bluuebunny/crossref_metadata_2025 huggingface dataset:
Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility
Authors: Le Phuong U. , Benlimame Naciba , Lagana Annick , Raz Avraham , Nabi Ivan R.
Published: April 2021

Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-β-cyclodextrin (mβCD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin receptor-negative, LAMP-1/lgpA-negative endocytic compartment identified by electron microscopy as a multivesicular body (MVB). Endocytosed AMF recycles to cell surface fibrillar structures which colocalize with fibronectin; AMF-R recycling is inhibited at 20°C, which blocks endocytosis past the early endosome, but not by mβCD demonstrating that AMF-R recycling to fibronectin fibrils is mediated by clathrin-dependent endocytosis to MVBs. Microtubule disruption with nocodazole did not affect delivery of bAMF to cell surface fibrils indicating that recycling bAMF traverses the MVB but not a later endocytic compartment. Plating NIH-3T3 cells on an AMF coated substrate did not specifically affect cell adhesion but prevented bAMF delivery to cell surface fibronectin fibrils and reduced cell motility. AMF-R internalization and recycling via the clathrin-mediated pathway are therefore rate-limiting for cell motility. This recycling pathway to the site of deposition of fibronectin may be implicated in the de novo formation of cellular attachments or the remodeling of the extracellular matrix during cell movement.
Biochemical characterization and localization of the dual specificity kinase CLK1
Authors: Menegay Harry J. , Myers Michael P. , Moeslein Fred M. , Landreth Gary E.
Published: April 2021

CLK1 was one of the first identified dual specificity kinases and is the founding member of the ‘LAMMER’ family of kinases. We have established the substrate site specificity of CLK1. We report here that truncation of the N terminus of CLK1 resulted in a dramatic increase in CLK1 enzymatic activity, indicating that the N terminus acts as a negative regulatory domain. The N-terminal truncation resulted in a 45-fold increase in Vmax, suggesting that this domain does not contain a pseudo-substrate motif, but may act to conformationally constrain the catalytic activity of CLK1. Tyrosine phosphorylation has been proposed to be critical for CLK1 activity, however, CLK1 activity was unaffected by exposure to tyrosine phosphatases. Treatment of CLK1 with the serine/threonine specific phosphatase PP2A, resulted in a 2-to 6-fold increase in enzymatic activity. Incubation of CLK1 with tyrosine phosphatases in combination with PP2A abolished CLK1 activity. These data suggest that CLK1 is regulated by three distinct mechanisms that serve to both positively and negatively regulate CLK1 activity. CLK1 activity is positively regulated by phosphorylation on either tyrosine residues or serine/threonine residues, and is negatively regulated by steric constraints mediated by the N-terminal domain, as well as, by phosphorylation on a subset of serine/threonine residues within the catalytic domain. CLK1 mRNA is expressed at low levels in all tissues and cell lines examined. The full-length and truncated splice forms are expressed at roughly equivalent levels in most tissues. The ratio of the two splice variants of CLK1 can be altered by treatment with cycloheximide. CLK1 protein expression is limited to a small subset of highly localized neuronal populations in the rat brain. Contrary to previous studies using overexpression systems, we show that CLK1 protein is primarily found in the cytoplasm of these cells, with only a small fraction localized to the nucleus.
A myosin family tree
Authors: Hodge Tony , Cope M. Jamie T. V.
Published: April 2021

This unrooted phylogenetic tree of the myosin superfamily (see insert) is derived from an alignment of 139 members of the myosin superfamily. The alignment compared the core motor domains (equivalent to residues 88-780 of chicken skeletal myosin II) of each myosin, using distance matrix analysis performed with the Clustal-W package. The exceptions, shown with a dotted line, are SsVIIa, which is a partial sequence as reported in the databases, and Hs MysPDZ. The latter is reported as a complete coding sequence but has a truncated N terminus starting some 52 residues into the core motor region (i.e. residue 140 of chicken skeletal myosin II). These shorter sequences have no significant effect on the branching order of the rest of the tree.
Positive and negative regulation of telomerase access to the telomere
Authors: Evans Sara K. , Lundblad Victoria
Published: April 2021

