bluuebunny/crossref_metadata_2025 huggingface dataset:
Chaperoning signaling pathways: molecular chaperones as stress-sensing`heat shock' proteins
Authors: Nollen Ellen A. A. , Morimoto Richard I.
Published: April 2021

Heat shock proteins interact with multiple key components of signaling pathways that regulate growth and development. The molecular relationships between heat shock proteins, various signaling proteins and partner proteins appear to be critical for the normal function of signal transduction pathways. The relative levels of these proteins may be important, as too little or too much Hsp70 or Hsp90 can result in aberrant growth control, developmental malformations and cell death. Although the functions of heat shock proteins as molecular chaperones have been well characterized, their complementary role as a `stress-induced' proteins to monitor changes and alter the biochemical environment of the cell remains elusive. Genetic and molecular interactions between heat shock proteins, their co-chaperones and components of signaling pathways suggest that crosstalk between these proteins can regulate proliferation and development by preventing or enhancing cell growth and cell death as the levels of heat shock proteins vary in response to environmental stress or disease.
Prolonged arrest of mammalian cells at the G1/S boundary results in permanent S phase stasis
Authors: Borel Franck , Lacroix Françoise B. , Margolis Robert L.
Published: April 2021

Mammalian cells in culture normally enter a state of quiescence during G1 following suppression of cell cycle progression by senescence, contact inhibition or terminal differentiation signals. We find that mammalian fibroblasts enter cell cycle stasis at the onset of S phase upon release from prolonged arrest with the inhibitors of DNA replication, hydroxyurea or aphidicolin. During arrest typical S phase markers remain present, and G0/G1 inhibitory signals such as p21WAF1 and p27 are absent. Cell cycle stasis occurs in T-antigen transformed cells, indicating that p53 and pRB inhibitory circuits are not involved. While no DNA replication is evident in arrested cells, nuclei isolated from these cells retain measurable competence for in vitro replication. MCM proteins are required to license replication origins, and are put in place in nuclei in G1 and excluded from chromatin by the end of replication to prevent rereplication of the genome. Strikingly, MCM proteins are strongly depleted from chromatin during prolonged S phase arrest,and their loss may underlie the observed cell cycle arrest. S phase stasis may thus be a `trap' in which cells otherwise competent for S phase have lost a key component required for replication and thus can neither go forward nor retreat to G1 status.
The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised
Authors: Huang Jun-yong , Raff Jordan W.
Published: April 2021

In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularised embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos,only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction,but its subcellular localisation remains controversial. We constructed GFP fusions of two core APC/C subunits, Cdc16 and Cdc27. These fusion proteins were incorporated into the endogenous APC/C and were largely localised in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulated in the nucleus prior to nuclear envelope breakdown but only weakly associated with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly,we noticed that GFP-Cdc27 associated with mitotic chromosomes, whereas GFP-Cdc16 did not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90%in tissue culture cells led to a transient mitotic arrest that was both biochemically and morphologically distinct. Taken together, our results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localised and perform distinct functions.
Functional consequence of targeting protein kinase B/Akt to GLUT4 vesicles
Authors: Ducluzeau Pierre-Henri , Fletcher Laura M. , Welsh Gavin I. , Tavaré Jeremy M.
Published: April 2021

We have investigated the role of protein kinase B (Akt) in the insulin-stimulated translocation of vesicles containing the insulin-responsive isoform of glucose transporter (GLUT4) to the plasma membrane of adipocytes. Previous reports have suggested that protein kinase B can bind to intracellular GLUT4 vesicles in an insulin-dependent manner, but the functional consequence of this translocation is not known. In this study we have artificially targeted constitutively active and kinase-inactive mutants of protein kinase B to intracellular GLUT4 vesicles by fusing them with the N-terminus of GLUT4 itself. We examined the effect of these mutants on the insulin-dependent translocation of the insulin-responsive amino peptidase IRAP(a bona fide GLUT4-vesicle-resident protein). A kinase-inactive protein kinase B targeted to GLUT4 vesicles was an extremely effective dominant-negative inhibitor of insulin-stimulated IRAP translocation to the plasma membrane. By contrast, a kinase-inactive protein kinase B expressed in the cytoplasm did not have an effect. The results suggest that protein kinase B has an important functional role at, or in the vicinity of, GLUT4 vesicles in the insulin-dependent translocation of those vesicles to the plasma membrane of adipocytes.
The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation
Authors: Saliba Richard S. , Munro Peter M. G. , Luthert Philip J. , Cheetham Michael E.
Published: April 2021

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with ΔF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.
Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts
Authors: Faccio Roberta , Grano Maria , Colucci Silvia , Villa Antonello , Giannelli Gianluigi , Quaranta Vito , Zallone Alberta
Published: April 2021

