bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of gene expression by microarrays: cell biologist’s gold mine or minefield?
Authors: Schulze Almut , Downward Julian
Published: April 2021

The development of DNA microarrays to study simultaneously the level of mRNA expressed from thousands of genes offers great promise to cell biologists. Microarrays can be used to gain detailed information about transcriptional changes involved in a specific pathway, potentially leading to the identification of novel components of the signalling system. They can also be used to obtain a fingerprint of the transcriptional status of the cell under a given condition, which may be useful for characterising the pathways used in response to novel stimuli. The use of microarrays will generate huge amounts of expression data, contributing to the transformation of biology from a data-poor to a data-rich science. Whether this leads to real advances in the understanding of cell biological problems will depend on the development of methodologies, both in experimental biology and in bioinformatics, that allow meaningful knowledge to be extracted from all this information.
LIF transduces contradictory signals on capillary outgrowth through induction of Stat3 and P41/43MAP kinase
Authors: Paradis Hélène , Gendron Robert L.
Published: April 2021

The signaling pathways regulating blood vessel growth and development are not well understood. In the present report, an in vitro model was used to identify signaling pathways regulating capillary formation in embryonic endothelial cells. Basic fibroblast growth factor (bFGF) plus leukemia inhibitory factor (LIF) optimally stimulate the formation of capillary-like structures of the embryonic endothelial cell line IEM. LIF stimulation of IEM cells leads to activation of the Stat3 as well as the P41/43mitogen-activated protein kinase (P41/43MAPK) cascade, while bFGF does not activate Stat3 but does induce the P41/43MAPK cascade. Inhibition of Stat3 DNA-binding activity by expression of a dominant inhibitory Stat3 mutant increases the capillary outgrowth of the IEM cells induced by LIF. Increased Stat3 activity by overexpression of the wild-type Stat3 greatly reduced capillary outgrowth. In contrast, inhibition of the P41/43MAPK cascade using a MEK-1 inhibitor dramatically inhibits the LIF-induced capillary outgrowth. Moreover, the increased formation of capillary-like structures of the IEM cells mediated by Stat3 inhibition does not overcome the requirement for activation of the P41/43MAPK pathway for capillary outgrowth. Stat3 activity correlates with the LIF-induced expression of the negative feedback regulators of the Janus (JAK) family of tyrosine kinases, SOCS-1 and SOCS-3. These results provide evidence that Stat3 acts as a negative regulator of capillary outgrowth, possibly by increasing SOCS-1 or SOCS-3 expression. The contradictory signals stimulated by LIF could be necessary to control the intensity of the response leading to capillary outgrowth in vivo.
The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner
Authors: Zhao Jian , Tenev Tencho , Martins Luis M. , Downward Julian , Lemoine Nicholas R.
Published: April 2021

Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G2/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G1 phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level.

In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G1 phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein.
How motor proteins influence microtubule polymerization dynamics
Authors: Hunter Andrew W. , Wordeman Linda
Published: April 2021

The interplay between microtubules and microtubule-based motors is fundamental to basic aspects of cellular function, such as the intracellular transport of organelles and alterations in cellular morphology during cell locomotion and division. Motor proteins are unique in that they couple nucleotide hydrolysis to force production that can do work. The force transduction by proteins belonging to the kinesin and dynein superfamilies has been thought only to power movement of these motors along the surface of microtubules; however, a growing body of evidence, both genetic and biochemical, suggests that motors can also directly influence the polymerization dynamics of microtubules. For example, at the vertebrate kinetochore, motors interact directly with microtubule ends and modulate polymerization dynamics to orchestrate chromosome movements during mitosis. Although a role for motors in regulating microtubule length has been established, the mechanisms used by motors to promote microtubule growth or shrinkage are unclear, as is an understanding of why cells might choose motors to control dynamics rather than a variety of non-motor proteins known to affect microtubule stability. Elucidation of the exact mechanisms by which motors alter the exchange of tubulin subunits at microtubule ends in vitro may shed light on how microtubule stability is regulated to produce the array of dynamic behavior seen in cells.
Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose
Authors: Lee Hon Cheung , Aarhus Robert
Published: April 2021

