bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reorganization of actin cytoskeleton at the growing end of the cleavage furrow of Xenopus egg during cytokinesis
Authors: Noguchi Tatsuhiko , Mabuchi Issei 
Published: April 2021

ABSTRACT
We studied reorganization of actin-myosin cytoskeleton at the growing ends of the cleavage furrow of Xenopus eggs in order to understand how the contractile ring is formed during cytokinesis.
Reorganization of F-actin structures during the furrow formation was demonstrated by rhodamine-phalloidin staining of the cleavage furrow and by time-lapse scanning with laser scanning microscopy of F-actin structures in the cleavage furrow of live eggs to which rhodamine-G-actin had been injected. Actin filaments assemble to form small clusters that we call ‘F-actin patches’ at the growing end of the furrow. In live recordings, we observed emergence and rapid growth of F-actin patches in the furrow region. These patches then align in tandem, elongate and fuse with each other to form short F-actin bundles. The short bundles then form long F-actin bundles that compose the contractile ring.
During the furrow formation, a cortical movement towards the division plane occurs at the growing ends of the furrow, as shown by monitoring wheatgerm agglutinin-conjugated fluorescent beads attached to the egg surface. As a result, wheatgerm agglutinin-binding sites accumulate and form ‘bleb-like’ structures on the surface of the furrow region. The F-actin patch forms and grows underneath this structure. The slope of F-actin accumulation in the interior region of the furrow exceeds that of accumulation of the cortex transported by the cortical movement. In addition, rhodamine-G-actin microinjected at the growing end is immediately incorporated into the F-actin patches. These data, together with the rapid growth of F-actin patches in the live image, suggest that actin polymerization occurs in the contractile ring formation.
Distribution of myosin II in the cleavage furrow was also examined by immunofluorescence microscopy. Myosin II assembles as spots at the growing end underneath the bleb-like structure. It was suggested that myosin is transported and accumulates as spots by way of the cortical movement. F-actin accumulates at the position of the myosin spot a little later as the F-actin patches. The myosin spots and the F-actin patches are then simultaneously reorganized to form the contractile ring bundles
Experimental observations of a nuclear matrix
Authors: Nickerson Jeffrey A. 
Published: April 2021

ABSTRACT
Nuclei are intricately structured, and nuclear metabolism has an elaborate spatial organization. The architecture of the nucleus includes two overlapping and nucleic-acid-containing structures-chromatin and a nuclear matrix. The nuclear matrix is observed by microscopy in live, fixed and extracted cells. Its ultrastructure and composition show it to be, in large part, the ribonucleoprotein (RNP) network first seen in unfractionated cells more than 30 years ago. At that time, the discovery of this RNP structure explained surprising observations that RNA, packaged in proteins, is attached to an intranuclear, non-chromatin structure. Periodic and specific attachments of chromatin fibers to the nuclear matrix create the chromatin loop domains that can be directly observed by microscopy or inferred from biochemical experiments. The ultrastructure of the nuclear matrix is well characterized and consists of a nuclear lamina and an internal nuclear network of subassemblies linked together by highly structured fibers. These complex fibers are built on an underlying scaffolding of branched 10-nm filaments that connect to the nuclear lamina. The structural proteins of the nuclear lamina have been well characterized, but the structural biochemistry of the internal nuclear matrix has received less attention. Many internal matrix proteins have been identified, but far less is known about how these proteins assemble to make the fibers, filaments and other assemblies of the internal nuclear matrix. Correcting this imbalance will require the combined application of biochemistry and electron microscopy. The central problem in trying to define nuclear matrix structure is to identify the proteins that assemble into the 10-nm filaments upon which the interior architecture of the nucleus is constructed. Only by achieving a biochemical characterization of the nuclear matrix will we advance beyond simple microscopic observations of structure to a better understanding of nuclear matrix function, regulation and post-mitotic assembly.
Boursin, a sea urchin bimC kinesin protein, plays a role in anaphase and cytokinesis
Authors: Touïtou Irit , Lhomond Guy , Pruliére Gérard 
Published: April 2021

