bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain derived versican V2 is a potent inhibitor of axonal growth
Authors: Schmalfeldt Michael , Bandtlow Christine E. , Dours-Zimmermann María T. , Winterhalter Kaspar H. , Zimmermann Dieter R.
Published: April 2021

In this paper, we identify the chondroitin sulfate proteoglycan versican V2 as a major inhibitor of axonal growth in the extracellular matrix of the mature central nervous system. In immunohistochemical and in situ hybridization experiments we show that this tissue-specific splice variant of versican is predominantly present in myelinated fiber tracts of the brain and in the optic nerve, most likely being expressed by oligodendrocytes. We demonstrate that isolated versican V2 strongly inhibits neurite outgrowth of central and peripheral neurons in stripe-choice assays using laminin-1 as permissive substrate.

The inhibitory character of versican V2 is maintained after removal of chondroitin sulfate and N- and O-linked oligosaccharide side chains, but it is abolished after core protein digestion with proteinase-K. Our data support the notion, that intact versican V2 prevents excessive axonal growth during late phases of development and hereby participates in the structural stabilization of the mature central nervous system.
Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres
Authors: Aagaard Louise , Schmid Manfred , Warburton Peter , Jenuwein Thomas
Published: April 2021

Centromeres of eukaryotes are frequently associated with constitutive heterochromatin and their activity appears to be coregulated by epigenetic modification of higher order chromatin. Recently, we isolated murine (Suv39h1) and human (SUV39H1) homologues of the dominant Drosophila suppressor of position effect variegation Su(var)3-9, which is also related to the S. pombe silencing factor Clr4. We have shown that mammalian Su(var)3-9 homologues encode novel centromeric proteins on metaphase-arrested chromosomes. Here, we describe a detailed analysis of the chromatin distribution of human SUV39H1 during the cell cycle. Although there is significant heterochromatic overlap between SUV39H1 and M31 (HP1β) during interphase, mitotic SUV39H1 displays a more restricted spatial and temporal association pattern with metaphase chromosomes than M31 (HP1β), or the related HP1α gene product. SUV39H1 specifically accumulates at the centromere during prometaphase but dissociates from centromeric positions at the meta-to anaphase transition. In addition, SUV39H1 selectively associates with the active centromere of a dicentric chromosome and also with a neocentromere. Interestingly, SUV39H1 is shown to be a phosphoprotein with modifications at serine and, to a lesser degree, also at threonine residues. Whereas SUV39H1 steady-state protein levels appear constant during the cell cycle, two additional phosphorylated isoforms are detected in mitotic extracts. This intriguing localisation and modification pattern would be consistent with a regulatory role(s) for SUV39H1 in participating in higher order chromatin organisation at mammalian centromeres.
Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression
Authors: Pujuguet Philippe , Simian Marina , Liaw Jane , Timpl Rupert , Werb Zena , Bissell Mina J.
Published: April 2021

Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express β-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone, to mammary epithelial cells induced β-casein production. We asked whether recombinant nidogen-1 alone could signal directly for β-casein. Nidogen-1 did not induce β-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate β-casein expression. Addition of full-length nidogen-1 to the mixed cultures had no effect on β-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on β-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.
Import of proteins into the trypanosome nucleus and their distribution at karyokinesis
Authors: Marchetti Maria A. , Tschudi Christian , Kwon Helen , Wolin Sandra L. , Ullu Elisabetta
Published: April 2021

In all eukaryotic organisms proteins are targeted to the nucleus via a receptor-mediated mechanism that requires a specific nuclear localization sequence (NLS) in the protein. Little is known about this process in trypanosomatid protozoa that are considered amongst the earliest divergent eukaryotes. We have used the green fluorescent protein (gfp) and β-galactosidase reporters to identify the NLS of two trypanosomal proteins, namely the Trypanosoma brucei La protein homologue and histone H2B of T. cruzi. A monopartite NLS was demonstrated at the C terminus of the La protein, whereas a bipartite NLS was identified within the first 40 amino acids of histone H2B. Treatment of live trypanosomes with poisons of ATP synthesis resulted in exit of the La NLS-gfp fusion from the nucleus. Interestingly, this fusion protein accumulated at several discrete sites in the cytoplasm, rather than equilibrating between the nucleus and the cytoplasm. When ATP levels returned to normal, the protein reentered the nucleus, demonstrating that the process was energy dependent. Finally, using fusion proteins that localize to the nucleoplasm or the nucleolus, we identified a subpopulation of mitotic cells in which the chromosomes have segregated but the daughter nuclei remain connected by a thin thread-like structure. We propose that cells containing this structure represent a late stage in nuclear division that can be placed after chromosome segregation, but before completion of karyokinesis.
VEGF nuclear accumulation correlates with phenotypical changes in endothelial cells
Authors: Li Wenlu , Keller Gilbert-A.
Published: April 2021

