bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier
Authors: Nico Beatrice , Frigeri Antonio , Nicchia Grazia Paola , Quondamatteo Fabio , Herken Rainer , Errede Mariella , Ribatti Domenico , Svelto Maria , Roncali Luisa
Published: April 2021

In this study, we have investigated the expression of aquaporin 4 during blood-brain barrier development in the optic tectum of chick embryos and newly hatched chicks, by means of western-blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and freeze-fracture and high-resolution immunogold electron microscopy.

In the optic tecta of day-14 embryos, western blot analysis revealed an approx. 30 kDa band, immunoreactive for aquaporin-4, which was increased in day-20 embryos and in chicks. Semi-quantitative reverse transcriptase chain reaction experiments showed that there was already a high level of aquaporin-4 mRNA in day-9 embryos as well as in the subsequent stages and in newly hatched chicks. Immunohistochemically, reactivity for aquaporin-4 was detected in the optic tectum of day-14 embryos; similar results were obtained in telencephalon and cerebellum. Ultrastructurally, the microvessels of the tectum showed immunoreactivity for aquaporin-4 on the astroglial endfeet, which discontinuously surrounded endothelial cells joined by immature tight junctions. In the tectum, telencephalon and cerebellum of 20-day embryos and chicks, aquaporin-4 strongly labeled the ependymal cells and the subpial glial membranes, as well as the bodies and processes of astroglial cells. A continuous aquaporin-4 staining was found around the microvessel endothelial cells, which were sealed off from one another by extensive tight junctions. A complete astrocytic sheath, labeled by anti-aquaporin-4 gold particles, enveloped the endothelium-pericyte layer. Orthogonal arrays of particles were observed on fractured astrocytic membranes, starting from embryonic day 14 when the aquaporin-4 immunogold staining revealed clusters of gold particles, often forming square or rectangular clusters. The results showed that aquaporin-4 expression and organization of the intramembrane particles in orthogonal arrays followed the same temporal sequence. Finally, the lipopolysaccharide, a substance that induces blood-brain barrier distruption, determines a remarkable reduction in aquaporin-4 labeling, expressed by a few aquaporin-4 gold particles attached on swollen perivascular glial membranes.

All these data show that aquaporin-4 expression occurs in the chick embryonic brain, in parallel with maturation and functioning of the blood-brain barrier and suggest that there is a close relationship between water transport regulation and brain development.
Differential galactosylation of neuronal and haematopoietic signal regulatory protein-α determines its cellular binding-specificity
Authors: Nieuwenhof Ingrid M. van den , Lavalette Chantal Renardel de , Diaz Niubel , Die Irma van , van den Berg Timo K.
Published: April 2021

Signal regulatory protein-α (SIRPα) is a member of the Ig superfamily selectively expressed by neuronal and myeloid cells. The molecule mediates functional interactions with CD47/integrin-associated protein. Here we provide evidence for the tissue-specific glycosylation of neuronal and haematopoietic SIRPα. We demonstrate a major difference in the galactosylation of N-linked glycans isolated from neuronal (i.e. brain-derived) SIRPα as compared to myeloid (i.e. spleen-derived) SIRPα, with neuronal SIRPα almost completely lacking galactose. p4-galactosyltransferase assays demonstrated that this is most likely due to a low galactosylation capacity of the brain. In order to investigate the role of galactosylation of SIRPα in cellular interactions, soluble recombinant SIRPα glycoforms containing galactose (SIRPα-Fc) or lacking galactose (SIRPα(ΔGal)-Fc) were produced. Binding studies demonstrated superior binding of SIRPα(ΔGal)-Fc to cerebellar neurons and isolated lymphocytes. In contrast, SIRPα-Fc bound relatively strong to macrophages. These data show that the galactosylation of SIRPα determines its cellular binding specificity.
BMP-2 augments FGF-induced differentiation of PC12 cells through upregulation of FGF receptor-1 expression
Authors: Hayashi Hisaki , Ishisaki Akira , Suzuki Masashi , Imamura Toru
Published: April 2021

When exposed to various neurotrophic factors, including fibroblast growth factors (FGF)-1 and -2, rat pheochromocytoma-derived PC12 cells differentiate into sympathetic neuron-like cells possessing elongated neurites. We found that while bone morphogenetic protein-2 (BMP-2) exerted little effect by itself on the differentiation of PC12 cells, in combination with FGF it strongly induced neurite outgrowth, even at subthreshold concentrations of FGF. Analysis of gene expression revealed that FGF receptor-1 (FGFR-1) mRNA was abundantly expressed in PC12 cells and that its expression was upregulated by pretreating the cells with BMP-2. Crosslinking the receptors with 125I-FGF-2 and then immunoprecipitating them confirmed that expression of FGFR-1, but not other FGF receptor types, was enhanced by BMP-2. Furthermore, Scatchard analyses revealed that the numbers of FGF-2 binding sites were increased by approximately 40% after BMP-2 treatment. Pretreatment with BMP-2 also enhanced peak and sustained levels of FGF-induced ERK1/2 phosphorylation in PC12 cells. Finally, the augmentation of neurotrophic activity by BMP-2 was inhibited by SU5402, an FGFR-1 inhibitor. These findings indicate that BMP-2 augments FGF-induced differentiation of PC12 cells through selective upregulation of FGFR-1 expression, and suggest that BMP-2 and FGF act in concert to regulate cell differentiation in the nervous system.
Structural analysis of the role of the p3 subunit of the αVβ3 integrin in IGF-I signaling
Authors: Maile Laura A. , Badley-Clarke Jane , Clemmons David R.
Published: April 2021

