bluuebunny/crossref_metadata_2025 huggingface dataset:
Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection: functional characterization of a novel aquaporin
Authors: Cahérec F. Le , Bron P. , Verbavatz J. M. , Garret A. , Morel G. , Cavalier A. , Bonnec G. , Thomas D. , Gouranton J. , Hubert J. F.
Published: April 2021

Xenopus laevis oocytes are widely used as an expression system for plasma membrane proteins, achieved by cyto-plasmic microinjection of messenger RNA. In the present study, we propose an alternative system allowing functional insertion of exogenous proteins into the plasma membrane of Xenopus oocytes. We microinjected proteoliposome sus-pensions into the cytoplasm and then analyzed membrane protein function. The proteins used in this work were members of the MIP family: the human erythrocyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MIP26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-sucking insect (Cicadella viridis). Proteoliposomes containing either AQP1, MIP26, or P25 were injected into Xenopus oocytes. The subsequent insertion of these proteins into the plasma membrane of oocytes was demonstrated by immunocytochemistry.

Oocytes microinjected with either AQP1 or P25-pro-teoliposomes exhibited significantly increased osmotic membrane water permeabilities (Pf = 3.16±026 and 4.03±0.26 ×10−3 cm/second, respectively) compared to those measured for oocytes injected with liposomes alone or with

MIP26-proteoliposomes (Pf = 1.39±0.07 and 1.44±0.10×10−3 cm/second, respectively). These effects were inhibited by HgCl2 in a reversible manner. Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte plasma membranes (Ea = 2.29 and 3.01 kcal/mol, respectively, versus Ea = 11.75 kcal/mol for liposome injected oocytes). The properties observed here for AQP1 are identical to those widely reported following AQP1 cRNA expression in oocytes.

From the present study, we conclude that: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequently found in the plasma membrane of the oocytes in a functional state; and (2) in this system, the P25 polypeptide from the MIP family found in the digestive tract of Cicadella viridis exhibits properties similar to those described for the archetype of water channels AQP1, and thus is a new member of the aquaporin family.
Remodeling of cytoskeleton and triads following activation of v-Src tyrosine kinase in quail myotubes
Authors: Castellani L. , Reedy M. , Airey J. A. , Gallo R. , Ciotti M. T. , Falcone G. , Alemà S.
Published: April 2021

To study the cellular signals underlying the regulatory mechanisms involved in maintenance of sarcomeric integrity, we have used quail skeletal muscle cells that reach a high degree of structural maturation in vitro, and also express a temperature-sensitive mutant of the v-Src tyrosine kinase that allows the control of differentiation in a reversible manner. By immunofluorescence and electron microscopy we show that v-Src activity in myotubes leads to an extensive cellular remodeling which affects components of the sarcomeres, the cytoskeleton network and the triad junctions. We have previously shown that activation of v-Src causes a selective dismantling of the I-Z-I segments coupled to the formation of aggregates of sarcomeric actin, α-actinin and vinculin, called actin bodies. We now show that intermediate filaments do not participate in the formation of actin bodies, while talin, a component of costameres, does. The I-Z-I segments are completely dis-mantled within 24 hours of v-Src activity, but the A-bands persist for a longer time, implying distinct pathways for the turnover of sarcomeric subdomains. Immunofluorescence labeling of markers of the triad junctions demonstrates that the localization of the α1 subunit of the dihydropyri-dine receptor is disrupted earlier than that of the ryanodine receptor after tyrosine kinase activation. Furthermore, the location of junctional sarcoplasmic reticulum and trans-verse tubule membranes is maintained in myotubes in which the I-Z-I have been removed and the regular dispo-sition of the intermediate filaments is disrupted, supporting a role for sarcoplasmic reticulum in the proper pos-itioning of triad junctions. Altogether these results point to a tyrosine kinase signaling cascade as a mechanism for selectively destabilizing sarcomere subdomains and their tethering to the cytoskeleton and the sarcolemma.
A serine-kinase associated with the p127-l(2)gl tumour suppressor of Drosophila may regulate the binding of p127 to nonmuscle myosin II heavy chain and the attachment of p127 to the plasma membrane
Authors: Kalmes Andreas , Merdes Gunter , Neumann Beate , Strand Dennis , Mechler Bernard M.
Published: April 2021

