bluuebunny/crossref_metadata_2025 huggingface dataset:
mph1, a member of the Mps1-like family of dual specificity protein kinases, is required for the spindle checkpoint in S. pombe
Authors: He Xiangwei , Jones Michele H. , Winey Mark , Sazer Shelley
Published: April 2021

The spindle assembly checkpoint pathway is not essential for normal mitosis but ensures accurate nuclear division by blocking the metaphase to anaphase transition in response to a defective spindle. Here, we report the isolation of a new spindle checkpoint gene, mphl (Mps1p-like pombe homolog), in the fission yeast Schizosaccharomyces pombe, that is required for checkpoint activation in response to spindle defects. mphl functions upstream of mad2, a previously characterized component of the spindle checkpoint. Overexpression of mphl, like overexpression of mad2, mimics activation of the checkpoint and imposes a metaphase arrest. mph1 protein shares sequence similarity with Mps1p, a dual specificity kinase that functions in the spindle checkpoint of the budding yeast Saccharomyces cerevisiae. Complementation analysis demonstrates that mph1 and Mps1p are functionally related. They differ in that Mps1p, but not mph1, has an additional essential role in spindle pole body duplication. We propose that mphl is the MPS1 equivalent in the spindle checkpoint pathway but not in the SPB duplication pathway. Overexpression of mad2 does not require mphl to impose a metaphase arrest, which indicates a mechanism of spindle checkpoint activation other than mph1/Mps1p kinase-dependent phosphorylation. In the same screen which led to the isolation of mad2 and mphl, we also isolated dphl, a cDNA that encodes a protein 46% identical to an S. cerevisiae SPB duplication protein, Dsk2p. Our initial characterization indicates that S.p. dphl and S.c. DSK2 are functionally similar. Together these results suggest that the budding and fission yeasts share common elements for SPB duplication, despite differences in SPB structure and the timing of SPB duplication relative to mitotic entry.
Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein
Authors: Hofmann Martin , Fieber Christina , Assmann Volker , Göttlicher Martin , Sleeman Jonathan , Plug Ria , Howells Norma , Stein Oliver von , Ponta Helmut , Herrlich Peter
Published: April 2021

The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.
Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions
Authors: Constantin Bruno , Meerschaert Kris , Vandekerckhove Joël , Gettemans Jan
Published: April 2021

Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actinfragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actinfragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.
Partial denaturation of small chromatin fragments: direct evidence for the radial distribution of nucleosomes in folded chromatin fibers
Authors: Bermúdez Antonio , Bartolomé Salvador , Daban Joan-Ramon
Published: April 2021

To examine the internal structure of chromatin fibers, we have developed procedures for partial denaturation of small chromatin fragments (8-30 nucleosomes) from chicken erythrocytes. Electron micrographs of samples prepared under conditions that cause nucleosome dissociation show rods and loops projecting from short compact fibers fixed by glutaraldehyde in 1.7 mM Mg2+. According to previous studies in our laboratory, these images correspond to the top view of partially denatured fibers. Our results indicate that rods and loops consist of extended duplex DNA of different lengths. DNA in loops is nicked, as demonstrated by experiments performed in the presence of high concentrations of ethidium bromide. Length measurements indicate that the radial projections of DNA are produced by unfolding of nucleosomal units. Loops are formed by DNA from denatured nucleosomes in internal positions of the fiber; DNA from denatured nucleosomes in terminal positions form rods. Our micrographs show clearly a radial distribution of DNA loops and rods projecting from fibers. Rods are orthogonal to the surface of the chromatin fragments. Considering that the high ionic strength used in this study (0.8-2.0 M NaCl) neutralizes the electrostatic repulsions between rods and fiber, this observation suggests that rods are extensions of nucleosomes radially organized inside the fiber. The position of the entry points of DNA loops into the fiber could be influenced by constraint on loops, but our results showing that the arc that separates these points in dinucleosome loops is relatively short suggest that consecutive nucleosomes are relatively close to each other in the folded fiber.
The S. cerevisiae CLU1 and D. discoideum cluA genes are functional homologues that influence mitochondrial morphology and distribution
Authors: Fields Stephen D. , Conrad Michael N. , Clarke Margaret
Published: April 2021

