bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential assembly of α- and γ-filagenins into thick filaments in : Caenorhabditis elegans
Authors: Liu Feizhou , Ortiz Irving , Hutagalung Alex , Bauer Christopher C. , Cook Richard G. , Epstein Henry F.
Published: April 2021

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized α- and γ-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that α-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas γ-filagenin is localized in the flanking regions, and that α-filagenin is expressed in 1.5-twofold embryos while γ-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of α- and γ-filagenins into thick filaments of distinct lengths may be developmentally regulated.
The breakdown of autophagic vesicles inside the vacuole depends on Aut4p
Authors: Suriapranata Ivet , Epple Ulrike D. , Bernreuther Daniela , Bredschneider Monika , Sovarasteanu Katja , Thumm Michael
Published: April 2021

Autophagy is a degradative transport pathway that delivers cytosolic proteins to the lysosome (vacuole). Cytosolic proteins appear inside the vacuole enclosed in autophagic vesicles. These autophagic vesicles are broken down in the vacuole together with their cytosolic content. The breakdown of vesicular transport intermediates is a unique feature of autophagy. We here identify Aut4p, a component essential for the disintegration of autophagic vesicles, inside the vacuole of S. cerevisiae cells. Aut4p is a putative integral membrane protein with limited homologies to permeases. Chromosomal deletion of AUT4 has no obvious influence on growth, vacuolar acidification and the activities of vacuolar proteinases. Like proteinase B-deficient cells, aut4-deleted cells show a partial reduction in total protein breakdown during nitrogen starvation. A biologically active fusion protein of Aut4p and the green fluorescent protein is visualized at the vacuolar membrane and in punctate structures attached to the vacuole.
Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide
Authors: Chen Qin M. , Tu Victoria C. , Catania Jeffrey , Burton Maggi , Toussaint Olivier , Dilley Tarrah
Published: April 2021

Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H2O2. We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H2O2 treatment. H2O2 treatment causes transient elevation of p53 protein and prolonged inhibition of Rb hyperphosphorylation. Expression of human papillomaviral E6 gene prevented elevation of p53 but did not affect senescent morphogenesis. Expression of human papillomaviral E7 gene reduced the level of Rb protein and prevented induction of senescent morphology by H2O2. The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H2O2 from inducing senescent morphology. Senescent-like cells showed enhanced actin stress fibers. In untreated cells, vinculin and paxillin preferentially distributed along the edge of the cells. In contrast, vinculin and paxillin distributed randomly and sporadically throughout senescent-like cells. E7 expression prevented enhancement of actin filament formation and redistribution of vinculin or paxillin. Neither wild-type nor E7 cells showed changes in the protein level of actin, vinculin or paxillin measured by western blot after H2O2 treatment. Finally, depletion of methionine in the culture medium after H2O2 treatment prevented senescent morphogenesis without affecting dephosphorylation of Rb protein. Our results suggest that senescent morphology likely develops by a program involving activated Rb family proteins, enhancement of actin stress fibers, redistribution of focal adhesion proteins and de novo protein synthesis.
Loss of cation-independent mannose 6-phosphate receptor expression promotes the accumulation of lysobisphosphatidic acid in multilamellar bodies
Authors: Reaves Barbara J. , Row Paula E. , Bright Nicholas A. , Luzio J. Paul , Davidson Howard W.
Published: April 2021

A number of recent studies have highlighted the importance of lipid domains within endocytic organelles in the sorting and movement of integral membrane proteins. In particular, considerable attention has become focussed upon the role of the unusual phospholipid lysobisphosphatidic acid (LBPA). This lipid appears to be directly involved in the trafficking of cholesterol and glycosphingolipids, and accumulates in a number of lysosomal storage disorders. Antibody-mediated disruption of LBPA function also leads to mis-sorting of cation-independent mannose 6-phosphate receptors. We now report that the converse is also true, and that spontaneous loss of cation-independent mannose 6-phosphate receptors from a rat fibroblast cell line led to the formation of aberrant late endocytic structures enriched in LBPA. Accumulation of LBPA was directly dependent upon the loss of the receptors, and could be reversed by expression of bovine cation-independent mannose 6-phosphate receptors in the mutant cell line.