The p127 tumour suppressor protein encoded by the lethal(2)giant larvae, [l(2)gl], gene of Drosophila melanogaster is a component of a cytoskeletal network dis-tributed in both the cytoplasm and on the inner face of the plasma membrane. The p127 protein forms high molecular mass complexes consisting mainly of homo-oligomerized p127 molecules and at least ten additional proteins. One of these proteins has been recently identified as nonmuscle myosin type II heavy chain. To determine the functional interactions between p127 and other proteins present in the p127 complexes, we analyzed p127 for posttranslational modifications and found that p127 can be phosphorylated at serine residues. In this report we describe the charac-teristics of a serine kinase which is associated with p127, as judged by its recovery in p127 complexes purified by either gel filtration or immuno-affinity chromatography. This kinase phosphorylates p127 in vitro and its activation by supplementing ATP results in the release of p127 from the plasma membrane. Moreover, similar activation of the kinase present in immuno-purified p127 complexes dissociates nonmuscle myosin II from p127 without affecting the homo-oligomerization of p127. This dissociation can be inhibited by staurosporine and a 26mer peptide covering amino acid positions 651 to 676 of p127 and containing five serine residues which are evolutionarily conserved from Drosophila to humans. These results indicate that a serine-kinase tightly associated with p127 regulates p127 binding with components of the cytoskeleton present in both the cytoplasm and on the plasma membrane.
Cell cycle regulators in Drosophila: downstream and part of developmental decisions
Authors: Lehner Christian F. , Lane Mary Ellen
Published: April 2021

The molecular identification of an evolutionarily conserved set of cell cycle regulators in yeast, Xenopus egg extracts, and vertebrate cell culture has opened up a new perspective for understanding the mechanisms that regulate cell proliferation during metazoan development. Now we can study how the crucial regulators of eukaryotic cell cycle progression, the various cyclin/cdk complexes (for a recent review see Nigg (1995)BioEssays 17, 471-480), are turned on or off during development. In Drosophila, this analysis is most advanced, in particular in the case of the rather rigidly programmed embryonic cell cycles that generate the cells of the larvae. In addition, this analysis has revealed how the mitotic cycle is transformed into an endocycle which allows the extensive growth of larvae and oocytes. In contrast, we know little about cyclin/cdk regulation during the imaginal proliferation that generates the cells of the adult. Nevertheless, we will also consider this second developmental phase with its conspicuous regulative character, because it will be of great interest for the analysis of the molecular mechanisms that integrate growth and proliferation during development.
Tension-sensitive kinetochore phosphorylation and the chromosome distribution checkpoint in praying mantid spermatocytes
Authors: Li Xiaotong , Nicklas R. Bruce
Published: April 2021

Improper chromosome attachment to the spindle can lead to daughter cells with missing or extra chromosomes. Such mishaps are avoided in many cells by a checkpoint that detects even a single improperly attached chromosome. What is detected? A misattached chromosome is not under tension from opposed mitotic forces, and in praying mantid spermatocytes, direct experiments show that the absence of tension is what the checkpoint detects. How is the absence of tension detected? Tension-sensitive kinetochore protein phosphorylation is the most likely possibility. We combined micromanipulation with immunostaining for phosphoproteins in order to study the effect of tension on kinetochore phosphorylation in mantid spermatocytes. We confirm earlier observations on mammalian cells and grasshopper spermatocytes that misattached chromosomes have phosphorylated kinetochore proteins. We also confirm experiments in grasshopper spermatocytes showing that tension alters kinetochore chemistry: tension from a micromanipulation needle causes kinetochore protein dephosphorylation, and relaxation of tension causes kinetochore protein rephosphorylation. Beyond confirmation, our results provide fresh evidence for phosphorylation as the signal to the checkpoint. First, mantid cells are the only ones in which an effect of tension on the checkpoint has been directly demonstrated; by equally direct experiments, we now show that tension affects kinetochore phosphorylation in these same cells. Second, sex chromosome behavior in mantids provides a natural experiment to test the relationship between phosphorylation and the checkpoint. In grasshoppers, an unpaired sex chromosome is normal, its kinetochore is under-phosphorylated, and the checkpoint is not activated. In mantids, exactly the opposite is true: an unpaired sex chromosome is abnormal, its kinetochore is phosphorylated and, as predicted, the checkpoint is activated. We conclude that tension-sensitive kinetochore protein phosphorylation very likely is the essential link between proper chromosome attachment and the checkpoint, the link that permits potential errors in chromosome distribution to be detected and avoided.
The role of γ-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans
Authors: Martin Mary Ann , Osmani Stephen A. , Oakley Berl R.
Published: April 2021