The cluA gene, encoding a novel 150 kDa protein, was recently characterized in Dictyostelium discoideum; disruption of cluA impaired cytokinesis and caused mitochondria to cluster at the cell center. The genome of Saccharomyces cerevisiae contains an open reading frame (CLU1) that encodes a protein that is 27% identical, 50% similar, to this Dictyostelium protein. Deletion of CLU1 from S. cerevisiae did not affect cell viability, growth properties, sporulation efficiency, or frequency of occurrence of cells lacking functional mitochondria. However, in clu1▵cells the mitochondrial reticulum, which is normally highly branched, was condensed to one side of the cell. Transformation of cluA-Dictyostelium mutants with the yeast CLU1 gene yielded amoebae that divided normally and had dispersed mitochondria. The mitochondria in cluA-Dictyostelium cells complemented with CLU1 were not as widely scattered as in cluA+Dictyostelium cells, but formed loose clusters throughout the cytoplasm. These results indicate that the products of the CLU1 and cluA genes, in spite of their limited homology, are functional homologues.
Identification of connexin43 as a functional target for Wnt signalling
Authors: Heyden Marcel A. G. van der , Rook Martin B. , Hermans Monique M. P. , Rijksen Gert , Boonstra Johannes , Defize Libert H. K. , Destrée Olivier H. J.
Published: April 2021

Wnt mediated signal transduction is considered to regulate activity of target genes. In Xenopus embryos, ectopic Wnt1 and Wnt8 expression induces gap-junctional communication. During murine brain formation, Wnt1 and the gap-junctional protein connexin43 (Cx43) are co-expressed at the mid/hindbrain border, while interference with Wnt1 or Cx43 expression during embryogenesis leads to severe brain defects in the mid/hindbrain region. In PC12 cells, Wnt1 expression leads to an apparent increase in cell-cell adhesion. We investigated the effects of Wnt1 overexpression on gap-junctional communication in PC12 cells. Wnt1 expressing clones displayed an increased electrical and chemical coupling. This coincides with an increased expression of Cx43 mRNA and protein, while other connexins, Cx26, Cx32, Cx37, Cx40 and Cx45, were not up-regulated. Also, induction of Wnt1 expression in a mammary epithelial cell line leads to an increase in gap-junctional communication and Cx43 protein expression. In transient transactivation assays in P19 EC cells we found that Wnt1 and Li+, an ion that mimics Wnt signalling, increased transcription from the rat Cx43 promoter, potentially via TCF/LEF binding elements, in a pathway separate from cAMP-induced Cx43 transactivation. The results demonstrate that Cx43 acts as a functional target of Wnt1 signalling, and Cx43 expression can be regulated by Wnt1 at the transcriptional level. Our data suggest that Wnt1-induced cell fate determination is likely to involve regulation of gap-junctional communication.
The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs
Authors: Abrieu Ariane , Brassac Thierry , Galas Simon , Fisher Daniel , Labbé Jean-Claude , Dorée Marcel
Published: April 2021

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.
Compressive force promotes Sox9, type II collagen and aggrecan and inhibits IL-1β expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells
Authors: Takahashi Ichiro , Nuckolls Glen H. , Takahashi Katsu , Tanaka Osamu , Semba Ichiro , Dashner Ralph , Shum Lillian , Slavkin Harold C.
Published: April 2021

The initial modeling and subsequent development of the skeleton is controlled by complex gene-environment interactions. Biomechanical forces may be one of the major epigenetic factors that determine the form and differentiation of skeletal tissues. In order to test the hypothesis that static compressive forces are transduced into molecular signals during early chondrogenesis, we have developed a unique three-dimensional collagen gel cell culture system which is permissive for the proliferation and differentiation of chondrocytes. Mouse embryonic day 10 (E10) limb buds were microdissected and dissociated into cells which were then cultured within a collagen gel matrix and maintained for up to 10 days. Static compressive forces were exerted onto these cultures. The time course for expression pattern and level for cartilage specific markers, type II collagen and aggrecan, and regulators of chondrogenesis, Sox9 and IL-1β, were analyzed and compared with non-compressed control cultures. Under compressive conditions, histological evaluation showed an apparent acceleration in the rate and extent of chondrogenesis. Quantitatively, there was a significant 2- to 3-fold increase in type II collagen and aggrecan expression beginning at day 5 of culture and the difference was maintained through 10 days of cultures. Compressive force also causes an elevated level of Sox9, a transcriptional activator of type II collagen. In contrast, the expression and accumulation of IL-1β, a transcriptional repressor of type II collagen was down-regulated. We conclude that static compressive forces promote chondrogenesis in embryonic limb bud mesenchyme, and propose that the signal transduction from a biomechanical stimuli can be mediated by a combination of positive and negative effectors of cartilage specific extracellular matrix macromolecules.
XMAP230 is required for the assembly and organization of acetylated microtubules and spindles in Xenopus oocytes and eggs
Authors: Cha Byeong-Jik , Error Brett , Gard David L.
Published: April 2021

