bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells
Authors: Kato Takayuki , Watanabe Naoki , Morishima Yosuke , Fujita Akiko , Ishizaki Toshimasa , Narumiya Shuh
Published: April 2021

mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu434 and Leu455 in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.
Keratinocyte migration requires α2β1 integrin-mediated interaction with the laminin 5 γ2 chain
Authors: Decline Françoise , Rousselle Patricia
Published: April 2021

Keratinocyte migration is an absolute requirement for correct epithelialization during the process of wound healing. This process requires changes in extracellular matrix ligand expression as well as changes in ligand-binding affinity of the corresponding cellular integrins. In this study, we attempt to understand the role of laminin 5 in migration by investigating the integrin-mediated interactions of migrating keratinocytes with their newly synthesized laminin 5. We chose to induce migration of freshly isolated NHK in vitro by exposing them to TGF-β1 which, in addition to promoting epithelial cell migration, is also known to prevent cell proliferation. This important feature allowed the study to be focused on cell migration without interfering with cell proliferation. We confirm that keratinocyte migration on plastic, fibronectin or collagen IV substrates requires endogenous laminin 5 deposition, which is predominantly detected under its unprocessed form. Despite a crucial role for laminin 5 in migration, we show that this process is accompanied by a significant decrease in adhesion to purified laminin 5. Moreover, we provide evidence that the α3β1 integrin interaction with newly synthesized laminin 5 renders the cells more adherent and retards migration. Conversely, we provide evidence that the α2β1 integrin-laminin 5 interaction is absolutely required for keratinocyte migration and that the α2β1 integrin is responsible for cell spreading on laminin 5. Finally, we demonstrate that the α2β1 integrin binding to laminin 5 occurs within the short arm of the γ2 subunit.
Hypoxia in combination with FGF-2 induces tube formation by human microvascular endothelial cells in a fibrin matrix: involvement of at least two signal transduction pathways
Authors: Kroon Marielle E. , Koolwijk Pieter , Van der Vecht Bea , Van Hinsbergh Victor W. M.
Published: April 2021

Hypoxia in combination with a growth factor is a strong inducer of angiogenesis. Among several effects, hypoxia can activate endothelial cells directly, but the mechanism by which it acts is not fully elucidated. In vitro, human microvascular endothelial cells (hMVEC) form capillary-like tubules in fibrin solely after stimulation with a combination of fibroblast growth factor (FGF)-2 or vascular endothelial growth factor (VEGF) and the cytokine tumour necrosis factor (TNF)α. We show in this paper that in hypoxic conditions, FGF-2-stimulated hMVEC form tube-like structures in a fibrin matrix in the absence of TNFα. Hypoxia/FGF-2-stimulated cells express more urokinase-type plasminogen activator (u-PA) receptor than normoxia/FGF-2-stimulated cells and display a slightly higher turnover of u-PA. This small increase in u-PA activation probably cannot fully explain the hypoxia/FGF-2-induced tube formation. Hypoxia activated at least two signal pathways that may contribute to the enhanced angiogenic response. In hypoxia/FGF-2-stimulated hMVEC the transcription factor p65 was activated and translocated to the nucleus, whereas in normoxia/FGF-2-stimulated cells p65 remained inactive. Furthermore, in hypoxic conditions, the amounts of phosphorylated mitogen-activated protein kinases ERK1/2 were increased compared to normoxic conditions. We conclude that hypoxia is able to activate different signal pathways in FGF-2-stimulated human endothelial cells, which may be involved in hypoxia-induced angiogenesis.
Illuminating the secretory pathway: when do we need vesicles?
Authors: Stephens David J. , Pepperkok Rainer
Published: April 2021

Recent studies using GFP-tagged markers and time-lapse microscopy have allowed direct visualisation of membrane traffic in the secretory pathway in living mammalian cells. This work shows that larger membrane structures, 300-500 nm in size, are the vehicles responsible for long distance, microtubule-dependent ER-to-Golgi and trans-Golgi to plasma membrane transport of secretory markers. At least two retrograde transport pathways from the Golgi to the ER exist, both of which are proposed to involve a further class of long, tubular membrane carrier that forms from the Golgi and fuses with the ER. Together, this has challenged established transport models, raising the question of whether larger pleiomorphic structures, rather than small 60-80 nm transport vesicles, mediate long-range transport between the ER and Golgi and between the Golgi and plasma membrane.

