bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peroxisomal targeting of mammalian hydroxyacid oxidase 1 requires the C-terminal tripeptide SKI
Authors: Recalcati Stefania , Menotti Eric , Kühn Lukas C. 
Published: April 2021

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.
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.
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.
Gap junction proteins are not interchangeable in development of neural function in theDrosophilavisual system
Authors: Curtin Kathryn D. , Zhang Zhan , Wyman Robert J. 
Published: April 2021

Gap junctions (GJs) are composed of proteins from two distinct families. In vertebrates, GJs are composed of connexins; a connexin hexamer on one cell lines up with a hexamer on an apposing cell to form the intercellular channel. In invertebrates, GJs are composed of an unrelated protein family, the innexins. Different connexins have distinct properties that make them largely non-interchangeable in the animal. Innexins are also a large family with high sequence homology, and some functional differences have been reported. The biological implication of innexin differences, such as their ability to substitute for one another in the animal, has not been explored.Recently, we showed that GJ proteins are necessary for the development of normal neural transmission in the Drosophila visual system. Mutations in either of two Drosophila GJ genes (innexins), shakB and ogre, lead to a loss of transients in the electroretinogram (ERG),which is indicative of a failure of the lamina to respond to retinal cell depolarization. Ogre is required presynaptically and shakB(N)postsynaptically. Both act during development.Here we ask if innexins are interchangeable in their role of promoting normal neural development in flies. Specifically, we tested several innexins for their ability to rescue shakB2 and ogremutant ERGs and found that, by and large, innexins are not interchangeable. We mapped the protein regions required for this specificity by making molecular chimeras between shakB(N) and ogre and testing their ability to rescue both mutants. Each chimera rescued either shakB or ogre but never both. Sequences in the first half of each protein are necessary for functional specificity. Potentially crucial residues include a small number in the intracellular loop as well as a short stretch just N-terminal to the second transmembrane domain.Temporary GJs, possibly between the retina and lamina, may play a role in final target selection and/or chemical synapse formation in the Drosophila visual system. In that case, specificity in GJ formation or function could contribute, directly or indirectly, to chemical synaptic specificity by regulating which neurons couple and what signals they exchange. Cells may couple only if their innexins can mate with each other. The partially overlapping expression patterns of several innexins make this `mix and match' model of GJ formation a possibility.
The cell cycle dependent mislocalisation of emerin may contribute to the Emery-Dreifuss muscular dystrophy phenotype
Authors: Fairley Elizabeth A. L. , Riddell Andrew , Ellis Juliet A. , Kendrick-Jones John 
Published: April 2021

Emerin is the nuclear membrane protein defective in X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). The majority of X-EDMD patients have no detectable emerin. However, there are cases that produce mutant forms of emerin, which can be used to study its function. Our previous studies have shown that the emerin mutants S54F, P183T, P183H, Del95-99, Del236-241 (identified in X-EDMD patients) are targeted to the nuclear membrane but to a lesser extent than wild-type emerin. In this paper, we have studied how the mislocalisation of these mutant emerins may affect nuclear functions associated with the cell cycle using flow cytometry and immunofluorescence microscopy. We have established that cells expressing the emerin mutant Del236-241 (a deletion in the transmembrane domain), which was mainly localised in the cytoplasm, exhibited an aberrant cell cycle length. Thereafter, by examining the intracellular localisation of endogenously expressed lamin A/C and exogenously expressed wild-type and mutant forms of emerin after a number of cell divisions, we determined that the mutant forms of emerin redistributed endogenous lamin A/C. The extent of lamin A/C redistribution correlated with the amount of EGFP-emerin that was mislocalised. The amount of EGFP-emerin mislocalized, in turn, was associated with alterations in the nuclear envelope morphology. The nuclear morphology and redistribution of lamin A/C was most severely affected in the cells expressing the emerin mutant Del236-241.It is believed that emerin is part of a novel nuclear protein complex consisting of the barrier-to-autointegration factor (BAF), the nuclear lamina, nuclear actin and other associated proteins. The data presented here show that lamin A/C localisation is dominantly directed by its interaction with certain emerin mutants and perhaps wild-type emerin as well. These results suggest that emerin links A-type lamins to the nuclear envelope and that the correct localisation of these nuclear proteins is important for maintaining cell cycle timing.
Three-dimensional reconstruction of cytoplasmic membrane networks in parietal cells
Authors: Duman Joseph G. , Pathak Nimesh J. , Ladinsky Mark S. , McDonald Kent L. , Forte John G. 
Published: April 2021

