bluuebunny/crossref_metadata_2025 huggingface dataset:
The TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215
Authors: Charrasse Sophie , Schroeder Marianne , Gauthier-Rouviere Cécile , Ango Fabrice , Cassimeris Lynne , Gard David. L. , Larroque Christian
Published: April 2021

We have recently identified a 6,449 bp cDNA, termed colonic, hepatic tumor over-expressed gene (ch-TOG), that is highly expressed in human tumors and brain. Its single open reading frame encodes a putative 218,000 Da polypeptide, TOGp. Antibodies generated against a bacterially expressed TOGp fragment specifically recognize a 218,000 Da polypeptide in two human cell lines and in brain. Immunofluorescence microscopy using affinity-purified TOGp antibodies revealed that the distribution of TOGp was dependent upon the cell cycle. During interphase, TOGp was found concentrated in the perinuclear cytoplasm, where it co-localized with ER markers. In contrast anti-TOGp antibodies stained centrosomes and spindles in mitotic cells. TOGp co-sedimented with taxol-stabilized microtubules in vitro. Moreover, a TOGp enriched fraction promotes microtubule assembly both in solution and from nucleation centers. Finally, sequence comparison and immunologic cross-reaction suggest that TOGp is homologous to XMAP215, a previously described microtubule associated protein (MAP) from Xenopus eggs. These results suggest that TOGp is a MAP and that TOGp/XMAP215 may be necessary for microtubules rearrangements and spindle assembly in rapidly dividing cells.
LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis
Authors: Duband-Goulet Isabelle , Courvalin Jean-Claude , Buendia Brigitte
Published: April 2021

Chromatin condensation and apposition to the nuclear envelope is an important feature of the execution phase of apoptosis. During this process, lamin proteins that are located between the inner nuclear membrane and heterochromatin are proteolyzed by the apoptosis-specific protease caspase 6. We have investigated the fate of nuclear membranes during apoptosis by studying the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane. LBR interacts through its nucleoplasmic amino-terminal domain with both heterochromatin and B-type lamins, and is phosphorylated throughout the cell cycle, but on different sites in interphase and mitosis. We report here that: (i) the aminoterminal domain of LBR is specifically cleaved during apoptosis to generate an ∼20 kDa soluble fragment; (ii) the cleavage of LBR is a late event of apoptosis and occurs subsequent to lamin B cleavage; (iii) the phosphorylation of LBR during apoptosis is similar to that occurring in interphase. As the association of condensed chromatin with the inner nuclear membrane persists until the late stages of apoptosis, we suggest that the chromatin binding protein LBR plays a major role in maintaining this association.
BIMAAPC3, a component of the Aspergillus anaphase promoting complex/cyclosome, is required for a G2 checkpoint blocking entry into mitosis in the absence of NIMA function
Authors: Lies C. Mark , Cheng Jijun , James Steven W. , Morris N. Ronald , O’Connell Matthew J. , Mirabito P. M.
Published: April 2021

Temperature sensitive (ts) nimA mutants of Aspergillus nidulans arrest at a unique point in G2 which is post activation of CDC2. Here we show that this G2 arrest is due to loss of nimA function and that it is dependent on BIMAAPC3, a component of the anaphase promoting complex/cyclosome (APC/C). Whereas nimA single mutants arrested in G2 with decondensed chromatin and interphase microtubule arrays, nimA, bimAAPC3 double mutants arrested growth with condensed chromatin and aster-like microtubule arrays. nimA, bimAAPC3 double mutants entered mitosis with kinetics similar to bimAAPC3 single mutants and wild-type cells, indicating a checkpoint-like role for BIMAAPC3 in G2. Even cells which had been depleted for NIMA protein and which contained insignificant levels of NIMA kinase activity entered mitosis on inactivation of bimAAPC3. BIMAAPC3 was present in a >25S complex containing BIMEAPC1, and bimAAPC3 mutants were sensitive to elevated CYCLIN B expression, consistent with BIMAAPC3 being a component of the APC/C. Inactivation of bimAAPC3 had little affect on the steady state levels of the B-type cyclin, NIMECydin B. Our results indicate that B?MAAPC?, and most likely the APC/C itself, is activated in G2 in nimA mutants. We propose that APC/C activation is part of a novel, late G2 checkpoint, which responds to a defective process or structure in nimA mutants, and which prevents inappropriate entry into mitosis.
Ligand-independent role of the β4 integrin subunit in the formation of hemidesmosomes
Authors: Nievers Mirjam G. , Schaapveld Roel Q. J. , Oomen Lauran C. J. M. , Fontao Lionel , Geerts Dirk , Sonnenberg Arnoud
Published: April 2021