Cells possess multiple Ca2+ stores and their selective mobilization provides the spatial-temporal Ca2+ signals crucial in regulating diverse cellular functions. Except for the inositol trisphosphate (IP3)-sensitive Ca2+ stores, the identities and the mechanisms of how these internal stores are mobilized are largely unknown. In this study, we describe two Ca2+ stores, one of which is regulated by cyclic ADP-ribose (cADPR) and the other by nicotinic acid adenine dinucleotide phosphate (NAADP). We took advantage of the large size of the sea urchin egg and stratified its organelles by centrifugation. Using photolysis to produce either uniform or localized increases of cADPR and NAADP from their respective caged analogs, the two separate stores could be visually identified by Ca2+ imaging and shown to be segregated to the opposite poles of the eggs. The cADPR-pole also contained the IP3-sensitive Ca2+ stores, the egg nucleus and the endoplasmic reticulum (ER); the latter was visualized using Bodipy-thapsigargin. On the other hand, the mitochondria, as visualized by rhodamine 123, were segregated to the opposite pole together with the NAADP-sensitive calcium stores. Fertilization of the stratified eggs elicited a Ca2+ wave starting at the cADPR-pole and propagating toward the NAADP-pole. These results provide the first direct and visual evidence that the NAADP-sensitive Ca2+ stores are novel and distinct from the ER. During fertilization, communicating signals appear to be transmitted from the ER to NAADP-sensitive Ca2+ stores, leading to their activation.
Human Cdc5, a regulator of mitotic entry, can act as a site-specific DNA binding protein
Authors: Lei Xiang-He , Shen Xun , Xu Xiao-Qin , Bernstein Harold S.
Published: April 2021

G2/M progression requires coordinated expression of many gene products, but little is known about the transcriptional regulators involved. We recently identified human Cdc5, a positive regulator of G2/M in mammalian cells. We also demonstrated the presence of a latent activation domain in its carboxyl terminus, suggesting that human Cdc5 regulates G2/M through transcriptional activation. Despite the presence of a DNA binding domain, studies by others have failed to identify a preferential binding site for Cdc5 family members. In addition, Cdc5 recently has been associated with the splicesome in several organisms, suggesting that it may not act through DNA binding. We now report the identification of a 12 bp sequence to which human Cdc5 binds specifically and with high affinity through its amino terminus. We show that this DNA-protein interaction is capable of activating transcription. We also used a selection system in yeast to identify human genomic fragments that interact with human Cdc5. Several of these contained sequences similar to the binding site. We demonstrate that these bind human Cdc5 with similar specificity and affinity. These experiments provide the first evidence that Cdc5 family members can act as site-specific DNA binding proteins, and that human Cdc5 may interact with specific, low abundance sequences in the human genome. This raises the possibility that Cdc5 proteins may participate in more than one process necessary for regulated cell division.
Yeast myosin light chain, Mlc1p, interacts with both IQGAP and Class II myosin to effect cytokinesis
Authors: Boyne James R. , Yosuf Hirzun Mohd , Bieganowski Pawel , Brenner Charles , Price Clive
Published: April 2021

MLC1 (myosin light chain) acts as a dosage suppressor of a temperature sensitive mutation in the gene encoding the S. cerevisiae IQGAP protein. Both proteins localize to the bud neck in mitosis although Mlc1p localisation precedes Iqg1p. Mlc1p is also found at the incipient bud site in G1 and the growing bud tip during S and G2 phases of the cell cycle. A dominant negative GST-Mlc1p fusion protein specifically blocks cytokinesis and prevents Iqg1p localisation to the bud neck, as does depletion of Mlc1p. These data support a direct interaction between the two proteins and immunoprecipitation experiments confirm this prediction. Mlc1p is also shown to interact with the class II conventional myosin (Myo1p). All three proteins form a complex, however, the interaction between Mlc1p and Iqg1p can be separated from the Mlc1p/Myo1p interaction. Mlc1p localisation and maintenance at the bud neck is independent of actin, Myo1p and Iqg1p. It is proposed that Mlc1p therefore functions to recruit Iqg1p and in turn actin to the actomyosin ring and that it is also required for Myo1p function during ring contraction.
Identification of an α-tubulin mutant of fission yeast from γ-tubulin-interacting protein screening: genetic evidence for α-/γ-tubulin interaction
Authors: Takeoka Aya , Shimizu Miyuki , Horio Tetsuya
Published: April 2021

