bluuebunny/crossref_metadata_2025_split huggingface dataset:
PUMA1: a novel protein that associates with the centrosomes, spindle and centromeres in the nematode Parascaris
Authors: Esteban M. R. , Giovinazzo G. , Hera A. de la , Goday C. 
Published: April 2021

ABSTRACT
We have identified a 227 kDa spindle- and centromere-associated protein in Parascaris, designated PUMA1 (Parascaris univalens mitotic apparatus), using a monoclonal antibody (mAb403) generated against Parascaris embryonic extracts. PUMA1 distribution was studied by immunofluorescence microscopy in mitotic and meiotic Parascaris cells, where centromere organization differs greatly. In mitosis, PUMA1 associates throughout cell division with the centrosomes and kinetochore-microtubules, and it concentrates at the continuous centromere region of the holocentric chromosomes. PUMA1 also localizes to the spindle mid-zone region during anaphase and at the midbody during telophase. In meiosis, PUMA1 associates with the centrosomes and with the discrete centromeric regions lacking kinetochore structures. The analysis of colchicine-treated embryos indicated that the association of PUMA1 with the centromeric region depends on microtubule integrity. mAb403 also recognizes spindle components in Drosophila. A series of overlapping cDNAs encoding the gene were isolated from a Parascaris embryonic expression library. Analysis of the nucleotide sequence identified an open reading frame capable of encoding a protein of 227 kDa. Analysis of the protein sequence indicated that PUMA1 is predicted to be a coiled-coil protein containing a large central α-helical domain flanked by nonhelical terminal domains. The structural features and cellular distribution of PUMA1 suggest that it may play a role in the organization of the spindle apparatus and in its interaction with the centromere in Parascaris.
Oligomerization of the extracellular domain of Boss enhances its binding to the Sevenless receptor and its antagonistic effect on R7 induction
Authors: Sevrioukov Evgueni A. , Walenta Jason H. , Sunio Arisa , Phistry Meridee , Krämer Helmut 
Published: April 2021

ABSTRACT
In the developing compound eye of Drosophila, neuronal differentiation of the R7 photoreceptor cell is induced by the interaction of the receptor tyrosine kinase Sevenless with its ligand Bride of sevenless (Boss), which is expressed on the neighboring R8 cell. Boss is an unusual ligand of a receptor tyrosine kinase: it is composed of a large extracellular domain, a transmembrane domain with seven membrane-spanning segments and a cytoplasmic tail. Expression of a monomeric, secreted form of the extracellular domain of Boss is not sufficient for Sevenless activation, and instead acts as a weak antagonist. Because oligomerization appears to be a critical step in the activation of receptor tyrosine kinases, we used oligomerized forms of the Boss extracellular domain to test their ability to bind to Sevenless in vivo and restore R7 induction in vivo. Oligomerization was achieved by fusion to the leucine zipper of the yeast transcription factor GCN4 or to the tetramerization helix of Lac repressor. Binding of these multivalent proteins to Sevenless could be detected in vitro by immunoprecipitation of cross-linked ligand/receptor complexes and in vivo by receptor-dependent ligand localization. However, neither R8-specific or ubiquitous expression of multivalent Exboss ligands rescued the boss phenotype. Instead, these ligands acted as competitive inhibitors for wild-type Boss protein and thereby suppressed R7 induction. Therefore the role of the transmembrane or cytoplasmic domains of Boss in the activation of the Sev receptor cannot be replaced by oligomerization.
Mutations in the bimC box of Cut7 indicate divergence of regulation within the bimC family of kinesin related proteins
Authors: Drummond Douglas R. , Hagan Iain M. 
Published: April 2021