Ultrastructural analysis indicated that the abnormal organelles were electron-dense, had a multi-lamellar structure, accumulated endocytosed probes, and were distinct from dense-core lysosomes present within the same cells. The late endocytic structures present at steady state within any particular cell likely reflect the balance of membrane traffic through the endocytic pathway of that cell, and the rate of maturation of individual endocytic organelles. Moreover, there is considerable evidence which suggests that cargo receptors also play a direct mechanistic role in membrane trafficking events. Therefore, loss of such a protein may disturb the overall equilibrium of the pathway, and hence cause the accumulation of aberrant organelles. We propose that this mechanism underlies the phenotype of the mutant cell line, and that the formation of inclusion bodies in many lysosomal storage diseases is also due to an imbalance in membrane trafficking within the endocytic pathway.
Microtubule-dependent formation of podosomal adhesion structures in primary human macrophages
Authors: Linder Stefan , Hüfner Katharina , Wintergerst Uwe , Aepfelbacher Martin
Published: April 2021

Podosomes are unique actin-rich adhesion structures of monocyte-derived cells such as macrophages and osteoclasts. They clearly differ from other substratum-contacting organelles like focal adhesions in morphological and functional regards. Formation of podosomes has been shown to be dependent on the small GTPase CDC42Hs and its effector Wiskott-Aldrich syndrome protein (WASp). In this study, we investigated the functional relation between podosomes and the microtubule system in primary human macrophages. We demonstrate that, in contrast to focal adhesions, assembly of podosomes in macrophages and their monocytic precursors is dependent on an intact microtubule system. In contrast, experiments using Wiskott-Aldrich syndrome (WAS) macrophages indicate that the microtubule system is not reciprocally dependent on podosomes. A potential linker between podosomes and microtubules may be WASp itself, considering that microinjection of the WASp polyproline domain prevents podosome reassembly. This polyproline domain is thought to link WASp to microtubules via CDC42 interacting protein 4 (CIP4). Consistently, macrophages microinjected with CIP4 constructs deficient in either the microtubule-or the WASp-binding domain also fail to reassemble podosomes. In sum, our findings show that microtubules are essential for podosome formation in primary human macrophages and that WASp and CIP4 may be involved in this phenomenon.
Supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes
Authors: Magin Thomas M. , Kaiser H.-W. , Leitgeb Sabine , Grund Christine , Leigh Irene M. , Morley Susan M. , Lane E. Birgitte
Published: April 2021

Mutations in keratin genes give rise to a number of inherited skin fragility disorders, demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder caused by mutations in keratins K5 or K14, which are expressed in the basal layer of stratified epithelia. Using a keratinocyte cell line established from an EBS patient, we investigated whether the muscle-specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes. We show that in stably transfected EBS cells, desmin forms an extended keratin-independent cytoskeleton. Immunogold-EM analysis demonstrated that in the presence of numerous keratin filaments attached to desmosomes, desmin could nevertheless interact with desmosomes in the same cell, indicating the dynamic nature of the filament-desmosome association. When desmin-transfected cells were subjected to heat shock, the mutant keratin filaments showed a transient collapse while desmin filaments were maintained. Thus the defective keratin filaments and the wild-type desmin filaments appear to coexist in cells without interference. Expression of a type III intermediate filament protein like desmin may offer a strategy for the treatment of patients suffering from epidermal keratin mutations.
Reversible induction of rat hepatoma cell polarity with bile acids
Authors: Ng Kim Heng , Goascogne Claude Le , Amborade Ernest , Stieger Bruno , Deschatrette Jean
Published: April 2021

A dynamic model for inducing and isolating polarized cell colonies from differentiated rat hepatoma was established with chenodeoxycholic acid (CDCA). Cells were treated with 75 μM CDCA in a 1% solvent mix (DMSO/ethanol: 0.5%/0.5%) for 11 days and positive Fao-BA1 and C2rev7-BA1 clones were isolated, respectively, from Fao and C2rev7. Cell polarization in these two clones was demonstrated by (i) the detection of γ-glutamyl transpeptidase activity (γ-GT) and the presence of specific proteins, namely aminopeptidase N (APN), bile acid export pump (Bsep), multidrug resistance-associated protein 2 (Mrp2) at the canalicular pole, (ii) the expression of tight junction (ZO-1) and basolateral (1-18) marker proteins, (iii) the presence of regular microvilli in the cavities sealed by tight junctions, and (iv) functional bile canaliculi-like structures with the capacity to metabolise and secrete carboxyfluorescein diacetate dye. The polarized phenotype was maintained for more than 200 cell generations in the presence of CDCA and could be modulated by cell density or omitting the inducing agent. Hence this cellular model is well suited for studies on hepatic differentiation, polarization and bile salt trafficking with therapeutic implications.
Dynein light chain interacts with NRF-1 and EWG, structurally and functionally related transcription factors from humans and Drosophila
Authors: Herzig Ronald P. , Andersson Ulf , Scarpulla Richard C.
Published: April 2021

