bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II
Authors: Zeng Qi , Lagunoff David , Masaracchia Ruthann , Goeckeler Zoe , Côté Graham , Wysolmerski Robert
Published: April 2021

The p21-activated kinase (PAK) family includes several enzyme isoforms regulated by the GTPases Rac1 and Cdc42. PAK1, found in brain, muscle and spleen, has been implicated in triggering cytoskeletal rearrangements such as the dissolution of stress fibers and reorganization of focal complexes. The role of the more widely distributed PAK2 in controlling the cytoskeleton has been less well studied. Previous work has demonstrated that PAK2 can monophosphorylate the myosin II regulatory light chain and induce retraction of permeabilized endothelial cells. In this report we characterize PAK2’s morphological and biochemical effect on intact endothelial cells utilizing microinjection of constitutively active PAK2. Under these conditions we observed a modification of the actin cytoskeleton with retraction of endothelial cell margins accompanied by an increase in monophosphorylation of myosin II. Selective inhibitors were used to analyze the mechanism of action of PAK2. Staurosporine, a direct inhibitor of PAK2, largely prevented the action of microinjected PAK2 in endothelial cells. Butanedione monoxime, a non-specific myosin ATPase inhibitor, also inhibited the effects of PAK2 implicating myosin in the changes in cytoskeletal reorganization. In contrast, KT5926, a specific inhibitor of myosin light chain kinase was ineffective in preventing the changes in morphology and the actin cytoskeleton. The additional finding that endogenous PAK2 associates with myosin II is consistent with the proposal that cell retraction and cytoskeletal rearrangements induced by microinjected PAK2 depend on the direct activation of myosin II by PAK2 monophosphorylation of the regulatory light chain.
Control of extracellular matrix assembly by syndecan-2 proteoglycan
Authors: Klass Carmen M. , Couchman John R. , Woods Anne
Published: April 2021

Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably transfected with full length (S2) or truncated syndecan-2 lacking the C-terminal 14 amino acids of the cytoplasmic domain (S2ΔS). No differences in the amount of matrix assembly were noted with S2 cells, but those expressing S2ΔS could not assemble laminin or fibronectin into a fibrillar matrix. The loss of matrix formation was not caused by a failure to synthesize or externalize ECM components as determined by metabolic labeling or due to differences in surface expression of α5 or β1 integrin. The matrix assembly defect was at the cell surface, since S2ΔS cells also lost the ability to rearrange laminin or fibronectin substrates into fibrils and to bind exogenous fibronectin. Transfection of activated αIibαLΔβ3 integrin into α5-deficient CHO B2 cells resulted in reestablishment of the previously lost fibronectin matrix. However, cotransfection of this cell line with S2ΔS could override the presence of activated integrins. These results suggest a regulatory role for syndecan-2 in matrix assembly, along with previously suggested roles for activated integrins.
A critical reader's guide to science-speak
Authors: A Caveman
Published: April 2021

Some time ago, I complained about the use of the phrase ‘data not shown’ in lieu of a full presentation of data and results in a paper, and provided some interpretations of why this phrase is used … Oh, and one more. Critical reader. This should be cynical reader.
Evidence that actin and myosin are involved in the poleward flux of tubulin in metaphase kinetochore microtubules of crane-fly spermatocytes
Authors: Silverman-Gavrila R. V. , Forer A.
Published: April 2021

We studied the effects of various drugs on the poleward flux of tubulin in kinetochore microtubules in metaphase-I crane-fly spermatocytes. We used as a measure of tubulin flux a ‘gap’ in acetylation of kinetochore microtubules immediately poleward from the kinetochore; the ‘gap’ is caused by a time lag between incorporation of new tubulin subunits at the kinetochore and subsequent acetylation of those subunits as they flux to the pole. We confirmed that the ‘gap’ is due to flux by showing that the ‘gap’ disappeared when cells were treated briefly with the antitubulin drug nocodazole, which decreases microtubule dynamics. The ‘gap’ disappeared when cells were treated for 10 minutes with anti-actin drugs (cytochalasin D, latrunculin B, swinholide A), or with the anti-myosin drug 2,3-butanedione 2-monoxime. The ‘gap’ did not disappear when cells were treated with the actin stabilizing drug jasplakinolide.