The disintegrin echistatin inhibits ligand occupancy of the αVβ3 integrin and reduces Insulin-like growth factor I (IGF-I) stimulated migration, DNA synthesis, and receptor autophosphorylation in smooth muscle cells. This suggests that ligand occupancy of the αVp3 receptor is required for full activation of the IGF-I receptor.

Transfection of the full-length β3 subunit into CHO cells that have no endogenous p3 and do not migrate in response to IGF-I was sufficient for IGF-I to stimulate migration of these anchorage dependent cells. In contrast, transfection of either of two truncated mutant forms of p3 (terminating at W715 or E731) or a mutant with substitutions for Tyr747 Tyr759 (YY) into either CHO or into porcine smooth muscle cells did not restore the capacity of these cells to migrate across a surface in response to IGF-I. This effect was not due to loss of IGF-I receptor autophosphorylation since the response of the receptor to IGF-I was similar in cells expressing either the full-length or any of the mutant forms of the β3 subunit. Echistatin reduced IGF-I receptor phosphorylation in cells expressing the full-length or the YY mutant forms of β3 subunit, but it had no effect in cells expressing either of two truncated forms of β3. A cell-permeable peptide homologous to the C-terminal region of the β3 subunit (amino acids 747-762) reduced IGF-I stimulated migration and receptor autophosphorylation of non-transfected porcine smooth muscle cells. These results demonstrate that the full-length β3 with intact tyrosines at positions 747 and 759 is required for CHO cells to migrate in response to IGF-I. Furthermore, a region of critical amino acids between residues 742-762 is required for echistatin to induce its regulatory effect on receptor phosphorylation. Since the IGF-I receptor does not bind to αVβ3 the results suggest that specific but distinct regions of the β3 subunit interact with intermediary proteins to facilitate IGF-I stimulated cell migration and echistatin induced inhibition of IGF-I signal transduction.
Peroxisomal targeting of mammalian hydroxyacid oxidase 1 requires the C-terminal tripeptide SKI
Authors: Recalcati Stefania , Menotti Eric , Kühn Lukas C.
Published: April 2021

Peroxisomal proteins are post-translationally imported into peroxisomes after recognition by specific receptors. The best-defined peroxisomal targeting signal is a C-terminal tripeptide SKL. Different functional variants of this tripeptide have been defined, but mutants with a SKI sequence were recognized as being inefficiently targeted to peroxisomes. Recently, we have cloned a cDNA for the mouse hydroxyacid oxidase 1 (Hao1), a protein that seems to be localized in peroxisomes. Interestingly, the mouse Hao1 sequence comprises a C-terminal SKI tripeptide. We have analyzed the subcellular localization of Hao1 and tested whether its SKI sequence acts as a targeting signal. Ltk− and Cos-7 cells were transfected with vectors expressing a fusion protein of green fluorescence protein and Hao1, as well as mutants thereof. Targeting to peroxisomes of the fusion protein with the wild-type SKI sequence was highly selective and as complete as with the peroxisome-specific SKL sequence. By contrast, targeting was lost in a mutant with the sequence CKM. The data show that mammalian Hao1 is a peroxisomal protein and that the C-terminal sequence SKI acts as the targeting signal.
Overexpression of the Csk homologous kinase (Chk tyrosine kinase) induces multinucleation: a possible role for chromosome-associated Chk in chromosome dynamics
Authors: Yamaguchi Naoto , Nakayama Yuji , Urakami Takeo , Suzuki Saeko , Nakamura Tatsunosuke , Suda Toshio , Oku Naoto
Published: April 2021

The Csk family of non-receptor-type tyrosine kinases consists of Csk and the Csk homologous kinase Chk. Each enzyme suppresses the catalytic activity of Src family kinases by phosphorylating their C-terminal negative regulatory tyrosine residues. Ectopic and transient expression of Chk in COS-1 cells showed nuclear localization of Chk and growth inhibition. To further explore the role of Chk in cell growth, we overexpressed Chk in human immature myeloid KMT-2 cells. Chk overexpression brought about growth retardation and aberrant chromosome movement leading to multinucleation, and these events were accompanied by insufficient formation of mitotic spindles. In vitro kinase assays showed that Chk overexpression suppressed the tyrosine kinase activity of Lyn, a member of the Src family, immunoprecipitated from Triton X-100 lysates. Subcellular fractionation studies revealed that fractions of Chk and Lyn, resistant to Triton X-100 solubilization, are associated with mitotic chromosome scaffolds and spindles. Chk overexpression induced a decrease in autophosphorylation of Lyn and concomitant changes in levels of tyrosine phosphorylation of proteins associated with both fractions. These results indicate that Chk, Lyn and the tyrosine-phosphorylated proteins localize to mitotic chromosomes and spindles, suggesting that Chk-dependent tyrosine phosphorylation, presumably through Lyn, may be involved in chromosome dynamics.
Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress
Authors: Dastoor Zubin , Dreyer Jean-Luc
Published: April 2021