Nuclear respiratory factor-1 is a transcriptional activator that has been implicated in the nuclear control of respiratory chain expression. Yeast two-hybrid screens were performed to identify proteins that physically interact with nuclear respiratory factor-1. Saturation screening of both mouse embryo and mouse testis libraries yielded 14 independent clones, all of which represented two different isoforms of dynein light chain. In addition to using the two-hybrid method, the specificity of the nuclear respiratory factor-1/dynein light chain interaction was established by chemical crosslinking of the purified native proteins and by co-immunoprecipitation of nuclear respiratory factor-1 and dynein light chain from mammalian cells. Both two-hybrid and chemical crosslinking assays demonstrated that binding of dynein light chain required the first 26 amino acids of nuclear respiratory factor-1. Although dynein light chain is associated with dynein, a cytoplasmic motor molecule, immunolocalizations showed substantial nuclear staining using several different anti-dynein light chain antibodies. Moreover, fluorescence overlays of confocal images established that nuclear respiratory factor-1 and dynein light chain displayed a very similar nuclear staining pattern. The significance of the nuclear respiratory factor-1/dynein light chain interaction was investigated further by determining whether a similar interaction was conserved between dynein light chain and the erect wing gene product of Drosophila, a protein related to nuclear respiratory factor-1 through its DNA binding domain. Here, we establish that the erect wing gene product can bind and trans-activate transcription through authentic nuclear respiratory factor-1 binding sites. Moreover, the erect wing gene product, like nuclear respiratory factor-1, interacted specifically with dynein light chain both in vitro and in transfected cells. Thus, the interaction with dynein light chain is conserved between transcription factors that are structurally and functionally similar between humans and Drosophila.
Fibronectin polymerization stimulates cell growth by RGD-dependent and-independent mechanisms
Authors: Sottile Jane , Hocking Denise C. , Langenbach Kurt J.
Published: April 2021

Many aspects of cell behavior are regulated by cell-extracellular matrix interactions, including cell migration and cell growth. We previously showed that the addition of soluble fibronectin to collagen-adherent fibronectin-null cells enhances cell growth. This growth-promoting effect of fibronectin depended upon the deposition of fibronectin into the extracellular matrix; occupancy and clustering of fibronectin-binding integrins was not sufficient to trigger enhanced cell growth. To determine whether the binding of integrins to fibronectin’s RGD site is required for fibronectin-enhanced cell growth, the ability of fibronectin lacking the integrin-binding RGD site (FNΔRGD) to promote cell growth was tested. FNΔRGD promoted cell growth when used as an adhesive substrate or when added in solution to collagen-adherent fibronectin-null cells. Addition of FNΔRGD to collagen-adherent fibronectin-null cells resulted in a 1.6-1.8× increase in cell growth in comparison with cells grown in the absence of fibronectin. The growth-promoting effects of FNΔRGD and wild-type fibronectin were blocked by inhibitors of fibronectin polymerization, including the anti-fibronectin antibody, L8. In addition, FNΔRGD-induced cell growth was completely inhibited by the addition of heparin, and was partially blocked by either heparitinase-treatment or by addition of recombinant fibronectin heparin-binding domain. Heparin and heparitinase-treatment also partially blocked the growth-promoting effects of wild-type fibronectin, as well as the deposition of wild-type fibronectin into the extracellular matrix. These data suggest that cell surface heparan-sulfate proteoglycans contribute to the growth-promoting effects of FNΔRGD and wild-type fibronectin. Addition of heparin, treatment with heparitinase, or incubation with monoclonal antibody L8 all inhibited the formation of short linear FNΔRGD fibrils on the cell surface. Inhibitory β1 integrin antibodies had no effect on FNΔRGD fibril formation, FNΔRGD-induced cell growth, or cell adhesion on FNΔRGD-coated substrates. These data suggest that fibronectin fibril formation can promote cell growth by a novel mechanism that is independent of RGD-integrin binding, and that involves cell surface proteoglycans.
Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and-independent pathways
Authors: Genersch Elke , Hayeß Katrin , Neuenfeld Yvette , Haller Hermann
Published: April 2021