γ-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of γ-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a γ-tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that γ-tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that γ-tubulin is involved in spindle microtubule nucleation. In the absence of functional γ-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that γ-tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.
Rac is required for growth cone function but not neurite assembly
Authors: Lamoureux Phillip , Altun-Gultekin Zeynep F. , Lin Chingju , Wagner John A. , Heidemann Steven R.
Published: April 2021

Recent work has suggested that racl and other members of the rho family of small GTP-binding proteins play an important role in the formation of neural processes. We have explored the mechanism of this effect by comparing the spontaneous, growth cone-mediated growth and experimental tension-induced growth of axons in normal PC12 cells and in mutant cells expressing a dominant negative form of rac. PC12 that have been primed by exposure to NGF, but not naive PC12 cells, initiate a microtubule-rich process de novo in response to tension applied to cell body. As in chick sensory neurons, neurite elongation rate is proportional to applied tension above a threshold. Addition of cyclic AMP, which has been shown to rapidly augment NGF-induced neurite outgrowth in PC12, causes a rapid increase in the rate of neurite elongation at a given tension level. Expression of a dominant negative form of rac1 inhibits spontaneous, growth cone-mediated neurite elongation in response to NGF, but does not substantially affect tension-induced neurite elongation. That is, rac-deficient cells show a normal linear relationship between applied tension and elongation rate and the elongations contain a normal density of axial microtubules by immuno-fluorescent assay. Thus, rac1 is apparently required for the mechanisms that normally generate tension in an elongating neurite, but if this tension is provided from an outside source, then axonal elongation can proceed normally in rac1-deficient cells. We conclude that rac1 is required for the adhesive and motile function of growth cones rather than the assembly of neurites per se.
Intracellular acidification induces apoptosis by stimulating ICE-like protease activity
Authors: Furlong Isla J. , Ascaso Rosalia , Rivas Abelardo Lopez , Collins Mary K. L.
Published: April 2021

ICE-like protease activation and DNA fragmentation are preceded by a decrease in intracellular pH (pHi) during apoptosis in the IL-3 dependent cell line BAF3. Acidification occurs after 7 hours in cells deprived of IL-3 and after 4 hours when cells are treated with etoposide, close to the time of detection of ICE-like protease activity. Increasing extracellular pH reduces ICE-like protease activation and DNA fragmentation. Bcl-2 over-expression both delays acidification and inhibits ICE-like protease activation. Generation of a rapid intracellular pH decrease, using the ionophore nigericin, induces ICE-like protease activation and apoptosis. ZVAD, a cell permeable inhibitor of ICElike proteases, does not affect acidification but inhibits apoptosis induced by IL-3 removal or nigericin treatment. These data suggest that intracellular acidification triggers apoptosis by directly or indirectly activating ICE-like proteases.
Internalized Listeria monocytogenes modulates intracellular trafficking and delays maturation of the phagosome
Authors: Alvarez-Dominguez C. , Roberts R. , Stahl P.D.
Published: April 2021