We studied whether these drugs altered spindle actin. We used fluorescent phalloidin to visualize spermatocyte F-actin, which was associated with kinetochore spindle fibers as well as the cell cortex, the contractile ring and finger-like protrusions at the poles. Spindle F-actin was no longer seen after cells were treated with cytochalasin D, swinholide A or a high concentration of latrunculin B, whereas a low concentration of latrunculin B, which did not completely remove the ‘gap’, caused reduced staining of spindle actin. Neither 2,3-butanedione 2-monoxime nor jasplakinolide altered spindle actin. These data suggest that an actomyosin mechanism drives the metaphase poleward tubulin flux.
Dictyostelium myosin IK is involved in the maintenance of cortical tension and affects motility and phagocytosis
Authors: Schwarz Eva C. , Neuhaus Eva M. , Kistler Claudia , Henkel Andreas W. , Soldati Thierry
Published: April 2021

Dictyostelium discoideummyosin Ik (MyoK) is a novel type of myosin distinguished by a remarkable architecture. MyoK is related to class I myosins but lacks a cargo-binding tail domain and carries an insertion in a surface loop suggested to modulate motor velocity. This insertion shows similarity to a secondary actin-binding site present in the tail of some class I myosins, and indeed a GST-loop construct binds actin. Probably as a consequence, binding of MyoK to actin was not only ATP-but also salt-dependent. Moreover, as both binding sites reside within its motor domain and carry potential sites of regulation, MyoK might represent a new form of actin crosslinker. MyoK was distributed in the cytoplasm with a significant enrichment in dynamic regions of the cortex. Absence of MyoK resulted in a drop of cortical tension whereas overexpression led to significantly increased tension. Absence and overexpression of MyoK dramatically affected the cortical actin cytoskeleton and resulted in reduced initial rates of phagocytosis. Cells lacking MyoK showed excessive ruffling, mostly in the form of large lamellipodia, accompanied by a thicker basal actin cortex. At early stages of development, aggregation of myoKnull cells was slowed due to reduced motility. Altogether, the data indicate a distinctive role for MyoK in the maintenance and dynamics of the cell cortex.
4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death
Authors: Liu Wei , Kato Masashi , Akhand Anwaral A, , Hayakawa Akemi , Suzuki Haruhiko , Miyata Toshio , Kurokawa Kiyoshi , Hotta Yoshihiro , Ishikawa Naohisa , Nakashima Izumi
Published: April 2021

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 μM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.
Tyrosine 766 in the fibroblast growth factor receptor-1 is required for FGF-stimulation of phospholipase C, phospholipase D, phospholipase A2, phosphoinositide 3-kinase and cytoskeletal reorganisation in porcine aortic endothelial cells
Authors: Cross Michael J. , Hodgkin Matthew N. , Roberts Sally , Landgren Eva , Wakelam Michael J. O. , Claesson-Welsh Lena
Published: April 2021

Fibroblast growth factor-mediated signalling was studied in porcine aortic endothelial cells expressing either wild-type fibroblast growth factor receptor-1 or a mutant receptor (Y766F) unable to bind phospholipase C-γ. Stimulation of cells expressing the wild-type receptor resulted in activation of phospholipases C, D and A2and increased phosphoinositide 3-kinase activity. Stimulation of the wild-type receptor also resulted in stress fibre formation and a cellular shape change. Cells expressing the Y766F mutant receptor failed to stimulate phospholipase C, D and A2as well as phosphoinositide 3-kinase. Furthermore, no stress fibre formation or shape change was observed. Both the wild-type and Y766F receptor mutant activated MAP kinase and elicited proliferative responses in the porcine aortic endothelial cells. Thus, fibroblast growth factor receptor-1 mediated activation of phospholipases C, D and A2and phosphoinositide 3-kinase was dependent on tyrosine 766. Furthermore, whilst tyrosine 766 was not required for a proliferative response, it was required for fibroblast growth factor receptor-1 mediated cytoskeletal reorganisation.
Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies
Authors: Wang Jin , Lenardo Michael J.
Published: April 2021

