bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational analysis of Hsp90α dimerization and subcellular localization: dimer disruption does not impede ‘in vivo’ interaction with estrogen receptor
Authors: Meng Xia , Devin Jocelyne , Sullivan William P. , Toft David , Baulieu Etienne-Emile , Catelli Maria-Grazia 
Published: April 2021

ABSTRACT
The molecular chaperone Hsp90 has been found ubiquitously as a predominantly cytoplasmic dimer. By interacting with cytoplasmic or nuclear proteins such as pp60v-src or steroid receptors, Hsp90 helps its targets to become competent for full biological activity.
Mutational deletion analysis of some properties of chicken Hsp90α was undertaken after transient transfection of the constructs in COS7 cells. First, Hsp90 mutants were analyzed for their ability to behave as cytosolic dimers. We confirmed that the C-terminal Hsp90 region (amino acids 446-728) was sufficient for dimerization, and found that deletion of three small subregions in the 200 C-terminal residues precluded Hsp90 dimer formation. Moreover, we demonstrated that the N-terminal region of the protein (1-442) was not involved in dimerization. Second, the subcellular localization of the wild-type (WT) protein and mutants was analyzed by specific immunodetection and confocal microscopy. Most of the mutants were cytoplasmic like Hsp90WT, a nuclear localization being barely detectable in the WT protein or in mutants with a C-terminal truncation equal to or shorter than 286 residues. Surprisingly a mutant encoding the N-terminal region (1-285) was nuclear localized.
In addition, the in vivo interaction between the cytoplasmic Hsp90 and the nuclear ER was documented after coexpression of both proteins in the same cells: some Hsp90 was shifted into the nucleus via its interaction with ER. From an analysis of dimeric or monomeric cytoplasmic Hsp90 mutants, we found that disruption of Hsp90 dimer did not systematically impede its interaction with ER.
Finally, Hsp90WT and cytoplasmic mutants were tested for their ability to rescue from lethality a yeast strain deleted of both Hsp90 genes. Interestingly, the Δ661-677 mutant that showed an impaired dimerization but interacted with ER was able to confer viability, while the mutant deleted of the 30 C-terminal residues (NC6) was monomeric, did not confer viability and did not interact with ER. We therefore suggest that Hsp90 properties analyzed here are not necessarily interdependent.
Nuclear localization of DP and E2F transcription factors by heterodimeric partners and retinoblastoma protein family members
Authors: Magae Junji , Wu Chin-Lee , Illenye Sharon , Harlow Ed , Heintz Nicholas H. 
Published: April 2021

ABSTRACT
E2F is a family of transcription factors implicated in the regulation of genes required for progression through G1 and entry into the S phase. The transcriptionally active forms of E2F are heterodimers composed of one polypeptide encoded by the E2F gene family and one polypeptide encoded by the DP gene family. The transcriptional activity of E2F/DP heterodimers is influenced by association with the members of the retinoblastoma tumor suppressor protein family (pRb, p107, and p130). Here the intracellular distribution of E2F and DP proteins was investigated in transiently transfected Chinese hamster and human cells. In transfected cells, DP-1 did not accumulate in the nucleus unless it was coexpressed with the heterodimeric partners E2F-1, E2F-2, or E2F-3. Domain mapping experiments showed that regions of E2F-1 and DP-1 that are required for stable association of the two proteins were also required for nuclear localization of DP-1. Unlike E2F-1, -2, and -3, E2F-4 did not accumulate in the nucleus unless it was coexpressed with DP-2. p107 and p130, but not pRb, stimulated nuclear localization of E2F-4, either alone or in combination with DP-2. These results indicate that DP proteins preferentially associate with specific E2F partners, and suggest that the ability of specific E2F/DP heterodimers to localize in the nucleus contributes to the regulation of E2F activity.
Vaccinia virus: a model system for actin-membrane interactions
Authors: Cudmore Sally , Reckmann Inge , Griffiths Gareth , Way Michael 
Published: April 2021