The protective caps on chromosome ends – known as telomeres – consist of DNA and associated proteins that are essential for chromosome integrity. A fundamental part of ensuring proper telomere function is maintaining adequate length of the telomeric DNA tract. Telomeric repeat sequences are synthesized by the telomerase reverse transcriptase, and, as such, telomerase is a central player in the maintenance of steady-state telomere length. Evidence from both yeast and mammals suggests that telomere-associated proteins positively or negatively control access of telomerase to the chromosome terminus. In yeast, positive regulation of telomerase access appears to be achieved through recruitment of the enzyme by the end-binding protein Cdc13p. In contrast, duplex-DNA-binding proteins assembled along the telomeric tract exert a feedback system that negatively modulates telomere length by limiting the action of telomerase. In mammalian cells, and perhaps also in yeast, binding of these proteins probably promotes a higher-order structure that renders the telomere inaccessible to the telomerase enzyme.
Exosome: from internal vesicle of the multivesicular body to intercellular signaling device
Authors: Denzer Kristin , Kleijmeer Monique J. , Heijnen Harry F. G. , Stoorvogel Willem , Geuze Hans J.
Published: April 2021

Exosomes are small membrane vesicles that are secreted by a multitude of cell types as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane. Depending on their origin, exosomes can play roles in different physiological processes. Maturing reticulocytes externalize obsolete membrane proteins such as the transferrin receptor by means of exosomes, whereas activated platelets release exosomes whose function is not yet known. Exosomes are also secreted by cytotoxic T cells, and these might ensure specific and efficient targeting of cytolytic substances to target cells. Antigen presenting cells, such as B lymphocytes and dendritic cells, secrete MHC class-I- and class-II-carrying exosomes that stimulate T cell proliferation in vitro. In addition, dendritic-cell-derived exosomes, when used as a cell-free vaccine, can eradicate established murine tumors. Although the precise physiological target(s) and functions of exosomes remain largely to be resolved, follicular dendritic cells (accessory cells in the germinal centers of secondary lymphoid organs) have recently been shown to bind B-lymphocyte-derived exosomes at their cell surface, which supports the notion that exosomes play an immunoregulatory role. Finally, since exosomes are derived from multivesicular bodies, their molecular composition might provide clues to the mechanism of protein and lipid sorting in endosomes.
Myosin light chain kinase plays an essential role in S. flexneri dissemination
Authors: Rathman Michelle , Lanerolle Primal de , Ohayon Hélène , Gounon Pierre , Sansonetti Philippe
Published: April 2021

Shigella flexneri, the causitive agent of bacillary dysentery, has been shown to disseminate in colonic epithelial cells via protrusions that extend from infected cells and are endocytosed by adjacent cells. This phenomenon occurs in the region of the eukaryotic cell’s adherens junctions and is inhibited by pharmacological reagents or host cell mutations that completely disrupt the junctional complex. In this study, inhibitors of the myosin light chain kinase (MLCK) were shown to dramatically decrease intercellular spread of S. flexneri but to have no inhibitory effect on bacterial entry, multiplication or actin-based motility within the host cell. Furthermore, cell-to-cell spread of Listeria monocytogenes, another bacterial pathogen that uses an actin-based mechanism to move within the eukaryotic cytoplasm and to spread from cell to cell, was not affected by the MLCK inhibitors, indicating that (1) the inhibition of S. flexneri cell-to-cell spread in treated cells is not due to a complete break down of cell-cell contacts, which was subsequently confirmed by confocal microscopy, and (2) MLCK plays a role in a S. flexneri-specific mechanism of dissemination. Myosin has been shown to play a role in a variety of membrane-based phenomena. The work presented here suggests that activation of this molecule via phosphorylation by MLCK, at the very least participates in the formation of the bacteria-containing protrusion, and could also contribute to the endocytosis of this structure by neighboring cells.
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

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

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 <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

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

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

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

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.
I. How to hook a PI. The science dating game
Authors: A Caveman
Published: April 2021

Are you considering a short-term relationship with a PI (Principal Investigator)â€¦?
Rab GTPases coordinate endocytosis
Authors: Rodman Jane Somsel , Wandinger-Ness Angela
Published: April 2021