There is general agreement that stimulation and consequent secretion of gastric parietal cells result in a great expansion of the apical canalicular membrane at the expense of an extensive intracellular network of membranes rich in the gastric proton pump (H,K-ATPase). However, there is ongoing controversy as to the precise nature of the intracellular membrane network,conventionally called tubulovesicles. At the heart of this controversy lies the question of whether tubulovesicles are a distinct membrane compartment or whether they are continuous with the apical plasma membrane.To address this controversy we used high-pressure, rapid freezing techniques to fix non-stimulated (resting) rabbit gastric glands for electron microscopy. Ultra-thin (60-70 nm) serial sections were used for conventional TEM; 400-500 nm sections were used for tomography. Images were digitized and models constructed using Midas and Imod software(http://bio3d.colorado.edu). Images were aligned and contours drawn on specific cellular structures. The contours from a stack of serial sections were arranged into objects and meshed into 3D structures. For resting parietal cells our findings are as follows:(1) The apical canaliculus is a microvilli-decorated, branching membrane network that extends into and throughout the parietal cell. This agrees well with a host of previous studies. (2) The plentiful mitochondria form an extensive reticular network throughout the cytoplasm. This has not previously been reported for the parietal cell, and the significance of this observation and the dynamics of the mitochondrial network remain unknown. (3)H,K-ATPase-rich membranes do include membrane tubules and vesicles; however,the tubulovesicular compartment is chiefly comprised of small stacks of cisternae. Thus a designation of tubulocisternae seems appropriate; however,in the resting cell there are no continuities between the apical canaliculus and the tubulocisternae or between tubulocisternae. These data support the recruitment-recycling model of parietal cell stimulation.
Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells
Authors: van Wetering Sandra , van Buul Jaap D. , Quik Safira , Mul Frederik P. J. , Anthony Eloise C. , Klooster Jean-Paul ten , Collard John G. , Hordijk Peter L. 
Published: April 2021

The integrity of the endothelium is dependent on cell-cell adhesion, which is mediated by vascular-endothelial (VE)-cadherin. Proper VE-cadherin-mediated homotypic adhesion is, in turn, dependent on the connection between VE-cadherin and the cortical actin cytoskeleton. Rho-like small GTPases are key molecular switches that control cytoskeletal dynamics and cadherin function in epithelial as well as endothelial cells. We show here that a cell-penetrating, constitutively active form of Rac (Tat-RacV12) induces a rapid loss of VE-cadherin-mediated cell-cell adhesion in endothelial cells from primary human umbilical veins (pHUVEC). This effect is accompanied by the formation of actin stress fibers and is dependent on Rho activity. However,transduction of pHUVEC with Tat-RhoV14, which induces pronounced stress fiber and focal adhesion formation, did not result in a redistribution of VE-cadherin or an overall loss of cell-cell adhesion. In line with this observation, endothelial permeability was more efficiently increased by Tat-RacV12 than by Tat-RhoV14.
The loss of cell-cell adhesion, which is induced by Tat-RacV12, occurred in parallel to and was dependent upon the intracellular production of reactive oxygen species (ROS). Moreover, Tat-RacV12 induced an increase in tyrosine phosphorylation of a component the VE-cadherin-catenin complex, which was identified as α-catenin. The functional relevance of this signaling pathway was further underscored by the observation that endothelial cell migration, which requires a transient reduction of cell-cell adhesion, was blocked when signaling through ROS was inhibited.
In conclusion, Rac-mediated production of ROS represents a previously unrecognized means of regulating VE-cadherin function and may play an important role in the (patho)physiology associated with inflammation and endothelial damage as well as with endothelial cell migration and angiogenesis.
Synthesis and secretion of growth hormone in the rat anterior pituitary: II. properties of the isolated growth hormone storage granules
Authors: Howell S. L. , Ewart R. B. L. 
Published: April 2021

ABSTRACT
Partially purified storage granule fractions obtained from rat anterior pituitaries have been utilized to study some properties of the isolated growth hormone granules during incubation in vitro. The isolated granules showed optimal stability at room temperature in the pH range 5·5-6·5 and dissolved progressively with time at a rate which was unaffected by the presence of EDTA, EGTA, ouabain, magnesium ions, or by resuspension in hypotonic media. However, the granules were partially stabilized by the presence of calcium, tyramine or adenosine phosphates ; ATP was most effective of the agents tested and induced almost complete stability. Agents known to stimulate the secretion of growth hormone from the intact cells, cyclic 3′,5′-AMP, dibutyryl cyclic 3′,5′-AMP, theophylline or high potassium concentrations, were each without significant effect on the isolated granules. The granule fraction was ineffective in degrading added 125I-growth hormone over a pH range of 4·5-8·5 and was unable specifically to bind exogenous 125I-growth hormone. Further experiments to elucidate a possible cytological role of nucleotides in stabilization of growth hormone during storage granule formation demonstrated that adenine nucleotides were not constituents of the isolated granules, nor were they secreted into the incubation medium concomitantly with growth hormone. Depletion of intracellular ATP levels has previously been shown to prevent the formation of growthhormone storage granules, and it is suggested that one role of the nucleotide may be to facilitate the formation and stabilization of storage granules in the Golgi complex by interacting with the growth hormone to induce its precipitation.