ABSTRACT
We have isolated and characterized Boursin, a kinesin-related protein of the bimC family, from Paracentrotus lividus sea urchin eggs. Boursin is expressed at high levels in eggs and embryos during early cleavage stages. Boursin was found to be associated with different parts of the mitotic spindle from early prophase to telophase. Expression of a form of the protein predicted to act as a dominant negative mutant caused severe defects in cell division and resulted in the formation of embryos with polyploid and multiastral blastomeres. Immunofluorescence analysis indicated that these defects did not arise from failure in either centrosome separation or bipolar spindle formation. Time-lapse observations showed rather that these perturbations in cell division resulted from abnormal anaphase and failure to complete cytokinesis. These phenotypes differ from the phenotype described following perturbation of the function of bimC family members in other organisms. Our study has thus uncovered roles for a bimC kinesin in late stages of cell division.
Tetraspanins in intercellular adhesion of polarized epithelial cells: spatial and functional relationship to integrins and cadherins
Authors: Yáñez-Mó María , Tejedor Reyes , Rousselle Patricia , Sánchez-Madrid Francisco 
Published: April 2021

ABSTRACT
The subcellular distribution of tetraspanin molecules and their functional relationship with integrins in cell-cell adhesion was studied in detail in different polarized epithelial cell models. CD9, CD81 and CD151 tetraspanins were localized at lateral cell-cell contact sites in a similar distribution to E-cadherin. Interestingly, CD9 was partially localized at the apical microvillae of Madin-Darby canine kidney cells forming multimolecular complexes distinct from those found on the basolateral membrane, suggesting the coexistence of differential tetraspanin webs with different subcellular localization. We found that tetraspanin-associated β1 integrins at cell-to-cell contacts were in a low-affinity conformational state, and that their localization at intercellular contacts was independent of cadherin expression and adhesion. Furthermore, integrin-tetraspanin complexes were functionally relevant in cell-cell adhesion in a cadherin-independent manner, without requiring a conformational change of the integrin moiety. Nevertheless, the integrin α3β1 was ligand-binding competent and this binding did not disrupt association to tetraspanins. Moreover, Chinese hamster ovary cells treated with anti-tetraspanin mAbs or activatory anti-β1 integrin mAbs were able to develop tubule-like structures. Together, these data support tetraspanin association as a new regulatory mechanism of integrin function and suggest a role for tetraspanins-integrin complexes in providing the cell with the spatial cues necessary for their proper polarization.
The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants
Authors: Hood Jennifer K. , Hwang William W. , Silver Pamela A. 
Published: April 2021

ABSTRACT
The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae. Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions. Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs. Wild-type Clb2p is primarily nuclear at all points of the cell. A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and Δyrb2 cells exhibit an apparent Clb2p nuclear export defect. Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the α and β importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators. Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck. These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.
Differential regulation of CENP-A and histone H3 phosphorylation in G2/M
Authors: Zeitlin Samantha G. , Barber Cynthia M. , Allis C. David , Sullivan Kevin E. 
Published: April 2021

ABSTRACT
After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.
Localization of two mammalian cyclin dependent kinases during mammalian meiosis
Authors: Ashley Terry , Walpita Deepika , Rooij Dirk G. de 
Published: April 2021

