bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle
Authors: Wu Xufeng , Rao Kang , Bowers M. Blair , Copeland Neal G. , Jenkins Nancy A. , Hammer John A. 
Published: April 2021

ABSTRACT
The peripheral accumulation of melanosomes characteristic of wild-type mouse melanocytes is driven by a cooperative process involving long-range, bidirectional, microtubule-dependent movements coupled to capture and local movement in the actin-rich periphery by myosin Va, the product of the dilute locus. Genetic evidence suggests that Rab27a, the product of the ashen locus, functions with myosin Va in this process. Here we show that ashen melanocytes, like dilute melanocytes, exhibit normal dendritic morphology and melanosome biogenesis, an abnormal accumulation of end-stage melanosomes in the cell center, and rapid, bidirectional, microtubule-dependent melanosome movements between the cell center and the periphery. This phenotype suggests that ashen melanocytes, like dilute melanocytes, are defective in peripheral melanosome capture. Consistent with this, introduction into ashen melanocytes of cDNAs encoding wild-type and GTP-bound versions of Rab27a restores the peripheral accumulation of melanosomes in a microtubule-dependent manner. Conversely, introduction into wild-type melanocytes of the GDP-bound version of Rab27a generates an ashen/dilute phenotype. Rab27a colocalizes with end-stage melanosomes in wild-type cells, and is most concentrated in melanosome-rich dendritic tips, where it also colocalizes with myosin Va. Finally, neither endogenous myosin Va nor an expressed, GFP-tagged, myosin Va tail domain fusion protein colocalize with melanosomes in ashen melanocytes, in contrast to that seen previously in wild-type cells. These results argue that Rab27a serves to enable the myosinVa-dependent capture of melanosomes delivered to the periphery by bidirectional, microtubule-dependent transport, and that it does so by recruiting the myosin to the melanosome surface. We suggest that Rab27a, in its GTP-bound and melanosome-associated form, predominates in the periphery, and that it is this form that recruits the myosin, enabling capture. These results argue that Rab27a serves as a myosin Va ‘receptor’, and add to the growing evidence that Rab GTPases regulate vesicle motors as well as SNARE pairing.
Translocation and accumulation of exogeneous hepatitis B virus preS surface proteins in the cell nucleus
Authors: Cho Eun-Wie , Park Jung-Hyun , Yoo Ook-Joon , Kim Kil Lyong 
Published: April 2021

ABSTRACT
Recurrent reports about protease-sensitive sites in the junction of the preS and S region of the hepatitis B virus large surface protein have raised the question about a possible biological role of S protein-depleted, independent preS protein fragments in the virus life cycle. In the present study, this question was addressed by exogenous introduction of fluorescence-labeled recombinant preS proteins into permeabilized HepG2 cells. While maltose-binding proteins (MBP) were evenly distributed throughout the cytoplasm, MBP-preS fusion proteins selectively accumulated in the nucleus. Using truncated preS proteins, the effective domain for this nuclear accumulation was localized around the preS2 region. The mode of this action differs from conventional nuclear translocation mechanism in its energy-and mediator-independency and in that it is not saturated regardless of the increase of preS protein concentration. The biological meaning of this phenomenon has to be further studied. However, in regard to hepatitis B virus infection, this observation might provide a clue for unveiling the still poorly characterized events after initial internalization of the virus, which might make use of the nuclear translocation effect of the preS2 region to facilitate the infection.
Shedding of c-Met is regulated by crosstalk between a G-protein coupled receptor and the EGF receptor and is mediated by a TIMP-3 sensitive metalloproteinase
Authors: Nath Deepa , Williamson Nicola J. , Jarvis Robert , Murphy Gillian 
Published: April 2021

ABSTRACT
A wide repertoire of transmembrane proteins are proteolytically released from the cell surface by a process known as ‘ectodomain shedding’, under both normal and pathophysiological conditions. Little is known about the physiological mechanisms that regulate this process. As a model system, we have investigated the metalloproteinase-mediated cleavage of the hepatocyte growth factor receptor, Met. We show that epidermal growth factor (EGF) receptor activation, either directly by EGF or indirectly via the G-protein coupled receptor (GPCR) agonist lysophosphatidic acid (LPA), induces cleavage of Met through activation of the Erk MAP kinase signalling cascade. The tyrosine kinase activity of the EGFR was a prerequisite for this stimulation, since treatment of cells with a synthetic inhibitor of this receptor, AG1478, completely abrogated shedding. The metalloproteinase mediating Met cleavage was specifically inhibited by the tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. Furthermore, the level of Met shedding could be modulated by different cell-matrix interactions. Our results indicate that ectodomain shedding is a highly regulated process that can be stimulated by EGFR signalling pathways and integrin ligation.
The postsynaptic NMDA-receptor–PSD-95 signaling complex in excitatory synapses of the brain
Authors: Sheng Morgan 
Published: April 2021

