bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells
Authors: Atkinson Michael M. , Lamper Paul D. , Lin Helen H. , Kollander Rahn , Li Xen-Ren , Kiang David T. 
Published: April 2021

ABSTRACT
Direct communication between cells via gap junctions is thought to be an important component of homeostasis and coordinated cellular responses to external signals. We investigated how the second messenger cAMP exerts its effects on junctional communication in a mouse mammary tumor cell line, MMT22. Junctional permeance was quantitatively assessed using dye microinjection and video microscopy. An increase of permeance was found after exposure to 8-bromo-cAMP, being detectable after 30 minutes of treatment and attaining a fourfold higher level of permeance by 24 hours. This elevated level was maintained with continuous exposure to 8-bromo-cAMP for seven days. The permeability change was accompanied by an increase in gap junctions as shown by freeze-fracture electron microscopy and by confocal microscopy using antibodies directed against the gap junction protein, connexin43. The amount of detergent-insoluble connexin43 also increased with 8-bromo-cAMP treatment, and most of the increase could be attributed to an increase of slower migrating (i.e. phosphorylated) species of connexin43. However, connexin43 mRNA and the total cellular content of connexin43 did not change over this period of exposure to 8-bromo-cAMP, as shown by densitometric analyses of northern and western blots. We conclude that 8-bromo-cAMP affects the distribution of connexin43 such that a greater proportion of the protein is utilized for channel formation. Since these changes were relatively slow to develop and persisted with prolonged exposure to 8-bromo-cAMP, it is possible that the junctional permeability of these mammary tumor cells is linked to the ‘basal’ level of cAMP, i.e. levels maintained by the cells in accordance with a particular cell state.
DNA polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast
Authors: D’Urso Gennaro , Grallert Beáta , Nurse Paul 
Published: April 2021

ABSTRACT
Genetic analysis in the yeast Schizosaccharomyces pombe has shown that three genes cdc18, cut5, and cdtl, are essential for DNA synthesis and also for the checkpoint control that couples completion of DNA replication to the onset of mitosis. To test whether assembly of the replication initiation complex is an important element in the checkpoint control pathway we have investigated if DNA polymerase alpha (poll), a component of the initiation complex, is essential for the S-phase checkpoint control. We show that germinating S. pombe spores disrupted for the pol1 gene enter mitosis despite defects in DNA synthesis. This is shown by monitoring septation index, DNA content, and by direct immunofluorescence of mitotic spindles using antibodies to alpha-tubulin. In addition we have isolated six temperature sensitive mutants in the pol1 gene that cause cell cycle arrest when grown at the non-permissive temperature. Our experiments support a model in which DNA polymerase alpha, in addition to being part of the initiation complex, is required for a checkpoint signal that is activated as cells traverse START, and is essential to prevent mitosis until S phase has been completed. In contrast, proteins responsible for the elongation of DNA may not be necessary for this checkpoint signal.
The carboxyl terminus of myosin binding protein C (MyBP-C, C-protein) specifies incorporation into the A-band of striated muscle
Authors: Gilbert Rénald , Kelly Michael G. , Mikawa Takashi , Fischman Donald A. 
Published: April 2021

ABSTRACT
Myosin binding protein-C (MyBP-C), also known as C-protein, is a major constituent of the thick filaments of vertebrate striated muscles. The protein, ∼130 kDa, consists of a series of 10 globular motifs (numbered I to X) each of ∼90-100 amino acids, bearing resemblance to the C2-set of immunoglobins (Ig C2) and to the fibronectin type III (FnIII) motifs. Using pure preparations of myosin and MyBP-C, it has been demonstrated that the major myosin binding domain of MyBP-C resides within the C-terminal Ig C2 motif (motif X). However, in the context of the in vivo thick filament, it is uncertain if the latter domain is sufficient to target MyBP-C correctly to the A-band or if other regions of the molecule are required for this process. To answer this question, cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding seven truncation mutants of MyBP-C, and their targeting to the A-band investigated by immunofluorescence microscopy. To distinguish the recombinant proteins from endogenous MyBP-C, a myc epitope was inserted at each amino terminus. Recombinant MyBP-C exhibited an identical distribution in the sarcomere to that of native MyBP-C; i.e. it was found exclusively in the C-zone of the A-band. A mutant encoding the C-terminal 372 amino acids, but lacking motifs I-VI (termed Δ1-6), also targeted correctly to the A-band. This fragment, which is composed of two Ig C2 and two FnIII motifs, was the minimal protein fragment required for correct A-band incorporation. Larger aminoterminal deletions or deletion of motif X, the myosin binding domain, abolished all localization to the A-band. One construct (Δ10) lacking only motif X strongly inhibited myofibril assembly. We conclude that the myosin binding domain of MyBP-C, although essential, is not sufficient for correct incorporation into the A-band and that motifs VII to IX are required for this process. The data suggest a topological model in which MyBP-C is associated with the thick filament through its C terminus.
Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring
Authors: Chang Fred , Woollard Alison , Nurse Paul 
Published: April 2021