Recent studies indicating a role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in apoptosis or oxidative stress has been reported. Using confocal laser-scanning microscopy, we have investigated the cellular distribution of GAPDH in central nervous system (CNS)-derived cells (neuroblastoma mNB41A3), in non-CNS derived cells (R6 fibroblast) and in an apoptosis-resistant Bcl2 overexpressing cell line (R6-Bcl2). Induction of apoptosis by staurosporine or MG132 and oxidative stress by H2O2 or FeCN enhanced the nuclear translocation of endogenous GAPDH in all cell types, as detected by immunocytochemistry. In apoptotic cells, GAPDH expression is three times higher than in non-apoptotic cells. Consistent with a role for GAPDH in apoptosis, overexpression of a GAPDH-green fluorescent protein (GAPDH-GFP) hybrid increased nuclear import of GAPDH-GFP into transfected cells and the number of apoptotic cells, and made them more sensitive to agents that induce apoptosis. Bcl2 overexpression prevents nuclear translocation of GAPDH and apoptosis in untransfected cells, but not in transfected cells that overexpress GAPDH-GFP. Our observations indicate that nuclear translocation of GAPDH may play a role in apoptosis and oxidative stress, probably related to the activity of GAPDH as a DNA repair enzyme or as a nuclear carrier for pro-apoptotic molecules.
A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis
Authors: Sutovsky Peter , Moreno Ricardo , Ramalho-Santos João , Dominko Tanja , Thompson Winston E. , Schatten Gerald
Published: April 2021

The normal structure and function of sperm are prerequisites for successful fertilization and embryonic development, but little is known about how defective sperm are eliminated during mammalian spermatogenesis. Here, we describe a ubiquitin-dependent, sperm quality control mechanism that resides in the mammalian epididymis, the site of sperm maturation and storage. We used immunofluorescence, electron microscopy, western blotting and pulse-chase experiments to show that ubiquitin is secreted by the epididymal epithelium and binds to the surface of defective sperm. Most of the ubiquitinated sperm are subsequently phagocytosed by the epididymal epithelial cells. A portion of defective sperm escapes phagocytosis and can be found in the ejaculate. Cultured epididymal cells maintain their ability to produce ubiquitin and phagocytose the defective sperm, as well as the ubiquitin-coated microspheres, in vitro. The surprising phenomenon of cell-surface ubiquitination in defective sperm provides a possible mechanism for sperm quality control in mammals and a new marker of semen abnormalities in men and animals.
A single internalization signal from the di-leucine family is critical for constitutive endocytosis of the type II TGF-β receptor
Authors: Ehrlich Marcelo , Shmuely Avi , Henis Yoav I.
Published: April 2021

Endocytosis has an important contribution to the regulation of the surface expression levels of many receptors. In spite of the central role of the transforming growth factor β (TGF-β) receptors in numerous cellular and physiological processes, their endocytosis is largely unexplored. Current information on TGF-β receptor endocytosis relies exclusively on studies with chimeric constructs containing the extracellular domain of the GM-CSF receptors, following the internalization of the GM-CSF ligand; the conformation and interactions of the chimeric receptors (and therefore their endocytosis) may differ considerably from those of the native TGF-β receptors. Furthermore, there are no data on the potential endocytosis motif(s) of the TGF-β receptors or other receptor Ser/Thr kinases. Here, we report the use of type II TGF-β receptors, myc-tagged at their extracellular terminus, to investigate their endocytosis. Employing fluorescent antibody fragments to label exclusively the cell surface myc-tagged receptors exposed to the external milieu, made it possible to follow the internalization of the receptors, without the complications that render labeling with TGF-β (which binds to many cellular proteins) unsuitable for such studies. The results demonstrate that the full-length type II TGF-β receptor undergoes constitutive endocytosis via clathrin-coated pits. Using a series of truncation and deletion mutants of this receptor, we identified a short peptide sequence (I218I219L220), which conforms to the consensus of internalization motifs from the di-leucine family, as the major endocytosis signal of the receptor. The functional importance of this sequence in the full-length receptor was validated by the near complete loss of internalization upon mutation of these three amino acids to alanine.
Kinesin: switch I & II and the motor mechanism
Authors: Kull F. Jon , Endow Sharyn A.
Published: April 2021

