bluuebunny/crossref_metadata_2025_split huggingface dataset:
The budding yeast Dbf2 protein kinase localises to the centrosome and moves to the bud neck in late mitosis
Authors: Frenz Lisa M. , Lee Sarah E. , Fesquet Didier , Johnston Leland H. 
Published: April 2021

ABSTRACT
Dbf2 is a multifunctional protein kinase in Saccharomyces cerevisiae that functions in transcription, the stress response and as part of a network of genes in exit from mitosis. By analogy with fission yeast it seemed likely that these mitotic exit genes would be involved in cytokinesis. As a preliminary investigation of this we have used Dbf2 tagged with GFP to examine intracellular localisation of the protein in living cells. Dbf2 is found on the centrosomes/ spindle pole bodies (SPBs) and also at the bud neck where it forms a double ring. The localisation of Dbf2 is cell cycle regulated. It is on the SPBs for much of the cell cycle and migrates from there to the bud neck in late mitosis, consistent with a role in cytokinesis. Dbf2 partly co-localises with septins at the bud neck. A temperature-sensitive mutant of dbf2 also blocks progression of cytokinesis at 37°C. Following cytokinesis some Dbf2 moves into the nascent bud. Localisation to the bud neck depends upon the septins and also the mitotic exit network proteins Mob1, Cdc5, Cdc14 and Cdc15. The above data are consistent with Dbf2 acting downstream in a pathway controlling cytokinesis.
Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin
Authors: Berg Jonathan S. , Derfler Bruce H. , Pennisi Christopher M. , Corey David P. , Cheney Richard E. 
Published: April 2021

ABSTRACT
Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein’s distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with &lt;45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil. Like several other unconventional myosins and a plant kinesin, myosin-X contains both a myosin tail homology 4 (MyTH4) domain and a FERM (band 4.1/ezrin/radixin/moesin) domain. The unique tail domain also includes three pleckstrin homology domains, which have been implicated in phosphatidylinositol phospholipid signaling, and three PEST sites, which may allow cleavage of the myosin tail. Most intriguingly, myosin-X in cultured cells is present at the edges of lamellipodia, membrane ruffles, and the tips of filopodial actin bundles. The tail domain structure, biochemical features, and localization of myosin-X suggest that this novel unconventional myosin plays a role in regions of dynamic actin.
Lamina-associated polypeptide 2α binds intranuclear A-type lamins
Authors: Dechat Thomas , Korbei Barbara , Vaughan O. Anthony , Vlcek Sylvia , Hutchison Christopher J. , Foisner Roland 
Published: April 2021

ABSTRACT
The nucleoskeletal protein lamina-associated polypeptide 2α (LAP2α) contains a large, unique C terminus and differs significantly from its alternatively spliced, mostly membrane-integrated isoforms, such as LAP2β. Unlike lamin B-binding LAP2β, LAP2α was found by confocal immunofluorescence microscopy to colocalize preferentially with A-type lamins in the newly formed nuclei assembled after mitosis. While only a subfraction of lamins A and C (lamin A/C) was associated with the predominantly nuclear LAP2α in telophase, the majority of lamin A/C colocalized with LAP2α in G1-phase nuclei. Furthermore, selective disruption of A-type lamin structures by overexpression of lamin mutants in HeLa cells caused a redistribution of LAP2α. Coimmunoprecipitation experiments revealed that a fraction of lamin A/C formed a stable, SDS-resistant complex with LAP2α in interphase cells and in postmetaphase cell extracts. Blot overlay binding studies revealed a direct binding of LAP2α to exclusively A-type lamins and located the interaction domains to the C-terminal 78 amino acids of LAP2α and to residues 319-566 in lamin A/C, which include the C terminus of the rod and the entire tail common to lamin A/C. These findings suggest that LAP2α and A-type lamins cooperate in the organization of internal nuclear structures.
Omega speckles – a novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila
Authors: Prasanth K. V. , Rajendra T. K. , Lal A. K. , Lakhotia S. C. 
Published: April 2021