Endothelial expression of matrix metalloproteinase-9 (MMP-9), which degrades native type IV collagen, was implicated as a prerequisite for angiogenesis. Therefore, the aim of this study was to determine signaling requirements that regulate MMP-9 expression in endothelial cells. Both, primary and permanent human umbilical vein endothelial cells (HUVEC and ECV304, respectively) were stimulated with phorbol 12-myristate 13-acetate (PMA) and the cytokine tumor necrosis factor-α (TNFα) to induce MMP-9 expression. While both cell types responded to PMA at the protein, mRNA and promoter level by induction of MMP-9, TNFα caused this response only in ECV304. Inhibitors specific for mitogen-activated protein/ERK kinase 1/2 (MEK1/2), protein kinase C (PKC), and Ras and co-transfections of wild-type and mutant Raf were used to elucidate the signaling cascades involved. Thus, we could show that the Raf/MEK/ERK cascade is mainly responsible for MMP-9 induction in endothelial cells and that this cascade is regulated independently of PKC and Ras subsequent to TNFα stimulation and in a PKC-dependent manner as a result of PMA treatment. In addition, PMA triggers a Ras-dependent signal transduction pathway bypassing the phosphorylation of ERK. Finally, we provide evidence that sustained phosphorylation of ERK1/2 is necessary but not sufficient for expression of MMP-9.
The SHR3 homologue from S. pombe demonstrates a conserved function of ER packaging chaperones
Authors: Martínez Paula , Ljungdahl Per O.
Published: April 2021

In Saccharomyces cerevisiae cells lacking SHR3, amino acid permeases do not enter into COPII transport vesicles and specifically accumulate in the membrane of the endoplasmic reticulum. Shr3p functions as a packaging chaperone to prime transport vesicle formation in the proximity of amino acid permeases. A genetic screen was developed that enabled the Schizosaccharomyces pombe SHR3 functional homologue, designated psh3+ (pombe SHR3), to be cloned. The psh3+ gene encodes a protein of 215 amino acids, which shares a high degree of structural and functional similarity with Shr3p. The heterologous expression of psh3+ complements many, but not all, shr3 null mutant phenotypes in S. cerevisiae in a temperature-dependent manner. Psh3p is localised to the endoplasmic reticulum of S. pombe cells, and strains lacking the psh3+gene exhibit decreased rates of amino acid uptake due to reduced levels of functional permeases in the plasma membrane. No packaging chaperones, or proteins exhibiting homology with packaging chaperones, have so far been identified in other eukayotic organisms. The findings reported here are the first to establish that specific packaging chaperones exist in divergent organisms, and demonstrate a conserved function of packaging chaperones in facilitating the export of large polytopic membrane proteins from the endoplasmic reticulum.
Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles
Authors: Kaksonen Marko , Peng H. Benjamin , Rauvala Heikki
Published: April 2021

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1893.html
Unique properties of lamp2a compared to other lamp2 isoforms
Authors: Cuervo A. M. , Dice J. F.
Published: April 2021

Lamp2a acts as a receptor in the lysosomal membrane for substrate proteins of chaperone-mediated autophagy. Using antibodies specific for the cytosolic tail of lamp2a and others recognizing all lamp2 isoforms, we found that in rat liver lamp2a represents 25% of lamp2s in the lysosome. We show that lamp2a levels in the lysosomal membrane in rat liver and fibroblasts in culture directly correlate with rates of chaperone-mediated autophagy in a variety of physiological and pathological conditions. The concentration of other lamp2s in the lysosomal membrane show no correlation under the same conditions. Furthermore, substrate proteins bind to lamp2a but not to other lamp2s. Four positively-charged amino acids uniquely present in the cytosolic tail of lamp2a are required for the binding of substrate proteins. Lamp2a also distributes to an unique subpopulation of perinuclear lysosomes in cultured fibroblasts in response to serum withdrawal, and lamp2a, more than other lamp2s, tends to multimerize. These characteristics may be important for lamp2a to act as a receptor for chaperone-mediated autophagy.
Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1)
Authors: Bakos Éva , Evers Raymond , Calenda Giulia , E. Tusnády Gábor , Szakács Gergely , Váradi András , Sarkadi Balázs
Published: April 2021