We used affinity-purified polyclonal antibodies to characterize the distribution and function of XMAP230, a heat-stable microtubule-associated protein isolated from Xenopus eggs, during oogenesis. Immunoblots revealed that XMAP230 was present throughout oogenesis and early development, but was most abundant in late stage oocytes, eggs, and early embryos. Immunofluorescence microscopy revealed that XMAP230 was associated with microtubules in oogonia, post-mitotic stage 0 oocytes, early stage I oocytes, and during stage IV-VI of oogenesis. However, staining of microtubules by anti-XMAP230 was not detectable during late stage I through stage III. In stage VI oocytes, anti-XMAP230 stained a large subset of microtubules that were also stained with monoclonal antibodies specific for acetylated α-tubulin. During oocyte maturation, XMAP230 was associated with the transient microtubule array that serves as the precursor of the first meiotic spindle, as well as both first and second meiotic spindles. The extensive array of cytoplasmic microtubules present throughout maturation was not detectably stained by anti-XMAP230. Microinjection of anti-XMAP230 locally disrupted the organization and acetylation of microtubules in stage VI oocytes, and reduced the reacetylation of microtubules during recovery from cold-induced microtubule disassembly. Subsequent maturation of oocytes injected with anti-XMAP230 resulted in defects in the assembly of the transient microtubules array and first meiotic spindle. These observations suggest that XMAP230 is required for the stabilization and organization of cytoplasmic and spindle microtubules in Xenopus oocytes and eggs.
Centrosomes and microtubule organisation during Drosophila development
Authors: González Cayetano , Tavosanis Gaia , Mollinari Cristiana
Published: April 2021

Are the microtubule-organising centers of the different cell types of a metazoan interchangeable? If not, what are the differences between them? Do they play any role in the differentiation processes to which these cells are subjected? Nearly one hundred years of centrosome research has established the essential role of this organelle as the main microtubule-organising center of animal cells. But only now are we starting to unveil the answers to the challenging questions which are raised when the centrosome is studied within the context of a pluricellular organism. In this review we present some of the many examples which illustrate how centrosomes and microtubule organisation changes through development in Drosophila and discuss some of its implications.
Assembly of nuclear pore complexes and annulate lamellae promotes normal pronuclear development in fertilized mammalian oocytes
Authors: Sutovsky Peter , Simerly Calvin , Hewitson Laura , Schatten Gerald
Published: April 2021

In addition to functional nuclear pore complexes engaged in nucleo-cytoplasmic transport, the cytoplasmic stacks of pore complexes, called annulate lamellae, exist in numerous cell types. Although both annulate lamellae and nuclear pore complexes are present in fertilized mammalian oocytes, their relative roles in the process of fertilization and preimplantation development are not known. Using epifluorescence and electron microscopy, we explored their fate during bovine fertilization. The assembly of annulate lamellae in bovine oocytes was triggered by sperm-oocyte binding and continued concomitantly with the incorporation of the nuclear pores in the nuclear envelopes of the developing male and female pronuclei. This process was also induced by the parthenogenetic activation of metaphase-II-arrested oocytes. Depletion of Ca2+, previously implicated in oocyte activation and in the insertion of pore complexes into the nuclear envelope, prevented the formation of nuclear pore complexes, but not the assembly of annulate lamellae in oocyte cytoplasm. Injection of the nuclear pore antagonist, wheat germ agglutinin, into the cytoplasm of mature oocytes that were subsequently fertilized caused the arrest of pronuclear development, indicating the requirement of nuclear pore complexes for normal pronuclear development. Treatment of the fertilized oocytes with the microtubule inhibitor, nocodazole, prevented gathering of annulate lamellae around the developing pronuclei, insertion of nuclear pores into their nuclear envelopes, and further pronuclear development. The formation of the male pronuclei was reconstituted in Xenopus egg extracts and reflected the behavior of nuclear pores during natural fertilization. These data suggest that nuclear pore complexes are required for normal pronuclear development from its beginning up until pronuclear apposition. Annulate lamellae may be involved in the turnover of nuclear pore complexes during fertilization, which is in turn facilitated by the reorganization of oocyte microtubules and influx of Ca2+ into oocyte cytoplasm.
Localisation of Nramp1 in macrophages: Modulation with activation and infection
Authors: Searle Susan , Bright Nicholas A. , Roach Tamara I. A. , Atkinson Peter G. P. , Barton C. Howard , Meloen Robert H. , Blackwell Jenefer M.
Published: April 2021