Previous studies have shown that early phagosome-endosome fusion events following phagocytosis of Listeria monocytogenes are modulated by the live organism. In the present study, we have characterized more fully the intracellular pathway of dead and live Listeria phagosomes. To examine access of endosomal and lysosomal markers to phagosomes containing live and dead Listeria, quantitative electron microscopy was carried out with intact cells using internalized BSA-gold as a marker to quantify transfer of solute from endosomal and lysosomal compartments to phagosomes. To monitor the protein composition of phagosomal membranes and to quantify transfer of HRP from endosomes and lysosomes to phagosomes, highly enriched phagosomes containing live and dead Listeria were isolated. Enriched phagosomal membranes were used for western blotting experiments with endosomal and lysosomal markers. In this study, we used a listeriolysin-deficient mutant, Listeria(hly-), that is retained within the phagosome following phagocytosis. Western blotting experiments indicate that early endosomal markers (mannose receptor, transferrin receptor) and key fusion factors necessary for early events (NSF, alpha/beta-SNAP) but not late endosomal markers (cation dependent mannose 6-phosphate receptor) or lysosomal proteins (cathepsin D or lamp-1) accumulate on the live-Listeria phagosomal membranes. On the contrary, phagosomes containing dead-Listeria are readily accessible by both endocytic and lysosomal markers. Studies with radiolabeled dead- and live-Listeria(hly-) indicate that, following phagocytosis, degradation of the live microorganism is substantially delayed. These findings indicate that dead-Listeria containing phagosomes rapidly mature to a phagolysosomal stage whereas live-Listeria(hly-) prevents maturation, in part, by avoiding fusion with lysosomes. The data suggest that by delaying phagosome maturation and subsequent degradation, Listeria prolongs survival inside the phagosome/endosome assuring bacterial viability as a prelude to escape into the cytoplasm.
Genome replication in early mouse embryos follows a defined temporal and spatial order
Authors: Ferreira João , Carmo-Fonseca Maria
Published: April 2021

The spatial and temporal organisation of replication sites during early mouse embryogenesis was analysed using high resolution confocal and video fluorescence microscopy. The results show that distinct replication patterns occur in the transcriptionally inactive pronuclei of 1-cell embryos as well as in the transcriptionally active nuclei from 2- and 16/32-cell embryos. This indicates that specific chromatin regions are replicated at different times during S-phase and provides the first evidence that mechanisms controlling the temporal and spatial replication of DNA are already present in the haploid pronuclei of the mammalian zygote. Furthermore the data demonstrate that the male and female pronuclei in one-cell embryos replicate their genomes asynchronously. Finally, we observe changes in the dynamics of embryonic genome replication during early development which correlate with gross chromatin structure transitions detected at the electron microscope level. Taken together these results indicate that DNA synthesis in the mouse zygote follows a defined four-dimensional order which may evolve during development and differentiation.
Defining the essential functional regions of the nucleoporin Nup145p
Authors: Emtage Jennifer L. T. , Bucci Mirella , Watkins Janis L. , Wente Susan R.
Published: April 2021

Studies of the essential nucleoporin Nup145p have shown that its depletion is coincident with a block in RNA export and that deletion of its amino-terminal domain results in clustering of nuclear pore complexes. To further define the functional domains of Nup145p, we have characterized a panel of nup145 mutants. Deletions from both the amino terminus and the carboxy terminus resulted in temperature sensitive mutants that accumulated polyadenylated RNA in the nucleus at the non-permissive temperature. In addition, these mutants also displayed constitutive clustering of nuclear pore complexes in localized patches of the nuclear envelope. These results suggested that an internal region of Nup145p consisting of amino acids 593-893 is essential for function. Accordingly, when this region was deleted, growth was not supported at any temperature, whereas the region alone was able to complement a null mutation when expressed on a high copy plasmid. Previous studies have suggested that Nup145p is cleaved into two polypeptides of approximately 65 and 80 kDa. Interestingly, our experiments suggest that cleavage occurs in vivo. However, a small internal deletion of 17 amino acid residues that abolished cleavage had no effect on cell growth. Therefore, cleavage is not necessary for Nup145p function. When a sequence harboring the Nup145p cleavage site required for Nup145p cleavage was inserted in a chimeric protein, it was not sufficient for mediating cleavage. Cleavage likely requires a second region from amino acid residues 247-524 in addition to the cleavage site.
Localization of a novel 210 kDa protein in Xenopus tight junctions
Authors: Merzdorf Christa S. , Goodenough Daniel A.
Published: April 2021

