bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transmembrane-4-superfamily proteins CD151 and CD81 associate with α3β1 integrin, and selectively contribute to α3β1-dependent neurite outgrowth
Authors: Stipp Christopher S. , Hemler Martin E. 
Published: April 2021

ABSTRACT
Proteins in the transmembrane-4-superfamily (TM4SF) form many different complexes with proteins in the integrin family, but the functional utility of these complexes has not yet been demonstrated. Here we show that TM4SF proteins CD151, CD81, and CD63 co-distribute with α3β1 integrin on neurites and growth cones of human NT2N cells. Also, stable CD151-α3β1 and CD81-α3β1 complexes were recovered in NT2N detergent lysates. Total NT2N neurite outgrowth on laminin-5 (a ligand for α3β1 integrin) was strongly inhibited by anti-CD151 and -CD81 antibodies either together (∼85% inhibition) or alone (∼45% inhibition). Notably, these antibodies had no inhibitory effect on NT2N neurites formed on laminin-1 or fibronectin, when α3β1 integrin was not engaged. Neurite number, length, and rate of extension were all affected by anti-TM4SF antibodies. In summary: (1) these substrate-dependent inhibition results strongly suggest that CD151 and CD81 associations with α3β1 are functionally relevant, (2) TM4SF proteins CD151 and CD81 make a strong positive contribution toward neurite number, length, and rate of outgrowth, and (3) NT2N cells, a well-established model of immature central nervous system neurons, can be a powerful system for studies of integrin function in neurite outgrowth and growth cone motility.
N-terminal targeting of guanine nucleotide exchange factors (GEF) for ADP ribosylation factors (ARF) to the Golgi
Authors: Lee Stella Y. , Pohajdak Bill 
Published: April 2021

ABSTRACT
B2-1 (cytohesin-1) is a member of a group of proteins (including ARNO and ARNO3) that are all of similar size and domain composition. The three proteins contain an N-terminal coiled-coil domain, followed by a Sec7 and a pleckstrin homology (PH) domain. While it is well established that the Sec7 domain functions as a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factors (ARFs) and the PH domain anchors the proteins to membrane phosphoinositols, the function of the N-terminal domain is unknown. Here we show that the N terminus of B2-1 (residues 1-54) is necessary and sufficient to target the protein to the Golgi. The Sec7+PH domains of B2-1 (residues 55-398) are not sufficient for Golgi localization. Further deletion analysis and point mutagenesis indicate that the coiled-coil domain within the N terminus is responsible for Golgi targeting. Furthermore, ARNO and ARNO3 N termini also have the same capability of targeting to the Golgi. We conclude that the N-terminal, α-helical, coiled-coil domain is used to target this family of proteins to the Golgi complex.
Centromere clustering is a major determinant of yeast interphase nuclear organization
Authors: Jin Quan-wen , Fuchs Jörg , Loidl Josef 
Published: April 2021

ABSTRACT
During interphase in the budding yeast, Saccharomyces cerevisiae, centromeres are clustered near one pole of the nucleus as a rosette with the spindle pole body at its hub. Opposite to the centromeric pole is the nucleolus. Chromosome arms extend outwards from the centromeric pole and are preferentially directed towards the opposite pole. Centromere clustering is reduced by the ndc10 mutation, which affects a kinetochore protein, and by the microtubule poison nocodazole. This suggests that clustering is actively maintained or enforced by the association of centromeres with microtubules throughout interphase. Unlike the Rabl-orientation known from many higher eukaryotes, centromere clustering in yeast is not only a relic of anaphase chromosome polarization, because it can be reconstituted without the passage of cells through anaphase. Within the rosette, homologous centromeres are not arranged in a particular order that would suggest somatic pairing or genome separation.
Yeast colonies synchronise their growth and development
Authors: Palková Zdena , Forstová Jitka 
Published: April 2021

