bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell cycle-dependent repetitive Ca2+ waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization
Authors: McDougall Alex , Levasseur Mark , O’Sullivan Antony J. , Jones Keith T. 
Published: April 2021

ABSTRACT
Sperm-triggered Ca2+ oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca2+ oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca2+ oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca2+ oscillations that are all waves in ascidians. The first of these Ca2+ waves begins at the point of sperm-egg fusion while a second phase of Ca2+ waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca2+ waves during meiosis and thereby ensuring meiotic exit.
We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca2+-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca2+ waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca2+ waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca2+ oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca2+ oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca2+ release even though they responded to microinjection of the Ca2+-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca2+-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca2+-releasing activity.
These data support a model of egg activation whereby the sperm introduces a Ca2+-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca2+ waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.
Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages
Authors: Duclos Sophie , Diez Roberto , Garin Jérome , Papadopoulou Barbara , Descoteaux Albert , Stenmark Harald , Desjardins Michel 
Published: April 2021

ABSTRACT
Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites.
Synthesis and sorting of proteoglycans
Authors: Prydz Kristian , Dalen Knut Tomas 
Published: April 2021

ABSTRACT
Proteoglycans are widely expressed in animal cells. Interactions between negatively charged glycosaminoglycan chains and molecules such as growth factors are essential for differentiation of cells during development and maintenance of tissue organisation. We propose that glycosaminoglycan chains play a role in targeting of proteoglycans to their proper cellular or extracellular location. The variability seen in glycosaminoglycan chain structure from cell type to cell type, which is acquired by use of particular Ser-Gly sites in the protein core, might therefore be important for post-synthesis sorting. This links regulation of glycosaminoglycan synthesis to the post-Golgi fate of proteoglycans.
The PI 3-kinase isoforms p110α and p110β have differential roles in PDGF- and insulin-mediated signaling
Authors: Hooshmand-Rad Roya , Hájková Lucie , Klint Peter , Karlsson Roger , Vanhaesebroeck Bart , Claesson-Welsh Lena , Heldin Carl-Henrik 
Published: April 2021

ABSTRACT
Phosphoinositide 3´-kinases constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Phosphoinositide 3´-kinases that bind to the platelet-derived growth factor receptor are composed of two subunits: the p85 subunit acts as an adapter and couples the catalytic p110 subunit to the activated receptor. There are different isoforms of p85 as well as of p110, the individual roles of which have been elusive. Using microinjection of inhibitory antibodies specific for either p110α or p110β we have investigated the involvement of the two p110 isoforms in platelet-derived growth factor- and insulin-induced actin reorganization in porcine aortic endothelial cells. We have found that antibodies against p110α, but not antibodies against p110β, inhibit platelet-derived growth factor-stimulated actin reorganization, whereas the reverse is true for inhibition of insulin-induced actin reorganization. These data indicate that the two phosphoinositide 3´-kinase isoforms have distinct roles in signal transduction pathways induced by platelet-derived growth factor and insulin.
c-Cbl localizes to actin lamellae and regulates lamellipodia formation and cell morphology
Authors: Scaife Robin M. , Langdon Wallace Y. 
Published: April 2021