Caspases are a group of cysteine proteases critical for apoptosis of eukaryotic cells. Deletion of genes that encode murine caspases suggests that caspases are involved not only in apoptosis but also in cytokine maturation and cell growth and differentiation. Among them, caspase-1 and caspase-11 are primarily involved in the processing of pro-inflammatory cytokines. Caspase-3 and caspase-9 are essential for apoptosis during brain development. Caspase-8 is required for the development of heart muscle, cell proliferation in the hematopoietic lineage and death-receptor-mediated apoptosis. These studies suggest that caspases function in cell signaling events including apoptosis, cell growth and differentiation.
Calcium regulation of microtubule sliding in reactivated sea urchin sperm flagella
Authors: Bannai Hiroko , Yoshimura Misako , Takahashi Keiichi , Shingyoji Chikako
Published: April 2021

The changes in the bending pattern of flagella induced by an increased intracellular Ca2+ concentration are caused by changes in the pattern and velocity of microtubule sliding. However, the mechanism by which Ca2+ regulates microtubule sliding in flagella has been unclear. To elucidate it, we studied the effects of Ca2+ on microtubule sliding in reactivated sea urchin sperm flagella that were beating under imposed head vibration. We found that the maximum microtubule sliding velocity obtainable by imposed vibration, which was about 170-180 rad/second in the presence of 250 μM MgATP and <10−9 M Ca2+, was decreased by 10−6-10−5 M Ca2+ by about 15-20%. Similar decrease of the sliding velocity was observed at 54 and 27 μM MgATP. The Ca2+-induced decrease of the sliding velocity was due mainly to a decrease in the reverse bend angle. When the plane of beat was artificially rotated by rotating the plane of vibration of the pipette that held the sperm head, the asymmetric bending pattern also rotated at 10−5 M Ca2+ as well as at <10−9 M Ca2+. The rotation of the bending pattern was observed at MgATP higher than 54 μM (∼100 μM ATP). These results indicate that the Ca2+-induced decrease of the sliding velocity is mediated by a rotatable component or components (probably the central pair) at high MgATP, but is not due to specific dynein arms on particular doublets. We further investigated the effects of a mild trypsin treatment and of trifluoperazine on the Ca2+-induced decrease in sliding velocity. Axonemes treated for 3 minutes with a low concentration (0.1 μg/ml) of trypsin beat with a more symmetrical waveform than before the treatment. Also, their microtubule sliding velocity and reverse bend angle were not affected by high Ca2+ concentrations. Trifluoperazine (25-50 μM) had no effect on the decrease of the sliding velocity in beating flagella at 10−5 M Ca2+. However, the flagella that had been ‘quiescent’ at 10−4 M Ca2+ resumed asymmetrical beating following an application of 10-50 μM trifluoperazine. In such beating flagella, both the sliding velocity and the reverse bend angle were close to their respective values at 10−5 M Ca2+. Trypsin treatment induced a similar recovery of beating in quiescent flagella at 10−4 M Ca2+, albeit with a more symmetrical waveform. These results provide first evidence that, at least at ATP concentrations higher than ∼100 μM, 10−6-10−5 M Ca2+ decreases the maximum sliding velocity of microtubules in beating flagella through a trypsin-sensitive regulatory mechanism which possibly involves the central pair apparatus. They also suggest that calmodulin may be associated with the mechanism underlying flagellar quiescence induced by 10−4 M Ca2+.
Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by α3β1, α6β1 and α6β4 integrins
Authors: Kikkawa Yamato , Sanzen Noriko , Fujiwara Hironobu , Sonnenberg Arnoud , Sekiguchi Kiyotoshi
Published: April 2021