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a prominent role in normal vascular biology and pathology. In an experimental wound model, the mechanical disruption of monolayers of cultured endothelial cells resulted in two phenotypically distinct cell subpopulations in which VEGF was internalized by alternative endocytotic pathways and delivered to different subcellular compartments. In the cells away from the wound, VEGF was internalized via the classical receptor-mediated endocytosis pathway and accumulated in the endosomal compartment, whereas in the cells situated at the edges of a wound, VEGF was rapidly taken up and translocated to the nucleus. VEGF internalization and subsequent nuclear accumulation only occurred for a short period of time after the wounding and was specifically abolished by antibodies that bind to the KDR binding site of VEGF.

In the cells with VEGF nuclear accumulation, the levels of wound healing related proteins, such as Factor VIII (FVIII), tissue factor (TF) and tissue plasminogen activator, rapidly and dramatically increased compared to the cells that internalized VEGF via the classical endocytotic pathway. The increase in FVIII and TF was abolished when the nuclear transport is blocked. These data suggest that nuclear VEGF accumulation may be involved in modulating the levels of the proteins of the coagulation and fibrinolysis pathways.
Cadherin repertoire determines partner-specific gap junctional communication during melanoma progression
Authors: Hsu Mei-Yu , Andl Thomas , Li Gang , Meinkoth Judith L. , Herlyn Meenhard
Published: April 2021

Reduced gap junction activity has long been implicated in tumorigenesis. To elucidate the potential role of intercellular communication in melanoma development, we examined gap junctional capability of melanocytic cells from various stages of tumor progression in coculture models using dye transfer assays. Normal melanocytes coupled with keratinocytes by gap junctional formation, whereas melanoma cells did not. Instead, melanoma cells communicated among themselves and with fibroblasts. This switch in communication partners coincided with a shift from E-cadherin to N-cadherin expression during melanoma development. Forced expression of E-cadherin by adenoviral gene transfer in N-cadherin-expressing melanoma cells restored gap junctional compatibility with keratinocytes. Our data suggest that (1) melanocyte transformation is associated with loss of the pre-existing gap junctional activity with keratinocytes but a concomitant gain of communication with a newly juxtaposed cell type, the fibroblasts, (2) the specificity of gap junctional formation during melanoma development is determined by the cadherin profile on the melanocytic cells and (3) the overall gap junctional activity of melanocytic cells is not reduced with transformation.
A new specialized cell-matrix interaction in actively resorbing osteoclasts
Authors: Stenbeck Gudrun , Horton Michael A.
Published: April 2021

We have identified a novel cell-matrix interaction in activated osteoclasts. Resorbing osteoclasts maintain a barrier adjacent to the bone surface that prevents the leakage of secreted protons and proteases from the resorption area. Using a series of fluorescent dyes of known molecular mass and different surface charge we established that negatively charged molecules with Mr up to 10,000 rapidly accumulate underneath actively resorbing osteoclasts. Live cell imaging shows that staining could be detected underneath the osteoclasts as early as 30 seconds after the addition of the low molecular mass markers. We provide evidence that the actin cytoskeleton and the adhesion substrate in contact with the cells are critically involved in the maintenance of the sealing barrier. These data taken together suggest that the accumulation under resorbing osteoclasts is by diffusion rather than transcytotic delivery. Our results indicate that the net concentration of secreted and resorbed components is a balance between generation rate and limited diffusion rather than the presence of an impermeable barrier as previously suggested. This dynamic osteoclast sealing zone may, thus, provide the mechanism by which osteoclast migration and resorption can occur simultaneously.
Senescence represses the nuclear localization of the serum response factor and differentiation regulates its nuclear localization with lineage specificity
Authors: Ding Wei , Gao Sizhi , Scott Robert E.
Published: April 2021

The differentiation of cultured 3T3T mesenchymal stem cells into adipocytes represses growth factor responsiveness by limiting the nuclear localization of the serum response factor (SRF) that binds to and activates the promoters of growth control genes that contain the serum response elements (SRE), such as junB and c-fos. The regulation of SRF nuclear localization by adipocyte differentiation is specific, because we show that adipocyte differentiation does not repress the nuclear localization of six other transacting factors. To determine if repression of growth factor responsiveness that occurs during senescence also represses the nuclear localization of SRF, we studied normal human WI-38 fibroblasts at low versus high population doublings. The results show that SRF localizes to the nucleus of proliferative cells whereas in senescent cells SRF can not be detected in the nucleus. This result is apparent in both immunofluorescence assays and in western blot analysis. We next evaluated the cellular distribution of SRF in selected human tissues to determine whether the loss of proliferative potential in vivo could have a different effect on SRF nuclear localization. We found that in cells of the small bowel mucosa, differentiation modulates SRF nuclear localization in an opposite manner. Minimal SRF expression and nuclear localization is evident in undifferentiated cells at the base of crypts whereas increased SRF expression and nuclear localization is evident in differentiated cells at the surface tip of the villus. These results together establish that regulation of SRF expression and nuclear localization is important in senescence and differentiation in a lineage specific manner.
Novel roles for mammalian septins: from vesicle trafficking to oncogenesis
Authors: Kartmann Bettina , Roth Dagmar
Published: April 2021

