bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of MMH (Met murine hepatocyte) cell differentiation by TGFβ is abrogated by pre-treatment with the heritable differentiation effector FGF1
Authors: Spagnoli Francesca M. , Cicchini Carla , Tripodi Marco , Weiss Mary C. 
Published: April 2021

ABSTRACT
MMH (Met murine hepatocyte) liver cells derived from transgenic mice expressing a truncated constitutively active form of human c-Met are non-transformed immortalized cell lines. We have previously shown that they harbor: (1) epithelial cells that express the liver-enriched transcription factors HNF4 and HNF1α, and that can be stably induced by FGF1 to express liver functions, and (2) fibroblast-like bi-potential palmate cells that can differentiate into bile duct-like structures in Matrigel cultures, or into epithelial cells competent to express hepatic functions. Low concentrations of TGFβ have been found to inhibit growth and differentiation of MMH cells. The factor stabilized the palmate cell phenotype, and it provoked epithelial cells to acquire palmate-like morphological characteristics, in parallel with down-regulation of expression of HNF4 and HNF1α and activation of Snail transcripts. The effects of TGFβ were dominant if it was added with FGF1, but the effects on differentiation were abrogated if cells had been pre-treated with FGF1. This work identifies TGFβ as a factor that could be implicated in maintaining bi-potential precursor cells in the liver, FGF1 as one that could over-ride the TGFβ effects and Snail as a candidate for mediation of the signal.
C-terminal inhibition of tau assembly in vitro and in Alzheimer’s disease
Authors: Abraha Aida , Ghoshal Nupur , Gamblin T. Chris , Cryns Vincent , Berry Robert W. , Kuret Jeff , Binder Lester I. 
Published: April 2021

ABSTRACT
Alzheimer’s disease (AD) is, in part, defined by the polymerization of tau into paired helical and straight filaments (PHF/SFs) which together comprise the fibrillar pathology in degenerating brain regions. Much of the tau in these filaments is modified by phosphorylation. Additionally, a subset also appears to be proteolytically truncated, resulting in the removal of its C terminus. Antibodies that recognize tau phosphorylated at S396/404 or truncated at E391 do not stain control brains but do stain brain sections very early in the disease process. We modeled these phosphorylation and truncation events by creating pseudo-phosphorylation and deletion mutants derived from a full-length recombinant human tau protein isoform (ht40) that contains N-terminal exons 2 and 3 and all four microtubule-binding repeats. In vitro assembly experiments demonstrate that both modifications greatly enhance the rates of tau filament formation and that truncation increases the mass of polymer formed, as well. Removal of as few as 12 or as many as 121 amino acids from the C terminus of tau greatly increases the rate and extent of tau polymerization. However, deletion of an additional 7 amino acids, 314DLSKVTS320, from the third microtubule-binding repeat results in the loss of tau’s ability to form filaments in vitro. These results suggest that only part of the microtubule-binding domain (repeats 1, 2 and a small portion of 3) is crucial for tau polymerization. Moreover, the C terminus of tau clearly inhibits the assembly process; this inhibition can be partially reversed by site-specific phosphorylation and completely removed by truncation events at various sites from S320 to the end of the molecule.
Identification and characterization of Caenorhabditis elegans γ-tubulin in dividing cells and differentiated tissues
Authors: Bobinnec Yves , Fukuda Makoto , Nishida Eisuke 
Published: April 2021

ABSTRACT
γ-Tubulin is an essential component of the microtubule-nucleation machinery and therefore plays a crucial role during mitosis. To gain further insights into the function of this protein in the events that take place during embryogenesis and differentiation, we carried out detailed studies on γ-tubulin during all the developmental stages of Caenorhabditis elegans. We identified the γ-tubulin gene from this organism and analyzed the localization of the protein by both immunofluorescence and GFP reporter construct. We show that γ-tubulin association with the centrosome is highly dynamic in mitotic cells, being massively recruited at prophase and released at anatelophase. This accumulation in mitotic centrosomes is dramatic during the first embryonic divisions. We provide the first description of the morphological changes at the centrosome level during the orientation of the mitotic spindle and the flattening of the posterior aster. Loss of function of the γ-tubulin gene by RNAi induces a strong polyploidization of mitotic germ cells and embryos, but does not affect meiosis and pronuclear migration. In addition, we demonstrate the prominent redistribution of γ-tubulin in adults at basal bodies of amphid and phasmid neurons, and at the apical membrane of polarized intestinal cells.
Nuclear and subnuclear targeting sequences of the protein phosphatase-1 regulator NIPP1
Authors: Jagiello Izabela , Eynde Aleyde Van , Vulsteke Veerle , Beullens Monique , Boudrez An , Keppens Stefaan , Stalmans Willy , Bollen Mathieu 
Published: April 2021