ABSTRACT
Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a β-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.
Coordinated movements between autosomal half-bivalents in crane-fly spermatocytes: evidence that ‘stop’ signals are sent between partner half-bivalents
Authors: Yin Beihong , Forer Arthur 
Published: April 2021

ABSTRACT
During anaphase-I in crane-fly spermatocytes, sister half-bivalents separate and move to opposite poles. When we irradiate a kinetochore spindle fibre with an ultraviolet microbeam, the associated half-bivalent temporarily stops moving and so does the partner half-bivalent with which it was paired during metaphase. To test whether a ‘signal’ is transmitted between partner half-bivalents we irradiated the spindle twice, once in the interzone (the region between separating partner half-bivalents) and once in a kinetochore fibre. For both irradiations we used light of wavelength 290 μm and a dose that, after irradiating a spindle fibre only, altered movement in 63% of irradiations (12/19); in 11 of the 12 cells both partner half-bivalents stopped moving after the irradiation. In control experiments we irradiated the interzone only: these irradiations generally did not stop chromosomal poleward motion but sometimes (14/29) caused poleward movement to each pole to be abruptly reduced to about half the velocity prior to irradiation.
In double irradiation experiments we varied the order of the irradiations. In some double irradiation experiments we irradiated the interzonal region first and the spindle fibre second; in 75% (9/12) of the cells the half-bivalent associated with the irradiated fibre stopped moving while the partner half-bivalent moved normally, i.e. in 9/12 cells the interzonal irradiations uncoupled the movements of the partner half-bivalents. In other double irradiation experiments we irradiated the spindle fibre first and the interzone second: in 80% (4/5) of the cells the half-bivalents not associated with the irradiated spindle fibre resumed movement immediately after the irradiation while the other half-bivalent remained stopped. Interzonal irradiations therefore uncouple the poleward movements of sister half-bivalents and the uncoupling does not depend on the order of the irradiation. Our experiments suggest therefore that the irradiation of a spindle fibre causes negative (‘stop’) signals to be transmitted across the interzone and that irradiation of the interzone blocks the transmission of the stop signal.
Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds
Authors: Nishimura Karen Y. , Isseroff R. Rivkah , Nuccitelli Richard 
Published: April 2021

ABSTRACT
Previous measurements of the lateral electric fields near skin wounds in guinea pigs have detected DC fields between 100-200 mV/mm near the edge of the wound. We have studied the translocation response of motile primary human keratinocytes migrating on a collagen substrate while exposed to similar physiological DC electric fields. We find that keratinocytes migrate randomly on collagen in fields of 5 mV/mm or less, but in larger fields they migrate towards the negative pole of the field, exhibiting galvanotaxis. Since these cells have an average cell length of 50 µm, this implies that they are able to detect a voltage gradient as low as 0.5 mV along their length. This cath-odally-directed movement exhibits increased directedness with increasing field strengths between 10 and 100 mV/mm. We observe a maximally directed response at 100 mV/mm with half of the cells responding to the field within 14 minutes. The average speed of migration tended to be greater in fields above 50 mV/mm than in smaller fields. We conclude that human keratinocytes migrate towards the negative pole in DC electric fields that are of the same magnitude as measured in vivo near wounds in mammalian skin.
Three-dimensional organization of rat hepatocyte cytoskeleton: relation to the asialoglycoprotein endocytosis pathway
Authors: Novikoff P. M. , Cammer M. , Tao L. , Oda H. , Stockert R. J. , Wolkoff A. W. , Satir P. 
Published: April 2021

