bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation
Authors: Ory S. , Munari-Silem Y. , Fort P. , Jurdic P. 
Published: April 2021

ABSTRACT
Multinucleated giant cells (MNGC) derived from avian blood monocytes present, like osteoclasts, an unusual cytoskeletal organization characterized by (1) cortical rings of actin filaments, (2) unique adhesion structures called podosomes and (3) vinculin containing focal complexes which are not visibly connected to F-actin structures. The Rho family of small GTPases plays an essential role in the regulation and organization of cellular cytoskeletal structures including F-actin and vinculin associated structures. Using bacterial toxins such as modified exoenzyme C3 (C3B) and toxin B or overexpression of constitutively active Rac and Rho proteins fused to the green fluorescent protein (GFP), we show that Rac and Rho play antagonistic roles in regulating the morphology of osteoclast-like cells. Inhibition of Rho by C3B triggered MNGC spreading whereas activated Rho promoted cell retraction. However, inhibition or activation of Rho led to complete disorganization of fibrillar actin structures, including podosomes. Toxin B inhibition of Rho, Rac and Cdc42 induced a time dependent F-actin and vinculin reorganization. Initially, actin fibers with associated adhesion plaques formed and disappeared subsequently. Finally, only small focal complexes remained at the MNGC periphery before retracting. At the time when actin fibers formed, we observed that Rac was already inhibited by toxin B. By combining C3B treatment and overexpression of a dominant negative form of Rac (N17Rac), we show that the formation of these focal adhesion and actin fiber structures required neither Rho nor Rac activity. Moreover, our results show that podosomes are extremely unstable structures since any modifications of Rho or Rac activity resulted in their dissociation.
A checkpoint that monitors cytokinesis in Schizosaccharomyces pombe
Authors: Liu Jianhua , Wang Hongyan , Balasubramanian Mohan K. 
Published: April 2021

ABSTRACT
Cell division in Schizosaccharomyces pombeis achieved through the use of a medially positioned actomyosin ring. A division septum is formed centripetally, concomitant with actomyosin ring constriction. Genetic screens have identified mutations in a number of genes that affect actomyosin ring or septum assembly. These cytokinesis-defective mutants, however, undergo multiple S and M phases and die as elongated cells with multiple nuclei. Recently, we have shown that a mutant allele of the S. pombe drc1+/cps1+gene, which encodes a 1,3-β-glucan synthase subunit, is defective in cytokinesis but displays a novel phenotype. drc1-191/cps1-191 cells are capable of assembling actomyosin rings and completing mitosis, but are incapable of assembling the division septum, causing them to arrest as binucleate cells with a stable actomyosin ring. Each nucleus in arrested cps1-191 cells is able to undergo S phase but these G2nuclei are significantly delayed for entry into the M phase. In this study we have investigated the mechanism that causes cps1-191 to block with two G2nuclei. We show that the inability of cps1-191 mutants to proceed through multiple mitotic cycles is not related to a defect in cell growth. Rather, the failure to complete some aspect of cytokinesis may prevent the G2/M transition of the two interphase-G2nuclei. The G2/M transition defect of cps1-191 mutants is suppressed by a mutation in the wee1gene and also by the dominant cdc2allele cdc2-1w, but not the cdc2-3w allele. Transient depolymerization of all F-actin structures also allowed a significant proportion of the cps1-191 cells to undergo a second round of mitosis. We conclude that an F-actin and Wee1p dependent checkpoint blocks G2/M transition until previous cytokinesis is completed.
Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei
Authors: Volpi Emanuela V. , Chevret Edith , Jones Tania , Vatcheva Radost , Williamson Jill , Beck Stephan , Campbell R. Duncan , Goldsworthy Michelle , Powis Stephen H. , Ragoussis Jiannis , Trowsdale John , Sheer Denise 
Published: April 2021

ABSTRACT
The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs4708.html & Video 2
Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin
Authors: McNally Karen Perry , Bazirgan Omar A. , McNally Francis J. 
Published: April 2021