Integrins are membrane receptors that mediate interactions between cells and the extracellular matrix. We recently showed that the osteoclast integrinα vβ3 exists in two different conformations,so-called `basal' and `activated', with each exhibiting a distinct function. In this study we demonstrate that, in non-resorbing osteoclasts, the`activated' form of αvβ3 accumulates in the motile areas of the plasma membrane. During bone resorption this conformation is prevalent in the ruffled membrane, whereas the `basal' form ofα vβ3 is also present in the sealing zone. Moreover, hepatocyte growth factor (HGF) and macrophage colony stimulating factor (M-CSF), two molecules involved in osteoclastogenesis and osteoclast survival, modulate αvβ3 conformation in vitro. Preincubation with HGF or M-CSF induces a shift of conformation ofα vβ3 in primary human osteoclasts (OCs) and in the osteoclast-like cell line (GCT 23). Activated integrin promotes osteoclast migration to the αvβ3 ligand osteopontin and enhances bone resorption. Thus, HGF and M-CSF modulate theα vβ3 conformational states required for osteoclast polarization and resorption. The capacity of growth factors to alter the affinity of αvβ3 toward its ligands offers a potential explanation for the diverse responses of osteoclasts to the same ligand.
Differential regulation of maternal and paternal chromosome condensation in mitotic zygotes
Authors: Bomar Jacqueline , Moreira Pedro , Balise John J. , Collas Philippe
Published: April 2021

A-kinase anchoring protein AKAP95 is implicated in somatic mitotic chromosome condensation by recruiting the condensin complex. Here, we report a differential regulation of condensation of maternal and paternal chromosomes mediated by AKAP95 in mitotic mouse zygotes. AKAP95 is synthesized upon oocyte activation, targeted to the female pronucleus and specifically associates with maternal chromosomes at mitosis. AKAP95 mRNA is highly restricted to the vicinity of the meiotic spindle in metaphase II oocytes. In vivo displacement of endogenous AKAP95 in female pronuclei by microinjection of competitor peptides and rescue experiments show that AKPA95 is required for recruitment of the mCAP-D2 condensin subunit to, and condensation of, maternal chromosomes. In contrast, AKAP95 is dispensable for mCAP-D2 recruitment to,and condensation of, paternal chromosomes. Our results indicate that at first embryonic mitosis, paternal chromosomes target condensins and condense independently of AKAP95, whereas maternal chromosomes require AKAP95 for condensin recruitment and condensation. We propose a concept whereby condensation of chromosomes in gametes, zygotes and somatic cells involves related but distinct mechanisms.
Vesicular transport: the core machinery of COPI recruitment and budding
Authors: Nickel Walter , Brügger Britta , Wieland Felix T.
Published: April 2021

Vesicular transport is the predominant mechanism for exchange of proteins and lipids between membrane-bound organelles in eukaryotic cells. Golgi-derived COPI-coated vesicles are involved in several vesicular transport steps, including bidirectional transport within the Golgi and recycling to the ER. Recent work has shed light on the mechanism of COPI vesicle biogenesis, in particular the machinery required for vesicle formation. The new findings have allowed us to generate a model that covers the cycle of coat recruitment, coat polymerization, vesicle budding and uncoating.
Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells
Authors: Duval Nathalie , Gomès Danielle , Calaora Viviane , Calabrese Alessandra , Meda Paolo , Bruzzone Roberto
Published: April 2021

Embryonic neural progenitors isolated from the mouse striatal germinal zone grow in vitro as floating cell aggregates called neurospheres, which, upon adhesion, can be induced to differentiate into the three main cell types of the central nervous system (CNS), that is, astrocytes, neurons and oligodendrocytes. To study the possible role of connexins and junctional communication during differentiation of neural progenitors, we assessed cell-to-cell communication by microinjecting Lucifer Yellow into neurospheres at various times after adhesion. Cells located in neurospheres were strongly coupled, regardless of the differentiation time. Microinjections performed on the cell layers formed by differentiated cells migrating out of the neurosphere established that only astrocytes were coupled. These observations suggest the existence of at least three distinct communication compartments:coupled proliferating cells located in the sphere, uncoupled cells undergoing neuronal or oligodendrocytic differentiation and coupled differentiating astrocytes. A blockade of junctional communication by 18-β-glycyrrhetinic acid (βGA) reduced, in a concentration-dependent manner, the viability of undifferentiated neural progenitor cells. This effect appeared to be specific,inasmuch as it was reversible and that cell survival was not affected in the presence of the inactive analog glycyrrhyzic acid. Addition of βGA to adherent neurospheres also decreased cell density and altered the morphology of differentiated cells. Cx43 was strongly expressed in either undifferentiated or differentiated neurospheres, where it was found both within the sphere and in astrocytes, the two cell populations that were dye coupled. Western blot analysis further showed that Cx43 phosphorylation was strongly increased in adherent neurospheres, suggesting a post-translational regulation during differentiation. These results point to a major role of cell-to-cell communication and Cx43 during the differentiation of neural progenitor cells in vitro.
The mechanism regulating the dissociation of the centrosomal protein C-Nap1 from mitotic spindle poles
Authors: Mayor Thibault , Hacker Ulrike , Stierhof York-Dieter , Nigg Erich A.
Published: April 2021

