bluuebunny/crossref_metadata_2025_split huggingface dataset:
AAA domains and organization of the dynein motor unit
Authors: King Stephen M. 
Published: April 2021

ABSTRACT
Dyneins contain one-three microtubule motor units that are each derived from the C-terminal globular head of a heavy chain. The N-terminal regions of the heavy chains form stems that are required for intra-dynein associations. The microtubule-binding sites are located at the terminus of a short stalk that emanates from each globular head. Recent electron microscopic analysis indicates that the dynein head has a heptameric toroidal organization. This finding is echoed by the identification of six AAA (ATPases associated with cellular activities) domains and a seventh unrelated unit within this heavy chain region. At least two of these AAA domains can bind nucleotide, although only one appears able to hydrolyze ATP. Several other AAA domain proteins exhibit a similar annular organization of six AAA units. Detailed structural information is available for several AAA proteins, including N-ethylmaleimidesensitive vesicle-fusion protein and the RuvB motor involved in DNA migration and resolution of Holliday junctions. The resulting structural parallels allow intriguing predictions to be made concerning dynein organization and motor function.
RecQ-like helicases: the DNA replication checkpoint connection
Authors: Frei Christian , Gasser Susan M. 
Published: April 2021

ABSTRACT
The eukaryotic homologues of the Escherichia coli RecQ DNA helicase play conserved roles in the maintenance of genome stability. Results obtained in yeast and mammalian systems are beginning to form a coherent picture about what these helicases do to ensure normal cell division and why humans who lack these enzymes are cancer prone. Recent data suggest that the yeast enzyme Sgs1p, as well as two human homologues, which are encoded by the Bloom’s and Werner’s syndrome genes, function during DNA replication and possibly in a replication checkpoint specific to S phase.
Assembly of multiple dystrobrevin-containing complexes in the kidney
Authors: Loh Nellie Y. , Newey Sarah E. , Davies Kay E. , Blake Derek J. 
Published: April 2021

ABSTRACT
Dystrophin is the key component in the assembly and maintenance of the dystrophin-associated protein complex (DPC) in skeletal muscle. In kidney, dystroglycan, an integral component of the DPC, is involved in kidney epithelial morphogenesis, suggesting that the DPC is important in linking the extracellular matrix to the internal cytoskeleton of kidney epithelia. Here, we have investigated the molecular architecture of dystrophin-like protein complexes in kidneys from normal and dystrophin- deficient mice. Using isoform-specific antibodies, we show that the different cell types that make up the kidney maintain different dystrophin-like complexes. These complexes can be broadly grouped according to their dystrobrevin content: β-dystrobrevin containing complexes are present at the basal region of renal epithelial cells, whilst α-dystrobrevin-1 containing complexes are found in endothelial and smooth muscle cells. Furthermore, these complexes are maintained even in the absence of all dystrophin isoforms. Thus our data suggest that the functions and assembly of the dystrophin-like complexes in kidney differ from those in skeletal muscle and implicate a protein other than dystrophin as the primary molecule in the assembly and maintenance of kidney complexes. Our findings also provide a possible explanation for the lack of kidney pathology in Duchenne muscular dystrophy patients and mice lacking all dystrophin isoforms.
Amino-terminal domain of classic cadherins determines the specificity of the adhesive interactions
Authors: Klingelhöfer Jörg , Troyanovsky Regina B. , Laur Oscar Y. , Troyanovsky Sergey 
Published: April 2021

ABSTRACT
Classic cadherins are transmembrane receptors involved in cell type-specific calcium-dependent intercellular adhesion. The specificity of adhesion is mediated by homophilic interactions between cadherins extending from opposing cell surfaces. In addition, classic cadherins can self- associate forming lateral dimers. Whereas it is widely excepted that lateral dimerization of cadherins is critical for adhesion, details of this process are not known. Yet, no evidence for physical association between different classic cadherins in cells expressing complex cadherin patterns has been reported. To study lateral and adhesive intercadherin interactions, we examined interactions between two classic cadherins, E- and P-cadherins, in epithelial A-431 cells co- producing both proteins. We showed that these cells exhibited heterocomplexes consisting of laterally assembled E- and P-cadherins. These complexes were formed by a mechanism involving Trp156 of E-cadherin. Removal of calcium ions from the culture medium triggered a novel Trp156-independent type of lateral E-cadherin-P-cadherin association. Notably, an antiparallel (adhesive) mode of interaction between these cadherins was negligible. The specificity of adhesive interaction was localized to the amino-terminal (EC1) domain of both cadherins. Thus, EC1 domain of classic cadherins exposes two determinants responsible for nonspecific lateral and cadherin type- specific adhesive dimerization.
The chemotactic and mitogenic effects of platelet-derived growth factor-BB on rat aorta smooth muscle cells are inhibited by basic fibroblast growth factor
Authors: Facchiano Antonio , Marchis Francesco De , Turchetti Elisabetta , Facchiano Francesco , Guglielmi Mario , Denaro Alessandra , Palumbo Roberta , Scoccianti Marco , Capogrossi Maurizio C. 
Published: April 2021