ABSTRACT
Excitatory synapses of the brain use as neurotransmitter the amino acid glutamate, which is released in packets from the presynaptic terminal. The postsynaptic membrane is specialized for the reception of glutamate signals and the transduction of these signals into the postsynaptic cell. Containing a high concentration of glutamate receptors and associated cytoskeletal and signaling proteins, the postsynaptic specialization is visible by electron microscopy as a thickening (∼30 nm thick) of the postsynaptic membrane known as the postsynaptic density (PSD). The NMDA subtype of glutamate receptor (NMDAR) is an abundant constituent of the PSD and functions as a glutamategated calcium-permeable ion channel. NMDARs are anchored in the PSD by interactions between the cytoplasmic C-terminal tails of their NR2 subunits and the PDZ domains (orange) of PSD-95, an abundant PSD protein that forms a two-dimensional lattice immediately under the postsynaptic membrane. The PDZ domains of PSD-95 also bind to other postsynaptic membrane proteins, including potassium channels (K+ Ch), tyrosine kinases (ErbB4) and cell adhesion molecules (neuroligin). Having multiple domains that bind to a variety of cytoplasmic proteins, PSD-95 functions as a scaffold to assemble a specific set of signaling proteins around the NMDAR. These proteins, such as neuronal nitric oxide synthase (nNOS), SynGAP [a GTPase-activating protein (GAP) for Ras] and SPAR (a GAP for Rap), may participate in downstream signaling by NMDARs. PSD-95, in turn, interacts with GKAP and Shank, two scaffold proteins lying in the deep part of the PSD. Shank binds to Homer, which interacts directly with the cytoplasmic tail of the metabotropic glutamate receptor (mGluR), a G-protein-coupled glutamate receptor. Homer forms multimers and additionally binds to the inositol 1,4,5-trisphosphate receptor [Ins(1,4,5)P3R] found in smooth endoplasmic reticulum (SER), thereby linking cell surface mGluRs to a downstream effector [Ins(1,4,5)P3R] in intracellular calcium stores. Thus an extensive network of protein-protein interactions within the PSD links together different classes of postsynaptic glutamate receptor and couples them to specific intracellular signaling pathways.
Characterization of residues and sequences of the carbohydrate recognition domain required for cell surface localization and ligand binding of human lectin-like oxidized LDL receptor
Authors: Shi Xiaohua , Niimi Setsuko , Ohtani Toshio , Machida Sachiko 
Published: April 2021

ABSTRACT
Lectin-like oxidized low-density lipoprotein receptor (LOX-1) has been cloned from human aortic endothelial cells, and has a sequence identical to that from human lung. Previous studies showed that human LOX-1 can recognize modified LDL, apoptotic cells and bacteria. To further explore the relationship between the structure and function of LOX-1, a mutagenesis study was carried out. Our results showed that the carbohydrate recognition domain (CRD) was the ligand-binding domain of human LOX-1. We also investigated the sequences and residues in CRD that were essential for protein cell surface localization and ligand binding. LOX-1s carrying a mutation on each of six Cys in CRD resulted in a variety of N-glycosylation and failed to be transported to the cell surface. This was strong evidence for the involvement of all six Cys in the intrachain disulfide bonds required for proper folding, processing and transport of LOX-1. The C-terminal sequence (KANLRAQ) was also essential for protein folding and transport, while the four final residues (LRAQ) were involved in maintaining receptor function. Both positive charged (R208, R209, H226, R229 and R231) and non-charged hydrophilic (Q193, S198, S199 and N210) residues were involved in ligand binding, suggesting that ligand recognition of LOX-1 is not merely dependent on the interaction of positively charged residues with negatively charged ligands.
p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium
Authors: Rupper Adam C. , Rodriguez-Paris Juan M. , Grove Bryon D. , Cardelli James A. 
Published: April 2021