New crystal structures of the kinesin motors differ from previously described motor-ADP atomic models, showing striking changes both in the switch I region near the nucleotide-binding cleft and in the switch II or ‘relay’ helix at the filament-binding face of the motor. The switch I region, present as a short helix flanked by two loops in previous motor-ADP structures, rearranges into a pseudo-β-hairpin or is completely disordered with melted helices to either side of the disordered switch I loop. The relay helix undergoes a rotational movement coupled to a translation that differs from the piston-like movement of the relay helix observed in myosin. The changes observed in the crystal structures are interpreted to represent structural transitions that occur in the kinesin motors during the ATP hydrolysis cycle. The movements of switch I residues disrupt the water-mediated coordination of the bound Mg2+, which could result in loss of Mg2+ and ADP, raising the intriguing possibility that disruption of the switch I region leads to release of nucleotide by the kinesins. None of the new structures is a true motor-ATP state, however, probably because conformational changes at the active site of the kinesins require interactions with microtubules to stabilize the movements.
Recycling ability of the mouse and the human neurotensin type 2 receptors depends on a single tyrosine residue
Authors: Martin Stéphane , Vincent Jean-Pierre , Mazella Jean
Published: April 2021

Receptor recycling plays a key role in the modulation of cellular responses to extracellular signals. The purpose of this work was to identify residues in G-protein coupled neurotensin receptors that are directly involved in recycling. Both the high affinity receptor-1 (NTR1) and the levocabastine-sensitive NTR2 are internalized after neurotensin binding. Here, we show that only the mouse NTR2 recycled to the plasma membrane, whereas the rat NTR1 and the human NTR2 did not. Using site-directed mutagenesis, we demonstrate that tyrosine 237 in the third intracellular loop is crucial for recycling of the mouse NTR2. We show that the mouse NTR2 is phosphorylated on tyrosine residues by NT. This phosphorylation is essential for receptor recycling since the tyrosine kinase inhibitor genistein blocks this process. The absence of recycling observed with the human NTR2 could be completely explained by the presence of a cysteine instead of a tyrosine in position 237. Indeed, substitution of this cysteine by a tyrosine gave a mutant receptor that has acquired the ability to recycle to the cell surface after neurotensin-induced internalization. This work demonstrates that a single tyrosine residue in the third intracellular loop of a G-protein-coupled receptor is responsible for receptor phosphorylation and represents an essential structural element for receptor recycling.
Syndecan-4 mediates antithrombin-induced chemotaxis of human peripheral blood lymphocytes and monocytes
Authors: Kaneider Nicole C. , Reinisch Christina M. , Dunzendorfer Stefan , Römisch Jürgen , Wiederman Christian J.
Published: April 2021

Antithrombin inhibits chemokine-induced migration of neutrophils by activating heparan sulfate proteoglycan-dependent signaling. Whether antithrombin affects migration of other types of leukocytes is not known. We investigated the effects of antithrombin on spontaneous and chemokine-triggered migration of lymphocytes and monocytes from human peripheral blood in modified Boyden chamber micropore filter assays. Lymphocyte and monocyte populations from human peripheral blood were purified using magnetic antibody cell sorting. The signaling mechanisms required for antithrombin-dependent migration were studied using signaling enzyme blockers. Expression of heparan sulfate proteoglycan core protein was studied by RT-PCR and flow cytometry. The antithrombins used were Kybernin®P from human plasma and a monoclonal-antibody-purified preparation from this plasma. Pretreatment of lymphocytes and monocytes with antithrombin inhibited chemotaxis toward optimal concentrations of interleukin-8 or Rantes (regulated upon activation normal T-cell expressed and activated) at concentrations of antithrombin as low as 10 nU/ml. In the absence of the chemokines, direct exposure of cells to gradients of antithrombin stimulated migration. Effects of antithrombin were abolished by pretreating cells with heparinase-1, chondroitinase, sodium chlorate and anti-syndecan-4 antibodies. Expression of syndecan-4 mRNA and protein in monocytes and lymphocytes was demonstrated in RT-PCR and anti-syndecan-4 immunoreactivity assays, respectively. In the presence of pentasaccharide, antithrombin lost its effect on cells. Data indicate that antithrombin directly inhibits chemokine-stimulated migration of monocytes and lymphocytes via the effects of its heparin-binding site on cell surface syndecan-4 by activation of protein kinase C and Rho signaling.
Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition
Authors: Gudjonsson Thorarinn , Rønnov-Jessen Lone , Villadsen René , Rank Fritz , Bissell Mina J. , Petersen Ole William
Published: April 2021

The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and β4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal.

Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce α-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumor-associated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be recapitulated in culture and that one reason for the ability of myoepithelial cells to induce polarity is because they are the only source of laminin-1 in the breast in vivo. A further conclusion is that a majority of tumor-derived/-associated myoepithelial cells are deficient in their ability to impart polarity because they have lost their ability to synthesize sufficient or functional laminin-1. These results have important implications for the role of myoepithelial cells in maintenance of polarity in normal breast and how they may function as structural tumor suppressors.
Myne-1, a spectrin repeat transmembrane protein of the myocyte inner nuclear membrane, interacts with lamin A/C
Authors: Mislow John M. K. , Kim Marian S. , Davis Dawn Belt , McNally Elizabeth M.
Published: April 2021