ABSTRACT
The ability to emit and receive signals over long distances is one of the characteristic attributes of multicellular organisms. Such communication can be mediated in different manners (by chemical compounds, light waves, acoustic waves etc.) and usually is reflected in the behaviour of the communicating organisms. Recently, we reported that individual yeast colonies, organised multicellular structures, can also communicate at long distance by means of volatile ammonia, which is produced by colonies in pulses separated by acidification of the medium. Here, we demonstrate that the colony that first reached the stage of intense ammonia production induces ammonia production response in surrounding colonies regardless of their age, causing the synchronisation of their NH3 pulses and, consequently, the mutual affection of their growth. Also an artificial source of ammonia (but neither NH4+ nor NaOH gradients) can immediately induce the ammonia production even in the colony starting its acidic stage of the development. The repeated transition of Candida mogii colonies from the acidic phase to the phase of intensive ammonia production is accompanied by dramatic changes in colony morphology and also in cell morphology and growth. Relatively smooth colonies in the acidic phase are formed by growing pseudohyphae. After ammonia induction, pseudohyphae decompose into non-dividing yeast-like cells, which rearrange themselves into ruffled spaghetti-like structures. The synchronisation of colony growth, that also exists between yeast colonies of different genera, could be important in establishing their optimal distribution in a natural habitat.
The NIMA-related kinase X-Nek2B is required for efficient assembly of the zygotic centrosome in Xenopus laevis
Authors: Fry Andrew M. , Descombes Patrick , Twomey Ciara , Bacchieri Rachid , Nigg Erich A. 
Published: April 2021

ABSTRACT
Nek2 is a mammalian cell cycle-regulated serine/threonine kinase that belongs to the family of proteins related to NIMA of Aspergillus nidulans. Functional studies in diverse species have implicated NIMA-related kinases in G2/M progression, chromatin condensation and centrosome regulation. To directly address the requirements for vertebrate Nek2 kinases in these cell cycle processes, we have turned to the biochemically-tractable system provided by Xenopus laevis egg extracts. Following isolation of a Xenopus homologue of Nek2, called X-Nek2B, we found that X-Nek2B abundance and activity remained constant through the first mitotic cycle implying a fundamental difference in Nek2 regulation between embryonic and somatic cell cycles. Removal of X-Nek2B from extracts did not disturb either entry into mitosis or the accompanying condensation of chromosomes providing no support for a requirement for Nek2 in these processes at least in embryonic cells. In contrast, X-Nek2B localized to centrosomes of adult Xenopus cells and was rapidly recruited to the basal body of Xenopus sperm following incubation in egg extracts. Recruitment led to phosphorylation of the X-Nek2B kinase. Most importantly, depletion of X-Nek2B from extracts significantly delayed both the assembly of microtubule asters and the recruitment of γ-tubulin to the basal body. Hence, these studies demonstrate that X-Nek2B is required for efficient assembly of a functional zygotic centrosome and highlight the possibility of multiple roles for vertebrate Nek2 kinases in the centrosome cycle.
An isoform of kinesin light chain specific for the Golgi complex
Authors: Gyoeva Fatima K. , Bybikova Ekaterina M. , Minin Alexander A. 
Published: April 2021

ABSTRACT
Conventional kinesin is a motor protein implicated in the transport of a variety of cytoplasmic organelles along microtubules. The kinesin molecule consists of two heavy chains with motor domains at their amino termini and two light chains, which, together with the carboxyl termini of the heavy chains, are proposed to mediate binding to cargoes. Since the light chains are represented by multiple isoforms diverging at their carboxyl termini they are presumed to specify kinesin targeting to organelles. Previously, we isolated five cDNAs, encoding hamster kinesin light chain isoforms, and found that one of them (B or C) preferentially associated with mitochondria. To obtain additional evidence proving the specific location of various kinesin light chain isoforms on organelles, we made an antibody against a 56 amino-acid sequence found at the carboxyl-terminal regions of the hamster D and E isoforms. By indirect immunofluorescence, this antibody specifically labeled the Golgi complex in cultured cells. In western blots of total cell homogenates, it recognized two close polypeptides, one of which co-purified with the Golgi membranes. Thus, the results of this and previous studies demonstrate that different kinesin light chains are associated with different organelles in cells.
LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity
Authors: Cuvillier Armelle , Redon Fabienne , Antoine Jean-Claude , Chardin Pierre , DeVos Theo , Merlin Gilles 
Published: April 2021