ABSTRACT
NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the ‘ForkHead-Associated’ domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.
Caspase-mediated cleavage of the chromosome-binding domain of lamina-associated polypeptide 2α
Authors: Gotzmann Josef , Vlcek Sylvia , Foisner Roland 
Published: April 2021

ABSTRACT
Lamina-associated polypeptide 2α (LAP2α) is a non-membrane-bound isoform of the LAP2 family involved in nuclear structure organization. Using various cell systems, including Jurkat, HL-60, and HeLa cells, and different death-inducing agents, such as anti-Fas antibody, topoisomerase inhibitors, and staurosporine, we found that LAP2α was cleaved during apoptosis as rapidly as lamin B in a caspase-dependent manner yielding stable N- and C-terminal fragments of approximately 50 and 28 kDa, respectively. Based on fragment size and localization of immunoreactive epitopes, four potential cleavage sites were mapped between amino acids 403-485. These sites were located within a domain that has previously been described to be essential and sufficient for association of LAP2α with chromosomes, suggesting that LAP2α cleavage impairs its chromatin-binding properties. Immunofluorescence microscopy demonstrated that, unlike full length protein, apoptotic fragments did not colocalize with condensed chromatin, but remained in the nuclear compartment as long as a single nucleus was visible. Subfractionation analyses showed that the N-terminal LAP2α fragment was extracted from intranuclear structures in detergent/ salt buffers, whereas the C-terminal fragment remained associated with a residual framework devoid of chromatin. Our data suggest that early cleavage of LAP2α is important for chromatin reorganization during apoptosis.
Alternative splicing of the mouse profilin II gene generates functionally different profilin isoforms
Authors: Nardo Alessia Di , Gareus Ralph , Kwiatkowski David , Witke Walter 
Published: April 2021

ABSTRACT
Profilins are a conserved family of proteins participating in actin dynamics and cell motility. In the mouse, two profilin genes are known. Profilin I is expressed universally at high levels, while profilin II is expressed mainly in the brain. Here we describe the occurrence of two mouse profilin II isoforms, A and B, which are derived by alternative splicing. They are identical through residue 107 of the protein, but then have distinct C-terminal sequences. Profilin IIA binds to poly-L-proline and actin with high affinity similar to profilin I. Profilin IIB on the other hand does not bind to actin and the affinity for poly-L-proline is greatly diminished. However, tubulin was found to bind to GST-profilin IIB, and in vivo GFP-profilin IIB was recruited to spindles and asters during mitosis in HeLa cells. Our results indicate unexpected diversity in the functions of the profilin family of proteins, and suggest that in mouse profilin IIA is intimately involved in actin dynamics, while profilin IIB associates with other cytoskeletal components.
TGF-β1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation
Authors: Batard Pascal , Monier Marie-Noëlle , Fortunel Nicolas , Ducos Karin , Sansilvestri-Morel Patricia , Phan Thi-Haï , Hatzfeld Antoinette , Hatzfeld Jacques A. 
Published: April 2021

ABSTRACT
Somatic stem cells are largely quiescent in spite of their considerable proliferative potential. Transforming growth factor-β1 (TGF-β1) appears to be a good candidate for controlling this quiescence. Indeed, various mutations in the TGF-β signalling pathway are responsible for neoplasic proliferation of primitive stem/progenitor cells in human tissues of various origins. In hemopoietic single cell culture assays, blocking autocrine and endogeneous TGF-β1 triggers the cell cycling of high proliferative potential undifferenciated stem/progenitor cells. However, it has never been demonstrated whether TGF-β1 has an apoptotic effect or a differentiating effect on these primitive cells, as already described for more mature cells.
Using single cell experiments both in liquid or semi-solid culture assays and dye tracking experiments by flow cytometry, we demonstrate that low, physiological concentrations of TGF-β1, which specifically maintain primitive human hemopoietic stem/progenitor cells in quiescence, have a reversible effect and do not induce apoptosis. We moreover demonstrate that these low concentrations prevent the rapid loss of the mucin-like protein CD34, a most common marker of immature hematopoietic stem/progenitor cells, which is progressively lost during differentiation. TGF-β1 not only up-modulated the CD34 antigen before S phase entry but also maintained a high level of CD34 expression on cells which had escaped cell cycle inhibition, suggesting that proliferation inhibition and differentiation control by TGF-β1 may be independent. These data provide additional evidence that TGF-β1 acts as a key physiological factor ensuring the maintenance of a stem cell reserve.
Low-affinity LFA-1/ICAM-3 interactions augment LFA-1/ICAM-1-mediated T cell adhesion and signaling by redistribution of LFA-1
Authors: Bleijs Diederik A. , Binnerts Minke E. , Vliet Sandra J. van , Figdor Carl G. , Kooyk Yvette van 
Published: April 2021