Mutations in the genes encoding the inner nuclear membrane proteins lamin A/C and emerin produce cardiomyopathy and muscular dystrophy in humans and mice. The mechanism by which these broadly expressed gene products result in tissue-specific dysfunction is not known. We have identified a protein of the inner nuclear membrane that is highly expressed in striated and smooth muscle. This protein, myne-1 (myocyte nuclear envelope), is predicted to have seven spectrin repeats, an interrupted LEM domain and a single transmembrane domain at its C-terminus. We found that myne-1 is expressed upon early muscle differentiation in multiple intranuclear foci concomitant with lamin A/C expression. In mature muscle, myne-1 and lamin A/C are perfectly colocalized, although colocalization with emerin is only partial. Moreover, we show that myne-1 and lamin A/C coimmunoprecipitate from differentiated muscle in vitro. The muscle-specific inner nuclear envelope expression of myne-1, along with its interaction with lamin A/C, indicates that this gene is a potential mediator of cardiomyopathy and muscular dystrophy.
The protein tyrosine kinase Hck is located on lysosomal vesicles that are physically and functionally distinct from CD63-positive lysosomes in human macrophages
Authors: Astarie-Dequeker Catherine , Carreno Sébastien , Cougoule Céline , Maridonneau-Parini Isabelle
Published: April 2021

In macrophages, lysosomes are suspected to have a heterogenous population of vesicles. This study was thus undertaken to identify and to characterize lysosomal compartments in human macrophages. Hck is a Src-family tyrosine kinase associated with secretory lysosomes in neutrophils and with cytoplasmic vesicles in macrophages that fuse with phagosomes. We identified these Hck-positive vesicles and compared them to CD63-positive, M6PR-negative vesicles known as classical lysosomes. Hck vesicles exhibited lysosomal features. Indeed, Hck-positive vesicles could be loaded with rhodamine-dextran, which has been shown to accumulate in lysosomal compartments. Hck was delivered to zymosan-containing phagosomes at a late stage of the maturation process, which occurs after the fusion with CD63-positive lysosomes. Finally, when mycobacteria were used to prevent phagolysosome biogenesis, Hck was not recruited to phagosomes. Moreover, Hck lysosomes were physically and functionally distinct from CD63-lysosomes. For instance, sucrose induced swelling of CD63-lysosomes without affecting Hck-positive ones. Only CD63-lysosomes fused with phagosomes in a microtubule-dependent manner. Entry of particles through the mannose receptor and Fcγ receptors drove the phagosome towards a fusion with CD63-lysosomes, whereas only Fcγ receptors induced the mobilisation of Hck-lysosomes. This study provides further evidence for the existence of sub-populations of lysosomes in macrophages: one stained by CD63 and another one characterized by the presence of Hck. Therefore, Hck represents a new tool to study the fusion dynamics of lysosomal compartments and their subversion by several intracellular pathogens.
Heat shock induces mini-Cajal bodies in theXenopusgerminal vesicle
Authors: Handwerger Korie E. , Wu Zheng'an , Murphy Christine , Gall Joseph G.
Published: April 2021

Cajal bodies are evolutionarily conserved nuclear organelles that are believed to play a central role in assembly of RNA transcription and processing complexes. Although knowledge of Cajal body composition and behavior has greatly expanded in recent years, little is known about the molecules and mechanisms that lead to the formation of these organelles in the nucleus. The Xenopus oocyte nucleus or germinal vesicle is an excellent model system for the study of Cajal bodies, because it is easy to manipulate and it contains 50-100 Cajal bodies with diameters up to 10 μm. In this study we show that numerous mini-Cajal bodies (less than 2 μm in diameter) form in the germinal vesicle after oocytes recover from heat shock. The mechanism for heat shock induction of mini-Cajal bodies is independent of U7 snRNA and does not require transcription or import of newly translated proteins from the cytoplasm. We suggest that Cajal bodies originate by self-organization of preformed components, preferentially on the surface of B-snurposomes.
Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains
Authors: Nielsen Finn C. , Nielsen Jacob , Kristensen Mette A. , Koch Grete , Christiansen Jan
Published: April 2021

The IGF-II mRNA-binding proteins (IMPs), which are composed of two RNA recognition motifs, (RRM) and four hnRNP K homology (KH) domains, have been implicated in subcytoplasmic localization of mRNAs during embryogenesis. The IMP family originated via two gene duplications before the divergence of vertebrates, and IMP homologues consisting of only the four KH motifs have been identified in Drosophila and Caenorhabditis elegans. Here we characterise the trafficking of GFP-IMP1 fusion proteins and determine the structural determinants for proper cytoplasmic localization. GFP-IMP1 is present in large 200-700 nm RNP granules, which are distributed along microtubules. In motile cells, GFP-IMP1 is transported towards the leading edge into the cortical region of the lamellipodia where it is connected to microfilaments. Granules travel in an ATP-dependent fashion at an average speed of 0.12 μm/s (range 0.04-0.22 μm/s), and cells switch from a delocalized to a localized pattern within 15-20 minutes. Both granule formation and localization are unaffected by removal of the two RRMs, whereas deletion of the KH domains, which mediate RNA-binding, impairs these functions. We conclude that IMP1 localization is associated with motility and that the major functions of IMP1 are carried out by the phylogenetically conserved KH domains.
Regulation, mechanisms and proposed function of ferritin translocation to cell nuclei
Authors: Thompson Khristy J. , Fried Michael G. , Ye Zheng , Boyer Phillip , Connor James R.
Published: April 2021