ABSTRACT
The small G protein-encoding LdARL-3A gene, a homologue of the human ARL-3 gene, was isolated from Leishmania donovani, and its protein product characterised. It is unique in the Leishmania genome and expressed only in the extracellular promastigote insect form, but not in the intracellular amastigote mammalian form, as shown by northern blots and western blots developed with a specific anti-C terminus immune serum. Indirect immunofluorescence microscopy revealed distinct labelled spots regularly distributed on the plasma membrane, including the part lining the flagellum and the flagellar pocket. By transfection experiments, it was found that wild-type LdARL-3A-overexpressing promastigotes reached higher densities in culture, but released significantly less secreted acid phosphatase in the extracellular medium than the parental strain. When LdARL-3A blocked under the GDP-bound ‘inactive’ form or with an inactivated potential myristoylation site was overexpressed, the cells displayed an apparent wild-type phenotype, but died earlier in the stationary phase; in contrast to parental cells, they showed a diffuse pattern of fluorescence labelling in the cytoplasm and on the cell membrane. Strikingly, when a constitutively ‘active’ form of LdARL-3A (blocked under the GTP-bound form) was overexpressed, the promastigotes were immobile with a very short flagellum, a slow growth rate and a low level of acid phosphatase secretion; the length of the flagellum was inversely proportional to mutant protein expression. We concluded that LdARL-3A could be an essential gene involved in flagellum biogenesis; it may provide new approaches for control of the parasite at the insect stage.
Caveolin proteins in signaling, oncogenic transformation and muscular dystrophy
Authors: Razani Babak , Schlegel Amnon , Lisanti Michael P. 
Published: April 2021

ABSTRACT
In adult animals and humans, signal transduction maintains homeostasis. When homeostatic mechanisms are interrupted, an illness or disease may ensue. Caveolae are plasma membrane specializations that contain the structural proteins caveolins, and appear to be important for normal signal transduction. The caveolin scaffolding domain interacts with several signaling molecules, sequestering them in the absence of activating signals, and thereby reducing the signal-to-noise ratio. Deletion and mutation of genes that encode caveolins is implicated in the pathogenesis of several human diseases. Down-regulation of caveolin-1 protein expression leads to deregulated signaling and consequently tumorigenesis, whereas naturally occurring dominant-negative caveolin-3 mutations cause muscular dystrophy.
Cdc7p-Dbf4p becomes famous in the cell cycle
Authors: Sclafani Robert A. 
Published: April 2021

ABSTRACT
Great insight into the molecular details of cell cycle regulation has been obtained in the past decade. However, most of the progress has been in defining the regulation of the family of cyclin-dependent kinases (CDKs). Recent studies of a myriad of eukaryotic organisms have defined both the regulation and substrates of Cdc7p kinase, which forms a CDK–cyclin-like complex with Dbf4p, is necessary for the initiation of DNA replication and has been conserved in evolution. This kinase is also required for the induction of mutations after DNA damage and for commitment to recombination in the meiotic cell cycle. However, less is known about the role of the kinase in these processes. In a manner similar to CDKs, Cdc7p is activated by a regulatory subunit, Dbf4, the levels of which fluctuate during the cell cycle. One or more subunits of the conserved MCM helicase complex at chromosomal origins of DNA replication are substrates for the kinase during S phase. Phosphorylation of the MCM complex by Cdc7p-Dbf4p might activate DNA replication by unwinding DNA. Therefore, activation of Cdc7p is required for DNA replication. Given that Cdc7p-Dbf4 kinase is overexpressed in many neoplastic cells and tumors, it might be an important early biomarker during cancer progression.
Dynein and plus-end microtubule-dependent motors are associated with specialized Sertoli cell junction plaques (ectoplasmic specializations)
Authors: Guttman Julian A. , Kimel Gil H. , Vogl A. Wayne 
Published: April 2021