ABSTRACT
Mammalian meiotic progression, like mitotic cell cycle progression, is regulated by cyclins and cyclin dependent kinases (CDKs). However, the unique requirements of meiosis (homologous synapsis, reciprocal recombination and the dual divisions that segregate first homologues, then sister chromatids) have led to different patterns of CDK expression. Here we show that Cdk4 colocalizes with replication protein A (RPA) on the synaptonemal complexes (SCs) of newly synapsed axes of homologously pairing bivalents, but disappears from these axes by mid-pachynema. The switch from the mitotic pattern of expression occurs during the last two spermatogonial divisions. Cdk2 colocalizes with MLH1, a mismatch repair protein at sites of reciprocal recombination in mid-late pachynema. In addition Cdk2 localizes to the telomeres of chromosomal bivalents throughout meiotic prophase. The mitotic pattern of expression of Cdk2 remains unchanged throughout the spermatogonial divisions, but is altered in meiosis of the spermatocytes.
p120 catenin affects cell motility via modulation of activity of Rho-family GTPases: a link between cell-cell contact formation and regulation of cell locomotion
Authors: Grosheva Inna , Shtutman Michael , Elbaum Michael , Bershadsky Alexander D. 
Published: April 2021

ABSTRACT
The molecular basis for contact inhibition of cell locomotion is still largely unknown. Cadherins, the major receptors mediating cell-cell adhesion, associate in the cytoplasm with armadillo family proteins, including β- and γ-catenin and p120 catenin (p120ctn). E-cadherin-mediated contact formation was shown to inhibit cellular motility. We examine whether p120ctn may have a role in this regulation. We show here that overexpression of p120ctn in fibroblasts and epithelial cells induces pronounced changes in cell shape, motility and adhesion to the extracellular matrix. p120ctn-transfected cells display increased filopodial/lamellipodial activity, decreased contractility and focal adhesion formation, and augmented migratory ability. These effects of p120ctn are mediated by small GTPases of the Rho family. Direct assessment of the activity of these GTPases in cells expressing a 5-fold higher level of p120ctn as compared to non-transfected control cells revealed significant augmentation of Cdc42 and Rac activity. Moreover, co-transfection of p120ctn with dominant-negative Cdc42 and Rac, or constitutively active Rho, suppressed morphological effects of p120ctn. Confocal immunofluorescence visualization of the distribution of endogenous p120ctn in dense cultures showed that formation of cadherin-mediated cell-cell contacts is accompanied by sequestering of p120ctn to the junction regions. In sparse cultures p120ctn is distributed over the cytoplasm. Co-transfection with an excess of E-cadherin leads to sequestration of exogenous p120ctn to cell-cell junctions or to small cadherin-containing vesicles, and abolishes p120ctn effects on cell morphology. Thus, p120ctn may couple the formation and disruption of cadherin-mediated contacts with regulation of cell motility by triggering pathway(s) affecting Rho family GTPases.
The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in early S-phase
Authors: ínský Jan Mal , Koberna Karel , ěk David Stan , šata Martin Ma , Votruba Ivan , ška Ivan Ra 
Published: April 2021

ABSTRACT
Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than in late S-phase and that the intracellular 2′-deoxyribonucleoside 5′-triphosphate pools grow during S-phase. In this study, the effect of the exogenous 2′-deoxyribonucleoside 5′-triphosphate (dNTP) supply on the average replication speed in a synchronised population of human HeLa cells was tested. The speed of replication fork movement was measured on extended DNA fibers labelled with 2′-deoxythymidine analogues 5-chloro-2′-deoxyuridine and 5-iodo-2′-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. In late S-phase, the administration of additional dNTPs has no effect on the speed of replication forks. The availability of 2′-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.
Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells
Authors: Kato Takayuki , Watanabe Naoki , Morishima Yosuke , Fujita Akiko , Ishizaki Toshimasa , Narumiya Shuh 
Published: April 2021

ABSTRACT
mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu434 and Leu455 in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.
Keratinocyte migration requires α2β1 integrin-mediated interaction with the laminin 5 γ2 chain
Authors: Decline Françoise , Rousselle Patricia 
Published: April 2021