Recently, we have shown that a region within the β4 cytoplasmic domain, encompassing the second fibronectin type III (FNIII) repeat and the first 27 amino acids of the connecting segment, is critical for the localization of α6β4 in hemidesmosomes. In addition, this region was shown to regulate the distribution of HD1/plectin in transfected cells. In order to investigate the function of the β4 extracellular and cytoplasmic domains in the assembly and integrity of hemidesmosomes, we have constructed chimeric receptors consisting of the extracellular and transmembrane domains of the interleukin 2 receptor (IL2R), fused to different parts of the β4 cytoplasmic domain. These chimeras are expressed as single subunits at the plasma membrane. The results show that the first and the second FNIII repeat, together with the first part of the connecting segment (in total a stretch of 241 amino acids spanning amino acids 1,115 to 1,356) are both essential and sufficient for the localization of β4 in pre-existing hemidesmosomes. Moreover, expression of the IL2R/β4 chimeric constructs in COS-7 and CHO cells, which do not express α6β4 or the bullous pemphigoid (BP) antigens but do express HD1/plectin, revealed that the stretch of 241 amino acids is sufficient for inducing the formation of type II hemidesmosomes. Expression of the IL2R/β4 chimeras in a keratinocyte cell line derived from a patient lacking β4 expression, showed that amino acids 1,115 to 1,356 can also induce the formation of type I hemidesmosomes. We further demonstrate that type I and II hemidesmosomes can also be formed upon adhesion of α6β4-expressing cells to fibronectin. These findings establish that the β4 extracellular domain is not essential for the induction of hemidesmosome assembly. Moreover, they demonstrate that binding of α6β4 to ligand, and heterodimerization of α6 with β4, are not required for hemidesmosome formation. This indicates that the assembly of hemidesmosomes can be regulated from within the cell.
The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells
Authors: Assmann Volker , Marshall John F. , Fieber Christina , Hofmann Martin , Hart Ian R.
Published: April 2021

The receptor for hyaluronan mediated motility (RHAMM) has been reported to mediate migration, transformation, and metastatic spread of murine fibroblasts. Here we describe the expression of two human RHAMM isoforms, which are generated by alternative splicing of the primary gene transcript, by a series of human breast carcinoma cell lines. A polyclonal antibody, raised against a bacterially expressed RHAMM fusion protein, detected an 85-90 kDa protein by western blot analysis. No correlation between the level of RHAMM mRNA and protein expression with known metastatic/malignant potential of the tumour cell lines was observed. Interestingly, we found that the antibody did not stain the cell surface but the cytoplasm of breast cancer cells. The intracellular localisation of RHAMM was confirmed by subcellular fractionation studies. RHAMM proteins were capable of binding to hyaluronan, but not to heparin or chondroitin sulphate, in an vitro binding assay. We also provide evidence that a potential hyaluronan-binding motif in the N terminus of the protein is not involved in the interaction of RHAMM with hyaluronan. Our findings lead us to conclude that RHAMM does not function as a conventional motility receptor for HA in human breast cancer cells and we suggest the term RHAMM be substituted by ‘intracellular hyaluronic acid binding protein’ (IHABP).
Cytoplasmic dynamics of myosin IIA and IIB: spatial ‘sorting’ of isoforms in locomoting cells
Authors: Kolega John
Published: April 2021

Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells’ trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to ‘sort’ within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, timelapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.
Spatiotemporal dynamics of actin concentration during cytokinesis and locomotion in Dictyostelium
Authors: Yumura Shigehiko , Fukui Yoshio
Published: April 2021

To study the spatial and temporal regulation of the actin cytoskeleton, we have analyzed the actin concentration dynamics in live Dictyostelium. The relative actin concentration was analyzed with respect to cell behavior by fluorescence morphometry. We electroporated rhodamineactin into Dictyostelium cells and acquired images with 200–300 millisecond temporal and ∼250 nm spatial resolutions. To convert fluorescence intensity into actin concentration, the observation was made on nearly two-dimensional cells, and the actin signal was ratioed over a volume marker (FITC-BSA or GFP). Since the emission of FITC and GFP is pH-dependent, we first measured the cytoplasmic pH in live cells and determined that the pHi in pseudopods is same as that of general cytoplasm. During cytokinesis, the relative concentration of actin in the cleavage furrow was significantly higher than in the general cytoplasm. In migrating cells, actin was recruited surprisingly rapidly, particularly in the pseudopod. We found that the region of high actin concentration moves relative to the leading edge when a pseudopod projects or retracts. When the pseudopod retracts, the actin density dissipates within 5 seconds. We have also found that actin accumulates in developing pseudopods in an oscillatory manner, and this timing coordinates with advancement of the centroid. This is the first study to reveal the dynamic changes in relative concentration of actin in live cells and to quantitatively correlate these changes with the locomotive behavior of the amoeba.
Assembly of connexins and MP26 in lens fiber plasma membranes studied by SDS-fracture immunolabeling
Authors: Dunia I. , Recouvreur M. , Nicolas P. , Kumar N. , Bloemendal H. , Benedetti E. L.
Published: April 2021