The centrosomal protein C-Nap1 is thought to play an important role in centrosome cohesion during interphase of the cell cycle. At the onset of mitosis, when centrosomes separate for bipolar spindle formation, C-Nap1 dissociates from centrosomes. Here we report the results of experiments aimed at determining whether the dissociation of C-Nap1 from mitotic centrosomes is triggered by proteolysis or phosphorylation. Specifically, we analyzed both the cell cycle regulation of endogenous C-Nap1 and the fate of exogenously expressed full-length C-Nap1. Western blot analyses suggested a reduction in the endogenous C-Nap1 level during M phase, but studies using proteasome inhibitors and destruction assays performed in Xenopus extracts argue against ubiquitin-dependent degradation of C-Nap1. Instead, our data indicate that the mitotic C-Nap1 signal is reduced as a consequence of M-phase-specific phosphorylation. Overexpression of full-length C-Nap1 in human U2OS cells caused the formation of large structures that embedded the centrosome and impaired its microtubule nucleation activity. Remarkably, however, these centrosome-associated structures did not interfere with cell division. Instead, centrosomes were found to separate from these structures at the onset of mitosis, indicating that a localized and cell-cycle-regulated activity can dissociate C-Nap1 from centrosomes. A prime candidate for this activity is the centrosomal protein kinase Nek2, as the formation of large C-Nap1 structures was substantially reduced upon co-expression of active Nek2. We conclude that the dissociation of C-Nap1 from mitotic centrosomes is regulated by localized phosphorylation rather than generalized proteolysis.
Annexin 6 is a putative cell surface receptor for chondroitin sulfate chains
Authors: Takagi Hidekazu , Asano Yasushi , Yamakawa Naomi , Matsumoto Isamu , Kimata Koji
Published: April 2021

Chondroitin sulfate proteoglycans, including PG-M/versican, inhibit cell-substratum adhesion. They achieve this through their chondroitin sulfate chains. In order to define the molecular mechanism for this inhibition, we investigated the influence of these chains on cell attachment to substratum,the first step in cell adhesion. Chondroitin sulfate chains did not prevent cell attachment. In fact, a variety of cells attached to chondroitin sulfate,implying the existence of putative receptors and/or binding proteins for this extracellular matrix glycosaminoglycan. Detergent-extracted human fibroblast membrane protein extracts were examined by affinity chromatography in the presence of Ca2+ on chondroitin sulfate immobilized on agarose CL-6B. A 68 kDa and a 35 kDa protein were isolated, sequenced and demonstrated to be annexin 6 and annexin 4, respectively. Next we used A431 cells devoid of annexin 6 expression to verify that annexin 6 is the receptor for this glycosaminoglycan. We confirmed that A431 cells were unable to attach to the chondroitin sulfate substratum and that the stable transfectants expressing annexin 6 conferred the ability to attach to chondroitin sulfate chains. Further, the presence of annexin 6 on the cell surface was confirmed by fluorescence-activated cell sorting analysis using the annexin 6 antibody;annexin 4 is not present on the cell surface. In summary, annexin 6 is a candidate receptor for chondroitin sulfate chains.
Sites of Ca2+ wave initiation move with caveolae to the trailing edge of migrating cells
Authors: Isshiki Masashi , Ando Joji , Yamamoto Kimiko , Fujita Toshiro , Ying Yunshu , Anderson Richard G. W.
Published: April 2021

The caveola is a membrane domain that compartmentalizes signal transduction at the cell surface. Normally in endothelial cells, groups of caveolae are found clustered along stress fibers or at the lateral margins in all regions of the cell. Subsets of these clusters appear to contain the signaling machinery for initiating Ca2+ wave formation. Here we report that induction of cell migration, either by wounding a cell monolayer or by exposing cells to laminar shear stress, causes caveolae to move to the trailing edge of the cell. Concomitant with the relocation of the caveolae,sites of Ca2+ wave initiation move to the same location. In as much as the relocated caveolae contain elements of the signaling machinery required for ATP-stimulated release of Ca2+ from the ER, these results suggest that caveolae function as containers that carry this machinery to different cellular locations.
Gelsolin — evidence for a role in turnover of junction-related actin filaments in Sertoli cells
Authors: Guttman Julian A. , Janmey Paul , Vogl A. Wayne
Published: April 2021