The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electrondense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-γ and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3-to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.
Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity approach
Authors: David Violaine , Gouin Edith , Troys Marleen Van , Grogan Ann , Segal Anthony W. , Ampe Christophe , Cossart Pascale
Published: April 2021

Actin assembly is involved in cell motility and intracellular movement of Listeria monocytogenes. Induction of Listeria actin tails is mediated by the surface protein ActA. The N-terminal domain of ActA is sufficient for this function. Cell components known to play a role in the actin-based motility of Listeria are VASP (vasodilatator-stimulated phosphoprotein), the multiprotein Arp2/3 complex and cofilin. VASP interacts with the central domain of ActA. Proteins interacting with the N-terminal domain of ActA have not been identified. To identify novel host cell components of ActA-induced actin tails, we used bovine brain extracts and an affinity approach with Listeria as matrix. Several known components of Listeria tails were isolated including VASP, Arp3 and cofilin. Cofilin was identified by peptide sequencing, and cofilin recruitment and Listeria tail length were found to be pH-dependent, in agreement with its recently reported role in enhancing actin filament turnover. In addition, three proteins not previously known to be associated with Listeria tails, coronin, Rac and capZ, were identified in our affinity approach. In infected cells, the localization of the identified proteins was studied by immunofluorescence. Our findings suggest that these latter proteins, which are known to play critical roles in cellular actin rearrangements, may also be involved in the dynamics of Listeria-induced actin assembly.
The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes
Authors: Logarinho Elsa , Sunkel Claudio E.
Published: April 2021

The MPM2 antibody is a valuable tool for studying the regulation of mitotic events since it specifically recognises a subset of mitosis-specific phosphoproteins. Some MPM2 epitopes have been shown to be phosphorylated by p34cdc2. However, recent results suggest that the newly emerging family of polo-like kinases (Plks) may also act as MPM2 kinases. In this study, we present evidence suggesting that the Drosophila POLO protein is required for the phosphorylation of MPM2 reactive epitopes. POLO displays a dynamic localisation pattern during mitosis, which parallels that of the MPM2 phosphoepitopes, since it is found in the centrosome and centromere from early prophase until late anaphase, the microtubule-overlapping region during anaphase, and the region on either side of the midbody during telophase. Centromere localisation is not dependent upon microtubules since it is retained in colchicine-arrested cells and is present in isolated chromosomes. Furthermore, the level of MPM2 immunoreactivity is directly correlated to the severity of the polo mutant alleles. In cells carrying a hypomorphic allele, the centrosomes of abnormal cells are small and fail to efficiently recruit MPM2 epitopes. In neuroblasts homozygous for a severe loss-of-function allele, the mitotic index is low and the MPM2 labelling is severely reduced or absent. Finally, rephosphorylation of MPM2 epitopes in detergent-extracted Schneider cells requires either POLO stably bound to the cytoskeletons or POLO present in soluble extracts. These results suggest that POLO is required for the phosphorylation of MPM2 epitopes in Drosophila, at the centrosomes, centromeres and the mitotic spindle, and thus might be involved in co-ordinating the mitotic changes of cellular architecture with the activity of the maturation promoting factor.
The actin-myosin cytoskeleton mediates reversible agonist-induced membrane blebbing
Authors: Torgerson Rochelle R. , McNiven Mark A.
Published: April 2021