ABSTRACT
Members of the bimC family of kinesin related proteins (KRPs) play vital roles in the formation and function of the mitotic spindle. Although they share little amino acid homology outside the highly conserved microtubule motor domain, several family members do contain a ‘bimC box’, a sequence motif around a p34cdc2 consensus phosphorylation site in their carboxy-terminal ‘tail’ region. One family member, Eg5, requires phosphorylation at this site for association with the mitotic spindle. We show that mutations in the Schizosaccharomyces pombe cut7+ gene that change the bimC box p34cdc2 consensus phosphorylation site at position 1,011 and a neighbouring MAP kinase consensus phosphorylation site at position 1,020 to non-phosphorylatable residues did not affect the ability of S. pombe cut7 genes to complement temperature sensitive cut7 mutants. Phosphorylation site mutants expressed as fusions to green fluorescent protein associated with the mitotic spindle with a localisation indistinguishable from similarly expressed wild-type Cut7. Cells in which cut7.T1011A replaced the genomic copy of cut7+ were viable and formed normal spindles. Deletion of the entire carboxy-terminal tail region did not affect the ability of Cut7 to associate with the mitotic spindle but did inhibit normal spindle formation. Thus, unlike Eg5, neither the p34cdc2 consensus phosphorylation site in the bimC box nor the entire tail region of Cut7 are required for association with the mitotic spindle.
Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: Localization to the Trans-Golgi network and recycling from the cell surface
Authors: Varlamov Oleg , Fricker Lloyd D. 
Published: April 2021

ABSTRACT
Carboxypeptidase D (CPD) is a recently discovered membrane-bound metallocarboxypeptidase that has been proposed to be involved in the post-translational processing of peptides and proteins that transit the secretory pathway. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitaryderived corticotroph. Antisera to CPD stain the same intracellular structures as those labeled with furin and wheat germ agglutinin. This distribution is distinct from carboxypeptidase E, which is localized to the secretory vesicles in the cell processes. The perinuclear distribution of CPD is detected even when the AtT-20 cells are treated with brefeldin A for 1-30 minutes, suggesting that CPD is present in the trans-Golgi network (TGN).
Although CPD is predominantly found in the TGN, an antiserum to the full length protein is internalized within 15-30 minutes of incubation at 37°C. In contrast, an antiserum raised against the C-terminal region of CPD does not become internalized, suggesting that this domain is cytosolic. The antiserum to the full length CPD is internalized to a structure that co-stains with furin and wheat germ agglutinin, but is distinct from transferrin recycling endosomes. The internalization of CPD is not substantially affected by treatment of the AtT-20 cells with brefeldin A. These data are consistent with the cycling of CPD to the cell surface and back to the TGN. The TGN localization of CPD raises the possibility of a role for this enzyme in the processing of proteins that transit the secretory pathway.
The Tomaj mutant alleles of αtubulin67c reveal a requirement for the encoded maternal specific tubulin isoform in the sperm aster, the cleavage spindle apparatus and neurogenesis during embryonic development in Drosophila
Authors: Máthé1 Endre , Boros’ Imre , Jósvay Katalin , Li Kaijun , Puro Jaakko , Kaufman Thomas C. , Szabad János 
Published: April 2021

ABSTRACT
The three dominant TomajD and their eleven revertant (TomajR) alleles have been localized to the αTubulin67C gene of Drosophila melanogaster. Although the meiotic divisions are normally completed in eggs laid by TomajD/+, TomajD/-, TomajR/-females, embryogenesis arrests prior to the gonomeric division. The arrest is caused by: (1) the failure of prominent sperm aster formation; and (2) a consequent lack of female pronuclear migration towards the male pronucleus. Concomitant with the sperm aster defect, the four female meiotic products fuse (tetra-fusion), similar to what is seen in eggs of wild-type virgin females. In eggs of females heterozygous for weaker TomajR alleles, embryogenesis comes to a cessation before or shortly after cortical migration of cleavage nuclei. The apparent source of embryonic defect is the cleavage spindle apparatus. One of the three TomajD alleles is cold-sensitive and its cold-sensitive period coincides with the completion of female meiosis and pronuclear migration. Disorganized central and peripheral nervous systems are also characteristic of embryos derived from the temperature-sensitive TomajD/+ females. The Tomaj mutant phenotypes indicate an involvement of the normal αTubulin67C gene product in: (1) the formation of the sperm aster; (2) cleavage spindle apparatus formation/function; and (3) the differentiation of the embryonic nervous system. The TomajD alleles encode a normal-sized αTubulin67C isotype. Sequence analyses of the TomajD alleles revealed the replacement in different positions of a single negatively charged or neutral amino acid with a positively charged one. These residues presumably identify important functional sites.
Human myosin-IXb is a mechanochemically active motor and a GAP for rho
Authors: Post Penny L. , Bokoch Gary M. , Mooseker Mark S. 
Published: April 2021