The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an ∼220 kDa protein in Xenopus, while mAb 19B1 identified an ∼210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.
Characterization of the interactions of α-catenin with α-actinin and β-catenin/plakoglobin
Authors: Nieset Jill E. , Redfield Ann R. , Jin Fang , Knudsen Karen A. , Johnson Keith R. , Wheelock Margaret J.
Published: April 2021

Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, α-catenin, β-catenin and γ-catenin (also known as plakoglobin), have been identified. β-catenin or plakoglobin is associated directly with the cadherin; α-catenin binds to β-catenin/plakoglobin and serves to link the cadherin/catenin complex to the actin cytoskeleton. The domains on the cadherin and βcatenin/plakoglobin that are responsible for proteinprotein interactions have been mapped. However, little is known about the molecular interactions between α-catenin and β-catenin/plakoglobin or about the interactions between α-catenin and the cytoskeleton. In this study we have used the yeast two-hybrid system to map the domains on α-catenin that allow it to associate with β-catenin/plakoglobin and with α-actinin. We also identify a region on αactinin that is responsible for its interaction with α-catenin. The yeast two-hybrid data were confirmed with biochemical studies.
The carboxy-terminal peptides of 46 kDa and 300 kDa mannose 6-phosphate receptors share partial sequence homology and contain information for sorting in the early endosomal pathway
Authors: Böker Christian , Figura Kurt von , Hille-Rehfeld Annette
Published: April 2021

Recycling of mannose 6-phosphate receptors was investigated by microinjection of Fab fragments against their carboxy-terminal peptides (residues 54-67 or 150-164 of the cytoplasmic domain of 46 kDa and 300 kDa mannose 6-phosphate receptor, respectively). For each receptor, masking the carboxy-terminal peptide by the corresponding Fab fragments resulted in complete depletion of the intracellular pool. Redistributed 300 kDa mannose 6-phosphate receptor was shown to accumulate at the plasma membrane and to internalize anti-ectodomain antibodies. Internalization of anti-ectodomain antibodies was also observed for redistributed 46 kDa mannose 6-phosphate receptor. Semiquantitative analysis suggested that for both redistributed receptors the amount of intracellularly accumulated anti-ectodomain antibodies was reduced. In addition, downstream transport along the endosomal pathway was slowed down. These data suggest that sorting information for early steps in the endocytic pathway is contained within the carboxy-terminal peptides of mannose 6-phosphate receptors.
Mutation of a gene for a Drosophila kinesin-like protein, Klp38B, leads to failure of cytokinesis
Authors: Ohkura Hiroyuki , Török Tibor , Tick Gabriella , Hoheisel Jörg , Kiss István , Glover David M.
Published: April 2021

Mutations in a gene (Klp38B) encoding a novel kinesin-like protein in Drosophila melanogaster lead to the formation of polyploid cells in the larval central nervous system and in the follicle cells of adult egg chambers. Some homozygous mutants survive to adulthood and also exhibit morphological defects indicative of abnormal cell cycle progression, including rough eyes, missing bristles, and abnormal abdominal cuticles. In larval brains, there is no accumulation of mitotic cells and the frequency of anaphase figures is comparable to wild type, suggesting that nuclear division is not affected. Such brains contain polyploid cells with metaphase and anaphase chromosomes associated with bipolar spindles. Such spindles have a number of unseparated centrosomes at their poles reflecting the degree of polyploidy of the cell. Follicle cells frequently contain two nuclei of roughly equal size. Taken together, we conclude that these Klp38B mutations lead to a failure of cytokinesis resulting in polyploidy, and discuss whether or not this is a direct effect of the mutation.
Contrasting effects of K8 and K18 on stabilizing K19 expression, cell motility and tumorigenicity in the BSp73 adenocarcinoma
Authors: Pankov Roumen , Simcha Inbal , Zöller Margot , Oshima Robert G. , Ben-Ze’ev Avri
Published: April 2021