Laminin-10/11, the laminin isoforms containing the α5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin α3β1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin β1 antibody inhibited the adhesion of all cell types tested, anti-α3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-α3 and anti-α6 antibodies, suggesting that both α3β1 and α6β1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin α3 or α6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the α3β1 and α6β1 transfectants, whereas laminin-5 was the preferred ligand for the α3β1 transfectants. Upon stimulation with the activating anti-integrin β1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with α6 and β4 subunits were also capable of adhering to laminin-10/11, indicating that integrin α6β4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the α6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both α3β1 and α6β1 integrins with high avidity and also to α6β4 integrin.
ARP1 in Golgi organisation and attachment of manchette microtubules to the nucleus during mammalian spermatogenesis
Authors: Fouquet Jean-Pierre , Kann Marie-Louise , Souès Sylvie , Melki Ronald
Published: April 2021

Actin related protein of vertebrate, Arp1, is a major component of the dynactin complex. To characterise and localise Arp1 during mammalian spermatogenesis, polyclonal antibodies were raised against a human recombinant Arp1. Anti-Arp1 antibodies were used for western-immunoblotting, indirect immunofluorescence and immunoelectron microscopy. In round spermatids, Arp1 was detected at the centrosome and at the Golgi apparatus. In elongated spermatids, Arp1 was predominantly found along microtubules of the manchette and at their site of attachment to the nuclear envelope. In maturing spermatids, Arp1 was still present in the pericentriolar material, but in testicular spermatozoa it was not detectable. These various localisations of Arp1 and their changes during spermatid differentiation suggest that the dynactin complex in association with dynein might contribute to several activities: the functional organisation of the centrosome and of the Golgi apparatus and the shaping of the nucleus by manchette microtubules.
Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei
Authors: Li Cong-Jun , Bogan Joseph A. , Natale Darren A. , DePamphilis Melvin L.
Published: April 2021

As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G1-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-β and β′) that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-β/β′ appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G1-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G1-phase. Third, selective activation of ori-β in late G1-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.
Magna Carta and the Bill of Rights - what about a Postdoc Charter?
Authors: A Caveman
Published: April 2021

Over the centuries, we have dealt with social inequities and injustices through grand charters that identify and annul problemsâ€¦
Lysosome-endosome fusion and lysosome biogenesis
Authors: Luzio J. Paul , Rous Brian A. , Bright Nicholas A. , Pryor Paul R. , Mullock Barbara M. , Piper Robert C.
Published: April 2021

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.
Interferon γ regulates accumulation of the proteasome activator PA28 and immunoproteasomes at nuclear PML bodies
Authors: Fabunmi Rosalind P. , Wigley W. Christian , Thomas Philip J. , DeMartino George N.
Published: April 2021

PA28 is an interferon γ (IFNγ inducible proteasome activator required for presentation of certain major histocompatibility (MHC) class I antigens. Under basal conditions in HeLa and Hep2 cells, a portion of nuclear PA28 is concentrated at promyelocytic leukemia oncoprotein (PML)-containing bodies also commonly known as PODs or ND10. IFNγ treatment greatly increased the number and size of the PA28- and PML-containing bodies, and the effect was further enhanced in serum- deprived cells. PML bodies are disrupted in response to certain viral infections and in diseases such as acute promyelocytic leukemia (APL). Like PML, PA28 was delocalized from PML bodies by expression of the cytomegalovirus protein, IE1, and in NB4 cells, an APL model line. Moreover, retinoic acid treatment, which causes remission of APL in patients and reformation of PML- containing bodies in NB4 cells, relocalized PA28 to this site. In contrast, the proteasome, the functional target of PA28, was not detected at PML bodies under basal conditions in HeLa and Hep2 cells, but IFNγ promoted accumulation of ‘immunoproteasomes’ at this site. These results establish PA28 as a novel component of nuclear PML bodies, and suggest that PA28 may assemble or activate immunoproteasomes at this site as part of its role in proteasome-dependent MHC class I antigen presentation.
CHMP1 functions as a member of a newly defined family of vesicle trafficking proteins
Authors: Howard Tiffani L. , Stauffer Daniel R. , Degnin Catherine R. , Hollenberg Stanley M.
Published: April 2021

