bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of nuclear positioning
Authors: Reinsch Sigrid , Gönczy Pierre 
Published: April 2021

ABSTRACT
The mechanisms underlying two types of microtubuledependent nuclear positioning are discussed. ‘MTOC-dependent nuclear positioning’ occurs when a nucleus is tightly associated with a microtubule organizing center (MTOC). ‘Nuclear tracking along microtubules’ is analogous to the motor-driven motility of other organelles and occurs when the nucleus lacks an associated MTOC. These two basic types of microtubule-dependent nuclear positioning may cooperate in many proliferating ani mal cells to achieve proper nuclear positioning. Microtubule polymerization and dynamics, motor proteins, MAPs and specialized sites such as cortical anchors function to control nuclear movements within cells.
Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1
Authors: Weed Scott A. , Du Yunrui , Parsons J. Thomas 
Published: April 2021

ABSTRACT
Small GTPases of the Rho family regulate signaling pathways that control actin cytoskeletal structures. In Swiss 3T3 cells, RhoA activation leads to stress fiber and focal adhesion formation, Rac1 to lamellipoda and membrane ruffles, and Cdc42 to microspikes and filopodia. Several downstream molecules mediating these effects have been recently identified. In this report we provide evidence that the intracellular localization of the actin binding protein cortactin, a Src kinase substrate, is regulated by the activation of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and this translocation is blocked by expression of dominant negative Rac1N17. Expression of constitutively active Rac1L61 evoked the translocation of cortactin from cytoplasmic pools into peripheral membrane ruffles. Expression of mutant forms of the serine/threonine kinase PAK1, a downstream effector of Rac1 and Cdc42 recently demonstrated to trigger cortical actin polymerization and membrane ruffling, also led to the translocation of cortactin to the cell cortex, although this was effectively blocked by coexpression of Rac1N17. Collectively these data provide evidence for cortactin as a putative target of Rac1-induced signal transduction events involved in membrane ruffling and lamellipodia formation.
The cdc25B phosphatase is essential for the G 2/M phase transition in human cells
Authors: Lammer1 Christiane , Wagerer Sybille , Saffrich Rainer , Mertens Daniel , Ansorge Wilhelm , Hoffmann Ingrid 
Published: April 2021

ABSTRACT
Cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. In human cells, cdc25 proteins are encoded by a multigene family, consisting of cdc25A, cdc25B and cdc25C. While cdc25A plays a crucial role at the G 1/S phase transition, cdc25C is involved in the dephosphorylation and activation of the mitotic kinase, cdc2/cyclinB. In addition, cdc25C itself is regulated by cdc2/cyclinB which then creates a positive feedback loop that controls entry into mitosis. In this study we show that the activity of cdc25B appears during late S phase and peaks during G 2 phase. Both in vitro and in vivo cdc25B is activated through phosphorylation during S-phase. Using a cell duplication, microinjection assay we show that ablation of cdc25B function by specific antibodies blocks cell cycle progression in Hs68 cells by inhibition of entry into mitosis. Cdc25B function neither plays a role in later stages of mitosis nor for the inititation of DNA replication. These results indicate that cdc25B is a mitotic regulator that might act as a ‘starter phosphatase’ to initiate the positive feedback loop at the entry into M phase.
Escape of mitochondrial DNA to the nucleus in ymel yeast is mediated by vacuolar-dependent turnover of abnormal mitochondrial compartments
Authors: Campbell Corey L. , Thorsness Peter E. 
Published: April 2021

ABSTRACT
Inactivation of Ymelp, a mitochondrially-localized ATP-dependent metallo-protease in the yeast Saccharomyces cerevisiae, causes a high rate of DNA escape from mitochondria to the nucleus as well as pleiotropic functional and morphological mitochondrial defects. The evidence presented here suggests that the abnormal mitochondria of a ymel strain are degraded by the vacuole. First, electron microscopy of Yme1p-deficient strains revealed mitochondria physically associated with the vacuole via electron dense structures. Second, disruption of vacuolar function affected the frequency of mitochondrial DNA escape from ymel and wild-type strains. Both PEP4 or PRC1 gene disruptions resulted in a lower frequency of mitochondrial DNA escape. Third, an in vivo assay that monitors vacuole-dependent turnover of the mitochondrial compartment demonstrated an increased rate of mitochondrial turnover in ymel yeast when compared to the rate found in wild-type yeast. In this assay, vacuolar alkaline phosphatase, encoded by PHO8, was targeted to mitochondria in a strain bearing disruption to the genomic PHO8 locus. Maturation of the mitochondrially localized alkaline phosphatase pro-enzyme requires proteinase A, which is localized in the vacuole. Therefore, alkaline phosphatase activity reflects vacuole-dependent turnover of mitochondria. This assay reveals that mitochondria of a ymel strain are taken up by the vacuole more frequently than mitochondria of an isogenic wild-type strain when these yeast are cultured in medium necessitating respiratory growth. Degradation of abnormal mitochondria is one pathway by which mitochondrial DNA escapes and migrates to the nucleus.
MAP kinase activitys increases during mitosis in early sea urchin embryos
Authors: Philipova Rada , Whitaker Michael 
Published: April 2021