ABSTRACT
The heavy chains of the class IX myosins, rat myr5 and human myosin-IXb, contain within their tail domains a region with sequence homology to GTPase activating proteins for the rho family of G proteins. Because low levels of myosin-IXb expression preclude purification by conventional means, we have employed an immunoadsorption strategy to purify myosin-IXb, enabling us to characterize the mechanochemical and rho-GTPase activation properties of the native protein. In this report we have examined the light chain content, actin binding properties, in vitro motility and rho-GTPase activity of human myosin-IXb purified from leukocytes.
The results presented here indicate that myosin-IXb contains calmodulin as a light chain and that it binds to actin with high affinity in both the absence and presence of ATP. Myosin-IXb is an active motor which, like other calmodulin-containing myosins, exhibits maximal velocity of actin filaments (15 nm/second) in the absence of Ca2+. Native myosin-IXb exhibits GAP activity on rho. Class IX myosins may be an important link between rho and rho-dependent remodeling of the actin cytoskeleton.
LRetrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol
Authors: Drecktrah Daniel , Figueiredo Paul de , Mason Roger M. , Brown William J. 
Published: April 2021

ABSTRACT
Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.
A NIMA homologue promotes chromatin condensation in fission yeast
Authors: Krien Michael J. E. , Bugg Sarah J. , Palatsides Manuela , Asouline Gigi , Morimyo Mitsuoki , O’Connell Matthew J. 
Published: April 2021

ABSTRACT
Entry into mitosis requires p34cdc2, which activates downstream mitotic events through phosphorylation of key target proteins. In Aspergillus nidulans, the NIMA protein kinase has been identified as a potential downstream target and plays a role in regulating chromatin condensation at mitosis. nimA-mutants arrest in a state that physically resembles interphase even though p34cdc2 is fully active. Despite evidence for the existence of NIMA-like activities in a variety of cell types, the only bona fide NIMA homologue that has been identified is the nim-1 gene of Neurospora crassa. We report here the isolation of a fission yeast NIMA homologue, and have designated this genefinl and the 83 kDa predicted protein p83fin1. Overexpression offinl promotes premature chromatin condensation from any point in the cell cycle independently of p34cdc2 function.
Like NIMA, p83fin1 levels fluctuate through the cell cycle, peaking in mitosis and levels are greatly elevated by removal of C-terminal PEST sequences. Deletion of finl results in viable but elongated cells, indicative of a cell cycle delay. Genetic analysis has placed this delay in G2 but, unlike in nimA mutants of Aspergillus, p34cdc2 activation appears to be delayed. Interaction of finlΔ mutants with other strains defective in chromatin organisation also support the hypothesis of p83fin1 playing a role in this process at the onset of mitosis. These data indicate that NIMA-related kinases may be a general feature of the cell cycle and chromatin organisation at mitosis.
Nuclear location of PLA2-I in proliferative cells
Authors: Fayard Jean-Michel , Tessier Christian , Pageaux Jean-François , Lagarde Michel , Laugier Christian 
Published: April 2021