ABSTRACT
Our understanding of the interactions between the actin cytoskeleton and cellular membranes at the molecular level is rudimentary. One system that offers an opportunity to examine these interactions in greater detail is provided by vaccinia virus, which induces the nucleation of actin tails from the outer membrane surrounding the virion. To further understand the mechanism of their formation and how they generate motility, we have examined the structure of these actin tails in detail. Actin filaments in vaccinia tails are organized so they splay out at up to 45° from the centre of the tail and are up to 0.74 μm in length, which is considerably longer than those reported in the Listeria system. Actin filaments show unidirectional polarity with their barbed filament ends pointing towards the surface of the virus particle. Rhodamine-actin incorporation experiments show that the first stage of tail assembly involves a polarized recruitment of G-actin, and not pre-formed actin filaments, to the membrane surrounding the virion. Incorporation of actin into the tail only occurs by nucleation from the viral surface, suggesting filament ends in the tail are blocked against further actin addition. As virus particles fuse with the plasma membrane during the extention of projections, actin nucleation sites previously in the viral membrane become localized to the plasma membrane, where they are able to nucleate actin polymerization in a manner analogous to the leading edge of motile cells.
Paradoxical accumulation of the cyclin-dependent kinase inhibitor p27kip1 during the cAMP-dependent mitogenic stimulation of thyroid epithelial cells
Authors: Depoortere Fabienne , Dumont Jacques E. , Roger Pierre P. 
Published: April 2021

ABSTRACT
In different systems, cAMP either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S pre-replicative phase progression by cyclic AMP (cAMP) as a second messenger for thyrotropin (TSH). We report here that TSH markedly increases the expression of p27kip1, the inhibitor of the cell cycle and cyclin-dependent kinases. This effect was prevented by the concomitant administration of the cAMP-independent mitogens, epidermal growth factor (EGF)+serum. EGF+serum also slightly inhibited the weak basal accumulation of p27kip1. Nevertheless, in the case of stimulation by TSH alone, the cAMP-dependent cell cycle progression was fully compatible with the enhanced expression of p27kip1. This observation is paradoxical since a decrease of p27kip1 is generally associated with growth stimulation in other systems, and since a similar cAMP-dependent increase of p27kip1 in macrophages has been found responsible for mid-G1 cell cycle arrest. The opposite regulation of p27kip1 in response to TSH or EGF+serum in dog thyroid epithelial cells suggests a major difference at mid to late G1 stages between cAMP-dependent and cAMP-independent mitogenic pathways.
Characterization and quantitation of three B-type lamins in Xenopus oocytes and eggs: increase of lamin LI protein synthesis during meiotic maturation
Authors: Lourim David , Kempf Anja , Krohne Georg 
Published: April 2021

ABSTRACT
We have previously shown that Xenopus oocytes, eggs, and early embryos contain lamins LII and LIII, and that portions of each are associated with distinct egg vesicle populations. We now report that a lamin similar or identical to the B-type lamin LI is also present in oocyte nuclei and in egg extracts. We have quantitated the three B-type lamins per oocyte nucleus, and have calculated relative ratios of LI:LIII = 1:100, and LII:LIII = 1:10. Similar to lamin LII, 5-15% of lamin LI is associated with egg membranes in a biochemically stable manner. Egg vesicles absorbed with lamin isoform-specific antibodies to magnetic beads indicate that lamin LI-associated egg membranes are of heterogenous morphology, and are independent from the lamin LII and LIII vesicle populations.
Compared to other nuclear envelope proteins, the synthesis of lamin LI protein is specifically elevated during meiotic maturation, resulting in a 4- to 12-fold higher amount of lamin LI in eggs than is present in oocyte nuclei. Immunofluorescence and immunoblot analysis demonstrated that lamins LI, LII, and LIII are associated with the nuclear envelope formed on demembranated sperm when added to activated egg extract. These results strongly suggest that three different lamin-associated vesicle populations are involved in the formation of a nuclear envelope in egg extracts.
Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants
Authors: Stoeckelhuber Mechthild , Noegel Angelika A. , Eckerskorn Christoph , Köhler Jana , Rieger Daniela , Schleicher Michael 
Published: April 2021

ABSTRACT
Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner. To further characterize this interaction we isolated hisactophilin over-expression (hisII+) and hisactophilin minus (his−) mutants. D. discoideum contains two hisactophilin isoforms; both genes are independently transcribed and carry a short intron at the same position of the coding region. The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of a total 118 amino acids. After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression. The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette. The properties of the hisII+ and his− cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions. However, the hisII+ transformants revealed a significant difference to wild-type cells and his− cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor. HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his− cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin. Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane. The fact that cells deficient in hisactophilin again lose resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration.
Non-neural agrin codistributes with acetylcholine receptors during early differentiation of Torpedo electrocytes
Authors: Cartaud A. , Ludosky M. A. , Haasemann M. , Jung D. , Campbell K. , Cartaud J. 
Published: April 2021