ABSTRACT
The mechanism responsible for spermatid translocation in the mammalian seminiferous epithelium was proposed to be the microtubule-based transport of specialized junction plaques (ectoplasmic specializations) that occur in Sertoli cell regions attached to spermatid heads. These plaques each consist of a cistern of endoplasmic reticulum, a layer of actin filaments and the adjacent plasma membrane. It is predicted that motor proteins function to move the junction plaques, and hence the attached spermatids, first towards the base and then back to the apex of the epithelium, along microtubules. If this hypothesis is true, motor proteins should be associated with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. In addition, isolated junction plaques should support microtubule movement both in the plus and minus directions to account for the bidirectional translocation of spermatids in vivo. To determine if cytoplasmic dynein is localized to the endoplasmic reticulum of the plaques, perfusion-fixed rat testes were immunologically probed, at the ultrastructural level, for the intermediate chain of cytoplasmic dynein (IC74). In addition, testicular fractions enriched for spermatid/junction complexes were incubated with and without gelsolin, centrifuged and the supernatants compared, by western blot analysis, for Glucose Regulated Protein 94 (a marker for endoplasmic reticulum) and IC74. At the ultrastructural level, the probe for IC74 clearly labelled material associated with the cytoplasmic face of the endoplasmic reticulum component of the junction plaques. In the gelsolin experiments, both probes reacted more strongly with appropriate bands from the gelsolin-treated supernatants than with corresponding bands from controls. To determine if the junction plaques support microtubule transport in both directions, polarity-labelled microtubules were bound to isolated spermatid/junction complexes and then assessed for motility in the presence of ATP and testicular cytosol (2 mg/ml). Of 25 recorded motility events, 17 were in a direction consistent with a plus-end directed motor being present, and 8 were in the minus-end direction. The results are consistent with the conclusion that the junction plaques have the potential for moving along microtubules in both the plus and minus directions and that both a kinesin-type and a dynein-type motor may be associated with the junction plaques. The data also indicate that cytoplasmic dynein is localized to the cytoplasmic face of the endoplasmic reticulum component of the plaques.
COPI-coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites
Authors: Stephens David J. , Lin-Marq Nathalie , Pagano Alessandra , Pepperkok Rainer , Paccaud Jean-Pierre 
Published: April 2021

ABSTRACT
Transport of proteins between the endoplasmic reticulum and Golgi apparatus is mediated by two distinct membrane coat complexes, COPI and COPII. Genetic, biochemical and morphological data have accumulated into a model which suggests a sequential mode of action with COPII mediating the selection of cargo and formation of transport vesicles at the ER membrane for ER-to-Golgi transport and COPI mediating recycling of the transport machinery from post-ER membranes.
To test this transport model directly in vivo, and to study the precise temporal sequence of COPI and COPII action in ER-to-Golgi transport, we have used time lapse microscopy of living cells to visualise simultaneously the dynamics of COPII and COPI, as well as COPII and GFP tagged secretory markers in living cells. The majority of COPII labelling appears tightly associated with ER membranes that move only within a limited area (less than 2 μm). Secretory cargo segregates from these sites and is then transported to the Golgi apparatus without any apparent association with COPII. COPI-coated transport complexes are seen to form adjacent to the COPII sites on the ER before segregating and moving directionally towards the Golgi apparatus. COPII is not present on these transport complexes and remains associated with the ER. These data demonstrate for the first time directly in vivo that ER-to-Golgi transport is organised in two steps characterised by a sequential mode of action of COPII and COPI.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1368.html
Immobilized dimers of N-cadherin-Fc chimera mimic cadherin-mediated cell contact formation: contribution of both outside-in and inside-out signals
Authors: Lambert Mireille , Padilla Françoise , Mège René Marc 
Published: April 2021