The gelsolin-phosphoinositide pathway may be part of the normal mechanism by which Sertoli cells regulate sperm release and turnover of the blood-testis barrier. The intercellular adhesion complexes (ectoplasmic specializations)involved with these two processes are tripartite structures consisting of the plasma membrane, a layer of actin filaments and a cistern of endoplasmic reticulum. Gelsolin is concentrated in these adhesion complexes. In addition,phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphoinositide-specific phospholipase C are found in the structures. Treatment of isolated spermatid/junction complexes with exogenous phosphoinositide-specific phospholipase C, or with a synthetic peptide consisting of the PtdIns(4,5)P2 binding region of gelsolin, results in the release of gelsolin and loss of actin from the adhesion complexes. We present a model for the disassembly of the actin layer of the adhesion complex that involves the hydrolysis of PtdIns(4,5)P2 resulting in the release of gelsolin within the plaque. Further, we speculate that the hydrolysis of PtdIns(4,5)P2 may result in a local Ca2+ surge via the action of inositol triphosphate on junctional endoplasmic reticulum. This Ca2+ surge would facilitate the actin severing function of gelsolin within the adhesion complex.
Pax3 regulates morphogenetic cell behavior in vitro coincident with activation of a PCP/non-canonical Wnt-signaling cascade
Authors: Wiggan O'Neil , Hamel Paul A.
Published: April 2021

Mutations to Pax3 and other Pax family genes in both mice and humans result in numerous tissue-specific morphological defects. Little is known, however, about the cellular and molecular mechanisms by which Pax genes regulate morphogenesis. We previously showed that Pax3 induces cell aggregation and a mesenchymal-to-epithelial transition in Saos-2 cells. We show here that Pax3-induced aggregates arise through the formation of distinct structures involving cell rearrangements and cell behaviors resembling those that occur during gastrulation and neurulation known as convergent extension. During these Pax3-induced processes, Dishevelled and Frizzled are localized to the actin cytoskeleton and both proteins coimmunoprecipitate focal adhesion components from detergent-insoluble cell fractions. We show further that these Pax3-induced cell movements are associated with activation of a Wnt-signaling cascade, resulting in induction and activation of c-Jun-N-terminal kinase/stress activated protein kinase (JNK/SAPK). All of these Wnt-signaling factors exhibit altered subcellular distribution in Pax3-expressing cells. In particular, we show the localization of JNK/SAPK to both the nucleus and to cytoplasmic multi-vesicular structures. These data show that Pax3 regulates morphogenetic cell behavior and that regulation of a conserved, planar cell polarity/noncanonical Wnt-signaling cascade entailing JNK activation is a function of Pax3 activity.
Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function
Authors: Fortugno Paola , Wall Nathan R. , Giodini Alessandra , O'Connor Daniel S. , Plescia Janet , Padgett Karen M. , Tognin Simona , Marchisio Pier Carlo , Altieri Dario C.
Published: April 2021

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala3-Ile19 exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence,nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys57-Trp67 identified a cytosolic pool of survivin,which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala3-Ile19,Met38-Thr48, Pro47-Phe58 and Cys57-Trp67 identified both survivin pools within the same mitotic cell. A ratio of ∼1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34cdc2, and was phosphorylated by p34cdc2 on Thr34, in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34cdc2 and was not phosphorylated on Thr34. Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.
Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere
Authors: Souchet Michel , Portales-Casamar Elodie , Mazurais David , Schmidt Susanne , Léger Isabelle , Javré Jean-Luc , Robert Philippe , Berrebi-Bertrand Isabelle , Bril Antoine , Gout Bernard , Debant Anne , Calmels Thierry P. G.
Published: April 2021

The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Db1-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Db1 homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology.
Actin filament turnover removes bundles fromDrosophilabristle cells
Authors: Guild Gregory M. , Connelly Patricia S. , Vranich Kelly A. , Shaw Michael K. , Tilney Lewis G.
Published: April 2021