Movie available on-line: http://www.biologists.com/JCS/movies/jcs2220.html, Movie 2A, Movie 2AA, Movie 2B, Movie 2BB, Movie 2C
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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
The role of IKK in constitutive activation of NF-κB transcription factor in prostate carcinoma cells
Authors: Gasparian Alexander V. , Yao Ya Juan , Kowalczyk Dariusz , Lyakh Ludmila A. , Karseladze Apollon , Slaga Thomas J. , Budunova Irina V.
Published: April 2021

Rel/NF-κB transcription factors are implicated in the control of cell proliferation, apoptosis and transformation. The key to NF-κB regulation is the inhibitory IκB proteins. During response to diverse stimuli, IκBs are rapidly phosphorylated by IκB kinases (IKKs), ubiquitinated and undergo degradation. We have investigated the expression and function of NF-κB, IκB inhibitors and IKKs in normal prostate epithelial cells and prostate carcinoma (PC) cell lines LNCaP, MDA PCa 2b, DU145, PC3, and JCA1. We found that NF-κB was constitutively activated in human androgen-independent PC cell lines DU145, PC3, JCA1 as well as androgen-independent CL2 cells derived from LNCaP. In spite of a strong difference in constitutive κB binding, Western blot analysis did not reveal any significant variance in the expression of p50, p65, IκBs, IKKα, and IKKβ between primary prostate cells, androgen-dependent and androgen-independent PC cells. However, we found that in androgen-independent PC cells IκBα was heavily phosphorylated and displayed a faster turnover. Using an in vitro kinase assay we demonstrated constitutive activation of IKK in androgen-independent PC cell lines. Blockage of NF-κB activity in PC cells by dominant-negative IκBα resulted in increased constitutive and TNF-α-induced apoptosis. Our data suggest that increased IKK activation leads to the constitutive activation of NF-κB ‘survival signaling’ pathway in androgen-independent PC cells. This may be important for the support of their androgen-independent status and growth advantage.
DNA damage-dependent interaction of the nuclear matrix protein C1D with translin-associated factor X (TRAX)
Authors: Erdemir Tuba , Bilican Bilada , Oncel Dilhan , Goding Colin R. , Yavuzer Ugur
Published: April 2021

The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination. C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon γ-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo. In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX. Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus. We show that C1D and TRAX interact specifically in both yeast and mammalian cells. Interestingly, however, interaction of these two proteins in mammalian cells only occur following γ-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX. Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization. These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation.
Mammalian nuclei become licensed for DNA replication during late telophase
Authors: Dimitrova Daniela S. , Prokhorova Tatyana A. , Blow J. Julian , Todorov Ivan T. , Gilbert David M.
Published: April 2021

Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.
The tubby-like proteins, a family with roles in neuronal development and function
Authors: Ikeda Akihiro , Nishina Patsy M. , Naggert Jürgen K.
Published: April 2021

The identification of a mutation at the tubby (Tub) locus, which causes obesity and neurosensory degeneration, led to the discovery of the tubby-like proteins (TULPs). Tub and the genes that encode three tubby-like proteins (TULP1- TULP3) form a novel, small gene family that plays an important role in maintenance and function of neuronal cells during development and post-differentiation. Although exploration of the molecular function of these genes is still in its infancy, recent biochemical studies have provided ‘entry points’ into pathways whose elucidation will further our understanding of TULP action. In addition, mRNA expression and translocation of the TUB protein have been shown to be regulated by thyroid hormone and by G-protein-coupled receptor signaling, respectively. These latter findings may help to link the cellular function of TUB to known mechanisms for energy homeostasis.
Cell-surface attachment of pedestal-forming enteropathogenicE. coliinduces a clustering of raft components and a recruitment of annexin 2
Authors: Zobiack Nicole , Rescher Ursula , Laarmann Sven , Michgehl Silke , Schmidt M. Alexander , Gerke Volker
Published: April 2021

Annexin 2 is a Ca2+-regulated membrane- and F-actin-binding protein implicated in the stabilization or regulation of membrane/cytoskeleton contacts, or both, at the plasma membrane and at early endosomal membranes. To analyze the dynamic nature of such action we investigated whether annexin 2 could be found at sites of localized actin rearrangements occurring at the plasma membrane of HeLa cells infected with noninvading enteropathogenic Escherichia coli (EPEC). We show that adherent EPEC microcolonies, which are known to induce the formation of actin-rich pedestals beneath them, specifically recruit annexin 2 to the sites of their attachment. Mutant EPEC (EPECtir), which lack a functional receptor for intimate attachment (Tir, translocated intimin receptor) and which fail to produce full pedestal formation, are still capable of recruiting annexin 2 to the bacterial contact sites. Accumulation of annexin 2 at sites of EPEC or EPECtir attachment is accompanied by a recruitment of the annexin 2 protein ligand S100A10. EPEC and EPECtir attachment also induces a concentration of cholesterol and glycosyl phosphatidylinositol-anchored proteins at sites of bacterial contact. This indicates that membrane components present in rafts or raft-like microdomains are clustered upon EPEC adherence and that annexin 2 is recruited to the cytoplasmic membrane surface of such clusters, possibly stabilizing raft patches and their linkage to the actin cytoskeleton beneath adhering EPEC.
Extracellular matrix histone H1 binds to perlecan, is present in regenerating skeletal muscle and stimulates myoblast proliferation
Authors: Henriquez Juan Pablo , Casar Juan Carlos , Fuentealba Luis , Carey David J. , Brandan Enrique
Published: April 2021

Heparan sulfate chains of proteoglycans bind to and regulate the function of a wide variety of ligands. In myoblasts, heparan sulfate proteoglycans modulate basic fibroblast growth factor activity and regulate skeletal muscle differentiation. The aim of this study was to identify endogenous extracellular ligands for muscle cell heparan sulfate proteoglycans.