Ferritin is traditionally considered a cytoplasmic iron-storage protein,but recent reports indicate that it is also found in cell nuclei. Nuclear ferritin has been proposed to be involved in both the protection of DNA and the exacerbation of iron-induced oxidative damage to DNA. We demonstrate that H-rich ferritin is present in the nucleus of human astrocytoma tumor cells. To study the mechanism and regulation of ferritin translocation to the nucleus,we developed a cell culture model using SW1088 human astrocytoma cells. Changes in cellular iron levels, cytokine treatments and hydrogen peroxide exposure affected the distribution of ferritin between the cytosol and the nucleus. Ferritin enters the nucleus via active transport through the nuclear pore and does not require NLS-bearing cytosolic factors for transport. Furthermore, H-rich ferritin is preferred over L-rich ferritin for uptake into the nucleus. Whole cell crosslinking studies revealed that ferritin is associated with DNA. Ferritin protected DNA from iron-induced oxidative damage in both in vitro and in cell culture models. These results strongly suggest a novel role for ferritin in nuclear protection. This work should lead to novel characterization of ferritin functions in the context of genomic stability and may have unparalleled biological significance in terms of the accessibility of metals to DNA. The knowledge generated as a result of these studies will also improve our understanding of iron-induced damage of nuclear constituents.
PLC-γ1 is required for IGF-I protection from cell death induced by loss of extracellular matrix adhesion
Authors: Chattopadhyay Ansuman , Carpenter Graham
Published: April 2021

Phospholipase C-γ1, a tyrosine kinase substrate, hydrolyses phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and diacylglycerol, which act as second messenger moleculesto mobilize intracellular calcium and activate protein kinase C, respectively. We have investigated the role of phospholipase C-γ1 in anoikis, or cell death,induced by the loss of extracellular matrix adhesion. Spontaneously immortalized mouse embryonic fibroblasts nullizygous at the Plcg1locus (Plcg1-/-), referred to as Null cells, were derived from targeted gene disruption experiments. Subsequently, phospholipase C-γ1 was re-expressed in these cells to derive Null+ cells. The Null and Null+ cells were then placed in suspension to induce cell death, which was measured directly as well as by the induction of caspase 3, as an index of programmed cell death or apoptosis. The results demonstrate that insulin-like growth factor can rescue Null+ cells but not Null cells from suspension-induced cell death. This demonstrates that phospholipase C-γ1 is required for insulin-like growth factor dependent cell survival under these conditions. Lastly, the data demonstrate that insulinlike growth factor stimulated tyrosine phosphorylation of phospholipase C-γ1 in both adherent and suspension cells.
Rho-binding kinase (LET-502) and myosin phosphatase (MEL-11) regulate cytokinesis in the earlyCaenorhabditis elegansembryo
Authors: Piekny Alisa J. , Mains Paul E.
Published: April 2021

Rho-binding kinase and myosin phosphatase regulate the contraction of actomyosin filaments in non-muscle and smooth muscle cells. Previously, we described the role of C. elegans genes encoding Rho-binding kinase(let-502) and myosin phosphatase targeting subunit (mel-11)in epidermal cell-shape changes that drive morphogenesis and in spermathecal contraction. Here we analyze their roles in a third contractile event,cytokinesis within early embryos. We demonstrate that these genes function together to regulate the rate of cleavage furrow contraction, with Rho-binding kinase/LET-502 mediating contraction, whereas myosin phosphatase/MEL-11 acts as a brake to contraction: early embryonic cleavage often fails or is slowed when let-502 is mutated, whereas mel-11 mutations result in ectopic furrowing and faster furrow ingression. These phenotypes correspond to changes in the levels of phosphorylated regulatory non-muscle myosin light chain (rMLC). The gene products of let-502 and mel-11colocalize at cleavage furrows, and their mutations alleviate one another's defects. rMLC is phosphorylated in let-502; mel-11 double mutants,indicating that a kinase is able to phosphorylate rMLC in the absence of both LET-502 and MEL-11. Genetic and molecular epistasis experiments place LET-502 and MEL-11 in a cytokinetic pathway. LET-502 and MEL-11 regulate the activity of non-muscle myosin after actin, non-muscle myosin heavy chain/NMY-2,regulatory non-muscle myosin light chain/MLC-4 and early formin/CYK-1 have formed a contractile ring. Proteins including Rho GTPase activating protein/CYK-4 and late CYK-1, which are required for late stages of cytokinesis, function downstream of LET-502 and MEL-11.
Humanmelanocortin 1 receptorvariants, receptor function and melanocyte response to UV radiation
Authors: Scott M. Cathy , Wakamatsu Kazumasa , Ito Shosuke , Kadekaro Ana Luisa , Kobayashi Nobuhiko , Groden Joanna , Kavanagh Renny , Takakuwa Takako , Virador Victoria , Hearing Vincent J. , Abdel-Malek Zalfa A.
Published: April 2021