The human multidrug resistance protein (MRP1) contributes to drug resistance in cancer cells. In addition to an MDR1-like core, MRP1 contains an N-terminal membrane-bound (TMD0) region and a cytoplasmic linker (L0), both characteristic of several members of the MRP family. In order to study the role of the TMD0 and L0 regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells. The function of the various proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C4 and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion. Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells. We found that chimeric proteins consisting of N-terminal fragments of MRP1 fused to the N terminus of MDR1 preserved the transport, nucleotide occlusion and apical membrane routing of wild-type MDR1. As shown before,MRP1 without TMD0L0 (ΔMRP1), was non-functional and localized intracellularly, so we investigated the coexpression of ΔMRP1 with the isolated L0 region. Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells. Interestingly, the L0 peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent. A 10-amino-acid deletion in a predicted amphipathic region of L0 abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experiments suggest that the L0 region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.
The role of transient ERK2 signals in fibronectin- and insulin-mediated DNA synthesis
Authors: Asthagiri Anand R. , Reinhart Cynthia A. , Horwitz Alan F. , Lauffenburger Douglas A.
Published: April 2021

Both the extracellular matrix and growth factors jointly regulate cell cycle progression via a complex network of signaling pathways. Applying quantitative assays and analysis, we demonstrate here that concurrent stimulation of Chinese hamster ovary (CHO) cells with fibronectin (Fn) and insulin elicits a DNA synthesis response that reveals a synergy far more complex than a simple additive enhancement of response magnitude. CHO cell adhesion to higher Fn density shifts the sensitivity of the DNA synthesis response to insulin concentration from smoothly graded to sharply ‘switch-like’ and dramatically decreases the insulin concentration required for half-maximal response by about 1000-fold. Conversely, treatment with insulin has a milder and less complex effect on the response to varying Fn concentrations. Governing this DNA synthesis response is a common requirement for a transient, cell area-independent extracellular signal-regulated kinase 2 (ERK2) signal. Moreover, we show that the time-integrated value of this ‘pulse’ signal provides an appropriate metric for quantifying the dependence of DNA synthesis on the degree of ERK2 activation. Indeed, in the absence of insulin, the adhesion-mediated response is linearly proportional to ERK2 activation over a broad range of stimulatory Fn and MEK inhibitor amounts. However, in the presence of both Fn and insulin, total integrated ERK2 activity (the sum of Fn- and insulin-mediated signals) no longer serves as a predictor of DNA synthesis, demonstrating that the signaling crosstalk underlying response synergism does not converge at ERK2 activation. Instead, adhesion to higher Fn density enhances insulin stimulation of DNA synthesis, not by increasing insulin-mediated ERK2 activation, but via parallel elevation of at least one other insulin-mediated signal such as IRS-1 phosphorylation.
The developmentally regulated ECM glycoprotein ISG plays an essential role in organizing the ECM and orienting the cells of Volvox
Authors: Hallmann Armin , Kirk David L.
Published: April 2021

Volvox is one of the simplest multicellular organisms with only two cell types, yet it has a surprisingly complex extracellular matrix (ECM) containing many regionspecific morphological components, making Volvox suitable as a model system for ECM investigations. ECM deposition begins shortly after inversion, which is the process by which the embryo turns itself right-side-out at the end of embryogenesis. It was previously shown that the gene encoding an ECM glycoprotein called ISG is transcribed very transiently during inversion. Here we show that the developmentally controlled ISG accumulates at the bases of the flagella right after inversion, before any morphologically recognizable ECM structures have yet developed. Later, ISG is abundant in the ‘flagellar hillocks’ that encircle the basal ends of all flagella, and in the adjacent ‘boundary zone’ that delimits the spheroid. Transgenic Volvox were generated which express a truncated form of ISG. These transgenics exhibit a severely disorganized ECM within which the cells are embedded in a highly chaotic manner that precludes motility. A synthetic version of the C-terminal decapeptide of ISG has a similar disorganizing effect, but only when it is applied during or shortly after inversion. We postulate that ISG plays a critical role in morphogenesis and acts as a key organizer of ECM architecture; at the very beginning of ECM formation ISG establishes an essential initial framework that both holds the somatic cells in an adaptive orientation and acts as the scaffold upon which the rest of the ECM can be properly assembled, assuring that somatic cells of post-inversion spheroids are held in orientations and locations that makes adaptive swimming behavior possible.
Reversible programming of pluripotent cell differentiation
Authors: Lake Julie-anne , Rathjen Joy , Remiszewski Jackie , Rathjen Peter D.
Published: April 2021

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors.

EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.
The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement
Authors: Chubb Jonathan R. , Wilkins Andrew , Thomas Geraint M. , Insall Robert H.
Published: April 2021

Endocytosis and cell migration both require transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. We have generated Dictyosteliumcells lacking one member of this family, RasS, and the mutant cells are perturbed in endocytosis and cell migration. Mutant amoebae are defective in phagocytosis and fluid-phase endocytosis and are impaired in growth. Conversely, the rasS−cells show an enhanced rate of cell migration, moving three times faster than wild-type controls. The mutant cells display an aberrant morphology, are highly polarised, carry many elongated actin protrusions and show a concomitant decrease in formation of pinocytic crowns on the cell surface. These morphological aberrations are paralleled by changes in the actin cytoskeleton, with a significant proportion of the cortical F-actin relocalised to prominent pseudopodia. Rapid migration and endocytosis appear to be mutually incompatible and it is likely that RasS protein is required to maintain the normal balance between these two actin-dependent processes.
TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of RhoG
Authors: Blangy A. , Vignal E. , Schmidt S. , Debant A. , Gauthier-Rouvière C. , Fort P.
Published: April 2021

Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of Rho regulators that contain two GEF domains of distinct specificities. We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1). In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoGGTPled to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.
Regulation of protein sorting at the TGN by plasma membrane receptor activation
Authors: Baldassarre Massimiliano , Dragonetti Antonella , Marra Pierfrancesco , Luini Alberto , Isidoro Ciro , Buccione Roberto
Published: April 2021

We show that in the rat basophilic leukemia cell line RBL, the physiological stimulation of the IgE receptor or direct activation of PKC leads to the missorting of proteins to the plasma membrane, diverting them from their normal intracellular destination. This is demonstrated for two classes of proteins that are normally targeted to the secretory lysosomes via completely different mechanisms, i.e. proteoglycans and the aspartic protease cathepsin D. In the latter case, normal processing of the enzyme is also affected, leading to secretion of the immature form of cathepsin. The present study shows how completely different sorting mechanisms, such as those for delivering proteoglycans and cathepsin D to secretory lysosomes, might share common regulatory signals and are similarly affected when the levels of these signals are perturbed. Finally, protein kinase C appears to be a major player in the signal transduction pathways, leading to proteoglycan and cathepsin D missorting.
Mammalian S-phase checkpoint integrity is dependent on transformation status and purine deoxyribonucleosides
Authors: Downes C. Stephen , Bachrati Csanád Z. , Devlin S. Jayne , Tommasino Massimo , Cutts Timothy J. R. , Watson James V. , Raskó stván , Johnson Robert T.
Published: April 2021

In eukaryotic cells arrested in S-phase, checkpoint controls normally restrain mitosis until after replication. We have identified an array of previously unsuspected factors that modulate this restraint, using transformed hamster cells in which cycle controls are known to be altered in S-phase arrest. Arrested cells accumulate cyclin B, the regulatory partner of the mitotic p34cdc2kinase, which is normally not abundant until late G2phase; treatment of arrested cells with caffeine produces rapid S-phase condensation. We show here that such S-phase checkpoint slippage, as visualised through caffeine-dependent S-phase condensation, correlates with rodent origin and transformed status, is opposed by reverse transformation, and is favoured by c-srcand opposed by wnt1overexpression. Slippage is also dependent on a prolonged replicative arrest, and is favoured by arrest with hydroxyurea, which inhibits ribonucleotide reductase. This last is a key enzyme in deoxyribonucleotide synthesis, recently identified as a determinant of malignancy. Addition of deoxyribonucleosides shows that rapid S-phase condensation is suppressed by a novel checkpoint mechanism: purine (but not pyrimidine) deoxyribonucleosides, like reverse transformation, suppress cyclin B/p34cdc2activation by caffeine, but not cyclin B accumulation. Thus, ribonucleotide reductase has an unexpectedly complex role in mammalian cell cycle regulation: not only is it regulated in response to cycle progression, but its products can also reciprocally influence cell cycle control kinase activation.
Cross-regulation of the Wnt signalling pathway: a role of MAP kinases
Authors: Behrens Jürgen
Published: April 2021