ABSTRACT
Adhesive and locomotive properties of cells have key roles in normal physiology and disease. Cell motility and adhesion require the assembly and organization of actin microfilaments into stress fibers, lamellipodia and filopodia, and the formation of these structures is mediated by signalling through Rho GTPases. Here we identify c-Cbl (a multi-adaptor proto-oncogene product involved in protein tyrosine kinase signalling) as an important regulator of the actin cytoskeleton. By immunofluorescence microscopy we have determined that c-Cbl co-localizes with the adaptor protein Crk to submembranous actin lamellae in NIH 3T3 fibroblasts and that c-Cbl’s actin localization requires specific SH3-binding sequences. Further, we have found that truncation of this SH3-binding domain in c-Cbl profoundly alters the morphology of NIH 3T3 fibroblasts by inhibiting the formation of actin lamellae, lamellipodia and membrane ruffles. The induction of lamellipodia and membrane ruffles are also inhibited during cell spreading and migration, conditions when these structures are normally most prominent. The inhibitory effect of truncated c-Cbl expression on lamellipodia formation can be reversed by mutational inactivation of its divergent SH2 domain, by the co-expression of constitutively active Rac or by the overexpression of c-Cbl. This study therefore identifies a cytoskeletal role for c-Cbl which may involve the regulation of Crk and Rac, and which is dependent on targeting of c-Cbl to actin lamellae and the ability to recruit signalling protein(s) associated with its divergent SH2 domain.
Increased recycling of α5β1 integrins by lung endothelial cells in response to tumor necrosis factor
Authors: Gao Baochong , Curtis Theresa M. , Blumenstock Frank A. , Minnear Fred L. , Saba Thomas M. 
Published: April 2021

ABSTRACT
Tumor necrosis factor α (TNF-α) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-α may influence cell-matrix interactions by altering the clustering as well as internalization of the α5β1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-α caused an increase in the intracellular staining of α5β1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37°C after antibody-labeling of their surface α5β1 integrins at 4°C confirmed an increase in the rate of α5β1 integrin internalization which was at least 3 times greater after TNF-α exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of α5β1 integrins was relatively constant after TNF-α exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized α5β1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P&lt;0.01) increase in the rate of recycling of the internalized integrins in TNF-treated endothelial cells. Enhanced internalization and subsequent recycling of α5β1 integrins by endothelial monolayers exposed to TNF-α may facilitate the redistribution of cell-surface integrins in response to this inflammatory cytokine and may also modify cell-matrix interactions leading to reduced integrity and increased protein permeability of the lung endothelial monolayers.
Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes
Authors: Khayat Zayna A. , Tong Peter , Yaworsky Karen , Bloch Robert J. , Klip Amira 
Published: April 2021

ABSTRACT
We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucose transporters in L6 myotubes expressing myc-tagged glucose transporter 4 (GLUT4myc). Scanning electron microscopy revealed a dynamic distortion of the dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimulated cells, phalloidin-labeled actin filaments ran parallel to the longitudinal axis of the cell. Immunostaining of the p85 regulatory subunit of phosphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuclear. After 3 minutes of insulin treatment, actin reorganized to form structures; these structures protruded from the dorsal surface of the myotubes by 10 minutes and condensed in the myoplasm into less prominent foci at 30 minutes. The p85 polypeptide colocalized with these structures at all time points. Actin remodeling and p85 relocalization to actin structures were prevented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the actin-rich projections was also observed, but only after 10 minutes of insulin treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble material that was also enriched with actin. In contrast, vp165, a transmembrane aminopeptidase that morphologically colocalized with GLUT4 vesicles, was fully soluble in Triton X-100 extracts of both insulin-treated and control myotubes. Transient transfection of dominant inhibitory Rac1 (N17) into L6 myotubes prevented formation of dorsal actin structures and blocked insulin-induced GLUT4myc translocation to the cell surface. We propose that insulin-dependent formation of actin structures facilitates the association of PI3-K (p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.
The path of RNA through nuclear pores: apparent entry from the sides into specialized pores
Authors: Iborra Francisco J. , Jackson Dean A. , Cook Peter R. 
Published: April 2021