Drosophila bristle cells form enormous extensions that are supported by equally impressive scaffolds of modular, polarized and crosslinked actin filament bundles. As the cell matures and support is taken over by the secreted cuticle, the actin scaffold is completely removed. This removal begins during cell elongation and proceeds via an orderly series of steps that operate on each module. Using confocal and electron microscopy, we found that the ∼500-filament modules are fractured longitudinally into 25-50-filament subbundles, indicating that module breakdown is the reverse of assembly. Time-lapse confocal analysis of GFP-decorated bundles in live cells showed that modules were shortened by subunit removal from filament barbed ends, again indicating that module breakdown is the reverse of assembly. Module shortening takes place at a fairly slow rate of ∼1μm/hour,implying that maximally crosslinked modules are not rapidly depolymerized. Barbed-end depolymerization was prevented with jasplakinolide and accelerated with cycloheximide, indicating that barbed-end maintenance requires continuous protein synthesis. Subbundle adhesion was lost in the presence of cytochalasin, indicating that continuous actin polymerization is required. Thus, these polarized actin filament bundles are dynamic structures that require continuous maintenance owing to protein and actin filament turnover. We propose that after cell elongation, maintenance falls behind turnover,resulting in the removal of this modular cytoskeleton.
Vid22p, a novel plasma membrane protein, is required for the fructose-1,6-bisphosphatase degradation pathway
Authors: Brown C. Randell , McCann Jameson A. , Hung Graham Guo-Chiuan , Elco Christopher P. , Chiang Hui-Ling
Published: April 2021

Fructose-1,6-bisphosphatase (FBPase), an important enzyme in the gluconeogenic pathway in Saccharomyces cerevisiae, is expressed when cells are grown in media containing a poor carbon source. Following glucose replenishment, FBPase is targeted from the cytosol to intermediate Vid(vacuole import and degradation) vesicles and then to the vacuole for degradation. Recently, several vid mutants that are unable to degrade FBPase in response to glucose were identified. Here, we present VID22, a novel gene involved in FBPase degradation. VID22encodes a glycosylated integral membrane protein that localizes to the plasma membrane. Newly synthesized Vid22p was found in the cytoplasm and then targeted to the plasma membrane independent of the classical secretory pathway. A null mutation of VID22 failed to degrade FBPase following a glucose shift and accumulated FBPase in the cytosol. Furthermore, the majority of FBPase remained in a proteinase K sensitive compartment in the Δvid22 mutant, implying that VID22 is involved in FBPase transport from the cytosol to Vid vesicles. By contrast,starvation-induced autophagy and peroxisome degradation were not impaired in the Δvid22 mutant. This strain also exhibited the proper processing of carboxypeptidase Y and aminopeptidase I in the vacuole. Therefore, Vid22p appears to play a specific role in the FBPase trafficking pathway.
Multiple forms of SNARE complexes in exocytosis from chromaffin cells: effects of Ca2+, MgATP and botulinum toxin type A
Authors: Lawrence Gary W. , Dolly J. Oliver
Published: April 2021

The changes that SNAREs undergo during exocytosis were studied in permeabilised chromaffin cells treated with Ca2+, MgATP or botulinum neurotoxin A. High-resolution 2D SDS-PAGE revealed multiple SDS-resistant SNARE complexes having a wide range of sizes and in which SNAP-25 and syntaxin predominate over synaptobrevin. Their formation increased upon Ca2+-stimulated exocytosis; notably, the 2D protocol proved much superior to 1D SDS-PAGE for the detection of large complexes and revealed that for forms with relative molecular mass greater than 100,000 stimulated induction was more significant than for smaller species. MgATP enhanced Ca2+-triggered catecholamine release but reduced the content of complexes. By contrast, botulinum neurotoxin type A inhibited exocytosis and altered the stoichiometry of the SNAP-25:syntaxin binary association, without lowering its abundance. The individual SNAREs were protected against trypsin proteolysis to varying extents in binary and ternary complexes of different sizes, suggestive of distinct folding intermediates. Our data suggest that Ca2+ triggers an early stage of SNARE complex formation causing an accumulation of partially folded intermediates, especially of binary forms, as well as their maturation into smaller, more protease resistant states. In addition, botulinum neurotoxin A inhibits exocytosis by perturbing the syntaxin:SNAP-25 ratio in binary intermediates.
Two members of the beige/CHS (BEACH) family are involved at different stages in the organization of the endocytic pathway inDictyostelium
Authors: Cornillon Sophie , Dubois Annick , Brückert Franz , Lefkir Yaya , Marchetti Anna , Benghezal Mohammed , De Lozanne Arturo , Letourneur François , Cosson Pierre
Published: April 2021