The Wnt signal transduction pathway regulates various aspects of embryonal development and is involved in cancer formation. Wnts induce the stabilisation of cytosolic β-catenin, which then associates with TCF transcription factors to regulate expression of Wnt-target genes. At various levels the Wnt pathway is subject to cross-regulation by other components. Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling. In particular, homologues of TAK1 and NLK, MOM-4 and LIT-1, negatively regulate Wnt-controlled cell fate decision in the early Caenorhabditis elegansembryo. Moreover, TAK1 activates NLK, which phosphorylates TCFs bound to β-catenin. This blocks nuclear localization and DNA binding of TCFs. Since TAK1 is activated by TGF-β and various cytokines, it might provide an entry point for regulation of the Wnt system by other pathways. In addition, alterations in TAK1-NLK might play a role in cancer.
DNA-binding activity of the N-terminal cleavage product of poly(ADP-ribose) polymerase is required for UV mediated apoptosis
Authors: Kim Jin Woo , Won Jungyeon , Sohn Seonghyang , Joe Cheol O.
Published: April 2021

The role of the N-terminal cleavage product of poly(ADP-ribose) polymerase (PARP) on UV mediated apoptosis was investigated in cultured HeLa cells. Ultrastructural analysis of cells expressing caspase-resistant PARP (PARPD214A) revealed the typical features of necrosis following UV treatment. However, cells co-expressing PARPD214Awith the N-terminal fragment of PARP containing the DNA-binding domain underwent apoptosis instead of necrosis. In this study, we have demonstrated that the DNA-binding activity of the N-terminal fragment of PARP is important for the execution of apoptosis. Point mutations were introduced in the DNA-binding sites of the N-terminal fragment. Cells co-expressing PARPD214Awith the mutated N-terminal fragments neither stimulated apoptosis nor prevented necrosis in response to UV irradiation. The present study proposes that the DNA-binding activity of the N-terminal fragment of PARP in UV treated cells prevents cellular ATP depletion, a mechanism by which necrotic cell death is triggered.
Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+
Authors: Meunier Frédéric A. , Mattei Cesar , Chameau Pascal , Lawrence Gary , Colasante Cesare , Kreger Arnold S. , Dolly J. Oliver , Molgó Jordi
Published: April 2021

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+influx through voltage-activated Ca2+channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.
Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model
Authors: Muraglia Anita , Cancedda Ranieri , Quarto Rodolfo
Published: April 2021

Bone marrow stromal cells can give rise to several mesenchymal lineages. The existence of a common stem/progenitor cell, the mesenchymal stem cell, has been proposed, but which developmental stages follow this mesenchymal multipotent progenitor is not known. Based on experimental evidence, a model of mesenchymal stem cell differentiation has been proposed in which individual lineages branch directly from the same progenitor. We have verified this model by using clonal cultures of bone marrow derived stromal fibroblasts. We have analyzed the ability of 185 non-immortalized human bone marrow stromal cell clones to differentiate into the three main lineages: osteo-, chondro- and adipogenic. All clones but one differentiated into the osteogenic lineage. About one third of the clones differentiated into all three lineages analyzed. Most clones (60-80%) displayed an osteo-chondrogenic potential. We have never observed clones with a differentiation potential limited to the osteo-adipo- or to the chondro-adipogenic phenotype, nor pure chondrogenic and adipogenic clones. How long the differentiation potential of a number of clones was maintained was assessed throughout their life span. Clones progressively lost their adipogenic and chondrogenic differentiation potential at increasing cell doublings.