ABSTRACT
A MBP kinase activity increases at mitosis during the first two embryonic cell cycles of the sea urchin embryo. The activity profile of the MBP kinase is the same both in whole cell extracts and after immunoprecipitation with an anti-MAP kinase antibody (2199). An in-gel assay of MBP activity also shows the same activity profile. The activity is associated with the 44 kDa protein that cross-reacts with anti-MAP kinase antibodies. The 44 kDa protein shows cross-reactivity to anti-phosphotyrosine and MAP kinase-directed anti-phosphotyrosine/phosphothreonine antibodies at the times that MBP kinase activity is high. The 2199 antibody co-precipitates some histone H1 kinase activity, but the MBP kinase activity cannot be accounted for by histone H1 kinase-dependent phosphorylation of MBP. The MAP kinase 2199 antibody was used to purify the MBP kinase activity. Peptide sequencing after partial digestion shows the protein to be homologous to MAP kinases from other species. These data demonstrate that MAP kinase activation during nuclear division is not confined to meiosis, but also occurs during mitotic cell cycles. MAP kinase activity in immunoprecipitates also increases immediately after fertilization, which in the sea urchin egg occurs at interphase of the cell cycle. Treating unfertilized eggs with the calcium ionophore A23187 stimulates the increase in MAP kinase activity, demonstrating that a calcium signal can activate MAP kinase and suggesting that the activation of MAP kinase at fertilization is due to the fertilization-induced increase in cytoplasmic free calcium concentration. This signalling pathway must differ from the pathway responsible for calcium-induced inactivation of MAP kinase activity that is found in eggs that are fertilized in meiotic metaphase.
Evidence for MAP kinase activation during mitotic division
Authors: Céline De Nadai Sandrine Chiri , Ciapa Brigitte 
Published: April 2021

ABSTRACT
MAP kinases have been implicated in the control of a broad spectrum of cellular events in many types of cells. In somatic cells, MAP kinase activation seems to be triggered after exit from a quiescent state (in G0 or G2) only and then inactivated by entry into a proliferative state. In oocytes of various species, a one-time activation of MAP kinase that is apparently not repeated during the succeeding mitotic cycles occurs after meiotic activation. However, several reports suggest that a myelin basic protein (MBP) kinase activity, unrelated to that of maturation promoting factor, can sometimes be detected during mitotic divisions in various types of cells and oocytes. We have reinvestigated this problem in order to determine the origin and the role of MBP kinase that is stimulated at time of mitosis in the fertilized eggs of the sea urchin Paracentrotus lividus. We used anti-ERK1 antibodies or substrates specific for different MAP kinases, and performed in-gel phosphorylation experiments. Our results suggest that an ERK1-like protein was responsible for part of the MBP kinase activity that is stimulated during the first mitotic divisions. Furthermore, we observed that wortmannin, an inhibitor of PI 3-kinase that arrests the fertilized sea urchin eggs at the prometaphase stage, inhibited the inactivation of MAP kinase normally observed when the eggs divide, suggesting a role for PI 3-kinase in the deactivation process of MAP kinase. We also discuss how the activities of MPF and MAP kinase may be interconnected to regulate the first mitotic divisions of the early sea urchin embryo.
Characterisation of α-dystrobrevin in muscle
Authors: Nawrotzki Ralph , Loh Nellie Y. , Ruegg Markus A. , Davies Kay E. , Blake Derek J. 
Published: April 2021