Proteins of the Chediak-Higashi/Beige (BEACH) family have been implicated in the function of lysosomes, as well as in signal transduction, but their molecular role is still poorly understood. In Dictyostelium, at least six members of the family can be identified. Here cells with mutations in two of these genes, LVSA and LVSB, were analyzed. Interestingly both mutants exhibited defects in the organization of the endocytic pathway,albeit at distinct stages. In lvsB mutant cells, the regulated secretion of lysosomal enzymes was enhanced, a phenotype reminiscent of the Chediak-Higashi syndrome. LvsA mutant cells exhibited alterations in the organization and function of the early endocytic and phagocytic pathway. The LvsA protein may participate in the signaling pathway, which links adhesion of a particle to the subsequent formation of a phagocytic cup. Further genetic analysis will be necessary to determine whether other members of the BEACH family of proteins are also involved in controlling the organization of the endocytic pathway.
The T-box transcription factorBrachyurymediates cartilage development in mesenchymal stem cell line C3H10T1/2
Authors: Hoffmann Andrea , Czichos Stefan , Kaps Christian , Bächner Dietmar , Mayer Hubert , Zilberman Yoram , Turgeman Gadi , Pelled Gadi , Gross Gerhard , Gazit Dan
Published: April 2021

The BMP2-dependent onset of osteo/chondrogenic differentiation in the acknowledged pluripotent murine mesenchymal stem cell line (C3H10T1/2) is accompanied by the immediate upregulation of Fibroblast Growth Factor Receptor 3 (FGFR3) and a delayed response by FGFR2. Forced expression of FGFR3 in C3H10T1/2 is sufficient for chondrogenic differentiation, indicating an important role for FGF-signaling during the manifestation of the chondrogenic lineage in this cell line. Screening for transcription factors exhibiting a chondrogenic capacity in C3H10T1/2 indentified that the T-box containing transcription factor Brachyury is upregulated by FGFR3-mediated signaling. Forced expression of Brachyury in C3H10T1/2 was sufficient for differentiation into the chondrogenic lineage in vitro and in vivo after transplantation into muscle. A dominant-negative variant of Brachyury, consisting of its DNA-binding domain (T-box), interferes with BMP2-mediated cartilage formation. These studies indicate that BMP-initiated FGF-signaling induces a novel type of transcription factor for the onset of chondrogenesis in a mesenchymal stem cell line. A potential role for this T-box factor in skeletogenesis is further delineated from its expression profile in various skeletal elements such as intervertebral disks and the limb bud at late stages (18.5 d.p.c.) of murine embryonic development.
UV induces tyrosine kinase-independent internalisation and endosome arrest of the EGF receptor
Authors: Oksvold Morten P. , Huitfeldt Henrik S. , Østvold Anne Carine , Skarpen Ellen
Published: April 2021

We have compared the activation and trafficking of epidermal growth factor receptor (EGFR) induced by UV light and EGF. Tyrosine phosphorylation of EGFR was not detected in UV-exposed cells by immunoblotting of whole cell lysates or EGFR immunoprecipitates with antibodies specific for each of the five activated autophosphorylation sites of EGFR. In addition, EGFR of UV-irradiated cells did not demonstrate increased 32P-incorporation. However, UV-exposed cells demonstrated a gel mobility shift of EGFR, which was not abolished by alkaline phosphatase treatment. UV-exposure did not induce dimerisation of EGFR. Furthermore, UV induced internalisation of EGFR without polyubiquitination or degradation. UV-exposed EGFR was transferred to early endosomes and arrested in transferrin-accessible endosomes close to the cell surface. Whereas inhibition of the EGFR tyrosine kinase effectively inhibited tyrosine phosphorylation and internalisation of EGF-activated EGFR, internalisation of UV-exposed EGFR was unaffected. UV induced neither relocalisation of Shc and Grb2 nor activation of Raf, but activation of MEK and MAPK was observed.

Our work indicates that UV induces internalisation of EGFR independent of its phosphorylation or receptor tyrosine kinase activation, and altered EGFR trafficking compared with ligand-activated receptor. In addition, MAPK activation by UV does not appear to be mediated by EGFR activation.
Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence inTrypanosoma brucei
Authors: Böhme Ulrike , Cross George A. M.
Published: April 2021