Suprastimulation of pancreatic acinar cells with specific agonists inhibits zymogen secretion and induces the formation of large basolateral blebs. Currently the molecular mechanisms that mediate this dramatic morphologic response are undefined. Further, it is unclear if blebbing represents a terminal or reversible event. Using computer-enhanced video microscopy of living acini we have found that these large blebs form rapidly (within 2-3 minutes) and exhibit ameboid undulations. They are induced by small increases in agonist concentration and require an energy-dependent phosphorylation event. Remarkably, the blebs are rapidly absorbed when agonist levels are reduced, indicating that blebbing is a reversible response to a physiological stimulus, not a terminal event. Morphological methods show that these dramatic changes in cell shape are accompanied by a marked reorganization of actin and myosin II at the basolateral domain. During 30 minutes of suprastimulation, both basolateral actin and myosin II gradually increase to form a ring centered at the necks of the blebs. Immunocytochemical and biochemical studies with a phospho-specific antibody to the myosin regulatory light chain reveal an activation of myosin II in suprastimulated acini that is completely absent in resting cells. Studies using cytoskeletal antagonistic drugs indicate that bleb formation and motility require actin remodeling concomitant with an activation of myosin II. This aberrant activation and reorganization of the actin-myosin cytoskeleton is likely to have detrimental effects on acinar cell function. Additionally, this mechanism of bleb formation may be conserved among other forms of physiological blebbing events.
Common and variant properties of intermediate filament proteins from lower chordates and vertebrates; two proteins from the tunicate Styela and the identification of a type III homologue
Authors: Riemer Dieter , Weber Klaus
Published: April 2021

The chordates combine the vertebrates and the invertebrate phyla of the cephalo- and urochordates (tunicates). Two cytoplasmic intermediate filament (IF) proteins of the urochordate Styela plicata are characterized by cDNA cloning, gene organization, tissue specific expression patterns in the adult animal and the self assembly properties of the recombinant proteins. In line with metazoan phylogeny St-A and St-B have the short length version of the coil 1b domain found in all vertebrate and cephalochordate IF proteins while protostomic IF proteins have the longer length version with an extra 42 residues. St-A is the first IF protein from a lower chordate which can be unambiguously related to a particular vertebrate IF subfamily. St-A shares 46% sequence identity with desmin, displays the N-terminal motif necessary for filament assembly of type III proteins and forms normal homopolymeric 10 nm filaments in vitro. St-A but not St-B is present in smooth muscle cells of the body wall musculature. St-A and St-B are found as separate networks in some interior epithelia. St-B shares 30 to 35% identity with keratin 8, St-A and desmin and does not form IF under in vitro assembly conditions. Its relation to a particular vertebrate IF type or to the eight currently known IF proteins from the cephalochordate Branchiostoma remains unresolved. The striking relation between St-A and desmin predicts that the common progenitor of the urochordate (tunicate) and the cephalochordate/vertebrate lineages already possessed a type III homologue. Unlike in vertebrates intron patterns cannot be used to classify the tunicate IF genes. Although St-A is a type III homologue its gene shows an intron position which in vertebrates is restricted to keratin type II genes.
Transforming growth factor-β dynamically regulates vascular smooth muscle differentiation in vivo
Authors: Grainger David J. , Metcalfe James C. , Grace Andrew A. , Mosedale David E.
Published: April 2021

Variations in the levels of smooth muscle-specific isoforms of contractile proteins have been reported to occur in many different vascular diseases. However, although much work has been done in vitro to investigate the regulation of smooth muscle cell differentiation, the molecular mechanisms which regulate the differentiation of vascular smooth muscle tissue in vivo are unknown. Using quantitative immunofluorescence, we show that in rat arteries levels of smooth muscle differentiation markers correlate with the levels of the cytokine TGF-β. In young mice with one allele of the TGF-β1 gene deleted, the levels of both TGF-β1 and smooth muscle differentiation markers are reduced compared to wild-type controls. This regulation of smooth muscle differentiation by TGF-β?during post-natal development also occurs dynamically in the adult animal. Following various pharmacological or surgical interventions, including treatment of mice with tamoxifen and balloon injury of rat carotid arteries, there is a strong correlation between the changes in the levels of TGF-β and changes in the levels of smooth muscle differentiation markers (r=0.9, P<0.0001 for n=26 experiments). We conclude that TGF-β dynamically regulates smooth muscle differentiation in rodent arteries in vivo.
Regulation of mammalian replication origin usage in Xenopus egg extract
Authors: Dimitrova Daniela S. , Gilbert David M.
Published: April 2021

Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.
Genes that cause aberrant cell morphology by overexpression in fission yeast: a role of a small GTP-binding protein Rho2 in cell morphogenesis
Authors: Hirata Dai , Nakano Kentaro , Fukui Mikiko , Takenaka Hiroshi , Miyakawa Tokichi , Mabuchi Issei
Published: April 2021