ABSTRACT
Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocytes in culture, establishing the three-dimensional disposition of the microtubule-based cytoskeleton, its relation to the actin-based cytoskeleton and to ligand-containing endosomes during receptor-mediated endocytosis and the alterations in its structure and disposition by the microtubule pertubant, Taxol. By co-localization studies, we have been able to demonstrate that the microtubules have a significant role in receptor-mediated endocytosis of asialoglycoproteins in this cell. Asialoorosomucoid-containing endosomes attach to widely spaced arrays of microtubules running under the baso-lateral surface of the hepatocytes 5-15 minutes after the initiation of endocytosis and then travel along microtubule paths to become concentrated with microtubules near the centrosome and at bile canaliculi after 30-60 minutes of receptor-mediated endocytosis. Receptor-mediated endocytosis is affected, but not abolished by Taxol, which inhibits the rate of asialoorosomucoid degradation at the same concentrations as those that disrupt microtubule and cytoplasmic dynein distribution, and that prevent the concentration of endosomes centrally. The results support suggestions that asialoorosomucoid-containing endosomes are captured by microtubules just below the actin layer at the cell periphery and these are actively transported centrally along microtubules, possibly by cytoplasmic dynein, so that the concentration of endosomes near the centrosome, and the subsequent efficient lysosomal degradation of ligand, are consequences of the confluence of microtubules in this region.
Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes
Authors: Weaver Valerie M. , Carson Christine E. , Walker P. Roy , Chaly Nathalie , Lach Boleslaw , Raymond Yves , Brown David L. , Sikorska Marianna 
Published: April 2021

ABSTRACT
In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occured within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferasemediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis.
Plant and animal profilins are functionally equivalent and stabilize microfilaments in living animal cells
Authors: Rothkegel Martin , Mayboroda Oleg , Rohde Manfred , Wucherpfennig Carmen , Valenta Rudolf , Jockusch Brigitte M. 
Published: April 2021

ABSTRACT
We have analyzed the degree of functional similarity between birch and mammalian profilins, two members of the profilin family which show only a moderate sequence homology (22%) in living animal cells. The plant profilin, derived from birch pollen, was stably expressed in BHK-21 cells. Plant and endogenous profilin synthesis and cellular distribution were monitored by specific monoclonal antibodies. Quantitation of profilin and actin on calibrated immunoblots showed that two stable clones contained in total 1.4 and 2.0 times as much profilin as the parental cells. Using double fluorescence and confocal laser scanning microscopy, it was seen that the endogenous and the plant profilin colocalized with dynamic microfilaments, in particular with F-actin-rich foci and cortical microfilament webs of spreading cells, with dynamic microfilament bundles induced by serum deprival, and with cytochalasin D- and latrunculininduced transient F-actin aggregates. The increase in the overall profilin concentration correlated with a significantly higher resistance of actin filaments to these drugs. Our data indicate that even profilins of highly distant evolutionary origin can functionally substitute for each other and support the hypothesis that in animal cells, profilins are engaged in regulating either the stability or the kinetic properties of actin filaments.
A single gamma-tubulin gene and mRNA, but two gamma-tubulin polypeptides differing by their binding to the spindle pole organizing centres
Authors: Lajoie-Mazenc Isabelle , Détraves Claire , Rotaru Victor , Garès Michèle , Tollon Yvette , Jean Catherine , Julian Monique , Wright Michel , Raynaud-Messina Brigitte 
Published: April 2021

ABSTRACT
Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of α- and β-tubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for α- and β-tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of γ-tubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for γ-tubulin were detected. They coded for a polypeptide of 454 amino acids, with a predicted molecular mass of 50,674, which presented 64-76% identity with other γ-tubulins. However, immunological studies identified two γ-tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two γ-tubulins, called γs- and γf-tubulin for slow and fast electrophoretic mobility, exhibited apparent molecular masses of 52,000 and 50,000, respectively. They were recognized by two antibodies (R70 and JH46) raised against two distinct conserved sequences of γ-tubulins. They were present both in the preparations of amoebal centrosomes possessing two centrioles and in the preparations of plasmodial nuclear metaphases devoid of structurally distinct polar structures. These two γ-tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, γs-tubulin was tightly bound to microtubule organizing centers (MTOCs) while γf-tubulin was loosely associated with these structures. This first demonstration of the presence of two γ-tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed.
Three homologs of rds/peripherin in Xenopus laevis photoreceptors that exhibit covalent and non-covalent interactions
Authors: Kedzierski Wojciech , Moghrabi Walid N. , Allen Ann C. , Jablonski-Stiemke Monica M. , Azarian Sassan M. , Bok Dean , Travis Gabriel H. 
Published: April 2021