γ-Tubulin has been determined to be a central element of microtubule nucleation and, thus, indispensable for cellular organization of the microtubule. Utilizing the fact that human γ-tubulin can function in the fission yeast Schizosaccharomyces pombe, we have generated a unique mutant screening procedure which can specifically select mutants of genes encoding γ-tubulin-interacting proteins. One of the isolated mutants, cs76, turned out to carry a mutation in the α1-tubulin gene (nda2+). This result suggests a direct interaction between the α- and γ-tubulins. We located the mutation site in the nda2 gene and characterized the mutant phenotype. Our results demonstrate the importance of the α-/γ-tubulin interaction in microtubule nucleation and should complement previous knowledge.
A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii
Authors: Ayad-Durieux Yasmina , Knechtle Philipp , Goff Stephen , Dietrich Fred , Philippsen Peter
Published: April 2021

Filamentous fungi grow by hyphal extension, which is an extreme example of polarized growth. In contrast to yeast species, where polarized growth of the tip of an emerging bud is temporally limited, filamentous fungi exhibit constitutive polarized growth of the hyphal tip. In many fungi, including Ashbya gossypii, polarized growth is reinforced by a process called hyphal maturation. Hyphal maturation refers to the developmental switch from slow-growing hyphae of young mycelium to fast-growing hyphae of mature mycelium. This process is essential for efficient expansion of mycelium. We report for the first time on the identification and characterization of a fungal gene important for hyphal maturation. This novel A. gossypii gene encodes a presumptive PAK (p21-activated kinase)-like kinase. Its closest homolog is the S. cerevisiae Cla4 protein kinase; the A. gossypii protein is therefore called AgCla4p. Agcla4 deletion strains are no longer able to perform the developmental switch from young to mature hyphae, and GFP (green fluorescent protein)-tagged AgCla4p localizes with much higher frequency in mature hyphal tips than in young hyphal tips. Both results support the importance of AgCla4p in hyphal maturation. AgCla4p is also required for septation, indicated by the inability of Agcla4 deletion strains to properly form actin rings and chitin rings. Despite the requirement of AgCla4p for the development of fast-growing hyphae, AgCla4p is not necessary for actin polarization per se, because tips enriched in cortical patches and hyphae with a fully developed network of actin cables can be seen in Agcla4 deletion strains. The possibility that AgCla4p may be involved in regulatory mechanisms that control the dynamics of the actin patches and/or actin cables is discussed.
Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells
Authors: Weise Frank , Stierhof York-Dieter , Kühn Christina , Wiese Martin , Overath Peter
Published: April 2021

The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densly packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75% of the two GPI-anchored proteins but only 13% of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% and the tubular cluster/translucent vesicle complex 3.6% of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.
Interactions between FcεRI and lipid raft components are regulated by the actin cytoskeleton
Authors: Holowka David , Sheets Erin D. , Baird Barbara
Published: April 2021