The SDS-fracture immunolabeling technique, unlike conventional freeze-fracture, provides direct evidence for the biochemical nature of membrane constituents. SDS-fracture immunolabeling shows that during differentiation of lens fiber cells the onset of junctional assembly is characterized by the presence of small clusters and linear arrays comprising connexins α3 and α8. At this initial stage MP26, a major fiber membrane constituent, appears to be colocalized with these two connexins. The application of double-immunogold labeling reveals that when large junctional plaques are assembled MP26 becomes mainly associated with the periphery of the junctional domains. This type of distribution suggests that MP26 may play a role in the clustering and gathering of connexons. In aged nuclear fiber membranes connexins, MP26 and their proteolytic derivatives form an orthogonal lattice of repeating subunits.
Stimulated release of fluorescently labeled IgE fragments that efficiently accumulate in secretory granules after endocytosis in RBL-2H3 mast cells
Authors: Xu Keli , Williams Rebecca M. , Holowka David , Baird Barbara
Published: April 2021

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcεRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated β-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcεRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcεRI, but more rapidly. Binding of Cy3-modified IgE to FcεRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcεRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.
Cell-type specific and estrogen dependent expression of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 during mammary gland morphogenesis
Authors: Nikolova Zariana , Djonov Valentin , Zuercher Gisela , Andres Anne-Catherine , Ziemiecki Andrew
Published: April 2021

Morphogenesis of the mammary gland occurs mainly during adult life and is dependent on a complex interplay of hormonal, cell-cell and cell-matrix interactions. The molecular mechanisms involved in pattern formation of the mammary epithelium in adult life are poorly understood. Recently, several members of the Eph family of receptor tyrosine kinases and their ligands have been shown to participate in pattern formation during embryogenesis and conceivably may fulfill similar functions during adult morphogenesis. We have investigated the expression of a member of this family, EphB4, and its cognate ligand, ephrin-B2, during normal and malignant mouse mammary morphogenesis. A spatially, temporarily and hormonally coordinated expression of both the receptor and ligand was observed. The receptor was predominantly localized in the myoepithelial cells surrounding the ducts and alveoli whereas ligand expression was limited to the luminal epithelial cells. Expression of both was induced at the onset of gland morphogenesis at puberty and was differentially regulated during the estrus cycle. Ovariectomy of pre-pubertal or adult females abolished the expression of both receptor and ligand and administration of estrogen alone was sufficient to restore their normal expression. Disruption of the balanced expression was observed during experimental mouse mammary carcinogenesis. Ligand expression was lost at the onset of tumorigenesis and receptor expression shifted from myoepithelial to epithelial cells with progressive malignancy. These results implicate both the EphB4 receptor and its ligand ephrin-B2 in the hormone dependent morphogenesis of the mammary gland. Furthermore, their deregulated expression may contribute to mammary carcinogenesis.
Dynamics of the sub-nuclear distribution of Modulo and the regulation of position-effect variegation by nucleolus in Drosophila
Authors: Perrin L. , Demakova O. , Fanti L. , Kallenbach S. , Saingery S. , Mal’ceva N. I. , Pimpinelli S. , Zhimulev I. , Pradel J.
Published: April 2021

modulo belongs to the class of Drosophila genes named ‘suppressor of position-effect variegation’, suggesting the involvement of the encoded protein in chromatin compaction/relaxation processes. Using complementary procedures of cell fractionation, immunolocalisation on mitotic and polytene chromosomes and cross-linking/immunoprecipitation of genomic DNA targets, we have analysed the sub-nuclear distribution of Modulo. While actually associated to condensed chromatin and heterochromatin sites, the protein is also abundantly found at nucleolus. From a comparison of Modulo pattern on chromosomes of different cell types and mutant lines, we propose a model in which the nucleolus balances the Modulo protein available for chromatin compaction and PEV modification. At a molecular level, repetitive elements instead of rDNA constitute Modulo DNA targets, indicating that the protein directly contacts DNA in heterochromatin but not at the nucleolus. Consistent with a role for Modulo in nucleolus activity and protein synthesis capacity, somatic clones homozygous for a null mutation express a cell-autonomous phenotype consisting of growth alteration and short slender bristles, characteristic traits of Minute mutations, which are known to affect ribosome biogenesis. The results provide evidence suggesting that Modulo participates in distinct molecular networks in the nucleolus and heterochromatin and has distinct functions in the two compartments.
Evidence for a novel MAPKKK-independent pathway controlling the stress activated Sty1/Spc1 MAP kinase in fission yeast
Authors: Shieh Jia-Ching , Martin Humberto , Millar Jonathan B. A.
Published: April 2021