ABSTRACT
We have isolated and characterized three homologs of mammalian rds/peripherin from Xenopus retinae. One (xrds38) is likely the Xenopus ortholog, while the other two (xrds36 and -35) are more distant relatives. By immunocytochemical analysis of retinal sections, xrds38 is distributed in both rod and cone photoreceptors, while xrds36 and xrds35 are present in rods only. At the EM level, xrds38 is present specifically in the rims and incisures of rod and cone outer segment discs. All are N-glycosylated and form covalent dimers. Immunoprecipitation analysis showed that in rods, these three proteins interact to form heterotetrameric or higher-order complexes. The pattern of sequence conservation among the xrds proteins, mammalian rds/peripherin, and mammalian rom-1 suggest that the central portion of the intradiscal D2 loop contains the interacting structural elements.
Elongation factor-1α is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma
Authors: Edmonds Brian T. , Wyckoff Jeffrey , Yeung Yee-Guide , Wang Yun , Stanley E. Richard , Jones Joan , Segall Jeffrey , Condeelis John 
Published: April 2021

ABSTRACT
Overexpression of elongation factor-1α (EF1α) mRNA has been correlated with increased metastatic potential in mammary adenocarcinoma; however, this relationship was not explored at the level of protein expression. As EF1α has been shown in other cell types to be a component of the actin cytoskeleton, a likely effector in metastasis, the actin binding activity of EF1α from metastatic and nonmetastatic rat breast tumors and cell lines was investigated. We have shown that EF1α protein is overexpressed in metastatic compared to nonmetastatic cells and whole tumors. Similarly to other EF1αs, both types of tumor EF1α bind to F-actin, but EF1α from metastatic cells has a reduced affinity for actin. In addition, there is a high correlation between the intracellular distribution of filamentous actin and EF1α in those cytoskeletal structures thought to be important for supporting the cellular motility required for metastasis. Following stimulation with EGF, there is a parallel increase in the amount of F-actin and EF1α associated with the cytoskeleton. The response to EGF can be blocked with cytochalasin D indicating that the binding of EF1α to the cytoskeleton is mediated by F-actin. We propose that a weakened association of EF1α with actin may be related to the metastatic process via an altered organization of the actin cytoskeleton and the differential translation of mRNAs associated with the cytoskeleton.
NRD convertase: a putative processing endoprotease associated with the axoneme and the manchette in late spermatids
Authors: Chesneau Valérie , Prat Annik , Segretain Dominique , Hospital Véronique , Dupaix Alain , Foulon Thierry , Jégou Bernard , Cohen Paul 
Published: April 2021

ABSTRACT
N-arginine dibasic convertase is a novel metalloendopeptidase which selectively cleaves at the N terminus of arginine residues in paired basic amino acids. Although present in brain and several other tissues, NRD convertase is particularly abundant in testis, where its expression appeared to be restricted to germ cells. Low levels of both mRNA and its corresponding protein were detected early in spermatogenesis. However, a marked accumulation of the protein was observed during late steps (14 to 19) of spermiogenesis. By electron microscopy, the NRD convertase immunoreactivity was localized in the cytoplasm of elongating and elongated spermatids, with a noticeable concentration at the level of two microtubular structures, i.e. the manchette and the axoneme. These observations strongly support the hypothesis that NRD convertase is involved in processing events potentially associated with the morphological transformations occurring during spermiogenesis.
The central Z-disk region of titin is assembled from a novel repeat in variable copy numbers
Authors: Gautel Mathias , Goulding David , Bullard Belinda , Weber Klaus , Fürst Dieter O. 
Published: April 2021

ABSTRACT
The giant sarcomeric protein titin (also described as connectin) is composed mainly of immunoglobulin (Ig)-like and fibronectin type III (fn3)-like domains arranged consecutively. At both ends of the molecule, these domains are interrupted by sequence insertions. The amino terminus of titin is localized in the Z-disk, a structure of great variability in different muscle types. We have determined the ultrastructural position of sequences in this region of the molecule in skeletal and cardiac muscle by immunoelectron microscopy using antibodies directed against unique epitopes. Titin molecules entering the Z-disk from two half sarcomeres do not significantly overlap, showing that the amino terminus is at the centre of the Z-disk. A serine/proline rich site, which can be phosphorylated by kinases in developing muscle tissues, was identified near the amino terminus of titin. Sequence analysis revealed the presence of a novel 45 residue repeat (‘Z-repeats’) in this region of the molecule. The number of titin Z-repeats varies due to differential splicing. We propose that this mechanism is a means of assembling Z-disks of variable thickness and mechanical strength.