A multivesicular body is a vesicle-filled endosome that targets proteins to the interior of lysosomes. We have identified a conserved eukaryotic protein, human CHMP1, which is strongly implicated in multivesicular body formation. Immunocytochemistry and biochemical fractionation localize CHMP1 to early endosomes and CHMP1 physically interacts with SKD1/VPS4, a highly conserved protein directly linked to multivesicular body sorting in yeast. Similar to the action of a mutant SKD1 protein, overexpression of a fusion derivative of human CHMP1 dilates endosomal compartments and disrupts the normal distribution of several endosomal markers. Genetic studies in Saccharomyces cerevisiae further support a conserved role of CHMP1 in vesicle trafficking. Deletion of CHM1, the budding yeast homolog of CHMP1, results in defective sorting of carboxypeptidases S and Y and produces abnormal, multi-lamellar prevacuolar compartments. This phenotype classifies CHM1 as a member of the class E vacuolar protein sorting genes. Yeast Chm1p belongs to a structurally-related, but rather divergent family of proteins, including Vps24p and Snf7p and three novel proteins, Chm2p, Chm5p and Chm6p, which are all essential for multivesicular body sorting. These observations identify the conserved CHMP/Chmp family as a set of proteins fundamental to understanding multivesicular body sorting in eukaryotic organisms.
The melanocortin 1 receptor is the principal mediator of the effects of agouti signaling protein on mammalian melanocytes
Authors: Abdel-Malek Zalfa A. , Scott M. Cathy , Furumura Minao , Lamoreux M. Lynn , Ollmann Michael , Barsh Greg S. , Hearing Vincent J.
Published: April 2021

The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with α-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E+/E+, e/e, or Eso/Eso mice to α-MSH and/or ASP. E+/E+ melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and Eso/Eso is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to α-melanocyte stimulating hormone. Mouse E+/E+ melanocytes, but not e/e or Eso/Eso melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E+/E+ melanocytes does agouti signaling protein abrogate the stimulatory effects of α-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.
At the crossroads of cell biology and immunology: DRiPs and other sources of peptide ligands for MHC class I molecules
Authors: Yewdell Jonathan W. , Schubert Ulrich , Bennink Jack R.
Published: April 2021

CD8+ T cells are a critical element of vertebrate immune responses to viruses and other intracellular parasites. They roam the body, monitoring cells for the presence of foreign peptides associated with MHC class I molecules of the major histocompatibility complex (MHC). Although it is clear that most of these peptides are generated through the action of proteasomes, the nature of the substrates degraded by proteasomes is an open question. Recent findings indicate that the major pool of substrates consists of a heterogeneous subset of proteins that are degraded within minutes of their synthesis. Evidence suggests that the fraction of newly synthesized proteins targeted for destruction is remarkably high – 30% or more, depending on cell type – possibly because they are defective in some way and cannot reach their intended conformation or location cellular in a time frame deemed appropriate by cells.
Regulation of the differentiation and behaviour of extra-embryonic endodermal cells by basement membranes
Authors: Murray Patricia , Edgar David
Published: April 2021

Both the extracellular matrix and parathyroid hormone-related peptide (PTHrP) have been implicated in the differentiation and migration of extra-embryonic endodermal cells in the pre-implantation mammalian blastocyst. In order to define the individual roles and interactions between these factors in endodermal differentiation, we have used embryoid bodies derived from Lamc1−/− embryonic stem cells that lack basement membranes. The results show that in the absence of basement membranes, increased numbers of both visceral and parietal endodermal cells differentiate, but they fail to form organised epithelia. Furthermore, although parietal endodermal cells only migrate away from control embryoid bodies in the presence of PTHrP, they readily migrate from Lamc1−/− embryoid bodies in the absence of PTHrP, and this migration is unaffected by PTHrP. Thus, the basement membrane between epiblast and extra-embryonic endoderm is required for the proper organisation of visceral and parietal endodermal cells and also restricts their differentiation to maintain the population of primitive endodermal stem cells. Moreover, this basement membrane inhibits migration of parietal endodermal cells, the role of PTHrP being to stimulate delamination of parietal endodermal cells from the basement membrane rather than promoting migration per se.
Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase
Authors: Campbell Michael S. , Chan Gordon K. T. , Yen Tim J.
Published: April 2021