Previous studies showed that crosslinking of IgE-FcεRI complexes on RBL-2H3 mast cells causes their association with isolated detergent-resistant membranes, also known as lipid rafts, in a cholesterol-dependent process that precedes initiation of signaling by these receptors. To investigate these interactions on intact cells, we examined the co-redistribution of raft components with crosslinked IgE-FcεRI using confocal microscopy. After several hours of crosslinking at 4°C, the glycosylphosphatidylinositol-linked protein Thy-1 and the Src-family tyrosine kinase Lyn co-redistribute with IgE-FcεRI in large patches at the plasma membrane. Under these conditions, F-actin also undergoes dramatic co-segregation with FcεRI and raft components but is dispersed following a brief warm-up to 37°C. When crosslinking of IgE-FcεRI is initiated at higher temperatures, co-redistribution of raft components with patched FcεRI is not readily detected unless stimulated F-actin polymerization is inhibited by cytochalasin D. In parallel, cytochalasin D converts transient antigen-stimulated tyrosine phosphorylation to a more sustained response. Sucrose gradient analysis of lysed cells reveals that crosslinked IgE-FcεRI remains associated with lipid rafts throughout the time course of the transient phosphorylation response but undergoes a time-dependent shift to higher density that is prevented by cytochalasin D. Our results indicate that interactions between Lyn and crosslinked IgE-FcεRI are regulated by stimulated F-actin polymerization, and this is best explained by a segregation of anchored raft components from more mobile ones.
Evidence for the coincident initiation of homolog pairing and synapsis during the telomere-clustering (bouquet) stage of meiotic prophase
Authors: Bass Hank W. , Riera-Lizarazu Oscar , Ananiev Evgueni V. , Bordoli Stefano J. , Rines Howard W. , Phillips Ronald L. , Sedat John W. , Agard David A. , Cande W. Zacheus
Published: April 2021

To improve knowledge of the prerequisites for meiotic chromosome segregation in higher eukaryotes, we analyzed the spatial distribution of a pair of homologs before and during early meiotic prophase. Three-dimensional images of fluorescence in situ hybridization (FISH) were used to localize a single pair of homologs in diploid nuclei of a chromosome-addition line of oat, oat-maize9b. The system provided a robust assay for pairing based on cytological colocalization of FISH signals. Using a triple labeling scheme for simultaneous imaging of chromatin, telomeres and the homolog pair, we determined the timing of pairing in relation to the onset of three sequential hallmarks of early meiotic prophase: chromatin condensation (the leptotene stage), meiotic telomere clustering (the bouquet stage) and the initiation of synapsis (the zygotene stage). We found that the two homologs were mostly unpaired up through middle leptotene, at which point their spherical cloud-like domains began to transform into elongated and stretched-out domains. At late leptotene, the homologs had completely reorganized into long extended fibers, and the beginning of the bouquet stage was conspicuously marked by the de novo clustering of telomeres at the nuclear periphery. The homologs paired and synapsed during the bouquet stage, consistent with the timing of pairing observed for several oat 5S rDNA loci. In summary, results from analysis of more than 100 intact nuclei lead us to conclude that pairing and synapsis of homologous chromosomes are largely coincident processes, ruling out a role for premeiotic pairing in this system. These findings suggest that the genome-wide remodeling of chromatin and telomere-mediated nuclear reorganization are prerequisite steps to the DNA sequence-based homology-search process in higher eukaryotes.
A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints
Authors: Griffiths Dominic , Uchiyama Masashi , Nurse Paul , Wang Teresa S.-F.
Published: April 2021

To further dissect the genetic differences between the checkpoint pathway following S-phase cdcarrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase ε mutant, cdc20-M10. One such checkpoint mutant, rad17-d14, was found to display the cutphenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1Δ mutant. Unlike chk1Δ, rad17-d14was not sensitive to UV irradiation. Interestingly, the ionising radiation sensitivity of rad17-d14was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G2phase. Biochemical analysis attributes the checkpoint defects of rad17-d14to the failure to phosphorylate the checkpoint effector Chk1p. To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed. Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase. This interaction occurred irrespective of the arrest with cdc20-M10and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.
Formation of hemidesmosome-like structures in the absence of ligand binding by the α6β4 integrin requires binding of HD1/plectin to the cytoplasmic domain of the β4 integrin subunit
Authors: Nievers Mirjam G. , Kuikman Ingrid , Geerts Dirk , Leigh Irene M. , Sonnenberg Arnoud
Published: April 2021