ABSTRACT
Fluorescence RNA:RNA in situ hybridization studies in various larval and adult cell types of Drosophila melanogaster showed that the noncoding hsr-omega nuclear (hsrω-n) transcripts were present in the form of many small speckles. These speckles, which we name ‘omega speckles’, were distributed in the interchromatin space in close proximity to the chromatin. The only chromosomal site where hsrω-n transcripts localized was the 93D locus or the hsrω gene itself. The number of nucleoplasmic speckles varied in different cell types. Heat shock, which inhibits general chromosomal transcription, caused the individual speckles to coalesce into larger but fewer clusters. In extreme cases, only a single large cluster of hsrω-n transcripts localizing to the hsrω locus was seen in each nucleus. In situ immunocytochemical staining using antibodies against heterogenous nuclear RNA binding proteins (hnRNPs) like HRB87F, Hrp40, Hrb57A and S5 revealed that, in all cell types, all the hnRNPs gave a diffuse staining of chromatin areas and in addition, were present as large numbers of speckles. Colocalization studies revealed an absolute colocalization of the hnRNPs and the omega speckles. Heat shock caused all the hnRNPs to cluster together exactly, following the hsrω-n transcripts. Immunoprecipitation studies using the hnRNP antibodies further demonstrated a physical association of hnRNPs and hsrω transcripts. The omega speckles are distinct from interchromatin granules since nuclear speckles containing serine/arginine-rich SR-proteins like SC35 and SRp55 did not colocalize with the omega speckles. The speckled distribution of hnRNPs was completely disrupted in hsrω nullosomics. We conclude that the hsrω-n transcripts play essential structural and functional roles in organizing and establishing the hnRNP-containing omega speckles and thus regulate the trafficking and availability of hnRNPs and other related RNA binding proteins in the cell nucleus.
Isoforms of caveolin-1 and caveolar structure
Authors: Fujimoto Toyoshi , Kogo Hiroshi , Nomura Ryuji , Une Tomoko 
Published: April 2021

ABSTRACT
The relationship between caveolin-1 isoforms (α and β) and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-αβ) were not labeled by another antibody specific for the α isoform (anti-α). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-α labeled deep caveolae preferentially over shallow ones, whereas anti-αβ labeled both forms with an equivalent frequency and intensity. The presence of the β isoform in deep caveolae was confirmed by labeling epitope-tagged β-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the α isoform formed caveolar depressions efficiently, but the β isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.
Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos
Authors: Leclerc Catherine , Webb Sarah E. , Daguzan Christiane , Moreau Marc , Miller Andrew L. 
Published: April 2021

ABSTRACT
Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.
Rab GTPases coordinate endocytosis
Authors: Rodman Jane Somsel , Wandinger-Ness Angela 
Published: April 2021

ABSTRACT
Endocytosis is characterized by vesicular transport along numerous pathways. Common steps in each pathway include membrane budding to form vesicles, transport to a particular destination, and ultimately docking and fusion with the target membrane. Specificity of vesicle targeting is rendered in part by associated Rab GTPases. This review summarizes current knowledge about Rab GTPase functions in the endocytic pathways and provides insight into the regulation of Rab GTPase activity and mechanisms of Rab protein function. Functional assays have identified some Rab proteins that operate on individual pathways, but Rab proteins in several pathways remain controversial or have not been identified. Control of Rab GTPase activity is exerted through multiple levels of regulation. Significant new information pertaining to Rab protein function in regulating transport has emerged. Remarkably, Rab5 GTPase links budding, cytoskeletal transport and docking/fusion activities. This paradigm will most likely be generally applicable to other Rab GTPase pathways. Together with the cross-talk between different Rab proteins and their effectors, this may provide an integrated system for the general coordination of endocytic pathways to maintain organelle homeostasis.
Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase
Authors: Johansson Nina , Ala-aho Risto , Uitto Veli-Jukka , Grénman Reidar , Fusenig Norbert E. , López-OtÍn Carlos , Kähäri Veli-Matti 
Published: April 2021