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

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

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

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

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

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

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

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.
Inhibition of MMH (Met murine hepatocyte) cell differentiation by TGFβ is abrogated by pre-treatment with the heritable differentiation effector FGF1
Authors: Spagnoli Francesca M. , Cicchini Carla , Tripodi Marco , Weiss Mary C.
Published: April 2021

MMH (Met murine hepatocyte) liver cells derived from transgenic mice expressing a truncated constitutively active form of human c-Met are non-transformed immortalized cell lines. We have previously shown that they harbor: (1) epithelial cells that express the liver-enriched transcription factors HNF4 and HNF1α, and that can be stably induced by FGF1 to express liver functions, and (2) fibroblast-like bi-potential palmate cells that can differentiate into bile duct-like structures in Matrigel cultures, or into epithelial cells competent to express hepatic functions. Low concentrations of TGFβ have been found to inhibit growth and differentiation of MMH cells. The factor stabilized the palmate cell phenotype, and it provoked epithelial cells to acquire palmate-like morphological characteristics, in parallel with down-regulation of expression of HNF4 and HNF1α and activation of Snail transcripts. The effects of TGFβ were dominant if it was added with FGF1, but the effects on differentiation were abrogated if cells had been pre-treated with FGF1. This work identifies TGFβ as a factor that could be implicated in maintaining bi-potential precursor cells in the liver, FGF1 as one that could over-ride the TGFβ effects and Snail as a candidate for mediation of the signal.
C-terminal inhibition of tau assembly in vitro and in Alzheimer’s disease
Authors: Abraha Aida , Ghoshal Nupur , Gamblin T. Chris , Cryns Vincent , Berry Robert W. , Kuret Jeff , Binder Lester I.
Published: April 2021

Alzheimer’s disease (AD) is, in part, defined by the polymerization of tau into paired helical and straight filaments (PHF/SFs) which together comprise the fibrillar pathology in degenerating brain regions. Much of the tau in these filaments is modified by phosphorylation. Additionally, a subset also appears to be proteolytically truncated, resulting in the removal of its C terminus. Antibodies that recognize tau phosphorylated at S396/404 or truncated at E391 do not stain control brains but do stain brain sections very early in the disease process. We modeled these phosphorylation and truncation events by creating pseudo-phosphorylation and deletion mutants derived from a full-length recombinant human tau protein isoform (ht40) that contains N-terminal exons 2 and 3 and all four microtubule-binding repeats. In vitro assembly experiments demonstrate that both modifications greatly enhance the rates of tau filament formation and that truncation increases the mass of polymer formed, as well. Removal of as few as 12 or as many as 121 amino acids from the C terminus of tau greatly increases the rate and extent of tau polymerization. However, deletion of an additional 7 amino acids, 314DLSKVTS320, from the third microtubule-binding repeat results in the loss of tau’s ability to form filaments in vitro. These results suggest that only part of the microtubule-binding domain (repeats 1, 2 and a small portion of 3) is crucial for tau polymerization. Moreover, the C terminus of tau clearly inhibits the assembly process; this inhibition can be partially reversed by site-specific phosphorylation and completely removed by truncation events at various sites from S320 to the end of the molecule.
Identification and characterization of Caenorhabditis elegans γ-tubulin in dividing cells and differentiated tissues
Authors: Bobinnec Yves , Fukuda Makoto , Nishida Eisuke
Published: April 2021

γ-Tubulin is an essential component of the microtubule-nucleation machinery and therefore plays a crucial role during mitosis. To gain further insights into the function of this protein in the events that take place during embryogenesis and differentiation, we carried out detailed studies on γ-tubulin during all the developmental stages of Caenorhabditis elegans. We identified the γ-tubulin gene from this organism and analyzed the localization of the protein by both immunofluorescence and GFP reporter construct. We show that γ-tubulin association with the centrosome is highly dynamic in mitotic cells, being massively recruited at prophase and released at anatelophase. This accumulation in mitotic centrosomes is dramatic during the first embryonic divisions. We provide the first description of the morphological changes at the centrosome level during the orientation of the mitotic spindle and the flattening of the posterior aster. Loss of function of the γ-tubulin gene by RNAi induces a strong polyploidization of mitotic germ cells and embryos, but does not affect meiosis and pronuclear migration. In addition, we demonstrate the prominent redistribution of γ-tubulin in adults at basal bodies of amphid and phasmid neurons, and at the apical membrane of polarized intestinal cells.
Nuclear and subnuclear targeting sequences of the protein phosphatase-1 regulator NIPP1
Authors: Jagiello Izabela , Eynde Aleyde Van , Vulsteke Veerle , Beullens Monique , Boudrez An , Keppens Stefaan , Stalmans Willy , Bollen Mathieu
Published: April 2021

NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the ‘ForkHead-Associated’ domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.
Caspase-mediated cleavage of the chromosome-binding domain of lamina-associated polypeptide 2α
Authors: Gotzmann Josef , Vlcek Sylvia , Foisner Roland
Published: April 2021

Lamina-associated polypeptide 2α (LAP2α) is a non-membrane-bound isoform of the LAP2 family involved in nuclear structure organization. Using various cell systems, including Jurkat, HL-60, and HeLa cells, and different death-inducing agents, such as anti-Fas antibody, topoisomerase inhibitors, and staurosporine, we found that LAP2α was cleaved during apoptosis as rapidly as lamin B in a caspase-dependent manner yielding stable N- and C-terminal fragments of approximately 50 and 28 kDa, respectively. Based on fragment size and localization of immunoreactive epitopes, four potential cleavage sites were mapped between amino acids 403-485. These sites were located within a domain that has previously been described to be essential and sufficient for association of LAP2α with chromosomes, suggesting that LAP2α cleavage impairs its chromatin-binding properties. Immunofluorescence microscopy demonstrated that, unlike full length protein, apoptotic fragments did not colocalize with condensed chromatin, but remained in the nuclear compartment as long as a single nucleus was visible. Subfractionation analyses showed that the N-terminal LAP2α fragment was extracted from intranuclear structures in detergent/ salt buffers, whereas the C-terminal fragment remained associated with a residual framework devoid of chromatin. Our data suggest that early cleavage of LAP2α is important for chromatin reorganization during apoptosis.
Alternative splicing of the mouse profilin II gene generates functionally different profilin isoforms
Authors: Nardo Alessia Di , Gareus Ralph , Kwiatkowski David , Witke Walter
Published: April 2021

Profilins are a conserved family of proteins participating in actin dynamics and cell motility. In the mouse, two profilin genes are known. Profilin I is expressed universally at high levels, while profilin II is expressed mainly in the brain. Here we describe the occurrence of two mouse profilin II isoforms, A and B, which are derived by alternative splicing. They are identical through residue 107 of the protein, but then have distinct C-terminal sequences. Profilin IIA binds to poly-L-proline and actin with high affinity similar to profilin I. Profilin IIB on the other hand does not bind to actin and the affinity for poly-L-proline is greatly diminished. However, tubulin was found to bind to GST-profilin IIB, and in vivo GFP-profilin IIB was recruited to spindles and asters during mitosis in HeLa cells. Our results indicate unexpected diversity in the functions of the profilin family of proteins, and suggest that in mouse profilin IIA is intimately involved in actin dynamics, while profilin IIB associates with other cytoskeletal components.
TGF-β1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation
Authors: Batard Pascal , Monier Marie-Noëlle , Fortunel Nicolas , Ducos Karin , Sansilvestri-Morel Patricia , Phan Thi-Haï , Hatzfeld Antoinette , Hatzfeld Jacques A.
Published: April 2021

Somatic stem cells are largely quiescent in spite of their considerable proliferative potential. Transforming growth factor-β1 (TGF-β1) appears to be a good candidate for controlling this quiescence. Indeed, various mutations in the TGF-β signalling pathway are responsible for neoplasic proliferation of primitive stem/progenitor cells in human tissues of various origins. In hemopoietic single cell culture assays, blocking autocrine and endogeneous TGF-β1 triggers the cell cycling of high proliferative potential undifferenciated stem/progenitor cells. However, it has never been demonstrated whether TGF-β1 has an apoptotic effect or a differentiating effect on these primitive cells, as already described for more mature cells.