The fission yeast Sty1/Spc1 MAP kinase, like the mammalian JNK/SAPK and p38/CSBP1 kinases, is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, heat shock, UV light and the protein synthesis inhibitor anisomycin. Sty1 is activated by a single MAPKK, Wis1. We demonstrate that the conserved MAPKKK phosphorylation sites Ser 469 and Thr 473 in the catalytic domain of Wis1 are normally essential for Sty1 activation. However, when mildly overexpressed, a mutant Wis1 kinase lacking these conserved phosphorylation sites is able to support stress inducible gene expression and activation of the Sty1 MAP kinase in response to an oxidative or osmotic stress or to a mild heat shock. We show that phosphorylation and activation of Sty1 under these conditions is not due to inactivation of the Pyp1 MAP kinase phosphatase. These results reveal a novel MAPKKK-independent pathway by which the Wis1 MAPKK can activate the Sty1 MAPK in response to stress in fission yeast.
SPC72: a spindle pole component required for spindle orientation in the yeast Saccharomyces cerevisiae
Authors: Souès Sylvie , Adams Ian R.
Published: April 2021

The monoclonal antibody 78H6 recognises an 85 kDa component of the yeast spindle pole body. Here we identify and characterise this component as Spc72p, the product of YAL047C. The sequence of SPC72 contains potential coiled-coil domains; its overexpression induced formation of large polymers that were strictly localised at the outer plaque and at the bridge of the spindle pole body. Immunoelectron microscopy confirmed that Spc72p was a component of these polymers. SPC72 was found to be non-essential for cell growth, but its deletion resulted in abnormal spindle positioning, aberrant nuclear migration and defective mating capability. Precisely, deletion of SPC72 resulted in a decreased number of astral microtubules: early in the cell cycle only few were detectable, and these were unattached to the spindle pole body in small-budded cells. Later in the cell cycle few, if any, remained, and they were unable to align the spindle properly. We conclude that Spc72p is not absolutely required for nucleation per se, but is needed for normal abundance and stability of astral microtubules.
Protein transport from the endoplasmic reticulum to the Golgi apparatus
Authors: Hong Wanjin
Published: April 2021

As the first step of protein transport along the biosynthetic (secretory/exocytotic) pathway, transport from the endoplasmic reticulum (ER) to the Golgi apparatus has received much attention over the past several decades. The general structural organization underlying this transport process is becoming more defined. The major protein components participating in the budding, pre-docking, and docking/fusion events have been identified and their mechanistic aspects investigated. Conceptually, it is now clear that protein export from the ER is a selective process. Although much remains to be defined or refined, the general picture of this transport step has now emerged.
Muscle-specific functions of ryanodine receptor channels in Caenorhabditis elegans
Authors: Maryon Ed B. , Saari Bonnie , Anderson Philip
Published: April 2021

Ryanodine receptor channels regulate contraction of striated muscle by gating the release of calcium ions from the sarcoplasmic reticulum. Ryanodine receptors are expressed in excitable and non-excitable cells of numerous species, including the nematode C. elegans. Unlike vertebrates, which have at least three ryanodine receptor genes, C. elegans has a single gene encoded by the unc-68 locus. We show that unc-68 is expressed in most muscle cells, and that the phenotypic defects exhibited by unc-68 null mutants result from the loss of unc-68 function in pharyngeal and body-wall muscle cells. The loss of unc-68 function in the isthmus and terminal bulb muscles of the pharynx causes a reduction in growth rate and brood size. unc-68 null mutants exhibit defective pharyngeal pumping (feeding) and have abnormal vacuoles in the terminal bulb of the pharynx. unc-68 is required in body-wall muscle cells for normal motility. We show that UNC-68 is localized in body-wall muscle cells to flattened vesicular sacs positioned between the apical plasma membrane and the myofilament lattice. In unc-68 mutants, the vesicles are enlarged and densely stained. The flattened vesicles in body-wall muscle cells thus represent the C. elegans sarcoplasmic reticulum. Morphological and behavioral phenotypes of unc-68 mutants suggest that intracellular calcium release is not essential for excitation-contraction coupling in C. elegans.
Fibronectin matrix assembly enhances adhesion-dependent cell growth
Authors: Sottile Jane , Hocking Denise C. , Swiatek Pamela J.
Published: April 2021