ABSTRACT
Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by transformed squamous epithelial cells, i.e. squamous cell carcinoma (SCC) cells in culture and in vivo. Here, we have elucidated the signaling pathways regulating MMP-13 expression in transformed human epidermal keratinocytes, i.e. ras-transformed HaCaT cell line A-5 and cutaneous SCC cell line (UT-SCC-7). Treatment with tumor necrosis factor-α (TNF-α) resulted in activation of extracellular signal-regulated kinase (ERK)1,2, Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) in both cell lines. In addition, transforming growth factor-β (TGF-β) activated p38 MAPK in both cell lines, and ERK2 in A-5 cells. Selective inhibition of p38 activity with SB 203580 abolished the enhancement of MMP-13, as well as collagenase-1 (MMP-1) and 92-kDa gelatinase (MMP-9) expression by TNF-α and TGF-β. Blocking the ERK1,2 pathway by PD 98059 had no effect on the induction of MMP-13 expression by TNF-α or TGF-β, but potently suppressed MMP-1 and MMP-9 production. Inhibition of p38 activity by SB 203580 also suppressed collagenolytic activity produced by both cell lines and inhibited invasion of TNF-α or TGF-β stimulated A-5 cells through type I collagen and reconstituted basement membrane (Matrigel). These results show that activation of p38 MAPK pathway plays a crucial role in the invasive phenotype of transformed squamous epithelial cells, suggesting p38 MAPK as a target to specifically inhibit their invasion.
BMP-2/ALK3 and HGF signal in parallel to regulate renal collecting duct morphogenesis
Authors: Gupta Indra R. , Macias-Silva Marina , Kim Sammy , Zhou Xiaopeng , Piscione Tino D. , Whiteside Catharine , Wrana Jeffrey L. , Rosenblum Norman D. 
Published: April 2021

ABSTRACT
Bone morphogenetic protein (BMP)-2 and hepatocyte growth factor (HGF) exert antagonistic effects on renal collecting duct formation during embryogenesis. A current model proposes HGF inhibits BMP-2 signaling at the level of Smad1 in a common target cell. Here, we show that BMP-2 and HGF control collecting duct formation via parallel pathways. We examined the interactions between BMP-2 and HGF in the mIMCD-3 model of collecting duct morphogenesis. During tubule formation, HGF rescued the inhibitory effects of BMP-2 and of a constitutive active form of the BMP-2 receptor, ALK3, stably expressed in mIMCD-3 cells. To determine whether the effect of HGF occurs through known mediators which act downstream of the BMP-2/ALK3 complex, we examined the effect of HGF on BMP-2-induced Smad1 phosphorylation, Smad1/Smad4 complex formation, and Smad1 nuclear translocation.
Neither HGF nor other receptor tyrosine kinase ligands (EGF, FGF-4) induced phosphorylation of endogenous Smad1 in mIMCD-3 cells or in Mv1Lu, MC3T3-E1 or P19 cells. Furthermore, none of these ligands blocked induction of the BMP-responsive promoter, Tlx2. Thus, HGF overcomes the inhibitory effects of BMP-2 on collecting duct morphogenesis without interrupting any of the known signaling events in the BMP-2 dependent Smad1 signaling pathway. We conclude that BMP-2/ALK3 and HGF function to control parallel pathways downstream of their respective cell surface receptors. Integration of these signals likely occurs at the level of transcriptional or post-transcriptional events.
Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization
Authors: Baciu P. C. , Saoncella S. , Lee S. H. , Denhez F. , Leuthardt D. , Goetinck P. F. 
Published: April 2021

ABSTRACT
Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.
Interaction of folate receptor with signaling molecules lyn and Gαi-3 in detergent-resistant complexes from the ovary carcinoma cell line IGROV1
Authors: Miotti Silvia , Bagnoli Marina , Tomassetti Antonella , Colnaghi Maria Ines , Canevari Silvana 
Published: April 2021