Using single cell experiments both in liquid or semi-solid culture assays and dye tracking experiments by flow cytometry, we demonstrate that low, physiological concentrations of TGF-β1, which specifically maintain primitive human hemopoietic stem/progenitor cells in quiescence, have a reversible effect and do not induce apoptosis. We moreover demonstrate that these low concentrations prevent the rapid loss of the mucin-like protein CD34, a most common marker of immature hematopoietic stem/progenitor cells, which is progressively lost during differentiation. TGF-β1 not only up-modulated the CD34 antigen before S phase entry but also maintained a high level of CD34 expression on cells which had escaped cell cycle inhibition, suggesting that proliferation inhibition and differentiation control by TGF-β1 may be independent. These data provide additional evidence that TGF-β1 acts as a key physiological factor ensuring the maintenance of a stem cell reserve.
Low-affinity LFA-1/ICAM-3 interactions augment LFA-1/ICAM-1-mediated T cell adhesion and signaling by redistribution of LFA-1
Authors: Bleijs Diederik A. , Binnerts Minke E. , Vliet Sandra J. van , Figdor Carl G. , Kooyk Yvette van
Published: April 2021

Although ICAM-3 is implicated in both adhesion and signal transduction events of leukocytes, its low affinity for LFA-1 compared to other ligands of LFA-1 has puzzled many investigators. Here we investigated the role of ICAM-3 in supporting LFA-1-mediated ICAM-1 binding and subsequently cell signaling. We observed that although ICAM-3 binds poorly to LFA-1 expressed on resting T cells, it specifically facilitates and increases LFA-1-mediated adhesion to the high affinity ligand of LFA-1, ICAM-1. We demonstrate that low-affinity binding of LFA-1 to ICAM-3 together with ICAM-1 alters the cell surface distribution of LFA-1 dramatically, inducing large clusters of LFA-1 that facilitate ICAM-1 binding after LFA-1 activation. We found that LFA-1-mediated ICAM-1 cell-cell interactions such as T cell proliferation greatly depend on low affinity LFA-1/ICAM-3 interactions that enhance stable LFA-1/ICAM-1 cell-cell contact. Taken together, these data demonstrate that low affinity LFA-1 binding to ICAM-3 regulates strong LFA-1/ICAM-1-mediated adhesion by driving LFA-1 into clusters to facilitate cell-cell interactions that take place in the immune system.
Functional interaction between E-cadherin and αv-containing integrins in carcinoma cells
Authors: Schlippe Maria von , Marshall John F. , Perry Philip , Stone Michael , Zhu Alan Jian , Hart Ian R.
Published: April 2021

We have demonstrated the possibility of cross-talk between E-cadherin and αv integrins in breast carcinoma cells. Using the function-blocking anti-αv monoclonal antibody 17E6, applied to monolayer cultures of breast cancer lines, it was found that treatment of cells possessing detergent-insoluble (implying attachment to the actin cytoskeleton) E-cadherin resulted in the adoption of a spheroid configuration of cell growth. This effect was dependent upon not just αv occupancy but also receptor aggregation. Thus in vitro αv-dependent adhesion suppresses E-cadherin-mediated morphological changes. To investigate whether manipulation of E-cadherin would, conversely, modulate integrin activity we introduced a dominant-negative E-cadherin construct into one of the lines, ZR75-1, giving rise to the cell line ZR-E2R1. Surface expression of endogenous E-cadherin was downregulated (by around 25%), whereas β-catenin levels were increased two- to threefold in ZR-E2R1 cells. There was also a highly significant increase in migration of ZR-E2R1 cells (relative to control cells) toward vitronectin (P<0.001), but not toward collagen type I, fibronectin or laminin. Such increased migration could be abrogated totally by antibody blockade of αvβ5 and αvβ1 integrins. There was no detectable change in αv integrin levels. These data suggest that the introduction of a dominant-negative E-cadherin mutant into ZR75-1, in addition to a loss of cohesion, generates a signal (or signals) which increases migration towards vitronectin through increased activity of αv integrins.
Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis
Authors: Callanan Mary , Kudo Nobuaki , Gout Stephanie , Brocard Marie-Paule , Yoshida Minoru , Dimitrov Stefan , Khochbin Saadi
Published: April 2021

In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis. The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human). The protein and mRNA are maternally expressed and are present during early embryogenesis. However, our data show that the activity of the protein is developmentally regulated. Embryonic development is insensitive to leptomycin B, a specific inhibitor of CRM1, until the neurula stage. Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the leptomycin B sensitivity. These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression. Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development. This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.