[35S]heparin ligand blot assays identified a 33/30 kDa doublet(p33/30) in detergent/high ionic strength extracts and heparin soluble fractions obtained from intact C2C12 myoblasts. p33/30 is localized on the plasma membrane or in the extracellular matrix where its level increases during muscle differentiation. Heparin-agarose-purified p33/30 was identified as histone H1. In vitro binding assays showed that histone H1 binds specifically to perlecan. Immunofluorescence microscopy showed that an extracellular pool of histone H1 colocalizes with perlecan in the extracellular matrix of myotube cultures and in regenerating skeletal muscle. Furthermore, histone H1 incorporated into the extracellular matrix strongly stimulated myoblast proliferation via a heparan-sulfate-dependent mechanism.

These results indicate that histone H1 is present in the extracellular matrix of skeletal muscle cells, where it interacts specifically with perlecan and exerts a strong proliferative effect on myoblasts, suggesting a role for histone H1 during skeletal muscle regeneration.
Sperm plasma-membrane-associated glutathione S-transferases as gamete recognition molecules
Authors: Hemachand Tummala , Gopalakrishnan Bagavathi , Salunke Dinakar M. , Totey Satish M. , Shaha Chandrima
Published: April 2021

Glutathione S-transferases (GSTs) are enzymes that detoxify electrophilic compounds. Earlier studies from our laboratory showed that anti-GST antibodies interfered with the fertilising ability of spermatozoa from Capra hircus (goat) in vitro, suggesting that GSTs are localised at the cell surface. In this study, we provide evidence for the presence of GSTs of 24 kDa on the sperm plasma membrane attached by non-covalent interactions. The GST activity associated with the spermatozoal plasma membrane was significantly higher than the activity present in the plasma membranes of brain cells,hepatocytes, spleenocytes and ventriculocytes. Analysis of GST isoforms demonstrates the presence of GST Pi and Mu on the sperm plasma membranes. Both isoforms were able to bind to solubilised as well as intact zona pellucida(ZP) through their N-terminal regions but failed to bind to ZP once the oocytes were fertilised. Solubilised goat ZP separates into three components,one of which, the ZP3-like component, bound to sperm GSTs. High concentrations of anti-GST antibodies or solubilised ZP led to aggregation of sperm GSTs,resulting in the release of acrosin. In contrast, inhibition of sperm GST binding to ZP, by saturation of binding sites for sperm GSTs on the solubilised ZP using peptides designed from the N-terminii of GST Pi or Mu or blocking of binding sites for ZP on sperm GSTs with antibodies raised against the N-terminal GST peptides, inhibited essential prefertilisation changes in sperm.

These data therefore demonstrate the strategic location of catalytically active defensive enzymes on the sperm surface that also act as zona-binding proteins. Therefore, sperm-surface GSTs serve as bifunctional molecules in a transcriptionally inactive cell whose requirement for cellular defense and economy of molecules that it can carry is greater than that of any somatic cell type.
Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein
Authors: Vallenius Tea , Mäkelä Tomi P.
Published: April 2021

In this report we have characterized a novel, ubiquitously expressed kinase, Clik1, that is predominantly nuclear and undergoes autophosphorylation. Yeast two-hybrid analysis indicated a highly specific association between Clik1 and CLP-36, which was identified in 36 out of 37 Clik1-interacting clones. CLP-36 is a PDZ-LIM protein that localizes to actin stress fibers in nonmuscle cells and associates with α-actinin via its PDZ-domain. The association of CLP-36 with Clik1, in turn, is mediated by the C-terminal part of CLP-36 containing the LIM domain, and association was not noted with the closely related ALP PDZ-LIM protein. Interestingly, the association with CLP-36 led to relocalization of the otherwise nuclear Clik1 kinase to actin stress fibers, where it disrupted the periodic staining pattern of CLP-36. Taken together these results establish the CLP-36 PDZ-LIM protein as an adapter, recruiting the Clik1 kinase to actin stress fibers in nonmuscle cells, and suggest that Clik1 represents a novel regulator of actin stress fibers.
Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo
Authors: Marchetti Sandrine , Gimond Clotilde , Iljin Kristiina , Bourcier Christine , Alitalo Kari , Pouysségur Jacques , Pagès Gilles
Published: April 2021

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation, reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study, we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter, tie-1. Using EGFP as a reporter gene, we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently, tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected, puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers, including CD31, CD34,VEGFR-1, VEGFR-2, Tie-1, VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of α-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-β1, two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 andα-smooth muscle actin markers. Finally, we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together, these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies.
Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis
Authors: Oostendorp Robert A. J. , Medvinsky Alexander J. , Kusadasi Nuray , Nakayama Naoki , Harvey Kirsty , Orelio Claudia , Ottersbach Katrin , Covey Todd , Ploemacher Rob E. , Saris Chris , Dzierzak Elaine
Published: April 2021

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers,isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.