Tankyrase is a human poly(ADP-ribose) polymerase that was initially identified through its interaction with the telomeric protein TRF1, a negative regulator of telomere length. In vitro poly(ADP-ribosyl)ation by tankyrase inhibits TRF1 binding to telomeric DNA suggesting a role for tankyrase in telomere function. We previously demonstrated that tankyrase co-localizes with TRF1 at the ends of human chromosomes in metaphase. Here we show that tankyrase localizes to additional subcellular sites in a cell cycle dependent manner. In interphase, tankyrase colocalized with TRF1 to telomeres, but in addition was found to reside at nuclear pore complexes, as evidenced by indirect immunofluorescence, subcellular fractionation and immunoelectron microscopy. At mitosis, concomitant with nuclear envelope breakdown and nuclear pore complex disassembly, tankyrase was found to relocate around the pericentriolar matrix of mitotic centrosomes. This complex staining pattern along with the observation that tankyrase did not contain a nuclear localization signal suggested that its telomeric localization might be regulated, perhaps by TRF1. Indeed, localization of exogenously-expressed tankyrase to telomeres was dependent upon co-transfection with TRF1. These data indicate that the subcellular localization of tankyrase can be regulated by both the cell cycle and TRF1.
A targeted gene silencing technique shows that Drosophila myosin VI is required for egg chamber and imaginal disc morphogenesis
Authors: Deng Wu-Min , Leaper Kevin , Bownes Mary
Published: April 2021

We report that Drosophila unconventional myosin VI, encoded by Myosin heavy chain at 95F (Mhc95F), is required for both imaginal disc and egg chamber morphogenesis. During oogenesis, Mhc95F is expressed in migrating follicle cells, including the border cells, which migrate between the nurse cells to lie at the anterior of the oocyte; the columnar cells that migrate over the oocyte; the centripetal cells that migrate between the oocyte and nurse cells; and the dorsal-anterior follicle cells, which migrate to secrete the chorionic appendages. Its function during development has been studied using a targeted gene silencing technique, combining the Gal4-UAS targeted expression system and the antisense RNA technique. Antibody staining shows that the expression of myosin 95F is greatly decreased in follicle cells when antisense Mhc95F RNA is expressed. Interfering with expression of Drosophila myosin VI at various developmental stages frequently results in lethality. During metamorphosis it results in adult flies with malformed legs and wings, indicating that myosin VI is essential for imaginal disc morphogenesis. During oogenesis, abnormal follicle cell shapes and aberrant follicle cell migrations are observed when antisense Mhc95F is expressed in follicle cells during stages 9 to 10, suggesting that the Drosophila myosin VI is required for follicle cell epithelial morphogenesis.
The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites
Authors: Severt W. L. , Biber T. U. L. , Wu X.-Q. , Hecht N. B. , DeLorenzo R. J. , Jakoi E. R.
Published: April 2021

Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing α-calmodulin dependent kinase II (α-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of α-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.
VAP-33 localizes to both an intracellular vesicle population and with occludin at the tight junction
Authors: Lapierre Lynne A. , Tuma Pamela L. , Navarre Jennifer , Goldenring James R. , Anderson James M.
Published: April 2021

Tight junctions create a regulated intercellular seal between epithelial and endothelial cells and also establish polarity between plasma membrane domains within the cell. Tight junctions have also been implicated in many other cellular functions, including cell signaling and growth regulation, but they have yet to be directly implicated in vesicle movement. Occludin is a transmembrane protein located at tight junctions and is known to interact with other tight junction proteins, including ZO-1. To investigate occludin’s role in other cellular functions we performed a yeast two-hybrid screen using the cytoplasmic C terminus of occludin and a human liver cDNA library. From this screen we identified VAP-33 which was initially cloned from Aplysia by its ability to interact with VAMP/synaptobrevin and thus was implicated in vesicle docking/fusion. Extraction characteristics indicated that VAP-33 was an integral membrane protein. Antibodies to the human VAP-33 co-localized with occludin at the tight junction in many tissues and tissue culture cell lines. Subcellular fractionation of liver demonstrated that 83% of VAP-33 co-isolated with occludin and DPPIV in a plasma membrane fraction and 14% fractionated in a vesicular pool. Thus, both immunofluorescence and fractionation data suggest that VAP-33 is present in two distinct pools in the cells. In further support of this conclusion, a GFP-VAP-33 chimera also distributed to two sites within MDCK cells and interestingly shifted occludin’s localization basally. Since VAP-33 has previously been implicated in vesicle docking/fusion, our results suggest that tight junctions may participate in vesicle targeting at the plasma membrane or alternatively VAP-33 may regulate the localization of occludin.
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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Yeast ER-Golgi v-SNAREs Bos1p and Bet1p differ in steady-state localization and targeting
Authors: Ossipov Dmitri , Schröder-Köhne Stephan , Schmitt Hans Dieter
Published: April 2021

Vesicle specific SNAP receptors (v-SNAREs) Bos1p and Bet1p are involved in targeting of anterograde vesicles between the endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors that influence the targeting of these proteins, α-factor tagged versions of Bos1p and Bet1p were employed. The α-factor can be cleaved off by the Kex2p protease as soon as the hybrid proteins reach the late Golgi compartment. The data obtained by monitoring of Kex2p cleavage, by immunofluorescence microscopy and cell fractionation showed that Bos1-α and Bet1-α have different cellular localization and dynamics. Bos1-α is an ER protein, which recycles between the Golgi and the ER in COPI-dependent manner. Bet1-α is an early Golgi protein and it does not change its localization under conditions when other recycling Golgi proteins can be trapped in the ER.
Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane
Authors: Romisch Karin
Published: April 2021

Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded. Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol. The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane. For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane. Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.