Cell growth control in non-transformed cells depends, in part, on adhesive interactions with the extracellular matrix. Following injury, excess or altered fibronectin deposition into the extracellular matrix may contribute to the pathogenesis of fibrosis and atherosclerosis by triggering changes in specific cell functions associated with wound repair, including cell proliferation and migration. To assess the role of fibronectin polymerization on cell growth, we isolated mouse embryonic cells that lack endogenous fibronectin (fibronectin-null cells) and established them in culture under serum-free conditions. These fibronectin-null cells do not produce any detectable fibronectin, but are capable of assembling a fibronectin matrix when cultured in the presence of exogenously added fibronectin. Our data indicate that adhesion-dependent growth in fibronectin-null cells is dramatically increased (>2-5×) by culturing cells in the presence of fibronectin. This fibronectin-induced cell growth was blocked by inhibiting fibronectin matrix assembly. Arg-Gly-Asp peptides or fragments of fibronectin that contain the Arg-Gly-Asp cell binding site promoted clustering of the α5β1 integrin in focal adhesions, but did not enhance cell growth. These data indicate that the polymerization of fibronectin into the extracellular matrix positively regulates cell growth, and that occupancy and clustering of fibronectin-binding integrins alone are not sufficient to trigger increased cell growth.
Embryoglycan ectodomains regulate biological activity of FGF-2 to embryonic stem cells
Authors: Dvořák P. , Hampl A. , Jirmanová L. , Pacholíková J. , Kusakabe M.
Published: April 2021

Basic fibroblast growth factor (FGF-2) functions as a natural inducer of mesoderm, regulator of cell differentiation and autocrine modulator of cell growth and transformation. The FGF-2 signals are transduced through receptors with intrinsic protein tyrosine kinase activity. However, receptor binding and activation is governed by extracellular matrix, cell surface or soluble proteoglycans. This paper focuses on the role of proteoglycans synthesized by embryonic cells, embryoglycans, in FGF-2 signaling via FGF receptor-1 (FGFR-1).

We found that embryoglycan ectodomain Lewis X, analog of developmentally regulated embryonic cell surface epitope TEC 1, promotes oligomerization of FGF-2 in the cell free chemical crosslinking. In vitro assays show that a large molar excess of extracellular Lewis X does not inhibit binding of FGF-2 to embryonic stem (ES) cells, but prevents the mitogenic effect of FGF-2. Western blot analysis of ES cells revealed the presence of abundant 52 kDa and trace amounts of 67 and 125 kDa isoforms of FGFR-1. However, none of these isoforms undergo any detectable changes in tyrosine phosphorylation under the conditions that modulate the mitogenic effect of FGF-2. Rather, a primary substrate of all receptor tyrosine kinases, phospholipase Cγ (PLCγ), is activated by both FGF-2 and Lewis X. The combination, FGF-2 plus Lewis X, leads to weak inhibition, when compared with the effects of FGF-2 and Lewis X, respectively. In accordance, the level of phosphorylation of non-receptor tyrosine kinase c-Src is reduced in a reversed pattern to PLCγ. Furthermore, in this particular cell type we show the presence of activated forms of extracellular signal-related kinase (ERK) in all nontreated and treated cells.

These findings demonstrate that embryoglycan ectodomains may act as negative regulators of FGF-2-induced ES cell proliferation, most likely through the FGFR-1-independent signaling pathway.
Suppression of tubulin tyrosine ligase during tumor growth
Authors: Lafanechère Laurence , Courtay-Cahen Céline , Kawakami Toshiaki , Jacrot Michèle , Rüdiger Manfred , Wehland Jürgen , Job Didier , Margolis Robert L.
Published: April 2021