ABSTRACT
The Dictyostelium p110-related PI 3-kinases, PIK1 and PIK2, regulate the endosomal pathway and the actin cytoskeleton, but do not significantly regulate internalization of particles in D. discoideum. Bacteria internalized into Δddpik1/ddpik2 cells or cells treated with PI 3-kinase inhibitors remained intact as single particles in phagosomes with closely associated membranes after 2 hours of internalization, while in control cells, bacteria appeared degraded in multi-particle spacious phagosomes. Addition of LY294002 to control cells, after 60 minutes of chase, blocked formation of spacious phagosomes, suggesting PI 3-kinases acted late to regulate spacious phagosome formation. Phagosomes purified from control and drug treated cells contained equivalent levels of lysosomal proteins, including the proton pump complex, and were acidic, but in drug treated cells and Δddpik1/ddpik2 cells phagosomal pH was significantly more acidic during maturation than the pH of control phagosomes. Inhibition of phagosomal maturation by LY294002 was overcome by increasing phagosomal pH with NH4Cl, suggesting that an increase in pH might trigger homotypic phagosome fusion. A pkbA null cell line (PKB/Akt) reproduced the phenotype described for cells treated with PI 3-kinase inhibitors and Δddpik1/ddpik2 cells. We propose that PI 3-kinases, through a PKB/Akt dependent pathway, directly regulate homotypic fusion of single particle containing phagosomes to form multi-particle, spacious phagosomes, possibly through the regulation of phagosomal pH.
Rho and Rac but not Cdc42 regulate endothelial cell permeability
Authors: Wójciak-Stothard Beata , Potempa Sandra , Eichholtz Thomas , Ridley Anne J. 
Published: April 2021

ABSTRACT
Endothelial permeability induced by thrombin and histamine is accompanied by actin stress fibre assembly and intercellular gap formation. Here, we investigate the roles of the Rho family GTPases Rho1, Rac1 and Cdc42 in regulating endothelial barrier function, and correlate this with their effects on F-actin organization and intercellular junctions. RhoA, Rac1 and Cdc42 proteins were expressed efficiently in human umbilical vein endothelial cells by adenovirus-mediated gene transfer. We show that inhibition of Rho prevents both thrombin- and histamine-induced increases in endothelial permeability and decreases in transendothelial resistance. Dominant-negative RhoA and a Rho kinase inhibitor, Y-27632, not only inhibit stress fibre assembly and contractility but also prevent thrombin- and histamine-induced disassembly of adherens and tight junctions in endothelial cells, providing an explanation for their effects on permeability. In contrast, dominant-negative Rac1 induces permeability in unstimulated cells and enhances thrombin-induced permeability, yet inhibits stress fibre assembly, indicating that increased stress fibre formation is not essential for endothelial permeability. Dominant-negative Cdc42 reduces thrombin-induced stress fibre formation and contractility but does not affect endothelial cell permeability or responses to histamine. These results demonstrate that Rho and Rac act in different ways to alter endothelial barrier function, whereas Cdc42 does not affect barrier function.
Alteration of the stability of Bag-1 protein in the control of olfactory neuronal apoptosis
Authors: Sourisseau Tony , Desbois Christine , Debure Laure , Bowtell David D. L. , Cato Andrew C. B. , Schneikert Jean , Moyse Emmanuel , Michel Denis 
Published: April 2021

ABSTRACT
Normal apoptosis occurs continuously in the olfactory neuroepithelium of adult vertebrates, making it a useful model for studying neuronal apoptosis. Here we demonstrate that overexpression of the anti-apoptotic Bag-1 gene in olfactory neuronal cells confers a strong resistance to apoptosis. Conversely decreased levels of Bag-1 were found to precede a massive wave of olfactory neuronal apoptosis triggered by synaptic target ablation. We show that the decrease is brought about by ubiquitination and subsequent degradation of the Bag-1 protein. The ring finger protein Siah-2 is a likely candidate for the ubiquitination reaction since Siah-2 mRNA accumulated in lesioned olfactory neuroepithelium and overexpression of Siah-2 stimulated Bag-1 ubiquitination and degradation in transient expression assays. These results together identify destabilization of Bag-1 as a necessary step in olfactory neuronal apoptosis.
An AAA family tree
Authors: Fröhlich Kai-Uwe 
Published: April 2021