Cutaneous pigmentation is determined by the amounts of eumelanin and pheomelanin synthesized by epidermal melanocytes and is known to protect against sun-induced DNA damage. The synthesis of eumelanin is stimulated by the binding of α-melanotropin (α-melanocyte-stimulating hormone)to the functional melanocortin 1 receptor (MC1R) expressed on melanocytes. The human MC1R gene is highly polymorphic and certain allelic variants of the gene are associated with red hair phenotype, melanoma and non-melanoma skin cancer. The importance of the MC1R gene in determining skin cancer risk led us to examine the impact of specific polymorphisms in this gene on the responses of human melanocytes to α-melanotropin and UV radiation. We compared the ability of human melanocyte cultures, each derived from a single donor, to respond to α-melanotropin with dose-dependent stimulation of cAMP formation, tyrosinase activity and proliferation. In each of those cultures the MC1R gene was sequenced, and the eumelanin and pheomelanin contents were determined. Human melanocytes homozygous for Arg160Trp, heterozygous for Arg160Trp and Asp294His, or for Arg151Cys and Asp294His substitutions, but not melanocytes homozygous for Val92Met substitution, in the MC1R demonstrated a significantly reduced response toα-melanotropin. Additionally, melanocytes with a non-functional MC1R demonstrated a pronounced increase in their sensitivity to the cytotoxic effect of UV radiation compared with melanocytes expressing functional MC1R. We conclude that loss-of-function mutations in the MC1R gene sensitize human melanocytes to the DNA damaging effects of UV radiation, which may increase skin cancer risk.
Calmodulin-containing substructures of the centrosomal matrix released by microtubule perturbation
Authors: Moisoi Nicoleta , Erent Muriel , Whyte Sheena , Martin Stephen , Bayley Peter M.
Published: April 2021

Calmodulin redistribution in MDCK and HeLa cells subjected to microtubule perturbations by antimitotic drugs was followed using a calmodulin-EGFP fusion protein that preserves the Ca2+ affinity, target binding and activation properties of native calmodulin. CaM-EGFP targeting to spindle structures in normal cell division and upon spindle microtubule disruption allows evaluation of the dynamic redistribution of calmodulin in cell division. Under progressive treatment of stably transfected mammalian cells with nocodazole or vinblastine, the centrosomal matrix at the mitotic poles subdivides into numerous small `star-like' structures, with the calmodulin concentrated centrally, and partially distinct from the reduced microtubule mass to which kinetochores and chromosomes are attached. Prolonged vinblastine treatment causes the release of localised calmodulin into a uniform cytoplasmic distribution, and tubulin paracrystal formation. By contrast,paclitaxel treatment of metaphase cells apparently causes limited disassembly of the pericentriolar material into a number of multipolar `ring-like'structures containing calmodulin, each one having multiple attached microtubules terminating in the partially disordered kinetochore/chromosome complex. Thus drugs with opposite effects in either destabilising or stabilising mitotic microtubules cause subdivision of the centrosomal matrix into two distinctive calmodulin-containing structures, namely small punctate`stars' or larger polar `rings' respectively. The `star-like' structures may represent an integral subcomponent for the attachment of kinetochore microtubules to the metaphase centrosome complex. The results imply that microtubules have a role in stabilising the structure of the pericentriolar matrix, involving interaction, either direct or indirect, with one or more proteins that are targets for binding of calmodulin. Possible candidates include the pericentriolar matrix-associated coiled-coil proteins containing calmodulin-binding motifs, such as myosin V, kendrin (PCNT2) and AKAP450.
β2-microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn
Authors: Praetor Asja , Hunziker Walter
Published: April 2021

FcRn is a heterodimer of an α-chain andβ 2-microglobulin (β2m) and differs from other IgG Fc receptors in that it is structurally related to MHC class I molecules. Several functions attributed to FcRn are affected inβ 2-microglobulin (β2m)-deficient mice,suggesting that the α-chain needs to assemble with β2m to form a functional receptor. However, the precise role ofβ 2m in FcRn function is not known. Here we expressed the human FcRn α-chain alone or in combination with β2m in human melanoma FO-1 cells. We show that β2m is important for cell surface expression of FcRn and that, in the absence of β2m,the receptor is retained in the endoplasmic reticulum. Furthermore, in the absence of β2m, IgG binding is decreased compared with that of native FcRn. Thus, assembly of the FcRn α-chain with β2m is important for both transport of FcRn from the ER to the cell surface and efficient pH-dependent IgG binding.
Ectopic expression ofDrosophilaELAV and human HuD inDrosophilawing disc cells reveals functional distinctions and similarities
Authors: Toba Gakuta , Qui Jan , Koushika Sandhya P. , White Kalpana
Published: April 2021

Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements(AREs) in their 3′ untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes,which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame(ORF); (2) with a constitutively spliced intron inserted into the ORF; and (3)with the intron nASI whose splicing is regulated in neurons by ELAV,inserted into the ORF. The two sets differed from each other only in their 3′UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. Our results show that:(1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3′UTR, but not of transgenes with Act5C 3′UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm,ELAV is localized to both the cytoplasm and the nucleus.
The plant Spc98p homologue colocalizes with γ-tubulin at microtubule nucleation sites and is required for microtubule nucleation
Authors: Erhardt Mathieu , Stoppin-Mellet Virginie , Campagne Sarah , Canaday Jean , Mutterer Jérôme , Fabian Tanja , Sauter Margret , Muller Thierry , Peter Christine , Lambert Anne-Marie , Schmit Anne-Catherine
Published: April 2021

The molecular basis of microtubule nucleation is still not known in higher plant cells. This process is better understood in yeast and animals cells. In the yeast spindle pole body and the centrosome in animal cells,γ-tubulin small complexes and γ-tubulin ring complexes,respectively, nucleate all microtubules. In addition to γ-tubulin,Spc98p or its homologues plays an essential role. We report here the characterization of rice and Arabidopsis homologues of SPC98. Spc98p colocalizes with γ-tubulin at the nuclear surface where microtubules are nucleated on isolated tobacco nuclei and in living cells. AtSpc98p-GFP also localizes at the cell cortex. Spc98p is not associated with γ-tubulin along microtubules. These data suggest that multiple microtubule-nucleating sites are active in plant cells. Microtubule nucleation involving Spc98p-containing γ-tubulin complexes could then be conserved among all eukaryotes, despite differences in structure and spatial distribution of microtubule organizing centers.
From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner?
Authors: Dorée Marcel , Hunt Tim
Published: April 2021

The idea that Cdc2 and cyclins play a key role in the control of the G2/M transition of the cell cycle came largely from genetic analysis of fission yeast and physiological studies of clam, frog, sea urchin and starfish eggs and oocytes. However, it took a long time to realise that Cdc2 and cyclins form a stoichiometric complex and that a cyclin subunit is necessary for the Cdc2 subunit to gain its protein kinase activity.

Cyclins were first recognized as proteins whose abundance oscillates during the early cell cycles of marine invertebrate eggs and their connection with MPF (maturation-promoting factor), the entity defined in frog and starfish oocytes whose activity controls entry into M phase, was far from clear at first. Indeed, it was a long time before MPF was shown to be a protein kinase,and direct proof that MPF is a heterodimer comprising one molecule of cyclin and one molecule of Cdc2 was finally obtained only when the Cdc2-associated component of purified starfish MPF was sequenced and found to be cyclin B. When this fundamental discovery was confirmed in vertebrates and mammalian members of the Cdc2 family were also shown to bind cyclins, Cdc2 became Cdk1,the first cyclin-dependent protein kinase.
Mechanisms for targeting of theSaccharomyces cerevisiaeGPI-anchored cell wall protein Crh2p to polarised growth sites
Authors: Rodriguez-Peña Jose M. , Rodriguez Cristina , Alvarez Alberto , Nombela César , Arroyo Javier
Published: April 2021

The cell wall is an essential structure that preserves the osmotic integrity of fungal cells and determines cellular morphology during developmental programs. The high number of different wall components demands a variety of processes to deliver precursors and synthetic proteins to the proper location at the right time for wall development and modification. Here,the specificity of the mechanisms that regulate the temporal and spatial localisation of cell wall proteins to sites of polarised growth in Saccharomyces cerevisiae is investigated. For this purpose, the localisation of Crh2p, a cell wall glycosylphosphatidylinositol (GPI)-anchored mannoprotein that we have recently described as involved in cell wall construction and localised to polarised growth sites, was followed using a Crh2p-GFP fusion protein. Crh2p distribution was studied in several genetic backgrounds affected in different steps of the cell polarity establishment machinery or/and bud morphogenesis. Crh2p is localised at the mother-bud neck in bud1 cells following the random budding pattern characteristic of this mutant. The Crh2p distribution was greatly altered in a cdc42-1mutant, indicating complete dependence on an organised actin cytoskeleton for polarised Crh2p distribution. The usual deposition of Crh2p in a ring at the base of growing buds was lacking in cdc10-11 cells growing under restrictive temperature conditions, whereas Crh2p deposition at the septum region was absent in both cdc10-11 and cdc15-lyt1 cells. These results point to the dependence of Crh2p localisation at the bud-neck on both septins and septum integrity. Furthermore, in the absence of Bni4p, a scaffold protein involved in the targeting of the chitin synthase III complex to the bud neck, Crh2p was not longer found at the neck in large-budded cells undergoing cytokinesis. Finally, Crh2p was not properly localised in cells deleted in CHS5, which encodes a protein involved in the transport of Chs3p, and was completely mislocalised in sbe2/sbe22 mutants,suggesting that the transport systems for Chs3p and Crh2p are to a certain extent coincident. The transport of other GPI-cell wall proteins, such as Cwp1p, however, does not depend on these systems as the localisation of the latter protein was not affected in either of these mutants.