ABSTRACT
Agrin, an extracellular matrix protein synthesized by nerves and muscles is known to promote the clustering of acetylcholine receptors and other synaptic proteins in cultured myotubes. This observation suggests that agrin may provide at least part of the signal for synaptic specialization in vivo. The extracellular matrix components agrin, laminin and merosin bind to α-dystroglycan, a heavily glycosylated peripheral protein part of the dystrophin-glycoprotein complex, previously characterized in the sarcolemma of skeletal and cardiac muscles and at the neuromuscular junction.
In order to understand further the function of agrin and αDG in the genesis of the acetylcholine receptor-rich membrane domain, the settling of components of the dystrophin-glycoprotein complex and agrin was followed by immunofluorescence localization in developing Torpedo marmorata electrocytes. In 40-45 mm Torpedo embryos, a stage of development at which the electrocytes exhibit a definite structural polarity, dystrophin, α/β-dystroglycan and agrin accumulated concomitantly with acetylcholine receptors at the ventral pole of the cells. Among these components, agrin appeared as the most intensely concentrated and sharply localized. The scarcity of the nerve-electrocyte synaptic contacts at this stage of development, monitored by antibodies against synaptic vesicles, further indicates that before innervation, the machinery for acetylcholine receptor clustering is provided by electrocyte-derived agrin rather than by neural agrin.
These observations suggest a two-step process of acetylcholine receptor clustering involving: (i) an instructive role of electrocyte-derived agrin in the formation of a dystrophin-based membrane scaffold upon which acetylcholine receptor molecules would accumulate according to a diffusion trap model; and (ii) a maturation and/or stabilization step controlled by neural agrin. In the light of these data, the existence of more than one agrin receptor is postulated to account for the action of agrin variants at different stages of the differentiation of the postsynaptic membrane in Torpedo electrocytes.
Structural requirements for α1β1 and α2β1 integrin mediated cell adhesion to collagen V
Authors: Ruggiero Florence , Comte Jane , Cabañas Carlos , Garrone Robert 
Published: April 2021

ABSTRACT
A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via α1β1 and α2β1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive α-chains versus the triple helical conformation in the recognition of collagen V binding sites.
To investigate this question, in vitro renaturation of the isolated α1(V) and α2(V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the α1(V) and α2(V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed.
We have previously shown that α1β1 and α2β1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing α2β1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of α1β1 and α2β1 integrins. We showed that both α1(V) and α2(V) homotrimers induced cell adhesion but refolded α2(V) chains were more efficient and promoted cell adhesion as well as native collagen V.
Thermal stability of refolded α-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn2+-dependent and Ca2+ ions alone were ineffective. Antibodies against α2 and β1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with α2β1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded α-chains. Antibody to β1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against α1 and α2 integrin subunits. However, only α2β1 was engaged obviously in HBL100 cell adhesion to refolded α-chains.
These data indicate that triple helical conformation is particularly critical for α2β1- and α1β1-dependent adhesion and that the integrin α2β1 is a dominant functional receptor for refolded α-chains. We conclude that α2β1-dependent adhesion seems to involve multiple different conformational binding sites while α1β1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.
Apical β1 integrin in polarized MDCK cells mediates tubulocyst formation in response to type I collagen overlay
Authors: Zuk Anna , Matlin Karl S. 
Published: April 2021

ABSTRACT
A number of epithelia form tubulocysts in vitro when overlaid with type I collagen gel. Because collagen receptors are generally believed to be expressed on the basolateral domain, the mechanism by which collagen elicits this morphogenetic response from the apical surface is unclear. To investigate the role of β1 integrins, the major receptor family for collagen, in this process, we overlaid polarized monolayers of MDCK II cells grown on permeable supports with type I collagen gel and correlated integrin polarity with the polarity of other apical and basolateral membrane markers during tubulocyst formation. Polarized monolayers of one clone of MDCK II cells, referred to as Heidelberg MDCK, initially respond to collagen overlay by stratifying; within 48 hours, lumena develop between the cell layers giving rise to tubulocysts. Tight junctions remain intact during tubulocyst formation because transepithelial electrical resistance does not significantly change. Major alterations are observed, however, in the expression and localization of apical and basolateral membrane markers. β1 integrins are necessary for tubulocyst morphogenesis because a function-blocking antibody administered to the apical pole of the cells completely inhibits the formation of these structures. To determine how apical-cell collagen interactions elicit tubulocyst formation, we examined whether β1 integrins are mobilized to apical plasma membranes in response to collagen overlay. We found that in the absence of collagen, polarized monolayers of Heidelberg MDCK cells endogenously express on apical plasma membranes a small pool of the β1 family, including α2β1 and α3β1. Collagen overlay does not mobilize additional β1 integrins to apical domains. If β1 integrins are not already apically expressed, as in the C6 MDCK cell line (Schoenenberger et al. (1994)J. Cell Biol. 107, 527-541), β1 integrins are not directed apically and tubulocysts do not develop in response to collagen. Thus, interaction of β1 integrins pre-existing on apical plasma membranes of polarized epithelia with type I collagen gel is the mechanism by which apical application of collagen elicits the formation of tubulocysts. Depolarized integrins on apical plasma membranes of polarized epithelia may be relevant to the pathogenesis of disease and injury.
Functional compartmentalization of the nucleus
Authors: Strouboulis John , Wolffe Alan P. 
Published: April 2021