ABSTRACT
Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of α-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce α-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct α-dystrobrevin isoforms. In myotubes, α-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, α-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of α-dystrobrevin-1 by agrin. Furthermore, we show that α-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus α-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that α-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.
A cytoplasmic dynein required for mitotic aster formation in vivo
Authors: Inoue Satoshi , Yoder O. C. , Turgeon B. Gillian , Aist James R. 
Published: April 2021

ABSTRACT
An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.
α-SNAP but not γ-SNAP is required for ER-Golgi transport after vesicle budding and the Rab1-requiring step but before the EGTA-sensitive step
Authors: Peter Frank , Wong Siew Heng , Subramaniam V. Nathan , Tang Bor Luen , Hong Wanjin 
Published: April 2021

ABSTRACT
N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) have been implicated in diverse vesicular transport events; yet their exact role and site of action remain to be established. Using an established in vitro system, we show that antibodies against α-SNAP inhibit vesicle transport from the ER to the cis-Golgi and that recombinant α-SNAP enhances/stimulates the process. Cytosol immunodepleted of α-SNAP does not support normal transport unless supplemented with recombinant α-SNAP but not γ-SNAP. In marked contrast, cytosol immunodepleted of γ-SNAP supports ER-Golgi transport to the normal level. Neither antibodies against γ-SNAP nor recombinant γ-SNAP have any effect on ER-Golgi transport. These results clearly establish an essential role for α-SNAP but not γ-SNAP in ER-Golgi transport. When the transport assay is performed with cytosol immunodepleted of α-SNAP, followed by incubation with cytosol immunodepleted of a COPII subunit, normal transport is achieved. In marked contrast, no transport is detected when the assay is first performed with cytosol depleted of the COPII subunit followed by α-SNAP-depleted cytosol, suggesting that α-SNAP is required after a step that requires COPII (the budding step). In combination with cytosol immunodepleted of Rab1, it is seen that α-SNAP is required after a Rab1-requiring step. It has been shown previously that EGTA blocks ER-Golgi transport at a step after vesicle docking but before fusion and we show here that α-SNAP acts before the step that is blocked by EGTA. Our results suggest that α-SNAP may be involved in the pre-docking or docking but not the fusion process.
Suppression of tumorigenicity in an aggressive cervical carcinoma induced by protein zero, a nervous system IgCAM
Authors: Spiryda Lisa B. , Colman David R. 
Published: April 2021

ABSTRACT
In mammals, protein zero (P0), a neural IgCAM, is expressed solely in the peripheral nervous system where it mediates self-adhesion of Schwann cell membranes as compact myelin is generated. We show that when P0 is expressed in HeLa, a cervical carcinoma cell line, cells regain adhesion-mediated growth control, including the acquisition of contact inhibition and loss of anchorage-independent growth. Additionally, P0-expressing HeLa cells lose the ability to invade an artificial matrix, which correlates with decreased secretion of matrix-degrading enzymes. Lastly, and of great interest, unlike the aggressively metastatic cell line from which they were derived, P0-HeLa cells are neither tumorigenic nor metastatic when injected into athymic nude mice. By all these criteria, P0 expression appears to efficiently suppress in the long term, the transformed state of this carcinoma cell line. N-cadherin and its intracellular partners plakoglobin, α- and β-catenin were significantly upregulated in the P0-HeLa cells. It appears therefore that P0 induces epithelialization and suppression of tumorigenicity in HeLa through the activation of the cadherin/catenin signaling systems. We conclude that the forced expression of bona fide adhesion molecules, such as P0, may serve as ‘upstream’ inducers of an essentially dormant but undamaged adhesion program in carcinoma cells that ultimately triggers the re-acquisition of normal epithelial characteristics, thereby suppressing tumorigenicity. Therapeutically, it may be that intercellular adhesion, no matter how it is induced, may serve as a single master event that is able to induce reversion of the carcinomatous state.
DNA replication-dependent intranuclear relocation of double minute chromatin
Authors: Itoh Nobuo , Shimizu Noriaki 
Published: April 2021