The C terminus of the tubulin α-subunit of most eukaryotic cells undergoes a cycle of tyrosination and detyrosination using two specific enzymes, a tubulin tyrosine ligase (TTL) and a tubulin carboxypeptidase. Although this enzyme cycle is conserved in evolution and exhibits rapid turnover, the meaning of this modification has remained elusive. We have isolated several NIH-3T3 derived clonal cell lines that lack TTL (TTL−). TTL− cells contain a unique tubulin isotype (Δ2-tubulin) that can be detected with specific antibodies. When injected into nude mice, both TTL− cells and TTL− cells stably transfected with TTL cDNA form sarcomas. But in tumors formed from TTL rescued cells, TTL is systematically lost during tumor growth. A strong selection process has thus acted during tumor growth to suppress TTL activity. In accord with this result, we find suppression of TTL activity in the majority of human tumors assayed with Δ2-tubulin antibody. We conclude there is a widespread loss of TTL activity during tumor growth in situ, suggesting that TTL activity may play a role in tumor cell regulation.
EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells
Authors: Chan Amanda Y. , Raft Steven , Bailly Maryse , Wyckoff Jeffrey B. , Segall Jeffrey E. , Condeelis John S.
Published: April 2021

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12±0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.
Agonistic monoclonal antibodies against the Met receptor dissect the biological responses to HGF
Authors: Prat Maria , Crepaldi Tiziana , Pennacchietti Selma , Bussolino Federico , Comoglio Paolo M.
Published: April 2021

Hepatocyte growth factor, also known as scatter factor, is a pleiotropic cytokine, which stimulates cell motility, invasion, proliferation, survival and morphogenesis, and induces the expression of specific genes by activating its receptor tyrosine kinase. In this work we have isolated, characterized and used as agonists two monoclonal antibodies (mAbs) directed against the extracellular domain of HGF receptor to investigate the requirements for receptor activation and for the different biological responses. The two mAbs display similar affinities, react with epitopes different from the hepatocyte growth factor binding site, and behave as either full or partial agonists. The full agonist mAb (DO-24) triggers all the biological effects elicited by hepatocyte growth factor, namely motility, proliferation, cell survival, invasion, tubulogenesis and angiogenesis. The partial agonist mAb (DN-30) induces only motility. Only the full agonist mAb is able to induce and sustain the expression of urokinase-type plasminogen activator receptor for prolonged periods of time, while both mAbs up-regulate the constitutive expression of urokinase-type plasminogen activator. Both mAbs activate receptor phosphorylation, which, being strictly dependent on mAb bivalence, requires receptor dimerization. Since simple receptor dimerization is not sufficient to trigger full biological responses, we propose that the region on the β chain of the receptor recognized by the full agonist mAb is crucial for optimal receptor activation.
Disruption of Dictyostelium PI3K genes reduces [32P]phosphatidylinositol 3,4 bisphosphate and [32P]phosphatidylinositol trisphosphate levels, alters F-actin distribution and impairs pinocytosis
Authors: Zhou Kemin , Pandol S. , Bokoch G. , Traynor-Kaplan A. E.
Published: April 2021

To understand how phosphatidylinositol 3-kinase (PI3K) modulates cell structure and function, we examined the molecular and cellular defects of a Dictyostelium mutant strain (pik1Δ2Δ) missing two (DdPIK1 and 2) of three PI3K genes, which are homologues of the mammalian p110 subunit. Levels of [32P]phosphatidylinositol 3,4 bisphosphate (PI(3,4)P2) and [32P]phosphatidylinositol trisphosphate (PIP3) were reduced in pik1Δ2Δ, which had major defects in morphological and functional correlates of macropinocytosis. This was accompanied by dramatic deficits in a subset of F-actin-enriched structures such as circular ruffles, actin crowns and pseudopodia. Although pik1Δ2Δ were mobile, they failed to aggregate into streams. Therefore we conclude that PIK1 and 2, possibly through modulation of the levels of PIP3 and PI(3,4)P2, regulate the organization of actin filaments necessary for circular ruffling during macropinocytosis, the extension of pseudopodia and the aggregation of cells into streams, but not the regulation of cell motility.
The role of the Ran GTPase in nuclear assembly and DNA replication: Characterisation of the effects of Ran mutants
Authors: Hughes Mike , Zhang Chuanmao , Avis Johanna M. , Hutchison Christopher J. , Clarke Paul R.
Published: April 2021

The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. Here, we have investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin α and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin β, Ran binding protein 1 and RCC1, an exchange factor for Ran. We show that Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system.
Developmental association of the β-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts
Authors: Choi Je-Yong , van Wijnen André J. , Aslam Fauzia , Leszyk John D. , Stein Janet L. , Stein Gary S. , Lian Jane B. , Penman Sheldon
Published: April 2021

The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the β-galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.
The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility
Authors: Faix Jan , Clougherty Chris , Konzok Angelika , Mintert Ursula , Murphy John , Albrecht Richard , Mühlbauer Bettina , Kuhlmann Jürgen
Published: April 2021