ABSTRACT
The assembly and function of the mitotic spindle requires the activity of a number of microtubule-binding proteins. Some microtubule-binding proteins bind microtubules in vitro but do not co-localize with microtubules in interphase cells. Instead these proteins associate with specific subregions of the mitotic spindle. Katanin, a heterodimeric microtubule-severing ATPase, is found localized at mitotic spindle poles. In this paper we demonstrate that human p60 katanin and the C-terminal domain of human p80 katanin both bind microtubules in vitro. Association of these two proteins results in an increased microtubule affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and targeting to spindle poles. The N-terminal WD40 domain of p80 katanin acts as a negative regulator of microtubule disassembly activity and is also required for spindle pole localization, possibly through interactions with another spindle-pole protein. These results support a model in which katanin is targeted to spindle poles through a combination of direct microtubule binding by the p60 subunit and through interactions between the WD40 domain and an unknown protein. We propose that both domains of p80 are essential in precisely regulating katanin’s activity in vivo.
Involvement of actin filaments and integrins in the binding step in collagen phagocytosis by human fibroblasts
Authors: Segal Gersana , Lee Wilson , Arora Pamela D. , McKee Marc , Downey Gregory , McCulloch Christopher A. G. 
Published: April 2021

ABSTRACT
In physiological conditions, collagen degradation by fibroblasts occurs primarily via phagocytosis, an intracellular pathway that is thought to require collagen receptors and actin assembly for fibril internalization and degradation. Currently it is unclear which specific steps of collagen phagocytosis in fibroblasts involve actin filament assembly. As studies of phagocytosis in fibroblasts are complicated by the relatively slow rate of particle internalization compared to professional phagocytes, we have examined the role of collagen receptors and actin only in the initial collagen binding step. Prior to the binding of collagen-coated fluorescent beads by human gingival fibroblasts, a cell type that is avidly phagocytic in vitro, cells were treated with cytochalasin D (actin filament barbed-end capping) or swinholide A (actin dimer sequestering and severing) or latrunculin B (actin monomer sequestering). Bead binding and immunostaining of α2β1 and α3β1 integrin collagen receptors were measured by flow cytometry. After 1-3 hours of coincubation with beads, cytochalasin D or swinholide A eliminated actin filaments stained by rhodamine-phalloidin and inhibited collagen bead binding (reductions of 25% and 50%, respectively), possibly because of cell rounding and restricted interactions with beads. In contrast, latrunculin enhanced binding dose-dependently over controls (twofold at 1 μM) and induced the formation of brightly staining aggregates of actin and the retention of long cytoplasmic extensions. Latrunculin also reduced surface β1, α2 and α3 integrin staining up to 40% in bead-free and bead-loaded cells, indicating that latrunculin enhanced collagen receptor internalization. As determined by fluorescence recovery after photobleaching, latrunculin increased the mobility of surface-bound β1 integrin. The stimulatory effect of latrunculin on collagen bead binding was reduced to control levels by treatment with a β1 integrin inactivating antibody while a β1 integrin blocking antibody abrogated both bead binding and the latrunculin-induced stimulation. Immunoblotting of bead-associated proteins showed that latrunculin completely eliminated binding of β-actin to collagen beads but did not affect β1 integrin binding. These data indicate that latrunculin-induced sequestration of actin monomers facilitates the disengagement of actin from β1 integrin receptors, increases collagen bead binding and enhances collagen receptor mobility. We suggest that these alterations increase the probability of adhesive bead-to-cell interactions.
Abnormal localisation and hyperclustering of αVβ3 integrins and associated proteins in Src-deficient or tyrphostin A9-treated osteoclasts
Authors: Lakkakorpi P. T. , Nakamura I. , Young M. , Lipfert L. , Rodan G. A. , Duong L. T. 
Published: April 2021

ABSTRACT
The non-receptor tyrosine kinase Src was shown to be essential for osteoclast function in vivo. We have previously reported that engagement of αvβ3 integrin in osteoclasts induces tyrosine phosphorylation and activation of the adhesion kinase PYK2 and the adaptor protein p130Cas in a Src-dependent manner. The objective of this study was to analyse the role of c-Src in the αvβ3 integrin-dependent recruitment of signalling and cytoskeletal molecules in osteoclasts during bone resorption. Using prefusion osteoclasts (pOCs) obtained from cocultures of osteoblasts and spleen cells isolated from Src−/− mice or their normal littermates, we found: (1) similar expression levels and ligand binding affinities of αvβ3 integrins in Src−/− and Src+/? pOCs, (2) reduced adhesion and spreading of Src−/− pOCs, (3) defective organisation of the microfilament proteins, F-actin, vinculin and paxillin, and of PYK2 and p130Cas in the sealing zone of Src−/− OCLs, and (4) hyperclustering of αvβ3 integrins together with microfilament and signalling proteins in the basal membrane of Src-deficient OCLs. In normal OCLs, the tyrosine kinase inhibitor tyrphostin A9 inhibits actin ring formation, bone resorption and tyrosine phosphorylation of several proteins, including c-Src. Furthermore, tyrphostin A9 induced similar hyperclustering of αvβ3 integrins in osteoclasts as observed in Src−/− OCLs. Taken together, these findings suggest that normal localisation of αvβ3 and recruitment of its downstream effectors to the appropriate compartments of the osteoclast during resorption depend on Src kinase activity.
Cdc28-Clb mitotic kinase negatively regulates bud site assembly in the budding yeast
Authors: Padmashree C. G. , Surana Uttam 
Published: April 2021