To identify the genes involved in cell morphogenesis in Schizosaccharomyces pombe, we screened for the genes that cause aberrant cell morphology by overexpression. The isolated genes were classified on the basis of morphology conferred. One of the genes causing a rounded morphology was identified as the rho2+ gene encoding a small GTP-binding protein. The overexpression of rho2+ resulted in a randomized distribution of cortical F-actin and formation of a thick cell wall. Analyses using cdc mutants suggested that the overexpression of rho2+ prevents the establishment of growth polarity in G1. The rho2+ gene was not essential, but among cells deleted for rho2+, those with an irregular shape were observed. The disruptant also showed a defect in cell wall integrity. An HA-Rho2 expressed in the cell was suggested to be present as a membrane-bound form by a cell fractionation experiment. A GFP-Rho2 was localized at the growing end(s) of the cell and the septation site. The localization of GFP-Rho2 during interphase was partially dependent on sts5+. These results indicate that Rho2 is involved in cell morphogenesis, control of cell wall integrity, control of growth polarity, and maintenance of growth direction. Analysis of functional overlapping between Rho2 and Rho1 revealed that their functions are distinct from each other, with partial overlapping.
Aminopeptidase B: a processing enzyme secreted and associated with the plasma membrane of rat pheochromocytoma (PC12) cells
Authors: Balogh Agnès , Cadel Sandrine , Foulon Thierry , Picart Renée , Garabedian Arsène Der , Rousselet Annie , Tougard Claude , Cohen Paul
Published: April 2021

Aminopeptidase B (Ap-B) is a Zn2+-dependent exopeptidase which selectively removes Arg and/or Lys residues from the N terminus of several peptide substrates. Isolated and characterized from rat testes, this ubiquitous enzyme may participate in the final stages of precursor processing mechanisms. To test this hypothesis, we have investigated the secretion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells). By using a combination of biochemical and immunocytochemical methods, the following observations were made: (i) the level of aminopeptidase B detectable in the cell culture medium increased with time; (ii) 8-bromo-adenosine 3′-5′-cyclic monophosphate and the Ca2+ ionophore A23187 both stimulated enzyme liberation in the culture medium; (iii) brefeldin A, an inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi apparatus, decreased enzyme secretion in a time-dependent manner; (iv) whereas nocodazole, a microtubule depolymerizing agent, inhibited enzyme secretion, cytochalasin D, a microfilament disruption agent, had no effect on released aminopeptidase B level; (v) immunofluorescence demonstrated the presence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescence, electron microscopy and tests of enzyme activity on intact cells showed an association of the peptidase with the external face of the plasma membrane. Together these data strongly argued in favour of the enzyme secretion by PC12 cells. It is concluded that aminopeptidase B may participate in processing events occurring either during its intracellular transport along the secretory pathway or at the plasma membrane level, or both.
Drosophila fascin mutants are rescued by overexpression of the villin-like protein, quail
Authors: Cant Kelly , Knowles Brenda A. , Mahajan-Miklos Shalina , Heintzelman Matthew , Cooley Lynn
Published: April 2021

Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells requires two actin bundling proteins, fascin encoded by the singed gene and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. We used P-element mediated germline transformation to overexpress quail in singed mutants and test whether these proteins have redundant functions in vivo. Overexpression of quail protein in a sterile singed background restores actin bundle formation in egg chambers. The degree of rescue by quail depends on the level of quail protein overexpression, as well as residual levels of fascin function. In nurse cells that contain excess quail but no fascin, the cytoplasmic actin network initially appears wild type but then becomes disorganized in the final stages of nurse cell cytoplasm transport. The ability of quail overexpression to compensate for the absence of fascin demonstrates that fascin is partially redundant with quail in the Drosophila germline. Quail appears to function as a bundle initiator while fascin provides bundle organization.
Intracellular trafficking of the vacuolar H+-ATPase accessory subunit Ac45
Authors: Jansen Eric J. R. , Holthuis Joost C. M. , McGrouther Collette , Burbach J. Peter H. , Martens Gerard J. M.
Published: April 2021