In recent years a convergence of various aspects of cell biology has become apparent, and yet investigators are only beginning to grasp the underlying unifying mechanisms. Among the proteins that participate in diverse aspects of cell biology are the septins. These are a group of novel GTPase proteins that are broadly distributed in many eukaryotes except plants. Although septins were originally identified as a protein family involved in cytokinesis in yeast, recent advances in the field have now ascribed additional functions to these proteins. In particular, the number of known mammalian septin family members has increased dramatically as more data has become available through genome analyses. We suggest a classification for the mammalian septins based on the sequence homologies in their highly divergent N- and C-termini. Recent work suggests novel functions for septins in vesicle trafficking, oncogenesis and compartmentalization of the plasma membrane. Given the ability of the septins to bind GTP and phosphatidylinositol 4,5-bisphosphate in a mutually exclusive manner, these proteins might be crucial elements for the spatial and/or temporal control of diverse cellular functions. As the functions of the septins become unraveled, our understanding of seemingly different cellular processes may move a step further.
The splice variants of vascular endothelial growth factor (VEGF) and their receptors
Authors: Robinson Christopher J. , Stringer Sally E.
Published: April 2021

Vascular endothelial growth factor (VEGF) is a secreted mitogen highly specific for cultured endothelial cells. In vivo VEGF induces microvascular permeability and plays a central role in both angiogenesis and vasculogenesis. VEGF is a promising target for therapeutic intervention in certain pathological conditions that are angiogenesis dependent, most notably the neovascularisation of growing tumours. Through alternative mRNA splicing, a single gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. Two VEGF receptor tyrosine kinases (VEGFRs) have been identified, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 seems to mediate almost all observed endothelial cell responses to VEGF, whereas roles for VEGFR-1 are more elusive. VEGFR-1 might act predominantly as a ligand-binding molecule, sequestering VEGF from VEGFR-2 signalling. Several isoform-specific VEGF receptors exist that modulate VEGF activity. Neuropilin-1 acts as a co-receptor for VEGF165, enhancing its binding to VEGFR-2 and its bioactivity. Heparan sulphate proteoglycans (HSPGs), as well as binding certain VEGF isoforms, interact with both VEGFR-1 and VEGFR-2. HSPGs have a wide variety of functions, such as the ability to partially restore lost function to damaged VEGF165 and thereby prolonging its biological activity.
Growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in Paramecium
Authors: Vayssié Laurence , Loubresse Nicole Garreau de , Sperling Linda
Published: April 2021

Paramecium trichocysts are voluminous secretory vesicles consisting of a spindle-shaped body surmounted by a tip that serves to anchor them at exocytotic sites in the plasma membrane. This constrained shape is conferred by the proteins stored in the vesicles, which form an insoluble three-dimensional crystalline array. The constituent polypeptides (Trichocyst Matrix Proteins, TMPs), which assemble during trichocyst biogenesis, are produced by proteolytic processing of soluble proproteins encoded by a large multigene family. In order to investigate the functional significance of the TMP multigene family, which assures the synthesis of a mixture of related polypeptides, we have designed synthetic genes for heterologous expression of three different mature polypeptides, which were used to obtain sequence-specific rabbit antisera. We used these antisera to carry out immunolocalization experiments with wild-type trichocysts at different stages of development and found that the trichocyst matrix consists of two concentric layers containing different TMPs, and that the assembly of each layer corresponds to a distinct phase of trichocyst growth. Examination of mutant trichocysts created by targeted gene silencing of different TMP genes showed that the layer containing the products of the silenced genes is specifically affected, as are all subsequently assembled parts of the structure, consistent with an ordered assembly pathway. This stepwise assembly is not controlled by differential sorting of the TMPs, as single and double label experiments provided evidence that the different TMPs are delivered together to post-Golgi vesicles and developing trichocysts. We present a model for trichocyst biogenesis in which TMP assembly is controlled by protein processing.
Dissection of receptor folding and ligand-binding property with functional minireceptors of LDL receptor-related protein
Authors: Obermoeller-McCormick Lynn M. , Li Yonghe , Osaka Hiroshi , FitzGerald David J. , Schwartz Alan L. , Bu Guojun
Published: April 2021