ABSTRACT
Cell adhesion receptors of the cadherin family are involved in various developmental processes, affecting cell adhesion and migration, and also cell proliferation and differentiation. In order to dissect the molecular mechanisms of cadherin-based cell-cell adhesion and subsequent signal transduction to the cytoskeleton and/or cytoplasm leading to adapted cell responses, we developed an approach allowing us to mimic and control cadherin activation. We produced a dimeric N-cadherin-Fc chimera (Ncad-Fc) which retains structural and functional properties of cadherins, including glycosylation, Ca2+-dependent trypsin sensitivity and the ability to mediate Ca2+-dependent self-aggregation of covered microbeads. Beads covered with either Ncad-Fc or anti-N-cadherin antibodies specifically bound to N-cadherin expressing cells. Both types of beads induced the recruitment of N-cadherin, β-catenin, α-catenin and p120, by lateral mobilization of preexisting cell membrane complexes.
Furthermore, cadherin clustering elicited by Ncad-Fc beads triggered local accumulations of tyrosine phosphorylated proteins, a recruitment and redistribution of actin filaments, as well as local membrane remodeling. These results support a model where the adhesion of cadherin ectodomains is followed by clustering of cadherin/catenin complexes allowing signal transduction affecting both cytoskeletal reorganization and cytoplasmic signal mobilization (outside-in signaling). Interestingly, bead-cell binding was altered by agents promoting microfilament and microtubule depolymerization or tyrosine phosphorylation, indicating a possible regulation of the adhesive properties of the extracellular domain of N-cadherin by intracellular factors (inside-out signaling).
Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium
Authors: Dumontier Michel , Höcht Petra , Mintert Ursula , Faix Jan 
Published: April 2021

ABSTRACT
The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling macropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs4711.html, Movie 2, Movie 3, Movie 4, Movie 5
Nerve growth factor stimulates coupling of β1 integrin to distinct transport mechanisms in the filopodia of growth cones
Authors: Grabham Peter W. , Foley Mathew , Umeojiako Anuli , Goldberg Daniel J. 
Published: April 2021

ABSTRACT
The cycling of membrane receptors for substrate-bound proteins via their interaction with the actin cytoskeleton at the leading edge of growth cones and other motile cells is important for neurite outgrowth and cell migration. Receptor delivered to the leading edge binds to its ligand, which induces coupling of the receptor to a rearward flowing network of actin filaments. This coupling is thought to facilitate advance. We show here that a soluble growth factor stimulates this cycling. We have used single particle tracking to monitor the effects of nerve growth factor (NGF) on the movements of β1 integrin in the plane of the plasma membrane of the filopodia of growth cones. β1 integrin was visualized by its binding of 0.2 µm beads coated with a monoclonal Ab directed against an extracellular epitope distant from the binding site for extracellular matrix ligands. The beads were observed by video microscopy. Beads coated with a low concentration of antibody, and therefore bound to unliganded receptor with little cross-linking, showed an increase in both diffusion and directed forward transport in response to NGF. Transport had a net velocity of 37 µm/minute and was characterized by brief periods of sustained forward excursions with a velocity of 75-150 µm/minute. There was a 2-fold increase in the number of beads accumulated at the tips of filopodia after 10 minutes, indicating that NGF enhanced the delivery of β1 integrin to the periphery. Forward transport was dependent on an intact actin cytoskeleton and myosin ATPase, since treatment with cytochalasin D or the myosin ATPase inhibitor butanedione monoxime inhibited the transport but not the diffusion of receptors. NGF also greatly increased the steady rearward migration of beads coated with a high density of β1 integrin antibody, indicating that coupling of cross-linked receptor to the retrograde flow of actin is also enhanced. The rate of the retrograde flow of actin was unaffected by NGF. These studies show that a soluble factor can stimulate the coupling of a receptor for substrate-bound factor to two actomyosin-based transport mechanisms and thus facilitate the response of the growth cone to the substrate-bound factor by increasing cycling of the receptor at the periphery.