Hemidesmosomes are adhesion structures that mediate anchorage of epithelial cells to the underlying basement membrane. We have previously shown that the α6β4 integrin can induce the assembly of these multi-protein structures independent of binding to its ligand laminin-5 (ligand-independent formation of hemidesmosomes). Our results suggested a role for HD1/plectin, which binds to the cytoplasmic domain of the β4 integrin subunit, in controlling the clustering of hemidesmosomal components at the basal side of the cell. Using keratinocytes derived from patients lacking HD1/plectin, we now show that ligand-independent formation of hemidesmosomal clusters indeed requires HD1/plectin, in contrast to the ligand-dependent assembly of hemidesmosomes. No clustering of the α6β4 integrin, or of the bullous pemphigoid antigens BP180 and BP230, was seen when HD1/plectin-deficient keratinocytes were plated on fibronectin or type IV collagen. In β4-deficient keratinocytes, expression of an interleukin 2 receptor (IL2R) transmembrane chimera containing the β4 cytoplasmic tail with the mutation R1281W, which abrogates HD1/plectin binding, resulted in a diffuse distribution of the chimeric receptor. In contrast, a β4R1281Wmutant that can associate with α6 and bind ligand, was found to be directed to the basal surface of the cells, at sites where laminin-5 was deposited. In addition, this mutant induced clustering of BP180 and BP230 at these sites. Together, these results show that the formation of hemidesmosomes requires binding of either ligand or HD1/plectin to the β4 integrin subunit. Intriguingly, we found that IL2R/β4 chimeras become localized in pre-existing hemidesmosomes of HD1/plectin-deficient keratinocytes, and that this localization requires a domain in the β4 cytoplasmic tail that is also required for HD1/plectin binding (residues 1115-1356). Because this part of β4 lacks the BP180 binding site, and since we show in this study that it is unable to interact with the same part on another β4 molecule, we suggest that the chimera becomes incorporated into hemidesmosomes of HD1/ plectin-deficient keratinocytes by interacting with an as yet unidentified hemidesmosomal component.
p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton
Authors: Humbert Sandrine , Dhavan Rani , Tsai Li-Huei
Published: April 2021

Cyclin-dependent kinase 5 (cdk5) is a small serine/ threonine kinase that displays close sequence homology to the mitotically active cyclin-dependent kinases. Cdk5 has been shown to play an essential role in the development of the nervous system, including neuronal migration and neurite outgrowth. Cdk5 activation requires the presence of a regulatory activator such as p35. cdk5 −/− mice have much more extensive defects in the development of the nervous system than p35 −/− mice, leading to the speculation that other regulatory activators of cdk5 exist. Indeed, p39 is a p35 related protein isolated by sequence homology to p35. We show here that p39 associates with cdk5 in brain lysates, and that this complex is active in phosphorylation of histone H1. By extensive characterization of p39 subcellular localization in different cell types, we demonstrate the presence of p39 in lamellipodial and fillopodial structures of cells and in growth cones of neurons. We show that p39 colocalizes with actin, and cofractionates with the detergent insoluble cytoskeleton from brain. Further, p39 coimmunoprecipitates with actin in brain lysates. Finally, disruption of the actin cytoskeleton alters p39 subcellular localization as well as kinase activity of the p39/cdk5 complex. Therefore, our results reveal the existence of the p39/cdk5 complex in vivo and suggest that it might play a role in regulating actin cytoskeletal dynamics in cells.
Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly
Authors: Troxell Megan L. , Gopalakrishnan Shobha , McCormack Joanne , Poteat Bradley A. , Pennington Janice , Garringer Sean M. , Schneeberger Eveline E. , Nelson W. James , Marrs James A.
Published: April 2021

Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.
Politics in science: do you have your own John Wayne riding shotgun for you?
Authors: A Caveman
Published: April 2021

A naive view of a scientific career is that success and promotion rest solely on the ability of an individual to teach, contribute to scientific knowledge and perform academic service (this was my view in the beginning)â€¦
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

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

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

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

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

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

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

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

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

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.

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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

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

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.