The LDL receptor-related protein (LRP) is a large, multifunctional endocytic receptor that binds and endocytoses a variety of structurally and functionally distinct ligands. LRP contains four putative ligand-binding domains. However, only domains II, III and IV, but not domain I, bind the receptor-associated protein (RAP), a molecular chaperone and universal antagonist for LRP. In order to dissect the function of RAP in LRP folding and to examine the ligand-binding properties of LRP, we generated LRP minireceptors that represent each of the four putative ligand-binding domains (termed mLRP1, mLRP2, mLRP3 and mLRP4, respectively). We found that proper folding and trafficking of mLRP2, mLRP3, mLRP4, but not mLRP1, is facilitated by coexpression of RAP. When these mLRPs were stably expressed in Chinese

Hamster Ovary cells that lack the endogenous LRP, we found that each of these receptors was processed and traffics through the secretory pathway. Cell surface expression of these minireceptors was quantitatively examined by flow cytometric analyses. Using these minireceptor cell lines to map the ligand-binding domains, we found that although the majority of LRP ligands bind to both domain II and domain IV, Pseudomonas exotoxin A utilizes only domain IV for its binding to LRP. We conclude that while domains II and IV of LRP share many ligand-binding properties, each of the putative ligand-binding domains of LRP is unique in its contribution to ligand binding.
Mechanism for the transcriptional repression by c-Myc on PDGF β-receptor
Authors: Izumi Hiroto , Molander Catrin , Penn Linda Z. , Ishisaki Akira , Kohno Kimitoshi , Funa Keiko
Published: April 2021

c-Myc plays a key role in the cell cycle dependent control of the PDGF β-receptor mRNA. The mouse platelet-derived growth factor (PDGF) β-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF β-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF β-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.
Effects of mutations in potential phosphorylation sites on transcytosis of FcRn
Authors: McCarthy Kevin M. , Lam Michael , Subramanian Laura , Shakya Reena , Wu Zhen , Newton Estelle E. , Simister Neil E.
Published: April 2021

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across intestinal epithelial cells of suckling rats and mice from the lumenal surface to the serosal surface. In cell culture models FcRn transports IgG bidirectionally, but there are differences in the mechanisms of transport in the two directions. We investigated the effects of mutations in the cytoplasmic domain of FcRn on apical to basolateral and basolateral to apical transport of Fc across rat inner medullary collecting duct (IMCD) cells. Basolateral to apical transport did not depend upon determinants in the cytoplasmic domain. In contrast, an essentially tailless FcRn was markedly impaired in apical to basolateral transport. Using truncation and substitution mutants, we identified serine-313 and serine-319 as phosphorylation sites in the cytoplasmic domain of FcRn expressed in Rat1 fibroblasts. Mutations at Ser-319 did not affect transcytosis across IMCD cells. FcRn-S313A was impaired in apical to basolateral transcytosis to the same extent as tailless FcRn, whereas FcRn-S313D transported at wild-type levels. FcRn-S313A recycled more Fc to the apical medium than the wild-type receptor, suggesting that Ser-313 is required to allow FcRn to be diverted from an apical recycling pathway to a transcytotic pathway.
Elastic fibres
Authors: Kielty Cay M. , Sherratt Michael J. , Shuttleworth C. Adrian
Published: April 2021

Elastic fibres are essential extracellular matrix macromolecules comprising an elastin core surrounded by a mantle of fibrillin-rich microfibrils. They endow connective tissues such as blood vessels, lungs and skin with the critical properties of elasticity and resilience. The biology of elastic fibres is complex because they have multiple components, a tightly regulated developmental deposition, a multi-step hierarchical assembly and unique biomechanical functions. However, their molecular complexity is at last being unravelled by progress in identifying interactions between component molecules, ultrastructural analyses and studies of informative mouse models.
Dynamics of the vacuolar H+-ATPase in the contractile vacuole complex and the endosomal pathway ofDictyosteliumcells
Authors: Clarke Margaret , Köhler Jana , Arana Quyen , Liu Tongyao , Heuser John , Gerisch Günther
Published: April 2021