ABSTRACT
We have previously demonstrated that pancreatic PLA2 (PLA2-I) stimulates the proliferation of UIII cells, a stromal cell line derived from normal rat uterus. In order to gain further insight into the mechanism of action of PLA2-I, we have investigated the intracellular processing of PLA2-I. Either highly proliferative or growth arrested UIII cells were analyzed. Growth arrested cells were obtained from a contact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, western blotting, immunocytochemistry and confocal microscopy, we demonstrate that endogenous PLA2-I was mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmic in non proliferative cells. When non confluent UIII cells were incubated with nanomolar amounts of exogenous PLA2-I, the enzyme was internalized and, in the majority of cells, appeared within the nucleus. Both internalization and nuclear location of exogenous PLA2-I were suppressed by the addition of aristolochic acid to the culture medium. Binding experiments performed on purified nuclear preparations showed the presence of specific cooperative binding sites for PLA2-I. Collectively our data suggest that the proliferative effect exerted by pancreatic PLA2 in UIII cells is mediated by a direct interaction of the enzyme at the nuclear level. Putative mechanisms and targets are discussed.
Proteasomes regulate the motility of salmonid fish sperm through modulation of cAMP-dependent phosphorylation of an outer arm dynein light chain
Authors: Inaba Kazuo , Morisawa Sachiko , Morisawa Masaaki 
Published: April 2021

ABSTRACT
Proteasomes are involved in ATP-dependent regulation of sperm motility in salmonid fish. We have demonstrated here by immunoelectron microscopy that proteasomes are located at the structure of the chum salmon sperm flagellum that attaches at the base of the outer arm dynein and extends toward the plasma membrane. Furthermore, substrates and inhibitors of proteasome inhibit the cAMPdependent phosphorylation of a 22 kDa axonemal protein in chum salmon sperm. The 22 kDa phosphoprotein was solubilized by treatment of the axoneme with a high salt solution and subsequent sucrose density gradient centrifugation of the extract revealed that it cosedimented with 19 S outer arm dynein, indicating that it is a dynein light chain. These results suggest that proteasomes modulate the activity of outer arm dynein by regulating cAMP-dependent phosphorylation of the 22 kDa dynein light chain.
Translocation of microtubules caused by the αβ, β and γ outer arm dynein subparticles of Chlamydomonas
Authors: Sakakibara Hitoshi , Nakayama Haruto 
Published: April 2021

ABSTRACT
Three kinds of subparticles of Chlamydomonas outer-arm dynein containing the αβ, β and γ heavy chains were isolated and assayed for their activities to translocate microtubules in vitro. All of them had activities to form bundles of microtubules in solution in an ATP-dependent manner and, when adsorbed on an appropriate glass surface, translocated microtubules. The αβ subparticle readily translocated microtubules on a silicone-coated glass surface with a velocity of 4.6 μm/second at 1 mM ATP. The β subparticle translocated microtubules after it had been preincubated with tubulin dimer and when the Brownian movement of microtubules was suppressed by addition of methylcellulose. The velocity was on average 0.7 μm/second. The γ subparticle translocated microtubules after being preincubated with tubulin dimer and adsorbed onto a silicone-coated glass surface. The velocity was about 3.8 μm/second. The tubulin dimer appeared to facilitate in vitro motility by blocking the ATP-insensitive binding of dynein subparticles to microtubule. The αβ, β and γ subparticles were thus found to have different properties as motor proteins. In addition, these subparticles showed different dependencies upon the potassium acetate concentration. Hence the outer-arm dynein of Chlamydomonas is a complex of motor proteins with different properties.
Roles of Ca2+, inositol trisphosphate and cyclic ADP-ribose in mediating intercellular Ca2+ signaling in sheep lens cells
Authors: Churchill Grant C. , Louis Charles F. 
Published: April 2021