Our data suggest a possible model of predetermined bone marrow stromal cells differentiation where the tripotent cells can be considered as early mesenchymal progenitors that display a sequential loss of lineage potentials, generating osteochondrogenic progenitors which, in turn, give rise to osteogenic precursors.
Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments
Authors: Bursch Wilfried , Hochegger Karin , Török Ladislaus , Marian Brigitte , Ellinger Adolf , Hermann Rolf Schulte
Published: April 2021

Programmed cell death comprises several subtypes, as revealed by electron microscopy. Apoptosis or type I programmed cell death is characterized by condensation of cytoplasm and preservation of organelles, essentially without autophagic degradation. Autophagic cell death or type II programmed cell death exhibits extensive autophagic degradation of Golgi apparatus, polyribosomes and endoplasmatic reticulum, which precedes nuclear destruction. In the present study, we analysed the fate of cytokeratin and F-actin during autophagic cell death in the human mammary carcinoma cell line MCF-7 because recent studies suggest that an intact cytoskeleton is necessary for autophagocytosis. Programmed cell death was induced by 10−6M tamoxifen. For quantitative light microscopic analysis, autophagic vacuoles were visualized by monodansyl cadaverin, which stains autophagic vacuoles as distinct dot-like structures. In control cultures, the number of monodansylcadaverin-positive cells did not exceed 2%. Tamoxifen induced a dramatic increase 2-4 days after treatment to a maximum of 60% monodansylcadaverin-positive cells between days 5 and 7. Cell death, as indicated by nuclear condensation, increased more gradually to about 18% of all cells on day 7. In cells with pyknotic nuclei cytokeratin appeared disassembled but retained its immunoreactivity; actin was still polymerized to filaments, as demonstrated by its reaction with phalloidin. Western blot analysis showed no significant cleavage of the monomeric cytokeratin fraction. For comparison, apoptotic or type I cell death was studied using the human colon cancer cell HT29/HI1 treated with the tyrosine kinase inhibitor tyrphostin A25 as a model. Cleavage of cytokeratin was already detectable in early morphological stages of apoptosis. F-actin was found to depolymerize; its globular form could be detected by antibodies; western blot analysis revealed no products of proteolytic cleavage. In conclusion, in our model of apoptosis, early stages are associated with depolymerization of actin and degradation of intermediate filaments. In contrast, during autophagic cell death intermediate and microfilaments are redistributed, but largely preserved, even beyond the stage of nuclear collapse. The present data support the concept that autophagic cell death is a separate entity of programmed cell death that is distinctly different from apoptosis.
Bidirectional transcytosis of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system to study protein transport across epithelia
Authors: McCarthy Kevin M. , Yoong Yinlee , Simister Neil E.
Published: April 2021

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across cellular barriers between mother and offspring. FcRn also protects circulating IgG from catabolism, probably during transport across the capillary endothelium. Only one cell culture model of transcytosis has been used extensively, the transport of IgA from the basolateral to the apical surface of Madin-Darby canine kidney cells by the polymeric immunoglobulin receptor (pIgR). We report that rat inner medullary collecting duct (IMCD) cells transfected with DNA encoding the α subunit of rat FcRn specifically and saturably transport Fc when grown as polarized monolayers. Using this system, we have found that transcytosis by FcRn, like transcytosis by the pIgR, depends upon an intact microtubule system. FcRn differs most strikingly from the pIgR in its ability to transport its ligand in both the apical to basolateral and basolateral to apical directions. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibited basolateral to apical transport by FcRn more than apical to basolateral transport, suggesting that there are differences in the mechanisms of transport in the two directions. Lastly, we found that transcytosis by FcRn depends upon vesicular acidification. We anticipate that the IMCD cell culture model will allow further elucidation of the mechanism of IgG transport by FcRn.
The p120 catenin family: Complex roles in adhesion, signaling and cancer
Authors: Anastasiadis Panos Z. , Reynolds Albert B.
Published: April 2021

p120 catenin (p120) is the prototypic member of a growing subfamily of Armadillo-domain proteins found at cell- cell junctions and in nuclei. In contrast to the functions of the classical catenins (α-catenin, β-catenin, and γ- catenin/plakoglobin), which have been studied extensively, the first clues to p120’s biological function have only recently emerged, and its role remains controversial. Nonetheless, it is now clear that p120 affects cell-cell adhesion through its interaction with the highly conserved juxtamembrane domain of classical cadherins, and is likely to have additional roles in the nucleus. Here, we summarize the data on the potential involvement of p120 both in promotion of and in prevension of adhesion, and propose models that attempt to reconcile some of the disparities in the literature. We also discuss the structural relationships and functions of several known p120 family members, as well as the potential roles of p120 in signaling and cancer.