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit enzyme that plays important roles in eukaryotic cells. In Dictyostelium, it is found primarily in membranes of the contractile vacuole complex, where it energizes fluid accumulation by this osmoregulatory organelle and also in membranes of endolysosomes, where it serves to acidify the endosomal lumen. In the present study, a fusion was created between vatM, the gene encoding the 100 kDa transmembrane subunit of the V-ATPase, and the gene encoding Green Fluorescent Protein (GFP). When expressed in Dictyostelium cells, this fusion protein, VatM-GFP, was correctly targeted to contractile vacuole and endolysosomal membranes and was competent to direct assembly of the V-ATPase enzyme complex. Protease treatment of isolated endosomes indicated that the GFP moiety, located on the C-terminus of VatM, was exposed to the cytoplasmic side of the endosomal membrane rather than to the lumenal side. VatM-GFP labeling of the contractile vacuole complex revealed clearly the dynamics of this pleiomorphic vesiculotubular organelle. VatM-GFP labeling of endosomes allowed direct visualization of the trafficking of vacuolar proton pumps in this pathway, which appeared to be entirely independent from the contractile vacuole membrane system. In cells whose endosomes were pre-labeled with TRITC-dextran and then fed yeast particles,VatM-GFP was delivered to newly formed yeast phagosomes with the same time course as TRITC-dextran, consistent with transfer via a direct fusion of endosomes with phagosomes. Several minutes were required before the intensity of the VatM-GFP labeling of new phagosomes reached the level observed in older phagosomes, suggesting that this fusion process was progressive and continuous. VatM-GFP was retrieved from the phagosome membrane prior to exocytosis of the indigestible remnants of the yeast particle. These data suggest that vacuolar proton pumps are recycled by fusion of advanced with newly formed endosomes.
The minor regulated pathway, a rapid component of salivary secretion, may provide docking/fusion sites for granule exocytosis at the apical surface of acinar cells
Authors: Castle Anna M. , Huang Amy Y. , Castle J. David
Published: April 2021

Recently, we reported that the minor regulated and constitutive-like pathways are the main source of resting secretion by parotid acinar cells. Using tissue lobules biosynthetically labeled with [35S]amino acids, we now show that discharge of the minor regulated pathway precedes granule exocytosis stimulated by isoproterenol (≥1 μM) or carbachol (2μM). Stimulation of the minor regulated pathway by 40 nM carbachol as well as altering its trafficking, either by adding brefeldin A or by incubating in K+-free medium, cause potentiation of amylase secretion stimulated by isoproterenol, suggesting that the minor regulated pathway contributes to the mechanism of potentiation. Both exocytosis of the minor regulated pathway and the potentiation-inducing treatments induce relocation of immunostained subapical puncta of the SNARE protein syntaxin 3 into the apical plasma membrane. Rab11 and possibly VAMP2 may be concentrated in the same relocating foci. These results suggest that the minor regulated pathway and granule exocytosis are functionally linked and that the minor regulated pathway has a second role beyond contributing to resting secretion — providing surface docking/fusion sites for granule exocytosis. In the current model of salivary protein export, discharge of the minor regulated pathway by eitherβ-adrenergic or cholinergic stimulation is an obligatory first step. Ensuing granule exocytosis is controlled mainly by β-adrenergic stimulation whereas cholinergic stimulation mainly regulates the number of surface sites where release occurs.
Granule-specific ATP requirements for Ca2+-induced exocytosis in human neutrophils. Evidence for substantial ATP-independent release
Authors: Theander Sten , Lew Daniel P. , Nüße Oliver
Published: April 2021

Ca2+-induced exocytosis in neuronal and neuroendocrine cells involves ATP-dependent steps believed to `prime' vesicles for exocytosis. Primed, docked vesicles are released in response to Ca2+ influx through voltage-gated Ca2+ channels. Neutrophils, however, do not possess voltage-gated Ca2+ channels and appear to have no docked vesicles. Furthermore, neutrophils have several types of granules with markedly different Ca2+ requirements for exocytosis. These differential Ca2+ dependencies were used as a tool to investigate the ATP dependence of different granule populations. Here we demonstrate distinct ATP requirements for release of neutrophil granule populations, with respect to rate as well as amplitude. Intracellular ATP was depleted to various levels, and exocytosis was stimulated with different Ca2+concentrations and measured with the patch-clamp capacitance technique or by detecting enzyme release. Primary granule exocytosis displayed a distinct ATP dependence with an apparent Km of ∼80 μM ATP and no ATP-independent exocytosis. Release of secondary and tertiary granules displayed a more shallow ATP dependence (Km∼330 μM), and more than 50% of secondary and tertiary granules appeared not to need ATP at all for their release. Individual granules in human neutrophils have distinct ATP requirements for exocytosis, suggesting that the ATP-sensitive elements are localised to the granules. Primary granule exocytosis has a very low affinity for ATP. Furthermore, substantial ATP-independent exocytosis of secondary and tertiary granule occurs despite the absence of docked granules. These characteristics should help neutrophils to fulfil their bactericidal functions at poorly irrigated sites of infection with low glucose supply.
The molecular mechanism of translocation through the nuclear pore complex is highly conserved
Authors: Feldherr Carl , Akin Debra , Littlewood Trevor , Stewart Murray
Published: April 2021