ABSTRACT
To further characterize how gap junction-dependent Ca2+ waves propagate between sheep lens cells, we examined the possible roles of inositol 1,4,5-trisphosphate (IP3), Ca2+ and cyclic ADP-ribose (cADPR) in mediating intercellular Ca2+ waves. Second messengers were microinjected into a single cell in a monolayer of sheep lens cells while monitoring cytosolic Ca2+ with fura-2 and fluorescence microscopy. All three compounds initiated intercellular Ca2+ waves, but more cells responded following the injection of either IP3 or cADPR than responded following the injection of Ca2+. When either IP3 or cADPR was co-injected with the Ca2+ chelator EGTA, cytosolic Ca2+ in the injected cell decreased but cytosolic Ca2+ in the adjacent cells increased, indicating that the intercellular messenger was IP3 or cADPR, rather than Ca2+. The phospholipase C inhibitor U73122 eliminated mechanically initiated intercellular Ca2+ waves, indicating that mechanical initiation probably requires IP3 production. In U73122-treated cells, injected IP3 initiated an intercellular Ca2+ wave in which the number of cells responding increased as the amount of IP3 injected increased, indicating that the distance traveled by the Ca2+ wave was dependent on cell-to-cell diffusion of IP3. In contrast, the ability of cADPR both to increase cytosolic Ca2+ in the injected cell and to initiate intercellular Ca2+ waves was greatly attenuated by U73122. In conclusion, Ca2+, IP3 and cADPR can all mediate intercellular Ca2+ waves by passing through gap junction channels, but both IP3 and cADPR are more effective intercellular messengers than Ca2+.
Actin mRNA isoforms are differentially sorted in normal osteoblasts and sorting is altered in osteoblasts from a skeletal mutation in the rat
Authors: Watanabe Hiroki , Kislauskis Edward H. , Mackay Carole A. , Mason-Savas April , Marks Sandy C. 
Published: April 2021

ABSTRACT
Actin isoform sorting has been shown to occur in a variety of cell types in culture. To this list we add osteoblasts, in which we show by in situ hybridization that β-actin is distributed primarily in cell processes and on one side of the nucleus and γ-actin has a perinuclear distribution. Osteoblasts from the skeletal mutation toothless (tl), evaluated under identical conditions, fail to sort these actin isoforms differentially and exhibit diffuse labeling as their major manifestation. Northern analyses of actin mRNAs showed no differences between normal and mutant cultures. Shortened osteoblast life span and an inability to direct osteoclast-mediated bone resorption have recently been demonstrated in tl mutants. The present results suggest that a failure of osteoblasts to sort actin mRNAs may be related to one or both of these pathological manifestations in this mutation and represent, to our knowledge, the first correlation of an actin mRNA-sorting abnormality with a mammalian disease.
Identification of the A-band localization domain of myosin binding proteins C and H (MyBP-C, MyBP-H) in skeletal muscle
Authors: Gilbert Rénald , Cohen Julie A. , Pardo Sherly , Basu Amartya , Fischman Donald A. 
Published: April 2021

ABSTRACT
Although major constituents of the thick filaments of vertebrate striated muscles, the myosin binding proteins (MyBP-C and MyBP-H) are still of uncertain function. Distributed in the cross-bridge bearing zone of the A-bands of myofibrils, in a series of transverse 43 nm stripes, the proteins are constructed of a tandem series of small globular domains, each composed of ∿90-100 amino acids, which have sequence similarities to either the C2-set of immunoglobulins (IgC2) and the fibronectin type III (FnIII) motifs. MyBP-C is composed of ten globular domains (∿130 kDa) whereas MyBP-H is smaller (∿58 kDa) and consists of a unique N-terminal segment followed by four globular domains, the order of which is identical to that of MyBP-C (FnIII-IgC2-FnIII-IgC2). To improve our understanding of this protein family we have characterized the domains in each of these two proteins which are required for targeting the proteins to their native site(s) in the sarcomere during myogenesis. Cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding mutant constructs of the MyBPs bearing an N-terminal myc epitope, and their localization to the A-band examined by immunofluorescence microscopy. Based on the clarity and intensity of the myc A-band signals we concluded that constructs encoding the four C-terminal motifs of MyBP-C and MyBP-H (∿360 amino acids) were all that was necessary to efficiently localize each of these peptides to the A-band. Truncation mutants lacking one of these 4 domains were less efficiently targeted to the C-zone of the sarcomere. Deletion of the last C-terminal motif of MyBP-H, its myosin binding domain, abolished all localization to the A-band. A chimeric construct, HU-3C10, in which the C-terminal motif of MyBP-H was replaced by the myosin binding domain of MyBP-C, efficiently localized to the A-band. Taken together, these observations indicate that MyBP-C and MyBP-H are localized to the A-band by the same C-terminal domain, composed of two IgC2 and two FnIII motifs. A model has been proposed for the interaction and positioning of the MyBPs in the thick filament through a ternary complex of the four C-terminal motifs with the myosin rods and titin.