ABSTRACT
The path that RNA takes through nuclear pores was mapped using two high-resolution techniques. Unexpectedly, no RNA in HL60 cells was detected by immunogold labelling in the central axis of the pore complex on its way to the transporter at the nuclear membrane; instead, it was distributed around the sides, apparently entering just before the membrane. In rat liver nuclei, poly(A)+ RNA, hnRNPs A1 and C, mrnp 41, ASF, and a phosphorylated subset of SR proteins were also distributed like mRNA, as were various transport factors and their cargoes (NTF2, Ran, RCC1, karyopherin β, Rch1, transportin α, m2,2,7-trimethylG). Many pores were associated with particular transport factors/cargoes to the exclusion of others; some were associated with poly(A)+ RNA or phosphorylated SR proteins (but not NTF2), others with NTF2 (but not poly(A)+ RNA or the SR proteins). Electron spectroscopic imaging confirmed these results. Some pores contained phosphorus-rich RNA apparently entering from the sides; others lacked any phosphorus, and were surrounded by a ribosome-free zone in the cytoplasm. The results also suggest that pores have different functional zones where SR proteins are dephosphorylated, and where hnRNP C is removed from messages.
Clathrin-coated lattices and buds on MHC class II compartments do not selectively recruit mature MHC-II
Authors: Ramm Georg , Pond Leslie , Watts Colin , Stoorvogel Willem 
Published: April 2021

ABSTRACT
Newly synthesized major histocompatibility complex class II molecules (MHC-II) are transported to MHC-II-containing endosomal and lysosomal compartments (MIICs) for the degradation of associated invariant chain and peptide loading. Subsequently MHC-II is transported to the plasma membrane, in part through direct fusion of MIICs with the plasma membrane. In search of potential alternative pathway(s) we studied the 3-dimensional structure of MIICs and the subcellular distribution of MHC-II by immuno electronmicroscopy on whole-mount preparations and cryosections of Mel JuSo cells. Intracellular MHC-II and invariant chain mainly localized to lamp-1 positive compartments suggesting that the majority of MHC-II exits the endocytic tract at lysosomes. Clathrin-coated lattices and buds were found to be associated with these organelles, but MHC-II was not found to be enriched in the clathrin-coated domains. Moreover, leupeptin, a drug that interferes with Ii-processing and delays delivery of newly synthesized MHC-II to the plasma membrane, was not found to decrease the relative amount of MHC-II in clathrin-coated areas. Together these data indicate clathrin-mediated exit site(s) from lysosomes but suggest that they do not selectively recruit mature MHC-II, consistent with the notion that transport to the plasma membrane occurs independently of the cytoplasmic domains of the MHC-II α and β chains.
Integrin cytoplasmic domain-binding proteins
Authors: Liu Shouchun , Calderwood David A. , Ginsberg Mark H. 
Published: April 2021

ABSTRACT
Integrins are a large family of cell surface receptors that mediate cell adhesion and influence migration, signal transduction, and gene expression. The cytoplasmic domains of integrins play a pivotal role in these integrin-mediated cellular functions. Through interaction with the cytoskeleton, signaling molecules, and other cellular proteins, integrin cytoplasmic domains transduce signals from both the outside and inside of the cell and regulate integrin-mediated biological functions. Identification and functional analyses of integrin cytoplasmic domain-binding proteins have been pursued intensively. In recent years, more cellular proteins have been reported to directly interact with integrin cytoplasmic domains and some of these interactions may play important roles in integrin-mediated biological responses. Integrin β chains, for example, interact with actin-binding proteins (e.g. talin and filamin), which form mechanical links to the cytoskeleton. These and other proteins (e.g. FAK, ILK and novel proteins such as TAP20) might also link integrins to signaling mechanisms and, in some cases (e.g. JAB1) mediate integrin-dependent gene regulation.
Regulation of the formation of tumor cell pseudopodia by the Na+/H+ exchanger NHE1
Authors: Lagana A. , Vadnais J. , Le P. U. , Nguyen T. N. , Laprade R. , Nabi I. R. , Noël J. 
Published: April 2021

ABSTRACT
The Na+/H+ exchanger NHE1 is involved in intracellular pH homeostasis and cell volume regulation and accumulates with actin in the lamellipodia of fibroblasts. In order to determine the role of NHE1 following epithelial transformation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) transformed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV). Expression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with β-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-mediated 22Na uptake to ethylisopropylamiloride, a specific inhibitor of NHE1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intracellular pH induced upon EIPA treatment were also of higher magnitude in MSV-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 μM ethylisopropylamiloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells, the effect of ethylisopropylamiloride treatment was more pronounced and associated with the increased reversible detachment of the cells from the substrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minutes of addition, ethylisopropylamiloride induced pseudopodial retraction and inhibited cell motility. The ability of ethylisopropylamiloride to prevent nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO3 medium, showing that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesion and pseudopodial protrusion of motile, invasive tumor cells.
Real time imaging reveals a peroxisomal reticulum in living cells
Authors: Schrader Michael , King Stephen J. , Stroh Tina A. , Schroer Trina A. 
Published: April 2021

