bluuebunny/crossref_metadata_2025_split 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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.
Genome replication in early mouse embryos follows a defined temporal and spatial order
Authors: Ferreira João , Carmo-Fonseca Maria 
Published: April 2021

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.