The co-expression of vimentin and keratin-type intermediate filaments in the same cell was often reported to correlate with increased invasiveness and a more aggressive tumorigenic phenotype. To address the possible physiological relevance of these observations, we transfected simple keratins (K8 and 18) either individually, or in combination, into a tumorigenic but non-metastatic pancreatic adenocarcinoma that expresses vimentin but no keratins. Expression of K8 resulted in the stabilization of endogenous K19 in these cells, and formation of keratin filaments containing K8 and K19. Transfection of K18 yielded unstable K18 protein, but K18 could be stabilized when K8 was co-expressed in the same cells. Clones expressing K18 alone, or together with K8, displayed a reduced ability to grow in soft agar and decreased motility when compared to control, or K8/19 expressing cells. Moreover, K18 expressing cells were dramatically inhibited in their ability to form tumors when injected into syngeneic animals. The extent of suppression in the tumorigenicity of these cells correlated with the level of K18 expressed by these cells. The results show that K18 expression in cells may result in the suppression of the motile and tumorigenic abilities of this adenocarcinoma.
Activation of the β2 integrin Mac-1 (CD11b/CD18) by an endogenous lipid mediator of human neutrophils and HL-60 cells
Authors: Klugewitz Katja , Ley Klaus , Schuppan Detlef , Nuck Rolf , Gaehtgens Peter , Walzog Barbara
Published: April 2021

β2 integrins (CD11/CD18) play a key role in the adhesion, activation, migration and phagocytosis of human neutrophils. In order to exert their functions, β2 integrins require activation, which results in an enhancement of ligand affinity. This functional up-regulation is probably due to a conformational change of the β2 integrins, but the mechanisms of inside-out signaling that trigger this activation are still under investigation. In the present study, the effect of cellular lipids on the affinity state of β2 integrins was investigated. Lipids were extracted from human neutrophils and HL-60 cells after stimulation with IL-8 or phorbol ester, respectively. The extracts were purified by anion exchange chromatography and/or HPLC fractionation. The lipid extracts induced the adhesion of neutrophils to fibrinogen and, in a cell-free assay system, the binding of C3bi-coated zymosan-particles by purified β2 integrin Mac-1 (CD11b/CD18). The integrin up-regulating activity was resistant to ester hydrolysis, eluted as one particular HPLC-fraction, and showed an absorption maximum at 194±2 nm. Taken together, these data support the concept that activated neutrophils and HL-60 cells can generate an endogenous lipid mediator, which up-regulates ligand binding activity of β2 integrins.
AND-1, a natural chimeric DNA-binding protein, combines an HMG-box with regulatory WD-repeats
Authors: Köhler Andreas , Schmidt-Zachmann Marion S. , Franke Werner W.
Published: April 2021

Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of ∼4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive ‘WD-repeats’, which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long ‘HMG-box’, which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a ‘natural chimera’ combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.
Analysis of Toxoplasma gondii stably transfected with a transmembrane variant of its major surface protein, SAG1
Authors: Seeber Frank , Dubremetz Jean-François , Boothroyd John C.
Published: April 2021

We have genetically engineered Toxoplasma gondii so that its major surface antigen SAG1 is anchored by a human transmembrane domain (SAG1-TM) instead of its natural GPI anchor (SAG1-GPI) in order to initiate studies to address the function of this protein anchor in parasitic protozoa as well as to get insights into the functional role of SAG1. Our results show that SAG1-TM is correctly folded (at least as judged by the presence of conformationally dependent epitopes) and targeted to the surface of the parasite, indicating that the GPI anchor does not determine its localization nor overall three-dimensional structure. No significant difference was seen in any aspect of the growth of the SAG1-TM mutant. However, compared to the natural SAG1-GPI, SAG1-TM does not form strong associations with itself and/or other molecules in high molecular weight complexes suggesting that allowing such complexes to form may be one role of the GPI anchor. The in vitro half-life of SAG1-TM of extracellular parasites is significantly lower than that of SAG1-GPI suggesting a stabilizing function of the glycolipid anchor against degradation and/or membrane release. Antibodies to SAG1 are shed from SAG1-TM parasites as they invade, just as they are stripped from SAG1-GPI bearing parasites. The stripping, therefore, is unlikely to be driven by the action of lipases.
A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes
Authors: Cao Fengli , Eckert Reiner , Elfgang Claudia , Nitsche Johannes M. , Snyder Scott A. , Hülser Dieter F. , Willecke Klaus , Nicholson Bruce J.
Published: April 2021

Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8-14 Å in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.
Identification of intracellular sites of superoxide production in stimulated neutrophils
Authors: Kobayashi Toshihiro , Robinson John M. , Seguchi Harumichi
Published: April 2021

In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.
Unidirectional movement of fluorescent microtubules on rows of dynein arms of disintegrated axonemes
Authors: Yamada Akira , Yamaga Takako , Sakakibara Hitoshi , Nakayama Haruto , Oiwa Kazuhiro
Published: April 2021

Tetramethylrhodamine-labelled microtubules were observed to move on rows of dynein arms of sea urchin sperm axonemes exposed by elastase-induced sliding disintegration. The microtubules moved towards the flagellar tip at a velocity of 3.1±2.1 μm second−1 (mean ± s.d., n=53) in the presence of 0.1 mM ATP at 22°C, but none moved towards the sperm head. We also examined the polarity of microtubule binding to axonemal doublet microtubules in the absence of ATP by using microtubules brightly labelled at their minus-ends. In 140 of 210 microtubules studied, they bound to axonemal microtubules with a parallel polarity. These results suggest that tightly packed dynein arms on the outer doublet microtubules of sperm axoneme preferentially bind microtubules to themselves with the same polarity as that of the axoneme and that they generate a force to move only these microtubules in the direction away from the sperm head.
Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos
Authors: Helps Nicholas R. , Brewis Neil D. , Lineruth Katrin , Davis Terence , Kaiser Kim , Cohen Patricia T. W.
Published: April 2021

The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localisation in Drosophila cells and embryos and possess a highly conserved (91% identical) amino acid sequence from humans to invertebrates. A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA, ∼25% of normal PP4 protein in early embryos and exhibits a semi-lethal phenotype with only 10% viability in certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of γ-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. These findings indicate that PP4 is required for nucleation, growth and/or stabilisation of microtubules at centrosomes/spindle pole bodies.
Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm
Authors: Traverso Valerie , Morris John F. , Flower Rod J. , Buckingham Julia
Published: April 2021

Lipocortin 1 (annexin I) is a calcium- and phospholipidbinding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.
Sequential silver staining and in situ hybridization reveal a direct association between rDNA levels and the expression of homologous nucleolar organizing regions: a hypothesis for NOR structure and function
Authors: Zurita F. , Jiménez R. , Burgos M. , Díaz de la Guardia R.
Published: April 2021

We have developed a procedure for sequential silver staining and in situ hybridization to analyze the relationship between the amount of rDNA present in nucleolar organizer regions, as estimated by in situ hybridization, and their level of expression, as estimated by the silver signal. For simplicity we used cells from the insectivorous mole Talpa occidentals, which have a single pair of nucleolar organizer regions in chromosome pair 3. The relative content of ribosomal cistrons was also related to the hierarchy of activation of the nucleolar organizer regions present in this chromosomal pair. Statistical analyses demonstrated that both the relative level of expression and the activation hierarchy depended mainly on the number of ribosomal cistrons in nucleolar organizer regions. We propose a functional two-step hypothesis, which is consistent with most known data concerning interchromosomal, intercellular and interindividual variation in a number of plant and animal species, including Talpa occidentals. In step one, the first available transcription factors bind randomly to the ribosomal promoters, such that larger nucleolar organizer regions are more likely to recruit them. In the second step the remaining transcription factors are recruited in a cooperative way, thus completing activation of one nucleolar organizer region, before the next one becomes active.
Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility
Authors: Palin Andrew H. , Critcher Ricky , Fitzgerald Daniel J. , Anderson John N. , Farr Christine J.
Published: April 2021