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.
Assembly into chromatin and subtype-specific transcriptional effects of exogenous linker histones directly introduced into a living Physarum cell
Authors: Thiriet Christophe , Hayes Jeffrey J.
Published: April 2021

The apparent diversity of linker histone subtypes may be related to their specific roles in defining functional states of chromatin in vivo. We have developed a novel method to study constitutive peptides throughout the cell cycle and have demonstrated that an exogenous linker histone could be introduced into a living cell of the slime mold Physarum polycephalum. Here, we have used this method to assess the functional differences between three somatic linker histone subtypes in vivo, and to demonstrate the general applicability of this method. Exogenous linker histone proteins H1°, H5 and H1 were directly absorbed into living cell segments of the naturally synchronous Physarum macroplasmodia at precise cell cycle stages. Fluorescence microscopy, native nucleoprotein gels and immunoblotting of nuclei and chromatin with subtype-specific antibodies revealed that exogenous linker histones were efficiently transported into nuclei and were integrated into chromatin. The immunoreactivity of a preparation of anti- H1° antibodies that are blocked from binding to specific H1° epitopes in native chromatin indicates that the exogenous linker histones were similarly associated into Physarum chromatin. Interestingly, linker histones were found to be less stably associated with Physarum chromatin during S-phase than during G2-phase. Furthermore, we show that exogenous linker histones incorporated in early G2-phase inhibited transcription and that the level of inhibition correlates with the apparent role of the linker histone subtype in regulating transcription in cells where it normally occurs.
Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii
Authors: Alberti-Segui Christine , Dietrich Fred , Altmann-Jöhl Regula , Hoepfner Dominic , Philippsen Peter
Published: April 2021

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 μm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1921.html
Inward rectifier potassium channel Kir2.2 is associated with Synapse-Associated Protein SAP97
Authors: Leonoudakis Dmitri , Mailliard William S. , Wingerd Kevin L. , Clegg Dennis O. , Vandenberg Carol A.
Published: April 2021

The strong inwardly rectifying potassium channels Kir2.x are involved in maintenance and control of cell excitability. Recent studies reveal that the function and localization of ion channels are regulated by interactions with members of the membrane-associated guanylate kinase (MAGUK) protein family. To identify novel interacting MAGUK family members, we constructed GST-fusion proteins with the C termini of Kir2.1, Kir2.2 and Kir2.3. GST affinity-pulldown assays from solubilized rat cerebellum and heart membrane proteins revealed an interaction between all three Kir2.x C-terminal fusion proteins and the MAGUK protein synapse-associated protein 97 (SAP97). A truncated form of the C-terminal GST-Kir2.2 fusion protein indicated that the last three amino acids (S-E-I) are essential for association with SAP97. Affinity interactions using GST-fusion proteins containing the modular domains of SAP97 demonstrate that the second PSD-95/Dlg/ZO-1 (PDZ) domain is sufficient for interaction with Kir2.2. Coimmunoprecipitations demonstrated that endogenous Kir2.2 associates with SAP97 in rat cerebellum and heart. Additionally, phosphorylation of the Kir2.2 C terminus by protein kinase A inhibited the association with SAP97. In rat cardiac ventricular myocytes, Kir2.2 and SAP97 colocalized in striated bands corresponding to T-tubules. In rat cerebellum, Kir2.2 was present in a punctate pattern along SAP97-positive processes of Bergmann glia in the molecular layer, and colocalized with astrocytes and granule cells in the granule cell layer. These results identify a direct association of Kir2.1, Kir2.2 and Kir2.3 with the MAGUK family member SAP97 that may form part of a macromolecular signaling complex in many different tissues.
Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes
Authors: Baron Carole L. , Raposo Graça , Scholl Suzy M. , Bausinger Huguette , Tenza Danielle , Bohbot Alain , Pouillart Pierre , Goud Bruno , Hanau Daniel , Salamero Jean
Published: April 2021