ABSTRACT
Keratinocyte migration is an absolute requirement for correct epithelialization during the process of wound healing. This process requires changes in extracellular matrix ligand expression as well as changes in ligand-binding affinity of the corresponding cellular integrins. In this study, we attempt to understand the role of laminin 5 in migration by investigating the integrin-mediated interactions of migrating keratinocytes with their newly synthesized laminin 5. We chose to induce migration of freshly isolated NHK in vitro by exposing them to TGF-β1 which, in addition to promoting epithelial cell migration, is also known to prevent cell proliferation. This important feature allowed the study to be focused on cell migration without interfering with cell proliferation. We confirm that keratinocyte migration on plastic, fibronectin or collagen IV substrates requires endogenous laminin 5 deposition, which is predominantly detected under its unprocessed form. Despite a crucial role for laminin 5 in migration, we show that this process is accompanied by a significant decrease in adhesion to purified laminin 5. Moreover, we provide evidence that the α3β1 integrin interaction with newly synthesized laminin 5 renders the cells more adherent and retards migration. Conversely, we provide evidence that the α2β1 integrin-laminin 5 interaction is absolutely required for keratinocyte migration and that the α2β1 integrin is responsible for cell spreading on laminin 5. Finally, we demonstrate that the α2β1 integrin binding to laminin 5 occurs within the short arm of the γ2 subunit.
Hypoxia in combination with FGF-2 induces tube formation by human microvascular endothelial cells in a fibrin matrix: involvement of at least two signal transduction pathways
Authors: Kroon Marielle E. , Koolwijk Pieter , Van der Vecht Bea , Van Hinsbergh Victor W. M. 
Published: April 2021

ABSTRACT
Hypoxia in combination with a growth factor is a strong inducer of angiogenesis. Among several effects, hypoxia can activate endothelial cells directly, but the mechanism by which it acts is not fully elucidated. In vitro, human microvascular endothelial cells (hMVEC) form capillary-like tubules in fibrin solely after stimulation with a combination of fibroblast growth factor (FGF)-2 or vascular endothelial growth factor (VEGF) and the cytokine tumour necrosis factor (TNF)α. We show in this paper that in hypoxic conditions, FGF-2-stimulated hMVEC form tube-like structures in a fibrin matrix in the absence of TNFα. Hypoxia/FGF-2-stimulated cells express more urokinase-type plasminogen activator (u-PA) receptor than normoxia/FGF-2-stimulated cells and display a slightly higher turnover of u-PA. This small increase in u-PA activation probably cannot fully explain the hypoxia/FGF-2-induced tube formation. Hypoxia activated at least two signal pathways that may contribute to the enhanced angiogenic response. In hypoxia/FGF-2-stimulated hMVEC the transcription factor p65 was activated and translocated to the nucleus, whereas in normoxia/FGF-2-stimulated cells p65 remained inactive. Furthermore, in hypoxic conditions, the amounts of phosphorylated mitogen-activated protein kinases ERK1/2 were increased compared to normoxic conditions. We conclude that hypoxia is able to activate different signal pathways in FGF-2-stimulated human endothelial cells, which may be involved in hypoxia-induced angiogenesis.
Illuminating the secretory pathway: when do we need vesicles?
Authors: Stephens David J. , Pepperkok Rainer 
Published: April 2021

ABSTRACT
Recent studies using GFP-tagged markers and time-lapse microscopy have allowed direct visualisation of membrane traffic in the secretory pathway in living mammalian cells. This work shows that larger membrane structures, 300-500 nm in size, are the vehicles responsible for long distance, microtubule-dependent ER-to-Golgi and trans-Golgi to plasma membrane transport of secretory markers. At least two retrograde transport pathways from the Golgi to the ER exist, both of which are proposed to involve a further class of long, tubular membrane carrier that forms from the Golgi and fuses with the ER. Together, this has challenged established transport models, raising the question of whether larger pleiomorphic structures, rather than small 60-80 nm transport vesicles, mediate long-range transport between the ER and Golgi and between the Golgi and plasma membrane.
Movie available on-line: http://www.biologists.com/JCS/movies/jcs2220.html, Movie 2A, Movie 2AA, Movie 2B, Movie 2BB, Movie 2C