Ac45 is a type I transmembrane protein associated with vacuolar H+-ATPase, a proton pump mediating the acidification of multiple intracellular organelles. In this study, we examined the intracellular routing of Ac45 in transfected CV-1 fibroblasts. Steady state immunolabeling showed that Ac45 is located on the plasma membrane and in a vacuolar compartment in the juxtanuclear region. Antibody internalization experiments revealed that Ac45 is rapidly retrieved from the cell surface and is targeted to the vacuolar structures. The 26-residue cytoplasmic tail of Ac45 was intrinsically capable of mediating endocytosis of the cell surface protein Tac, indicating that the tail contains an autonomous internalization signal. Immunolocalization studies on cells expressing carboxy-terminally truncated Ac45 mutants showed the presence of essential routing information in the membrane-distal region of the cytoplasmic tail. Further mutational analysis of this region, which lacks the recognized tyrosine- or di-leucine-based sorting motifs, suggested that multiple sites rather than a short linear sequence are responsible for the internalization. Collectively, our results indicate that the cytoplasmic tail of Ac45 contains autonomous targeting information distinct from previously described routing determinants.
Ye1013p (Vac8p), an armadillo repeat protein related to plakoglobin and importin α, is associated with the yeast vacuole membrane
Authors: Fleckenstein Diana , Rohde Manfred , Klionsky Daniel J. , Rüdiger Manfred
Published: April 2021

Proteins of the armadillo family are involved in diverse cellular processes in higher eukaryotes. Some of them, like armadillo, β-catenin and plakoglobins have dual functions in intercellular junctions and signalling cascades. Others, belonging to the importin-α-subfamily are involved in NLS recognition and nuclear transport, while some members of the armadillo family have as yet unknown functions.

Here, we introduce the Saccharomyces cerevisiae protein Ye1013p as a novel armadillo (arm) repeat protein. The ORF Ye1013w was identified in the genome project on chromosome V (EMBL: U18530) and codes for an acidic protein of 578 residues with 8 central arm-repeats, which are closely related to the central repeat-domain of Xenopus laevis plakoglobin. We show that Ye1013p (Vac8p) is constitutively expressed in diploid and haploid yeasts and that it is not essential for viability and growth. However, the vacuoles of mutant cells are multilobular or even fragmented into small vesicles and the processing of aminopeptidase I, representing the cytoplasm-to-vacuole transport pathway, is strongly impaired. Consistent with these observations, subcellular fractionation experiments, immunolocalization and expression of green fluorescent protein (GFP) fusion proteins revealed that Ye1013p (Vac8p) is associated with the vacuolar membrane.

Our data provide evidence for the involvement of an arm-family member in vacuolar morphology and protein targeting to the vacuole.
A factor required for nonsense-mediated mRNA decay in yeast is exported from the nucleus to the cytoplasm by a nuclear export signal sequence
Authors: Shirley Renee L. , Lelivelt Michael J. , Schenkman Laura R. , Dahlseid Jeffrey N. , Culbertson Michael R.
Published: April 2021

In Saccharomyces cerevisiae, Upf3p is required for nonsense-mediated mRNA decay (NMD). Although localized primarily in the cytoplasm, Upf3p contains three sequence elements that resemble nuclear localization signals (NLSs) and two sequence elements that resemble nuclear export signals (NESs). We found that a cytoplasmic reporter protein localized to the nucleus when fused to any one of the three NLS-like sequences of Upf3p. A nuclear reporter protein localized to the cytoplasm when fused to one of the NES-like sequences (NES-A). We present evidence that NES-A functions to signal the export of Upf3p from the nucleus. Combined alanine substitutions in the NES-A element caused a re-distribution of Upf3p to a subnuclear location identified as the nucleolus and conferred an Nmd− phenotype. Single mutations in NES-A failed to affect the distribution of Upf3p and were Nmd+. When an NES element from HIV-1 Rev was inserted near the C terminus of a mutant Upf3p containing multiple mutations in NES-A, the cytoplasmic distribution typical of wild-type Upf3p was restored but the cells remained phenotypically Nmd−. These results suggest that NES-A is a functional nuclear export signal. Combined mutations in NES-A may cause multiple defects in protein function leading to an Nmd− phenotype even when export is restored.
The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells
Authors: Lyle Stephen , Christofidou-Solomidou Melpo , Liu Yaping , Elder David E. , Albelda Steven , Cotsarelis George
Published: April 2021