The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (MΦ) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-α. These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.
Insulin signalling
Authors: Bevan Paul
Published: April 2021

Insulin binding to its receptor results in receptor autophosphorylation on tyrosine residues and the tyrosine phosphorylation of insulin receptor substrates (IRS-1, IRS-2 and IRS-3) by the insulin receptor tyrosine kinase. This allows association of IRSs with the regulatory subunit of phosphoinositide 3-kinase (PI3K) through its SRC homology 2 (SH2) domains. Once activated, the catalytic subunit phosphorylates phosphoinositides at the 3′ position of the inositol ring or proteins at serine residues. PI3K activates PtdIns(3,4)P2 / PtdIns(3,4,5)P3-dependent kinase 1 (PDK1), which activates PKB/Akt, a serine kinase. PKB in turn deactivates glycogen synthase kinase 3 (GSK-3), leading to activation of glycogen synthase and thus glycogen synthesis. Activation of PKB also results in the translocation of GLUT-4 vesicles from their intracellular pool to the plasma membrane, where they allow uptake of glucose into the cell. PKB also leads to mTOR-mediated activation of protein synthesis by PHAS/elf4 and p70s6k.
AKAP signaling complexes at the cytoskeleton
Authors: Diviani Dario , Scott John D.
Published: April 2021

Targeting of protein kinases and phosphatases to the cytoskeleton enhances the regulation of signal transduction events. The assembly of cytoskeletal signaling complexes facilitates the relay of messages from membrane receptors to specific sites on the actin cytoskeleton. These signals influence fundamental cell properties, such as shape, movement and division. Targeting of the cAMP-dependent kinase (PKA) to the cytoskeleton is achieved through interaction with A-kinase anchoring proteins (AKAPs). AKAPs maintain multivalent signaling complexes by binding additional enzymes, including kinases and phosphatases.
The life cycle of actin patches in mating yeast
Authors: Smith Michael G. , Swamy Sudhindra R. , Pon Liza A.
Published: April 2021

Actin patches are core components of the yeast actin cytoskeleton that undergo redistribution during establishment of cell polarity. Using 4D imaging, we observe the life cycle of actin patches in living yeast for the first time. We observe assembly of actin patches at sites of polarized growth, and disassembly of actin patches concomitant with movement away from those sites. The total lifetime of an actin patch is 10.9±4.2 seconds. These findings indicate that actin patches are labile structures, and that the localization of actin patches during establishment of cell polarity occurs by assembly of these structures at sites of polarized cell surface growth. These findings were confirmed and extended by analysis of myosin I proteins and their receptor, verprolin, proteins implicated in actin assembly in yeast. Deletion of type I myosins or their receptor has no effect on the velocity of actin patch movement. However, these mutants show a 65% reduction in number of patch movements and a three-fold increase in patch lifetime. Finally, the actin patch resident proteins Abp1p, fimbrin, and Arp2p show normal association with actin patches in myosin I and verprolin mutants. However, cofilin accumulates in abnormal ‘bars’ of G-actin in myo3Δ,myo5Δ and vrp1Δ strains, and Las17p/Bee1p is not associated with actin patches in vrp1Δ strains. These findings imply a multi-step process for actin patch assembly. Early events in this process, including assembly of Abp1p, fimbrin and Arp2p with F-actin, can occur throughout the cell and do not require myosin I proteins or their receptor. Later events in this process are myosin I-dependent, and are required for assembly of actin patches at sites of polarized cell surface growth.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1990.html
Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos
Authors: Vautier Dominique , Chesné Patrick , Cunha Celso , Calado Angelo , Renard Jean-Paul , Carmo-Fonseca Maria
Published: April 2021