ABSTRACT
Double minutes (DMs) seen in a substantial fraction of human tumors are the cytogenetic manifestation of gene amplification which renders the tumor cells advantageous for growth and survival. DMs are acentric and atelomeric chromatin composed of circular DNA. In this study, we found they showed a remarkable relocation inside the nucleus which was spatially and temporally coupled to DNA replication. Using the human COLO 320DM tumor line, we detected DMs by fluorescence in situ hybridization followed by confocal examination. The location of multi-copy DMs was evaluated statistically by an easy method developed in this study. By examination of a synchronized culture, we found that DMs preferentially located at the nuclear periphery during G1-phase of the cell cycle, which is consistent with the location at M-phase. The peripheral DMs were in contact with the nuclear lamina which was shown by the simultaneous detection of DMs and lamin protein. The peripheral location persisted until the cells reached the G1/S-boundary, then the DMs relocated promptly to inward once the DNA replication started. The relocation was obvious using two different probes that detect DMs, or using two different methods for the cell fixation. Furthermore, the simultaneous detection of DMs and the site of DNA replication suggested that the inward relocation of peripheral DMs initiated just prior to the onset of DNA replication at the periphery. On the other hand, if the same amplified sequences were placed in a chromosome as an homogeneously staining region, they did not show any significant relocation during S-phase. From these and reported results, there may exist a generalized inward motion of some kind of chromatin that precedes the replication of their DNA. DMs might magnify the motion by their acentric, atelomeric or small circular nature.
Synaptojanin family members are implicated in endocytic membrane traffic in yeast
Authors: Singer-Krüger Birgit , Nemoto Yasuo , Daniell Laurie , Ferro-Novick Susan , Camilli Pietro De 
Published: April 2021

ABSTRACT
The synaptojanins represent a subfamily of inositol 5’-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Δsjl1 Δsjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Δsjl2 Δsjl3 mutant, while endocytosis is normal in a Δsjl1 Δsjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.
The recycling pathway of protein ERGIC-53 and dynamics of the ER-Golgi intermediate compartment
Authors: Klumperman Judith , Schweizer Anja , Clausen Henrik , Tang Bor Luen , Hong Wanjin , Oorschot Viola , Hauri Hans-Peter 
Published: April 2021

ABSTRACT
To establish recycling routes in the early secretory pathway we have studied the recycling of the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 in HepG2 cells. Immunofluorescence microscopy showed progressive concentration of ERGIC-53 in the Golgi area at 15°C. Upon rewarming to 37°C ERGIC-53 redistributed into the cell periphery often via tubular processes that largely excluded anterograde transported albumin. Immunogold labeling of cells cultured at 37°C revealed ERGIC-53 predominantly in characteristic β-COP-positive tubulo-vesicular clusters both near the Golgi apparatus and in the cell periphery. Concentration of ERGIC-53 at 15°C resulted from both accumulation of ERGIC-53 in the ERGIC and movement of ERGIC membranes closer to the Golgi apparatus. Upon rewarming to 37°C the labeling of ERGIC-53 in the ERGIC rapidly returned to normal levels whereas ERGIC-53’s labeling in the cis-Golgi was unchanged. Temperature manipulations had no effect on the average number of ERGIC-53 clusters. Density gradient centrifugation indicated that the surplus ERGIC-53 accumulating in the ERGIC at 15°C was rapidly transported to the ER upon rewarming. These results suggest that the ERGIC is a dynamic membrane system composed of a constant average number of clusters and that the major recycling pathway of ERGIC-53 bypasses the Golgi apparatus.
The role of sequences unique to nuclear intermediate filaments in the targeting and assembly of human lamin B: evidence for lack of interaction of lamin B with its putative receptor
Authors: Mical Timothy I. , Monteiro Mervyn J. 
Published: April 2021

ABSTRACT
The mechanism by which human nuclear lamin B is targeted and assembled has been studied by transfecting into mammalian cells lamin mutants deleted of three sequences unique to lamins. Nuclear lamins contain an extra 42 amino acids (aa) in their rod domains and NLS and CAAX motifs in their tail domains, which distinguishes them from cytoplasmic IF proteins. These three sequences act in concert to ensure correct temporal and spatial assembly of lamin B. Deletion of any one of these three sequences from lamin B did not significantly disrupt nuclear lamina targeting, but when two or more of these sequences were deleted, targeting was severely compromised. The CAAX motif is necessary for the efficient integration of lamin B into an already formed nuclear lamina, since lamin B CAAX–mutants had reduced targeting to the lamina when arrested in S phase of the cell cycle. CAAX-deficient mutant lamin B proteins were soluble and not associated with membranes at mitosis, proving that the CAAX motif is responsible for association of human lamin B with membranes. In addition, CAAX–mutant lamin B proteins fractionated independently of the lamin B-receptor (LBR), indicating that these two proteins do not bind directly to each other.