Stem cells are vital for the homeostasis of self-renewing tissues such as the hair follicle. Epithelial stem cells have been implicated in tumorigenesis and wound healing, and their manipulation may have wide ranging applications including gene therapy and tissue transplantation. Rodent hair follicle stem cells have been localized to an area of the follicle called the bulge, however, the identification and characterization of human hair follicle stem cells has been hampered by a lack of cellular markers for this area. We have determined that the C8/144B monoclonal antibody, originally generated against a short intracytoplasmic peptide of CD8, preferentially immunostains hair follicle bulge keratinocytes without staining the remaining hair follicle. Using expression cloning, we identified cytokeratin 15 as the keratinocyte protein recognized by the C8/144B monoclonal antibody. By delineating the bulge using this antibody, we demonstrated that bulge cells possess a stem cell phenotype characterized by their slowly-cycling nature, preferential proliferation at the onset of new hair follicle growth, high level of β1 integrin expression, and expression of cytokeratin 19.
N-terminal domain of Gpa1 (G protein α subunit) is sufficient for plasma membrane targeting in yeast Saccharomyces cerevisiae
Authors: Gillen Kathy M. , Pausch Mark , Dohlman Henrik G.
Published: April 2021

G proteins play a central role in transmitting signals from cell surface receptors to effector proteins inside the cell. Signaling can only occur, however, if all these protein components are properly assembled and localized at the plasma membrane. Past studies have shown that certain segments within the N-terminal region of the G protein α subunit are necessary for membrane attachment. Here we identify a region within the yeast Gα (Gpa1) that is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane. Initially, we constructed chimeric proteins that replace the N terminus of mammalian Gsα with the corresponding sequence from Gpa1. Gsα is inefficiently targeted to the yeast plasma membrane and therefore cannot fully complement the loss of Gpa1. Gpa1-Gsα chimeras were assayed for proper membrane localization by functional complementation of a gpa1Δ mutant, and by sucrose density gradient fractionation of cell membranes. Most of the chimeras tested, including one with only the N-terminal 7 amino acids from Gpa1, exhibited normal membrane targeting and complementing activity. We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expressed in the cytoplasm. The first 67- 36- or 9-amino acids of Gpa1 were all sufficient to direct GST specifically to the plasma membrane in yeast. This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isolating and identifying G protein-targeting proteins within the plasma membrane.
Cell biology of transcription and pre-mRNA splicing: nuclear architecture meets nuclear function
Authors: Misteli Tom
Published: April 2021

Gene expression is a fundamental cellular process. The basic mechanisms involved in expression of genes have been characterized at the molecular level. A major challenge is now to uncover how transcription, RNA processing and RNA export are organized within the cell nucleus, how these processes are coordinated with each other and how nuclear architecture influences gene expression and regulation. A significant contribution has come from cell biological approaches, which combine molecular techniques with microscopy methods. These studies have revealed that the mammalian cell nucleus is a complex but highly organized organelle, which contains numerous subcompartments. I discuss here how two essential nuclear processes – transcription and pre-mRNA splicing – are spatially organized and coordinated in vivo, and how this organization might contribute to the control of gene expression. The dynamic nature of nuclear proteins and compartments indicates a high degree of plasticity in the cellular organization of nuclear functions. The cellular organization of transcription and splicing suggest that the morphology of nuclear compartments is largely determined by the activities of the nucleus.
Patching plasma membrane disruptions with cytoplasmic membrane
Authors: McNeil Paul L. , Vogel Steven S. , Miyake Katsuya , Terasaki Mark
Published: April 2021

Vesicle-vesicle fusion initiated in cell cytoplasm by high Ca2+ can rapidly erect large membrane boundaries. These might be used as a ‘patch’ for resealing plasma membrane disruptions. Three central predictions of this ‘patch’ hypothesis are here established in sea urchin eggs. First, we show that surface markers for plasma membrane protein and lipid are initially absent over disruption sites after resealing is complete. Second, we demonstrate that resealing capacity is strongly dependent upon local availability of fusion competent cytoplasmic organelles, specifically the reserve or yolk granule. Lastly, we demonstrate that the reserve granule is capable of rapid (t1/2 <1 second), Ca2+-regulated (high threshold) fusion capable of erecting large (>1000 μm2), continuous membrane boundaries. Production of patch vesicles for resealing may proceed by an ‘emergency’ fusion mechanism distinct from that utilized for the much slower, highly regulated, cytosol-requiring organelle-organelle fusion events typical of constitutive membrane trafficking pathways.