ABSTRACT
We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.
Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover
Authors: Ren Xiang-Dong , Kiosses William B. , Sieg David J. , Otey Carol A. , Schlaepfer David D. , Schwartz Martin Alexander 
Published: April 2021

ABSTRACT
Focal adhesion kinase (FAK) is activated and localized at focal adhesions upon cell adhesion to extracellular matrices. Cells lacking FAK show increased focal adhesion number and decreased cell migration, functions that are regulated by the small GTPase Rho. We now report that fibroblasts from FAK−/− mice failed to transiently inhibit Rho activity when plated on fibronectin. Re-expression of FAK restored normal Rho regulation. Turnover of focal adhesions correlated inversely with Rho activity. The presence or absence of FAK was mimicked by inhibiting or activating Rho, respectively. These data suggest that loss of FAK resulting in constitutive activation of Rho and inhibition of focal adhesion turnover can account for deficiencies in cell migration and embryonic lethality of the FAK knockout.
IRE1 and efferent signaling from the endoplasmic reticulum
Authors: Urano Fumihiko , Bertolotti Anne , Ron David 
Published: April 2021

ABSTRACT
Genetic analysis of the cellular adaptation to malfolded proteins in the endoplasmic reticulum (the unfolded protein response – UPR) has revealed a novel signaling pathway initiated by activation of IRE1, an ER-resident protein kinase and endonuclease. In yeast, Ire1p activates gene expression by promoting a non-conventional splicing event that converts the mRNA encoding the Hac1p transcription factor from an inefficiently translated inactive mRNA to an actively translated one. Hac1p binds to the promoters of genes encoding chaperones and other targets of the UPR and activates them. Recently, mammalian IRE1 homologues have been identified and their response to ER stress is regulated by binding to the ER chaperone BiP. The mechanisms by which mammalian IRE1 activates gene expression have not been completely characterized and mammalian HAC1 homologues have not been identified. Surprisingly, mammalian IRE1s are able to activate both JUN N-terminal kinases and an alternative ER-stress signaling pathway mediated by the transcription factor ATF6. This indicates that the mammalian UPR is more complex than that found in yeast.
The localized assembly of extracellular matrix integrin ligands requires cell-cell contact
Authors: Martin-Bermudo Maria D. , Brown Nicholas H. 
Published: April 2021

ABSTRACT
The assembly of an organism requires the interaction between different layers of cells, in many cases via an extracellular matrix. In the developing Drosophila larva, muscles attach in an integrin-dependent manner to the epidermis, via a specialized extracellular matrix called tendon matrix. Tiggrin, a tendon matrix integrin ligand, is primarily synthesized by cells distant to the muscle attachment sites, yet it accumulates specifically at these sites. Previous work has shown that the PS integrins are not required for tiggrin localization, suggesting that there is redundancy among tiggrin receptors. We have examined this by testing whether the PS2 integrin can recruit tiggrin to ectopic locations within the Drosophila embryo. We found that neither the wild type nor modified forms of the PS2 integrin, which have higher affinity for tiggrin, can recruit tiggrin to new cellular contexts. Next, we genetically manipulated the fate of the muscles and the epidermal muscle attachment cells, which demonstrated that muscles have the primary role in recruiting tiggrin to the tendon matrix and that cell-cell contact is necessary for this recruitment. Thus we propose that the inherent polarity of the muscle cells leads to a molecular specialization of their ends, and interactions between the ends produces an integrin-independent tiggrin receptor. Thus, interaction between cells generates an extracellular environment capable of nucleating extracellular matrix assembly.