ABSTRACT
Recent applications of cell biology and molecular genetics have built an image of nuclear organization in which the molecular machines involved in transcription, RNA processing and replication assemble morphologically distinct nuclear organelles with defined functional properties. These observations indicate a very high level of structural organization for the various metabolic activities occurring within the nucleus. We discuss the possible existence of novel regulatory functions inherent to nuclear architecture itself.
Granular cells are required for encapsulation of foreign targets by insect haemocytes
Authors: Pech Louis L. , Strand Michael R. 
Published: April 2021

ABSTRACT
Haemocytes play an essential role in defending invertebrates against pathogens and parasites that enter their haemocoel. A primary defense response is encapsulation; a process in which haemocytes attach to the foreign organism and kill it. Whether encapsulation requires cooperation between specific subpopulations of haemocytes is unknown. Using purified subpopulations of haemocytes and an in vitro encapsulation assay, we investigated the process of capsule formation in the insect Pseudoplusia includens. Immunocytochemical staining revealed that capsule formation involves a three step process. Encapsulation began when granular cells attached to the foreign target. This was followed by attachment of multiple layers of plasmatocytes. Termination of capsule formation occurred when a subpopulation of granular cells formed a monolayer around the periphery of the capsule. Neither granular cells nor plasmatocytes were capable of forming a capsule independently. However, plasmatocytes encapsulated targets if granular cells were present or if targets were preincubated in medium conditioned by granular cells. The effect of granular cell-conditioned medium could be blocked by the addition of the cell adhesion recognition sequence, RGDS, but not by RGES. These results demonstrate experimentally that granular cells are required for encapsulation of foreign targets by plasmatocytes in vitro, and that the role of granular cells in this process involves an RGD-dependent cell adhesion mechanism.
The intracompartmental sorting of myosin alkali light chain isoproteins reflects the sequence of developmental expression as determined by double epitope-tagging competition
Authors: Komiyama Masatoshi , Soldati Thierry , Arx Pierre von , Perriard Jean-Claude 
Published: April 2021

ABSTRACT
In order to compare within the same cell the various degrees of specificity of myosin alkali light chain (MLC) isoproteins sorting to sarcomeres, a competition assay was established using double epitope tagging. Various combinations of two different MLC isoform cDNAs tagged with either a vesicular stomatitis virus VSV-G (VSV) or a medium T (mT) protein epitope were co-expressed in cultured cardiomyocytes from adult and neonatal rat ventricles. Expressed isoproteins were detected by means of anti-VSV and anti-mT antibodies and their sorting patterns were analyzed by confocal microscopy. The sorting specificity of MLC isoforms to sarcomeric sites was shown to increase in the order MLC3nm, to ML1sa, to MLC1sb, to MLC1f and MLC3f following the sequence of developmental expression. Expressed fast skeletal muscle isoforms (MLC1f and MLC3f) were always localized at the A-bands of myofibrils, while nonmuscle type (MLC3nm) was distributed throughout the cytoplasm. The slow skeletal muscle type (MLC1sa) showed a weak sarcomeric pattern if it was co-expressed with MLC3nm, but it was distributed throughout the cytoplasm when expressed in combination with MLC1f, MLC3f or the slow skeletal/ventricular muscle isoform (MLC1sb). The MLC1sb was localized at the A-bands when it was co-expressed with MLC3nm or MLC1sa, while it was also distributed to the cytoplasm if co-expressed with MLC1f or MLC3f. Further, expression of chimeric cDNAs revealed that the N-terminal lobe of each isoprotein is responsible for the isoform-specific sorting pattern.