The variant surface glycoproteins (VSG) of Trypanosoma brucei are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. All GPI-anchored proteins are synthesized with a C-terminal signal sequence,which is replaced by a GPI-anchor in a rapid post-translational transamidation reaction. VSG GPI signal sequences are extraordinarily conserved. They contain either 23 or 17 amino acids, a difference that distinguishes the two major VSG classes, and consist of a spacer sequence followed by a more hydrophobic region. The ω amino acid, to which GPI is transferred, is either Ser,Asp or Asn, the ω+2 amino acid is always Ser, and the ω+7 amino acid is almost always Lys. In order to determine whether this high conservation is necessary for GPI anchoring, we introduced several mutations into the signal peptide. Surprisingly, changing the most conserved amino acids, at positions ω+1, ω+2 and ω+7, had no detectable effect on the efficiency of GPI-anchoring or on protein abundance. Several more extensive changes also had no discernable impact on GPI-anchoring. Deleting the entire 23 amino-acid signal sequence or the 15 amino-acid hydrophobic region generated proteins that were not anchored. Instead of being secreted, these truncated proteins accumulated in the endoplasmic reticulum prior to lysosomal degradation. Replacing the GPI signal sequence with a proven cell-surface membrane-spanning domain reduced expression by about 99%and resulted not in cell surface expression but in accumulation close to the flagellar pocket and in non-lysosomal compartments. These results indicate that the high conservation of the VSG GPI signal sequence is not necessary for efficient expression and GPI attachment. Instead, the GPI anchor is essential for surface expression of VSG. However, because the VSG is a major virulence factor, it is possible that small changes in the efficiency of GPI anchoring,undetectable in our experiments, might have influenced the evolution of VSG GPI signal sequences.
Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP2
Authors: Kheradmand Farrah , Rishi Kirtee , Werb Zena
Published: April 2021

Epithelial-mesenchymal interactions during lung development require extracellular signaling factors that facilitate branching morphogenesis. We show here that matrix metalloproteinases (MMPs) originating in the mesenchyme are necessary for epithelial branching and alveolization. We found that the delayed lung maturation characterized by abnormal branching and poor alveolization seen in mice deficient in epidermal growth factor receptor(Egfr-/-) is accompanied by aberrant expression of MMPs. By in situ zymography, the lungs from newborn Egfr-/- mice had low gelatinolytic activity compared with wildtype. Inhibition of MMPs in developing lungs in vivo or in vitro severely retarded morphogenesis. Egfr-/- mice had low expression of MT1-MMP/MMP14, which is a potent activator of gelatinase A/MMP2, in their lungs. Egf ligand increased MT1-MMP mRNA by tenfold in lung fibroblasts from wild type, but not from Egfr-/- mice. Extracts from lungs of Egfr-/- mice showed a tenfold reduction in active MMP-2, but only a slight decrease in proMMP-2 by zymography. At birth, MMP-2-/- mice had a lung phenotype characterized by abnormal lung alveolization which phenocopied that of Egfr-/- mice, albeit somewhat less severe. We conclude that proteolysis mediates epithelial/mesenchymal interactions during lung morphogenesis. From the phenotypes of the Egfr-/- mice, we identify MT1-MMP as a major downstream target of Egfr signaling in lung in vivo and in vitro. MT1-MMP is, in turn, necessary for activation of MMP-2, a mesenchymal enzyme that is required for normal lung morphogenesis.
TheCaenorhabditis eleganshistone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division
Authors: Pettitt Jonathan , Crombie Catriona , Schümperli Daniel , Müller Berndt
Published: April 2021

As in all metazoans, the replication-dependent histone genes of Caenorhabditis elegans lack introns and contain a short hairpin structure in the 3′ untranslated region. This hairpin structure is a key element for post-transcriptional regulation of histone gene expression and determines mRNA 3′ end formation, nuclear export, translation and mRNA decay. All these steps contribute to the S-phase-specific expression of the replication-dependent histone genes. The hairpin structure is the binding site for histone hairpin-binding protein that is required for hairpin-dependent regulation. Here, we demonstrate that the C. elegans histone hairpin-binding protein gene is transcribed in dividing cells during embryogenesis and postembryonic development. Depletion of histone hairpin-binding protein (HBP) function in early embryos using RNA-mediated interference leads to an embryonic-lethal phenotype brought about by defects in chromosome condensation. A similar phenotype was obtained by depleting histones H3 and H4 in early embryos, indicating that the defects in hairpin-binding protein-depleted embryos are caused by reduced histone biosynthesis. We have confirmed this by showing that HBP depletion reduces histone gene expression. Depletion of HBP during postembryonic development also results in defects in cell division during late larval development. In addition, we have observed defects in the specification of vulval cell fate in animals depleted for histone H3 and H4, which indicates that histone proteins are required for cell fate regulation during vulval development.
Laminin assembles into separate basement membrane and fibrillar matrices in Schwann cells
Authors: Tsiper Maria V. , Yurchenco Peter D.
Published: April 2021