In this report we investigated the activity of vertebrate nuclear transport factors in a primitive organism, Amoeba proteus, to better understand evolutionary changes in the transport mechanisms of organisms expected to have different requirements for nucleocytoplasmic exchange. It was initially determined that FxFG-containing nucleoporins and Ran, both of which are essential for nuclear import in vertebrates, as well as yeast, are also present and functional in amoebae. This suggests that there are fundamental similarities in the transport process; however, there are also significant differences. Transport substrates containing either the hnRNP A1 M9 shuttling signal (a GST/GFP/M9 fusion protein) or the classical bipartite NLS (colloidal gold coated with BSA-bipartite NLS conjugates), both of which are effectively transported in vertebrate cells, are excluded from the nucleus when microinjected into amoebae. However, when these substrates are injected along with transportin or importin α/β, respectively, the vertebrate receptors for these signals, they readily accumulate in the nucleoplasm. These results indicate that although the molecular recognition of substrates is not well conserved between vertebrates and amoebae, vertebrate transport receptors are functional in A. proteus, showing that the translocation machinery is highly conserved. Since selected nuclear import pathways can be investigated in the absence of competing endogenous transport, A. proteus might provide a useful in vivo system for investigating specific molecular interactions involved in trafficking.
A role for mammalian Ubc6 homologues in ER-associated protein degradation
Authors: Lenk Uwe , Yu Helen , Walter Jan , Gelman Marina S. , Hartmann Enno , Kopito Ron R. , Sommer Thomas
Published: April 2021

Integral membrane and secretory proteins which fail to fold productively are retained in the endoplasmic reticulum and targeted for degradation by cytoplasmic proteasomes. Genetic and biochemical analyses suggest that substrates of this pathway must be dislocated across the membrane of the endoplasmic reticulum (ER) by a process requiring a functional Sec61 complex and multiubiquitinylation. In yeast, the tail-anchored ubiquitin-conjugating enzyme Ubc6p, which is localized to the cytoplasmic surface of the ER,participates in ER-associated degradation (ERAD) of misfolded proteins. Here we describe the identification of two families of mammalian Ubc6p-related proteins. Members of both families are also located in the ER membrane and display a similar membrane topology as the yeast enzyme. Furthermore we show that expression of elevated levels of wild-type and dominant-negative alleles of these components affects specifically ERAD of the α subunit of the T-cell receptor and a mutant form of the CFTR protein. Similarly, we describe that the expression level of Ubc6p in yeast is also critical for ERAD,suggesting that the Ubc6p function is highly conserved from yeast to mammals.
Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract
Authors: Kobayashi Takayuki , Tada Shusuke , Tsuyama Takashi , Murofushi Hiromu , Seki Masayuki , Enomoto Takemi
Published: April 2021

The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI,discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.
Unbound E2F modulates TGF-β1-induced apoptosis in HuH-7 cells
Authors: Fan Guangsheng , Ma Xiaoming , Kren Betsy T. , Steer Clifford J.
Published: April 2021

E2F is an important target of the retinoblastoma protein (pRb) and plays a critical role in G1/S progression through the cell cycle. TGF-β1 arrests HuH-7 cells in G1 by suppressing phosphorylation of pRb and induces apoptosis by inhibiting its expression. In this study, we examined the downstream effects of TGF-β1-induced apoptosis and the potential roles for pRb and E2F. The results indicated that greater than 90% of the TGF-β1-induced preapoptotic cells were arrested in G1 phase of the cell cycle. This was associated with a significant increase in both E2F-DNA-binding activity and transcription of E2F-responsive reporter constructs. In contrast, no significant changes were observed in E2F mRNA and protein levels, and the overexpression of pRb partially inhibited E2F activation. Gel-shift assays identified more than four E2F complexes from preapoptotic and synchronized G1 HuH-7 cells,each exhibiting different patterns of E2F-associated proteins. The increased E2F activity did not affect the association patterns with pRb, p107 and p130,but altered the formation of an E2F—DP-1 complex. In contrast,E2F—DP-2 exhibited little change in the preapoptotic cells. Moreover,TGF-β1 induced apoptosis at G1 and inhibited entry into S phase irrespective of the increased E2F activity. The release of preapoptotic cells from TGF-β1 resulted in rapid S phase entry and subsequent apoptosis in 33% of cells over a 72 hour period. In conclusion, the results demonstrate that TGF-β1-induced apoptosis in HuH-7 cells is associated with a marked increase in activity of transcription factor E2F that is partially inhibited by overexpression of pRb. Preapoptotic changes are, in part, reversible upon removal of TGF-β1 and the majority of cells re-enter the normal cell cycle. Finally, TGF-β1-induced apoptosis with the associated increase in E2F activity can occur in both the G1and S phases of the cell cycle.
p38 mitogen-activated protein kinase is required for TGFβ-mediated fibroblastic transdifferentiation and cell migration
Authors: Bakin Andrei V. , Rinehart Cammie , Tomlinson Anne K. , Arteaga Carlos L.
Published: April 2021