ABSTRACT
In the budding yeast Saccharomyces cerevisiae, a prospective mother normally commences the formation of a daughter (the bud) only in the G1 phase of the cell division cycle. This suggests a strict temporal regulation of the processes that initiate the formation of a new bud. Using cortical localization of bud site components Spa2 and Bni1 as an indicator of bud site assembly, we show that cells assemble a bud site following inactivation of the Cdc28-Clb mitotic kinase but prior to START. Interestingly, an untimely inactivation of the mitotic kinase is sufficient to drive cells to assemble a new bud site inappropriately in G2 or M phases. The induction of Cdc28/Clb kinase activity in G1, on the other hand, dramatically reduces a cell’s ability to construct an incipient bud site. Our findings strongly suggest that the Cdc28-Clb kinase plays a critical role in the mechanism that restricts the timing of bud formation to the G1 phase of the cell cycle.
Evidence for separate ND10-binding and homo- oligomerization domains of Sp100
Authors: Negorev Dmitri , Ishov Alexander M. , Maul Gerd G. 
Published: April 2021

ABSTRACT
Nuclear domains called ND10 or PML nuclear bodies consist of an aggregation of several proteins, most notably PML and Sp100. PML is essential in the nucleation and formation of ND10 as well as in the recruitment of other ND10-associated proteins such as Daxx, pRb, BLM and Sp100. In cells induced to overexpress Sp100, ND10 binding of Sp100 was saturable and excess Sp100 formed new aggregation sites devoid of other ND10-asssociated proteins, suggesting that homo-oligomerization is the basis for aggregation. To determine whether Sp100 binds to ND10 through hetero- or oligomerization, Sp100 deletion variants fused with GFP were transfected into cells with and without endogenous Sp100, and the localization of the GFP-labeled fragments was determined relative to ND10. Amino acids 29-152 were sufficient for deposition of the GFP-labeled fragments at ND10 in the absence of endogenous Sp100 (heterologous binding) and for self- aggregation (formation of new Sp100 deposits). None of the shorter fragments was deposited at ND10 or self- aggregated. The 29-152 amino acid fragment and some larger fragments, but not the full-size Sp100, induced elongation of ND10, which at their ends contain only Sp100, probably due to self-aggregation. By fusing a peptide consisting of the p53-binding domain from hMDM2 to the Sp100(29-152) fragment, this self-aggregation could be blocked while retaining the limited ND10 binding capacity, indicating that the Sp100 self-aggregation domain and the ND10 binding domain are separate entities. This fusion peptide was used to demonstrate the potential of ND10 to recruit p53 as a protein not usually present at this site. Such deposited p53 was protected from turnover. The capacity of ND10 to recruit Sp100 may serve primarily to reduce its availability.
Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites
Authors: Nakagawa Masato , Fukata Masaki , Yamaga Masaki , Itoh Naohiro , Kaibuchi Kozo 
Published: April 2021

ABSTRACT
Rac1, a member of the Rho family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of Rac1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged Rac1, we demonstrate that EGFP-Rac1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca2+ chelation. Re-establishment of cell-cell adhesion by restoration of Ca2+ caused EGFP-Rac1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-Rac1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced Rac1 activation by measuring the amounts of GTP-bound Rac1 based on its specific binding to the Cdc42/Rac1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced Rac1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of Rac1, and EGFP-Rac1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. Rac1 activation was also confirmed by measuring the amounts of coimmunoprecipitated Rac1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that Rac1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase.
Movies available on-line: http://www/biologists.com/JCS/movies/jcs2094.html
Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication
Authors: Postberg Jan , Juranek Stefan A. , Feiler Sabine , Kortwig Heike , Jönsson Franziska , Lipps Hans J. 
Published: April 2021