ABSTRACT
Although ICAM-3 is implicated in both adhesion and signal transduction events of leukocytes, its low affinity for LFA-1 compared to other ligands of LFA-1 has puzzled many investigators. Here we investigated the role of ICAM-3 in supporting LFA-1-mediated ICAM-1 binding and subsequently cell signaling. We observed that although ICAM-3 binds poorly to LFA-1 expressed on resting T cells, it specifically facilitates and increases LFA-1-mediated adhesion to the high affinity ligand of LFA-1, ICAM-1. We demonstrate that low-affinity binding of LFA-1 to ICAM-3 together with ICAM-1 alters the cell surface distribution of LFA-1 dramatically, inducing large clusters of LFA-1 that facilitate ICAM-1 binding after LFA-1 activation. We found that LFA-1-mediated ICAM-1 cell-cell interactions such as T cell proliferation greatly depend on low affinity LFA-1/ICAM-3 interactions that enhance stable LFA-1/ICAM-1 cell-cell contact. Taken together, these data demonstrate that low affinity LFA-1 binding to ICAM-3 regulates strong LFA-1/ICAM-1-mediated adhesion by driving LFA-1 into clusters to facilitate cell-cell interactions that take place in the immune system.
Functional interaction between E-cadherin and αv-containing integrins in carcinoma cells
Authors: Schlippe Maria von , Marshall John F. , Perry Philip , Stone Michael , Zhu Alan Jian , Hart Ian R. 
Published: April 2021

ABSTRACT
We have demonstrated the possibility of cross-talk between E-cadherin and αv integrins in breast carcinoma cells. Using the function-blocking anti-αv monoclonal antibody 17E6, applied to monolayer cultures of breast cancer lines, it was found that treatment of cells possessing detergent-insoluble (implying attachment to the actin cytoskeleton) E-cadherin resulted in the adoption of a spheroid configuration of cell growth. This effect was dependent upon not just αv occupancy but also receptor aggregation. Thus in vitro αv-dependent adhesion suppresses E-cadherin-mediated morphological changes. To investigate whether manipulation of E-cadherin would, conversely, modulate integrin activity we introduced a dominant-negative E-cadherin construct into one of the lines, ZR75-1, giving rise to the cell line ZR-E2R1. Surface expression of endogenous E-cadherin was downregulated (by around 25%), whereas β-catenin levels were increased two- to threefold in ZR-E2R1 cells. There was also a highly significant increase in migration of ZR-E2R1 cells (relative to control cells) toward vitronectin (P&lt;0.001), but not toward collagen type I, fibronectin or laminin. Such increased migration could be abrogated totally by antibody blockade of αvβ5 and αvβ1 integrins. There was no detectable change in αv integrin levels. These data suggest that the introduction of a dominant-negative E-cadherin mutant into ZR75-1, in addition to a loss of cohesion, generates a signal (or signals) which increases migration towards vitronectin through increased activity of αv integrins.
Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis
Authors: Callanan Mary , Kudo Nobuaki , Gout Stephanie , Brocard Marie-Paule , Yoshida Minoru , Dimitrov Stefan , Khochbin Saadi 
Published: April 2021

ABSTRACT
In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis. The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human). The protein and mRNA are maternally expressed and are present during early embryogenesis. However, our data show that the activity of the protein is developmentally regulated. Embryonic development is insensitive to leptomycin B, a specific inhibitor of CRM1, until the neurula stage. Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the leptomycin B sensitivity. These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression. Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development. This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.
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

ABSTRACT
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

ABSTRACT
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.
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

ABSTRACT
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

ABSTRACT
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.