Transforming growth factor β (TGFβ) contributes to tumor progression by inducing an epithelial to mesenchymal transdifferentiation(EMT) and cell migration. We found that TGFβ-induced EMT was blocked by inhibiting activation of p38 mitogen-activated protein kinase (MAPK) with H-7,a protein kinase C inhibitor, and with SB202190, a direct inhibitor of p38MAPK. Inhibition of the p38MAPK pathway affected TGFβ-mediated phosphorylation of ATF2, but did not inhibit phosphorylation of Smad2. SB202190 impaired TGFβ-mediated changes in cell shape and reorganization of the actin cytoskeleton. Forced expression of dominant-negative (DN) MAPK kinase 3 (MKK3) inhibited TGFβ-mediated activation of p38MAPK and EMT. Expression of DN-p38α impaired TGFβ-induced EMT. Inhibition of p38MAPK blocked TGFβ-induced migration of non-tumor and tumor mammary epithelial cells. TGFβ induced activation of the p38MAPK pathway within 15 minutes. Expression of TGFβ type II (TβRII) and type I(TβRI/Alk5) kinase-inactive receptors blocked EMT and activation of p38MAPK, whereas expression of constitutively active Alk5-T204D resulted in EMT and phosphorylation of MKK3/6 and p38MAPK. Finally, dominant-negative Rac1N17 blocked TGFβ-induced activation of the p38MAPK pathway and EMT,suggesting that Rac1 mediates activation of the p38MAPK pathway. These studies suggest that the p38MAPK pathway is required for TGFβ-mediated EMT and cell migration.
NUANCE, a giant protein connecting the nucleus and actin cytoskeleton
Authors: Zhen Yen-Yi , Libotte Thorsten , Munck Martina , Noegel Angelika A. , Korenbaum Elena
Published: April 2021

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.
Stressful initiations
Authors: Anderson Paul , Kedersha Nancy
Published: April 2021

Stress granules (SGs) are phase-dense particles that appear in the cytoplasm of eukaryotic cells that have been exposed to environmental stress(e.g. heat, oxidative conditions, hyperosmolarity and UV irradiation). SG assembly is a consequence of abortive translational initiation: SGs appear when translation is initiated in the absence of eIF2-GTP-tRNAiMet, the ternary complex that normally loads tRNAiMet onto the small ribosomal subunit. Stress-induced depletion of eIF2-GTP-tRNAiMet allows the related RNA-binding proteins TIA-1 and TIAR to promote the assembly of eIF2-eIF5-deficient preinitiation complexes, the core constituents of SGs. The mRNP components that make up the SG are in a dynamic equilibrium with polysomes. As such, the SG appears to constitute a metabolic domain through which mRNPs are continually routed and subjected to triage — they are first monitored for integrity and composition, and then sorted for productive translational initiation or targeted degradation.
Staufen2 isoforms localize to the somatodendritic domain of neurons and interact with different organelles
Authors: Duchaîne Thomas F. , Hemraj Indradeo , Furic Luc , Deitinghoff Anke , Kiebler Michael A. , DesGroseillers Luc
Published: April 2021

Mammalian Staufen2 (Stau2) is involved in mRNA transport in neurons. Here,we report that Stau2 is a double-stranded RNA-binding protein that is mainly expressed in the brain. We show that Stau2 is found in the somatodendritic compartment of neurons. In dendrites, Stau2 is aligned on individual tracts and colocalizes with microtubules. Stau2 is expressed as at least three splice isoforms, which can be observed in several subcellular complexes. Although a 62 kDa isoform (Stau262) fractionates in ribosome-free fractions of light density, Stau259 and Stau252 are found in high-density complexes. These complexes are resistant to EDTA and to non-ionic detergent. For the first time, we also provide evidence for an interaction of some Stau2 isoforms with ribosomes, thus pointing to an interesting new role for Stau2 in translation. EDTA treatment, which dissociates ribosome subunits,does not release Stau2 from the subunits, suggesting that Stau2-ribosome associations are not mediated mainly by mRNA intermediates. Although Stau2 has many features in common with its paralogue Stau1, it does not colocalize with Stau1-containing particles, indicating that these proteins are components of different complexes in dendrites. Our findings suggest that members of the Staufen family share evolutionarily conserved properties and highlight the complexity of Staufen-mediated RNA transport in neurons.
Invasive behaviour of glioblastoma cell lines is associated with altered organisation of the cadherin-catenin adhesion system
Authors: Perego Carla , Vanoni Cristina , Massari Silvia , Raimondi Andrea , Pola Sandra , Cattaneo Maria Grazia , Francolini Maura , Vicentini Lucia Maria , Pietrini Grazia
Published: April 2021