DGAP1 of Dictyostelium discoideum is a cell cortex associated 95 kDa protein that shows homology to both RasGTPase-activating proteins (RasGAPs) and RasGAP-related proteins. When tested for RasGAP activity, recombinant DGAP1 protein did not promote the GTPase activity of human H-Ras or of Dictyostelium RasG in vitro. Instead, DGAP1 bound to Dictyostelium Rac1A and human Rac1, but not to human Cdc42. DGAP1 preferentially interacted with the activated GTP-bound forms of Rac1 and Rac1A, but did not affect the GTPase activities. Since Rho-type GTPases are implicated in the formation of specific F-actin structures and in the control of cell morphology, the microfilament system of mutants that either lack or overexpress DGAP1 has been analysed. DGAP1-null mutants showed elevated levels of F-actin that was organised in large leading edges, membrane ruffles or numerous large filopods. Expression of actin fused to green fluorescent protein (GFP) was used to monitor the actin dynamics in these cells, and revealed that the F-actin cytoskeleton of DGAP1-null cells was rapidly re-arranged to form ruffles and filopods. Conversely, in DGAP1-overexpressing cells, the formation of cellular projections containing F-actin was largely suppressed. Measurement of cell migration demonstrated that DGAP1 expression is inversely correlated with the speed of cell motility.
Regulation of mRNA localization by transmembrane signalling: Local interaction of HB-GAM (heparin-binding growth-associated molecule) with the cell surface localizes β-actin mRNA
Authors: Fages Carole , Kaksonen Marko , Kinnunen Tarja , Punnonen Eeva-Liisa , Rauvala Heikki
Published: April 2021

Localization of mRNAs is currently thought to be partially responsible for molecular sorting to specific compartments within the cell. In mammalian cells the best-studied example is the β-actin mRNA that is localized to the cell processes, and its localization is necessary in migratory responses of cells. It is reasonable to assume that mRNA localization within cells is coupled to transmembrane signalling due to extracellular factors, but little is known about such putative mechanisms. We show here that HB- GAM, an extracellular matrix-associated factor that enhances migratory responses in cells, is able to localize β- actin mRNA when locally applied to cells via microbeads. The HB-GAM-induced mRNA localization is specifically inhibited by low concentrations of heparin and by heparitinase treatment of cells, showing that cell-surface heparin-type glycans are required for the effect. The finding that soluble N-syndecan is also inhibitory suggests that the transmembrane proteoglycan N-syndecan, previously identified as an HB-GAM receptor, is involved in the mRNA-localizing effect of HB-GAM. Inhibition of the mRNA localization by the src-kinase inhibitor PP1 is compatible with an N-syndecan-mediated effect since the receptor function of N-syndecan has been recently found to depend on the src-kinase signalling pathway. The mRNA- localizing activity of N-syndecan is also suggested by the finding that affinity-purified anti-N-syndecan antibodies coated on microbeads are able to localize β-actin mRNA.
A cell cycle regulated MAP kinase with a possible role in cytokinesis in tobacco cells
Authors: Calderini Ornella , Bögre László , Vicente Oscar , Binarova Pavla , Heberle-Bors Erwin , Wilson Cathal
Published: April 2021

Mitogen-activated protein (MAP) kinases have been demonstrated to have a role in meiosis but their involvement in mitotic events is less clear. Using a peptide antibody raised against the tobacco MAP kinase p43Ntf6 and extracts from synchronized tobacco cell suspension cultures, we show that this kinase is activated specifically during mitosis. Entry into mitosis appears to be necessary for the activation of the kinase, which occurs as a post-translational event. The activation of the kinase occurs in late anaphase/early telophase. The p43Ntf6protein shows a transient localization to the cell plate in anaphase cells, in the middle of the two microtubule arrays characteristic of the phragmoplast, a plant-specific structure involved in laying down the new cell wall. The combined data support a role for the MAP kinase p43Ntf6 in cytokinesis in tobacco cells.
Integrin ligation and PKC activation are required for migration of colon carcinoma cells
Authors: Rigot Véronique , Lehmann Maxime , André Frédéric , Daemi Noucha , Marvaldi Jacques , José Luis
Published: April 2021