Fragile sites are reproducibly expressed and chemically induced decondensations on mitotic chromosomes observed under cytological conditions. They are classified both on the basis of the frequency with which they occur (rare and common) and in terms of the chemical agent used to induce expression in tissue culture cells. Aphidicolin-sensitive common fragile sites appear to be ubiquitous in humans and other mammals and have been considered as candidates of pathological importance. Recently DNA from FRA3B, the most highly expressed constitutive fragile site in the human genome, has been cloned although as yet the cause of the underlying fragility has not been identified. In this study we describe the isolation, using a direct cloning approach, of DNA from a region of the Chinese hamster genome associated with aphidicolin-inducible fragility. Cells of a human-hamster somatic cell hybrid were transfected with a pSV2HPRT vector while exposed to aphidicolin, an inhibitor of DNA polymerases α, δ and ε. FISH analysis of stable transfectant clones revealed that the ingoing plasmid DNA had preferentially integrated into fragile site-containing chromosomal bands. Plasmid rescue was used to recover DNA sequences flanking one such integration site in the hamster genome. We demonstrate by FISH analysis of metaphase cells induced with aphidicolin that the rescued DNA is from a region of fragility on Chinese hamster chromosome 2, distal to the DHFR locus. Analysis of the DNA sequences flanking the integration site revealed the overall A+T content of the 3,725 bp region sequenced to be 63.3%, with a highly [A]·[T]-RICH 156 bp region (86.5%) almost adjacent to the integration site. Computational analyses have identified strong homologies to Saccharomyces cerevisiae autonomous replicating sequences (ARS), polypyrimidine tracts, scaffold attachment site consensus sequences and a 24 bp consensus sequence highly conserved in eukaryotic replication origins, all of which appear to cluster around the [A]·[T]-rich sequences. This domain also possesses structural characteristics which are common to both prokaryotic and eukaryotic origins of replications, in particular an unusually straight conformation of low thermal stability flanked either side by highly bent DNA segments. Further isolation and characterisation of DNA sequences from common fragile sites will facilitate studies into the underlying nature of these enigmatic regions of the mammalian genome, leading to a greater understanding of chromatin structure.
Actin dynamics in living mammalian cells
Authors: Ballestrem Christoph , Wehrle-Haller Bernhard , Imhof Beat A.
Published: April 2021

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.
The small GTP-binding protein RacG regulates uroid formation in the protozoan parasite Entamoeba histolytica
Authors: Guillén Nancy , Boquet Patrice , Sansonetti Philippe
Published: April 2021

Entamoeba histolytica is a protozoan parasite that invades human intestine leading to ulceration and destruction of tissue. Amoebic movement and phagocytosis of human cells is accompanied by characteristic changes in cell morphology. Amoebae become polarized, developing a frontal pseudopod and a well-defined rear zone of membrane accumulation designated the uroid. In motile eukaryotic cells, a phenomenon that contributes to movement is the capping of receptors at the cell surface. During the capping process, E. histolytica concentrates ligand-receptor complexes in the uroid. Interestingly, some of these surface receptors are involved in the survival of the parasite. While looking for regulators of capping and uroid formation, we identified RacG, an E. histolytica protein that is homologous to human Rac1. This protein belongs to the Rac subfamily of small GTPases implicated in interactions between the actin cytoskeleton and the membrane of mammalian cells. Cloning of the EhracG gene and analysis of the protein activity either in murine fibroblasts or in E. histolytica revealed that EhRacG induces a characteristic Rac phenotype. When expressed in amoebae, an EhRacG-V12 mutant protein not only deregulated cell polarity, but also caused a defect in cytokinesis. Analysis of the cytoskeleton in amoebae bearing this mutant revealed that F-actin concentrated at the periphery of the cell. In addition, the number and localization of uroids were modified. These results suggest a role for EhRacG in amoebic morphogenesis and cytokinesis.