ABSTRACT
Using as a model the ovary carcinoma cell line IGROV1, we analyzed the partitioning of the glycosyl-phosphatidylinositol-anchored folate receptor into lipid rafts based on its relative detergent insolubility, with a focus on physically and functionally associated signaling molecules. A variable amount (40-60%) of folate receptor was found in low-density Triton X-100 insoluble complexes together with subunits of heterotrimeric G-proteins and the src-family non-receptor tyrosine kinases p53-56 lyn. In the same fraction the structural component of caveolae, caveolin, was not detected at the protein level, although the corresponding mRNA was detected in trace amounts. Comodulation of folate receptor and signalling molecules was observed in the detergent-insoluble complexes during cell proliferation or induced by phosphatidylinositol-specific phospholipase C treatment or by interaction with anti-folate receptor monoclonal antibodies. Moreover, complexes of folate receptor, lyn and the Gαi-3 subunit were immunoprecipitated using either anti-folate receptor or anti-lyn antibodies. In vitro kinase assay of the immunoprecipitates revealed stimulation of phosphorylation of common and specific proteins. In particular, the p53 form of lyn appeared to be enriched and phosphorylated in the anti-folate receptor MOv19 monoclonal antibody immunoprecipitate, whereas a 40 kDa band common to anti-folate receptor and anti-lyn immunoprecipitates was the phosphorylated form of the Gαi-3 subunit. These findings point to the functional interaction between folate receptor and associated signaling molecules.
The cytoplasmic domain of the interleukin-6 receptor gp80 mediates its basolateral sorting in polarized Madin-Darby canine kidney cells
Authors: Martens Astrid S. , Bode Johannes G. , Heinrich Peter C. , Graeve Lutz 
Published: April 2021

ABSTRACT
The IL-6 receptor complex is expressed in different polarized epithelial cells such as liver hepatocytes and intestinal cells. It consists of two subunits: gp80, which binds the ligand, and gp130, which is responsible for signal transduction. In stably transfected Madin-Darby canine kidney (MDCK) cells we have studied the localization of the human IL-6 receptor subunits and found that gp80 and gp130 are predominantly expressed at the basolateral membrane. Analysis of MDCK cells expressing truncated forms of gp80 or gp130 showed that loss of the cytoplasmic domains results in apical delivery. Expression of deletion mutants of gp80 in MDCK cells led to the identification of two discontinous motifs responsible for basolateral sorting: a membrane-proximal tyrosine-based motif (YSLG) and a more membrane-distal dileucine-type motif (LI). Activation of signal transducer and activator of transcription-3 (STAT-3) only occurred via basolaterally located gp80, suggesting that endogenous gp130 is also constrained to the basolateral plasma membrane. Our identification of a basolateral sorting signal within the cytoplasmic region of gp80 for the first time attributes a function to this domain.
Senescence-associated β-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells
Authors: Kurz David J. , Decary Stephanie , Hong Ying , Erusalimsky Jorge D. 
Published: April 2021

ABSTRACT
Senescence-associated β-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal β-galactosidase. Here we have investigated the origin of senescence-associated-β-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A1, as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular β-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of β-galactosidase activity three-to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in β-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in β-galactosidase. These findings demonstrate that senescence-associated β-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.
The p58-positive pre-Golgi intermediates consist of distinct subpopulations of particles that show differential binding of COPI and COPII coats and contain vacuolar H+-ATPase
Authors: Ying Ming , Flatmark Torgeir , Saraste Jaakko 
Published: April 2021

ABSTRACT
We have studied the structural and functional properties of the pre-Golgi intermediate compartment (IC) in normal rat kidney cells using analytical cell fractionation with p58 as the principal marker. The sedimentation profile (sediterm) of p58, obtained by analytical differential centrifugation, revealed in steady-state cells the presence of two main populations of IC elements whose average sedimentation coefficients, sH=1150±58S (‘heavy’ and sL=158±8S (‘light’, differed from the s-values obtained for elements of the rough and smooth endoplasmic reticulum. High resolution analysis of these subpopulations in equilibrium density gradients further revealed that the large difference in their s-values was mainly due to particle size. The ‘light’ particle population contained the bulk of COPI and COPII coats, and redistribution of p58 to these particles was observed in transport-arrested cells, showing that the two types of elements are also compositionally distinct and have functional counterparts in intact cells. Using a specific antibody against the 16 kDa proteolipid subunit of the vacuolar H+-ATPase, an enrichment of the Vo domain of the ATPase was observed in the p58-positive IC elements. Interestingly, these elements could contain both COPI and COPII coats and their density distribution was markedly affected by GTPγS. Together with morphological observations, these results demonstrate that, in addition to clusters of small tubules and vesicles, the IC also consists of large-sized structures and corroborate the proposal that the IC elements contain an active vacuolar H+-ATPase.