The activation of protein kinases C (PKCs) is an essential step in integrin-dependent cell adhesion and spreading. In this report we examined the effect of the phorbol ester PMA, a PKC activator, on adhesion, spreading and migration of a colon carcinoma cell line, HT29-D4. Treatment with PMA increased the rate of cell spreading and induced the migration of these cells towards purified matrix proteins in haptotaxis assays on Boyden chambers. PMA-induced effects were the result of PKCs activation, as shown by using the inactive isomer 4α-PMA and PKCs inhibitors. The involvement of integrins in the phorbol ester-induced cell migration was demonstrated both by the absence of migration of cells plated on membranes coated with poly-L-lysine and by the use of function blocking antibodies. Thus, interactions between α2β1, α3β1, α6β4, αvβ5, αvβ6 integrins and their specific ligands are necessary for the PKC-mediated migration. However, adhesion, immunoprecipitation and immunocytofluorometry experiments clearly showed that HT29-D4 cell haptotaxis induced by PKC activation is not a consequence of quantitative or qualitative changes in the cell surface integrins. We also demonstrated that PKCs were able to activate the MAP kinase pathway and that the impediment of MAP kinase activation resulted in the loss of cell migration. Moreover, stimulation of the insulin-like growth factor I signalling pathway led to MAP kinase activation and to the induction of cell migration. In addition, the growth factor-induced motility of HT29-D4 cells was affected both by PKC and MAP kinase cascade inhibitors. It thus appears that both integrin ligation and MAP kinase activation by PKCs are required to promote the migration of HT29-D4 cells.
ELAV protein HuA (HuR) can redistribute between nucleus and cytoplasm and is upregulated during serum stimulation and T cell activation
Authors: Atasoy Ulus , Watson Janice , Patel Dhavalkumar , Keene Jack D.
Published: April 2021

ELAV proteins are implicated in regulating the stability and translation of cytokine and growth regulatory mRNAs such as GM-CSF, IL-2, c-myc, c-fos and GLUT1 by binding to their AU-rich 3′UTRs. The tissue-specific ELAV protein HuB (aka. Hel-N1) is predominantly cytoplasmic and has been shown to stabilize GLUT1 and c-myc mRNAs and to increase their translation following ectopic expression in 3T3-L1 cells. We report that the most widely expressed mouse ELAV protein, mHuA, is predominately nuclear in cultured NIH-3T3 cells, but is localized in the cytoplasm during early G1 of the cell cycle. Therefore, much like the primarily cytoplasmic HuB, HuA becomes temporally localized in the cytoplasm where it can potentially regulate the stability or translation of bound mRNAs. Moreover, we report that stimulation of mouse spleen cells using either mitogenic or sub-mitogenic levels of anti-CD3/CD28 resulted in a dramatic increase in the level of HuA. Upregulation of HuA corresponds to previously documented increases in cytokine expression which are due to increased mRNA stability following T cell activation. Consistent with these findings, HuA was down regulated in quiescent cells and upregulated in 3T3 cells following serum stimulation. The increase of murine HuA during the cell cycle closely resembles that of cyclin B1 which peaks in G2/M. Together with our earlier studies, these data indicate that mammalian ELAV proteins function during cell growth and differentiation due in part to their effects on posttranscriptional stability and translation of multiple growth regulatory mRNAs. This supports the hypothesis that ELAV proteins can function as transacting factors which affect a default pathway of mRNA degradation involved in the expression of growth regulatory proteins.
Tension-sensitive kinetochore phosphorylation in vitro
Authors: Nicklas R. Bruce , Campbell Michael S. , Ward Suzanne C. , Gorbsky Gary J.
Published: April 2021

Many cells have a checkpoint that detects a single misattached chromosome and delays anaphase, allowing time for error correction. Detection probably depends on tension-sensitive kinetochore protein phosphorylation. Somehow, mechanical tension, or some consequence of tension, produces a chemical change, dephosphorylation. The mechanism of tension-mediated dephosphorylation can be approached using an in vitro system. Earlier work showed that the kinetochores of washed chromosomes from a mammalian cell line can be phosphorylated in vitro simply by incubation with ATP and a phosphatase inhibitor. We confirm this for chromosomes from insect meiotic cells. Thus, kinetochores of washed chromosomes from diverse sources contain a complete phosphorylation system: a kinase, a phosphatase and the substrate protein(s). We show that phosphorylation in vitro is sensitive to tension, as it is in living cells. This makes the conditions required for phosphorylation in vitro relevant to the process in living cells. The phosphatase is ruled out as the tension-sensitive component in vitro, leaving either the kinase or the substrate as the sensitive component. We show that a kinase extracted from mammalian cells in mitosis phosphorylates the kinetochores of insect meiotic chromosomes very effectively. The mammalian kinase under-phosphorylates the kinetochore of the insect’s X-chromosome, just as the native insect kinase does. This provides a clue to the evolution of a chromosome that is not detected by the checkpoint. The mammalian kinase is not tightly bound to the chromosome and thus functions primarily in solution. This suggests that the substrate’s phosphorylatable groups are freely available to outside constituents, e.g. regulators, as well as to the kinetochore’s own kinase and phosphatase.