ABSTRACT
Members of the AAA (for ATPases associated with various cellular activities) protein superfamily are characterized by the presence of one or two highly conserved modules of approximately 230 amino acid residues (the AAA box) that include an ATP-binding consensus. AAA proteins are found in all organisms and are essential for cell cycle functions, vesicular transport, mitochondrial functions, peroxisome assembly and proteolysis.
KSR: a MAPK scaffold of the Ras pathway?
Authors: Morrison Deborah K. 
Published: April 2021

ABSTRACT
Kinase Suppressor of Ras (KSR) is an intriguing component of the Ras pathway that was first identified by genetic studies performed in Drosophila melanogaster and Caenorhabditis elegans. In both organisms, inactivating mutations in KSR suppress the phenotypic effects induced by activated Ras. These findings together with the fact that KSR contains many structural features characteristic of a protein kinase led to early speculation that KSR is a kinase functioning upstream of the Ras pathway component Raf-1 or in a parallel Ras-dependent pathway. However, in the six years since its discovery, KSR has been found to lack several key properties of known protein kinases, which has cast doubt on whether KSR is indeed a functional enzyme.
A major breakthrough in our understanding of the role of KSR in signal transduction has come from recent findings that KSR interacts with several components of the MAP kinase cascade, including Raf-1, MEK1/2 and ERK1/2. The model now emerging is that KSR acts as a scaffolding protein that coordinates the assembly of a membrane-localized, multiprotein MAP kinase complex, a vital step in Ras-mediated signal transduction. Thus, while Kinase Suppressor of Ras may be its name, phosphorylation may not be its game.
Regulation of heparan sulfate proteoglycan nuclear localization by fibronectin
Authors: Richardson Thomas P. , Trinkaus-Randall Vickery , Nugent Matthew A. 
Published: April 2021

ABSTRACT
Heparan sulfate proteoglycans (HSPG) regulate multiple cellular processes and mediate the cellular uptake of numerous molecules. While heparan sulphate glycosaminoglycan chains are known to modulate receptor binding of several heparin-binding proteins, here we show that distinct extracellular matrices direct HSPG to the nucleus. We analyzed HSPG localization in primary corneal fibroblasts, cultured on fibronectin or collagen type I matrices, using confocal laser scanning microscopy and cell fractionation. Image analysis revealed that the nuclear localization of HSPG core proteins was greater when cells were cultured on fibronectin versus collagen. Matrices containing the heparin-binding domain of fibronectin, but not the integrin-activating domain, demonstrated increased nuclear staining of core proteins. Furthermore, activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited nuclear targeting of HSPG in cells on fibronectin, whereas inhibition of protein kinase C with Ro-31-8220 greatly enhanced nuclear localization of HSPG in cells on both collagen and fibronectin. We propose a matrix-dependent mechanism for nuclear localization of cell surface HSPG involving protein kinase C-mediated signaling. Nuclear localization of HSPG might play important roles in regulating nuclear function.
Apoptotic cell death of cultured salamander photoreceptors induced by cccp: CsA-insensitive mitochondrial permeability transition
Authors: Yang Jun-Hai , Gross Ronald L. , Basinger Scott F. , Wu Samuel M. 
Published: April 2021

ABSTRACT
Photoreceptor degeneration is mediated by apoptosis in several animal models, although the underlying mechanisms are yet to be elucidated. We present here an apoptotic model based on a primary cell culture of tiger salamander photoreceptors, in which treatment with carbonyl cyanide m-chlorophenylhydrazone (cccp), a protonophore, induced apoptosis. Cells exposed to cccp showed condensed nuclei and displayed positive TdT-dUTP terminal nick-end labeling (TUNEL). In addition, 10-100 μM cccp rapidly induced a reduction of ΔΨm and ≥30 μM cccp induced a significant leakage of calcein from mitochondria to cytosol and nucleus, indicating a change in mitochondrial inner membrane permeability. Cyclosporin A (CsA), a transition pore blocker, did not prevent the cccp-induced MPT or the cccp-evoked apoptotic cell death, suggesting that cccp-induced apoptotic process was mediated by a CsA-insensitive pathway. This cell model provides an in vitro tool for studying mechanisms of photoreceptor apoptosis in isolated photoreceptors and may provide clues to the etiology of retinal degeneration.
The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration
Authors: Buensuceso Charito S. , Woodside Darren , Huff Janice L. , Plopper George E. , O’Toole Timothy E. 
Published: April 2021