A unique feature of certain members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins is that they shuttle continuously between nucleus and cytoplasm and their accumulation in the nucleus is transcription-dependent. An extensively characterised protein of this group is hnRNP A1. To date, most studies addressing the transcription-dependent transport of hnRNP A1 have been performed on cultured cell lines treated with transcription inhibitors. Here we have analysed the nucleocytoplasmic distribution of hnRNP A1 in early mouse embryos, where the haploid pronuclei remain transcriptionally inactive for a period of several hours. Consistent with its small molecular size (36 kDa), the hnRNP A1 protein diffuses passively through the nuclear pores and equilibrates between the nucleus and the cytoplasm of transcriptionally inactive embryos. In contrast, following transcriptional activation the A1 protein becomes accumulated in the nucleus. This accumulation of the A1 protein in the nucleus is blocked by the lectin wheat germ agglutinin (WGA), which binds to nuclear pore proteins and prevents translocation of receptor-cargo complexes through the pores. This indicates that a carrier-mediated transport pathway is required for the concentration of A1 in transcriptionally active nuclei. To further analyse how transcription is coupled to nucleocytoplasmic transport, we transplanted transcriptionally inactive pronuclei into the cytoplasm of transcriptionally active embryos. The results show that the presence of newly synthesised RNAs in the cytoplasm is not sufficient to induce the accumulation of hnRNP A1 in the nucleus. Rather, the appearance of nascent transcripts in the nucleus appears to be the crucial event. Since hnRNP A1 is a shuttling protein, an increase in its steady state nuclear concentration could be the result of either faster nuclear import or slower export to the cytoplasm. We propose that binding of A1 to nascent transcripts retards its export to the cytoplasm and therefore contributes to its concentration in the nucleus.
N-WASP, WAVE and Mena play different roles in the organization of actin cytoskeleton in lamellipodia
Authors: Nakagawa Hiroyuki , Miki Hiroaki , Ito Masayo , Ohashi Kazuyo , Takenawa Tadaomi , Miyamoto Shigeaki
Published: April 2021

WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. We observed the localization of the WASP family proteins, N-WASP and WAVE, and the Ena/VASP family protein, Mena, in protruding lamellipodia. Rat fibroblast cell line 3Y1 protruded lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 were concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena were observed to localize at the lamellipodium edge. Interestingly, Mena tended to concentrate at the microspike tips but WAVE did not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, was much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, was localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE was distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42.
N-cadherin is developmentally regulated and functionally involved in early hematopoietic cell differentiation
Authors: Puch Sabine , Armeanu Sorin , Kibler Christine , Johnson Keith R. , Müller Claudia A. , Wheelock Margaret J. , Klein Gerd
Published: April 2021

The cadherins, an important family of cell adhesion molecules, are known to play major roles during embryonic development and in the maintenance of solid tissue architecture. In the hematopoietic system, however, little is known of the role of this cell adhesion family. By RT-PCR, western blot analysis and immunofluorescence staining we show that N-cadherin, a classical type I cadherin mainly expressed on neuronal, endothelial and muscle cells, is expressed on the cell surface of resident bone marrow stromal cells. FACS analysis of bone marrow mononuclear cells revealed that N-cadherin is also expressed on a subpopulation of early hematopoietic progenitor cells. Triple-color FACS analysis defined a new CD34+ CD19+ N-cadherin+ progenitor cell population. During further differentiation, however, N-cadherin expression is lost.

Treatment of CD34+ progenitor cells with function-perturbing N-cadherin antibodies drastically diminished colony formation, indicating a direct involvement of N-cadherin in the differentiation program of early hematopoietic progenitors. N-cadherin can also mediate adhesive interactions within the bone marrow as demonstrated by inhibition of homotypic interactions of bone-marrow-derived cells with N-cadherin antibodies. Together, these data strongly suggest that N-cadherin is involved in the development and retention of early hematopoietic progenitors within the bone marrow microenvironment.
Tensile stress stimulates microtubule outgrowth in living cells
Authors: Kaverina Irina , Krylyshkina Olga , Beningo Karen , Anderson Kurt , Wang Yu-Li , Small J. Victor
Published: April 2021

Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.