Laminins are important for Schwann cell basement membrane assembly and axonal function. In this study, we found that exogenous laminin-1, like neuromuscular laminins-2/4, formed two distinct extracellular matrices on Schwann cell surfaces, each facilitated by laminin polymerization. Assembly of one, a densely-distributed reticular matrix, was accompanied by a redistribution of cell-surface dystroglycan and cytoskeletal utrophin into matrix-receptor-cytoskeletal complexes. The other, a fibrillar matrix,accumulated in separate zones associated with pre-existing β1-integrin arrays. The laminin-1 fragment E3 (LG-modules 4-5), which binds dystroglycan and heparin, inhibited reticular-matrix formation. By contrast,β1-integrin blocking antibody (Ha2/5) prevented fibrillar assembly. Ultrastructural analysis revealed that laminin treatment induced the formation of a linear electron-dense extracellular matrix (lamina densa)separated from plasma membrane by a narrow lucent zone (lamina lucida). This structure was considerably reduced with non-polymerizing laminin, fully blocked by E3, and unaffected by Ha2/5. Although it formed in the absence of type IV collagen, it was nonetheless able to incorporate this collagen. Finally, cell competency to bind laminin and form a basement membrane was passage-dependent. We postulate that laminin induces the assembly of a basement membrane on competent cell surfaces probably mediated by anchorage through LG 4-5. Upon binding, laminin interacts with dystroglycan,mobilizes utrophin, and assembles a `nascent' basement membrane, independent of integrin, that is completed by incorporation of type IV collagen. However,the fibrillar β1-integrin dependent matrix is unlikely to be precursor to basement membrane.
Ephrin-A5 induces rounding, blebbing and de-adhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling
Authors: Lawrenson Isobel D. , Wimmer-Kleikamp Sabine H. , Lock Peter , Schoenwaelder Simone M. , Down Michelle , Boyd Andrew W. , Alewood Paul F. , Lackmann Martin
Published: April 2021

Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions,membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.
Forskolin-mediated G1 arrest in acute lymphoblastic leukaemia cells: phosphorylated pRB sequesters E2Fs
Authors: Gützkow Kristine Bjerve , Naderi Soheil , Blomhoff Heidi K.
Published: April 2021

Increased intracellular levels of cAMP, induced by forskolin, lead to permanent G1 arrest of Reh cells. As expected, we observed a rapid dephosphorylation of the retinoblastoma protein (pRB) within 2 hours of forskolin treatment concomitant with reduced activity of the pRB-specific kinases. Interestingly, however, the dephosphorylation of pRB, as well as the inhibition of the kinase activities, was only transient, despite the permanent arrest of cells in G1. Importantly, although the pRB-specific kinases were fully active after 48 hours, pRB became only partially rephosphorylated.

The transient dephosphorylation of pRB could be explained by the transient decrease in the activities of the pRB-specific kinases, but to understand why pRB became only partially rephosphorylated, despite fully activated kinases,we postulated that cAMP could activate a pRB-directed phosphatase. It was therefore interesting to find that the phosphatase inhibitor, tautomycin, was able to abolish the forskolin-mediated dephosphorylation of pRB, without increasing the activities of the pRB-specific kinases.

To understand how Reh cells expressing hyperphosphorylated forms of pRB can remain arrested in G1, we used three different methods to test for the ability of pRB to form functional complexes with the family of E2F transcription factors. As expected, we observed an increased complex formation between E2F-1, E2F-4 and pRB after 2 hours when pRB was in its most dephosphorylated state. Suprisingly, however, prolonged treatment with forskolin, which induced partial rephosphorylation of pRB, in fact further increased the complex formation between the E2Fs and pRB, and this also resulted in reduced E2F-promoter activity in vivo. These data imply that in Reh cells, partially phosphorylated forms of pRB retain the ability to inhibit E2F-promoter activity, and thereby prevent cells from entering into S-phase.
Katanin inhibition prevents the redistribution of γ-tubulin at mitosis
Authors: Buster Dan , McNally Karen , McNally Francis J.
Published: April 2021

Katanin is a microtubule-severing protein that is concentrated at mitotic spindle poles but katanin's function in the mitotic spindle has not been previously reported. Inhibition of katanin with either of two dominant-negative proteins or a subunit-specific antibody prevented the redistribution of γ-tubulin from the centrosome to the spindle in prometaphase CV-1 cells as assayed by immunofluorescence microscopy. Becauseγ-tubulin complexes can bind to pre-existing microtubule minus ends,these results could be explained by a model in which the broad distribution ofγ-tubulin in the mitotic spindle is in part due to cytosolicγ-tubulin ring complexes binding to microtubule minus ends generated by katanin-mediated microtubule severing. Because microtubules depolymerize at their ends, we hypothesized that a greater number of microtubule ends generated by severing in the spindle would result in an increased rate of spindle disassembly when polymerization is blocked with nocodazole. Indeed,katanin inhibition slowed the rate of spindle microtubule disassembly in the presence of nocodazole. However, katanin inhibition did not affect the rate of exchange between polymerized and unpolymerized tubulin as assayed by fluorescence recovery after photobleaching. These results support a model in which katanin activity regulates the number of microtubule ends in the spindle.