ABSTRACT
The scaffolding protein, Rack1, is a seven-WD-domain-containing protein that has been implicated in binding to integrin β subunit cytoplasmic domains and to members of two kinase families (src and protein kinase C, PKC) that mediate integrin bidirectional signaling. To explore the role of Rack1 in integrin function we have transfected this protein in Chinese hamster ovary (CHO) cells. We have observed no effect of Rack1 overexpression on inside-out signaling as the ligand binding properties of CHO cells also expressing constitutively active or inactive integrins were not affected. In contrast, we observed that cells stably or transiently overexpressing Rack1 had decreased migration compared to mock transfected cells. Stable Rack1 transfectants also demonstrated an increased number of actin stress fibers and focal contacts. These effects on motility and cytoskeletal organization did not appear to result from Rack1 inhibition of src function as downstream substrates of this kinase were phosphorylated normally. In addition, expression of an active src construct did not reverse the migratory deficit induced by Rack1 overexpression. On the other hand when we overexpressed a Rack1 variant with alanine substitutions in the putative PKC binding site in its third WD domain, we observed no deficit in migration. Thus the ability of Rack1 to bind, localize and stabilize PKC isoforms is likely to be involved in aspects of integrin outside-in signaling.
Phosphorylation of p190 on Tyr1105 by c-Src is necessary but not sufficient for EGF-induced actin disassembly in C3H10T1/2 fibroblasts
Authors: Haskell Michelle D. , Nickles Amanda L. , Agati Joyce M. , Su Ling , Dukes Bernard D. , Parsons Sarah J. 
Published: April 2021

ABSTRACT
p190 RhoGAP is a tyrosine phosphorylated protein that contains an N-terminal GTP binding domain, a middle domain (MD) that mediates interaction with p120 RasGAP and a C-terminal GTPase-activating protein (GAP) domain that is specific for the Rho family of small GTPases. Evidence is accumulating to suggest that p190 participates in actin cytoskeleton rearrangements that occur following transformation by v-Src or stimulation by growth factors, and that tyrosine phosphorylation of p190 by Src influences these processes. The current study was performed to establish whether p190RhoGAP directly participates in epidermal growth factor-induced actin stress fiber disassembly and how c-Src is involved in this process. Our results support a model in which the p190 MD negatively regulates the activity of the GAP domain and that c-Src phosphorylation of Y1105 is necessary, but insufficient on its own, for actin stress fiber disassembly.
Self-assembly and binding of a sorting nexin to sorting endosomes
Authors: Kurten Richard C. , Eddington Anthony D. , Chowdhury Parag , Smith Richard D. , Davidson April D. , Shank Brian B. 
Published: April 2021

ABSTRACT
The fate of endocytosed membrane proteins and luminal contents is determined by a materials processing system in sorting endosomes. Endosomal retention is a mechanism that traps specific proteins within this compartment, and thereby prevents their recycling. We report that a sorting nexin SNX1, a candidate endosomal retention protein, self-assembles in vitro and in vivo, and has this property in common with its yeast homologue Vps5p. A comparison of SNX1 expressed in bacterial and in mammalian systems and analyzed by size-exclusion chromatography indicates that in cytosol SNX1 tetramers are part of a larger complex with additional proteins. An endosomal retention function would require that SNX1 bind to endosomal membranes, yet the complexes that we analyzed were largely soluble and little SNX1 was found in pellet fractions. Using green fluorescent protein fusions, endocytic compartment markers and fluorescence recovery after photobleaching, we found that there is an equilibrium between free cytoplasmic and early/sorting endosome-bound pools of green fluorescent protein-SNX1. Fluorescence resonance energy transfer indicated that spectral variants of green fluorescent protein-SNX1 were oligomeric in vivo. In cell extracts, these green fluorescent protein-SNX1 oligomers corresponded to tetrameric and larger complexes of green fluorescent protein-SNX1. Using video microscopy, we observed small vesicle docking and tubule budding from large green fluorescent protein-SNX1 coated endosomes, which are features consistent with their role as sorting endosomes.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs2058.html