ABSTRACT
Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 108individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.
Nuclear dispositions of subtelomeric and pericentromeric chromosomal domains during meiosis in asynaptic mutants of rye (Secale cereale L.)
Authors: Mikhailova Elena I. , Sosnikhina Svetlana P. , Kirillova Galina A. , Tikholiz Oxana A. , Smirnov Victor G. , Jones R. Neil , Jenkins Glyn 
Published: April 2021

ABSTRACT
The nuclear dispositions of subtelomeric and pericentromeric domains in pollen mother cells (PMCs) were tracked during meiosis in wildtype and two asynaptic mutants of rye (Secale cereale L.) by means of fluorescence in situ hybridization (FISH). Homozygotes for sy1 and sy9 non-allelic mutations form axial elements during leptotene of male meiosis, but fail to form synaptonemal complexes. Consequently, recombination is severely impaired, and high univalency is observed at metaphase I. Simultaneous FISH with pSc200 subtelomeric tandem repeat and CCS1 centromeric sequence revealed that at pre-meiotic interphase the two domains are in a bipolar Rabl orientation in both the PMCs and tapetal cells. At the onset of meiotic prophase, the subtelomeric regions in PMCs of wildtype and sy9 cluster into a typical bouquet conformation. The timing of this event in rye is comparable with that in wheat, and is earlier than that observed in other organisms, such as maize, yeast and mammals. This arrangement is retained until later in leptotene and zygotene when the pericentromeric domains disperse and the subtelomeric clusters fragment. The mutant phenotype of sy9 manifests itself during leptotene to zygotene, when the pericentromeric regions become distinctly more distended than in wildtype, and largely fail to pair during zygotene. This indicates that difference in the nature or timing of chromosome condensation in this region is the cause or consequence of asynapsis. By contrast, sy1 fails to form comparable aggregates of subtelomeric regions at leptotene in only half of the nuclei studied. Instead, two to five aggregates are formed that fail to disperse at later stages of meiotic prophase. In addition, the pericentromeric regions disperse prematurely at leptotene and do not associate in pairs at any subsequent stage. It is supposed that the sy1 mutation could disrupt the nuclear disposition of centromeres and telomeres at the end of pre-meiotic interphase, which could cause, or contribute to, its asynaptic phenotype.
The Drosophila Dpit47 protein is a nuclear Hsp90 co-chaperone that interacts with DNA polymerase α
Authors: Crevel Gilles , Bates Helen , Huikeshoven Hella , Cotterill Sue 
Published: April 2021

Hsp90 is gaining increasing importance as a protein involved in controlling the normal functioning of the cell. To do this it apparently interacts with a battery of co-chaperone proteins that are involved in both substrate recognition and the progression of the Hsp90 catalytic pathway. In this report we have identified the Drosophila Dpit47 protein (DNA polymerase interacting tpr containing protein of 47 kDa) through its interaction with the DNA polymerase α. This protein is a predominantly nuclear protein, which forms a tight and stoichiometric interaction with Hsp90 and shows interaction with Hsp70. It also has substantial homology to other known Hsp90 co-chaperones, e.g. CNS1 and hop1, making it likely that this protein also functions as an Hsp90 co-chaperone.
The interaction with the DNA polymerase α is not related to the special situation in early embryos where there are large amounts of maternal protein stockpiles of the polymerase, as it occurs to the same level in early and late embryos and also in proliferating cell culture. However, it does not occur in quiescent cells, making it likely that the protein is related to proliferation. This is also consistent with Dpit47 expression being higher in proliferating cells. The interaction between the Dpit47 and the polymerase takes place predominantly in the nucleoplasm, and seems to involve several subunits of the polymerase in comparable amounts, making it unlikely that it is solely required for the assembly of the polymerase complex. The polymerase can also be seen to interact with Hsp90, and the interaction between Dpit47 and the polymerase is increased by the specific Hsp90 inhibitor geldanamycin. This suggests that a complex of the Dpit47, Hsp90 and DNA polymerase exists in the cell. The interaction between DNA polymerase α and Dpit47 completely inhibits the activity of the polymerase.
These results suggest that Hsp90 acts as a chaperone for DNA polymerase α and that this interaction is mediated through the novel co-chaperone Dpit47. This provides the first suggestion of a role for chaperones in DNA replication in higher eukaryotes.