As little is known about the role of cadherin-mediated cell-cell adhesion in astrocytes and its alteration in migrating and invasive glioblastomas, we investigated its molecular composition and organisation in primary cultured astrocytes and the T98G and U373MG glioblastoma cell lines. Biochemical and morphological analysis indicated that all three cell types express all of the structural components of the adhesion system, including the LIN-7 PDZ protein,a novel component involved in the organisation of the junctional domain in epithelia and neurons. However, only the astrocytes and T98G cells generated and maintained mature adhesive junctional domains to which LIN-7 was recruited. Alterations in the junctional domain of U373MG cells were associated with higher motility in a poly-L-lysine migration assay. When the T98G cells were cultured on Matrigel matrix, they acquired invasive properties but, despite unchanged cadherin adhesion system protein levels, the invasive T98G cell-cell contacts failed to accumulate LIN-7 and failed to mature. These results identify the LIN-7 PDZ protein as a marker of cell adhesion maturity and cell invasion and indicate that instability and disorganisation of cadherin-mediated junctions rather than reduced expression of cadherin-catenin system components are required to promote migration and invasiveness in glioblastoma cell lines.
Plasma membrane targeting of SNAP-25 increases its local concentration and is necessary for SNARE complex formation and regulated exocytosis
Authors: Koticha Darshan K. , McCarthy Ellen E. , Baldini Giulia
Published: April 2021

SNAP-25 is an integral protein of the plasma membrane involved in neurotransmission and hormone secretion. The cysteine-rich domain of SNAP-25 is essential for membrane binding and plasma-membrane targeting. However, this domain is not required for SNARE complex formation and fusion of membranes in vitro. In this paper, we describe an `intact-cell'-based system designed to compare the effect of similar amounts of membrane-bound and soluble SNAP-25 proteins on regulated exocytosis. In transfected neuroblastoma cells,Botulinum neurotoxin E (BoNT/E), a protease that cleaves SNAP-25, blocks regulated release of hormone. However, hormone release is rescued by expressing a wild-type SNAP-25 protein resistant to the toxin. BoNT/E-resistant SNAP-25 proteins lacking the cysteine-rich domain or with all the cysteines substituted by alanines do not form SNARE complexes or rescue regulated exocytosis when expressed at the same level as membrane-bound SNAP-25, which is approximately four-fold higher than the endogenous protein. We conclude that the cysteine-rich domain of SNAP-25 is essential for Ca2+-dependent hormone release because, by targeting SNAP-25 to the plasma membrane, it increases its local concentration, leading to the formation of enough SNARE complexes to support exocytosis.
The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals
Authors: Luttrell Louis M. , Lefkowitz Robert J.
Published: April 2021

β-arrestins are versatile adapter proteins that form complexes with most G-protein-coupled receptors (GPCRs) following agonist binding and phosphorylation of receptors by G-protein-coupled receptor kinases (GRKs). They play a central role in the interrelated processes of homologous desensitization and GPCR sequestration, which lead to the termination of G protein activation. β-arrestin binding to GPCRs both uncouples receptors from heterotrimeric G proteins and targets them to clathrin-coated pits for endocytosis. Recent data suggest that β-arrestins also function as GPCR signal transducers. They can form complexes with several signaling proteins,including Src family tyrosine kinases and components of the ERK1/2 and JNK3 MAP kinase cascades. By recruiting these kinases to agonist-occupied GPCRs,β-arrestins confer distinct signaling activities upon the receptor.β-arrestin-Src complexes have been proposed to modulate GPCR endocytosis,to trigger ERK1/2 activation and to mediate neutrophil degranulation. By acting as scaffolds for the ERK1/2 and JNK3 cascades, β-arrestins both facilitate GPCR-stimulated MAP kinase activation and target active MAP kinases to specific locations within the cell. Thus, their binding to GPCRs might initiate a second wave of signaling and represent a novel mechanism of GPCR signal transduction.
The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis
Authors: Bech-Otschir Dawadschargal , Seeger Michael , Dubiel Wolfgang
Published: April 2021

Recently the COP9 signalosome (CSN) has become a focus of interest for many researchers, because of its function at the interface between signal transduction and ubiquitin-dependent proteolysis. It is required for the proper progression of the cell cycle in Schizosaccharomyces pombe and is essential for development in plants and Drosophila. However, its function in mammalian cells remains obscure. Although the CSN shares structural similarities with the 26S proteasome lid complex (LID), its functions seem to be different from that of the LID. A variety of CSN-specific protein-protein interactions have been described in mammalian cells. However,it is currently unclear how many reflect true functions of the complex. Two activities associated with the CSN have been identified so far: a protein kinase and a deneddylase. The CSN-associated kinase phosphorylates transcription factors, which determines their stability towards the ubiquitin system. The associated deneddylase regulates the activity of specific SCF E3 ubiquitin ligases. The CSN thus appears to be a platform connecting signalling with proteolysis.