ABSTRACT
In response to endovascular injury, platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) are released locally and modulate vascular smooth muscle cells (SMC) proliferation and migration within the vascular wall. The aim of the present in vitro study was to determine how rat aorta SMC respond to the simultaneous exposure to PDGF-BB and bFGF. In a modified Boyden chamber assay bFGF exhibited a dose-dependent effect to inhibit the chemotactic action of PDGF-BB. A comparable result was observed in proliferation assays. In contrast, MIP-1 β, epidermal growth factor (EGF), fibronectin and acidic FGF (aFGF) did not inhibit the chemotactic effect of PDGF-BB. Denatured bFGF did not exert an inhibitory effect and neutralizing antibodies either to bFGF or to bFGF-receptor abolished the inhibition observed in the presence of bFGF. The role played by PDGF receptor α (PDGF-Rα) was investigated in PDGF-Rα-dominant negative-transfected SMC, by selectively blocking PDGF- BB-binding to PDGF-Rα with neomycin, by neutralizing PDGF-Rα with a monoclonal antibody and by selectively stimulating PDGF-Rα with PDGF-AA; in all cases the effect of bFGF to inhibit PDGF-BB-directed SMC migration was abolished.
These in vitro studies show that bFGF significantly inhibits PDGF-BB-induced SMC migration and proliferation and that this effect is mediated by both PDGF-Rα and bFGF receptor.
Tension on chromosomes increases the number of kinetochore microtubules but only within limits
Authors: King Jennifer M. , Nicklas R. Bruce 
Published: April 2021

ABSTRACT
When chromosomes attach properly to a mitotic spindle, their kinetochores generate force in opposite directions, creating tension. Tension is presumed to increase kinetochore microtubule number, but there has been no direct evidence this is true. We micromanipulated grasshopper spermatocyte chromosomes to test this assumption and found that tension does indeed affect the number of kinetochore microtubules. Releasing tension at kinetochores causes a drop to less than half the original number of kinetochore microtubules. Restoring tension onto these depleted kinetochores restores the microtubules to their original number. However, the effects of tension are limited. Prometaphase kinetochores, when under normal tension from mitotic forces, have about half as many microtubules as they will in late metaphase. We imposed a tension force of 6×10−5 dynes, three times the normal tension, on prometaphase kinetochores. The elevated tension did not drive kinetochore microtubule number above normal prometaphase values. Tension probably increases the number of kinetochore microtubules by slowing their turnover rate. The limited effect of tension at prometaphase kinetochores suggests that they have fewer microtubule binding sites than at late metaphase. The relatively few sites available in prometaphase may be the decisive sites whose binding of microtubules regulates the dynamics of transient kinetochore constituents, including checkpoint components.
Antisense oligonucleotide experiments elucidate the essential role of titin in sarcomerogenesis in adult rat cardiomyocytes in long-term culture
Authors: Person Veronika , Kostin Sawa , Suzuki Keisuke , Labeit Siegfried , Schaper Jutta 
Published: April 2021

ABSTRACT
An essential role of titin as a molecular ruler in sarcomerogenesis has been frequently discussed. In this study, we tested the hypothesis that the expression of titin is a prerequisite for thick filament incorporation into sarcomeres by using an antisense oligonucleotide approach to interfere with titin translation in the de-/ redifferentiation model of adult rat cardiomyocytes (ARC) in long-term culture. As a first step, the growth pattern ranging from rod shape to round and later to spreading cells and the cell surface area of ARC were quantitatively evaluated and standardized. This represents the basis for experiments interfering with sarcomere formation using three different antisense phosphorothioate oligonucleotides (S-ODN) at a dosage of 10 μM specific for titin mRNA. Presence of fluorescein labeled S-ODN in ARC indicated cellular uptake and both, antisense and random S-ODN, induced a significant increase in cell size as compared with control untreated ARC. At days 12 and 16 in culture, antisense S-ODN treatment resulted in reduced expression of titin and disturbance of myosin incorporation into sarcomeres, evident by diffuse myosin labeling and a significantly decreased area of regular myosin cross-striation (control 75%, day 12 S-ODN 20%, day 16 14%) shown by laser scanning confocal microscopy. Cellular integrity indicated by presence of α-actinin was not disturbed. These findings provide evidence for the role of titin as a template for myosin incorporation and therefore as a prerequisite for sarcomerogenesis.
Intact Ca2+-binding sites are required for targeting of annexin 1 to endosomal membranes in living HeLa cells
Authors: Rescher Ursula , Zobiack Nicole , Gerke Volker 
Published: April 2021

ABSTRACT
Annexin 1 is a Ca2+-regulated membrane binding protein and a major substrate of the epidermal growth factor receptor kinase. Because of its properties and intracellular distribution, the protein has been implicated in endocytic trafficking of the receptor, in particular in receptor sorting occurring in multivesicular endosomes. Up to now, however, the localization of annexin 1 to cellular membranes has been limited to subcellular fractionation and immunocytochemical analyses of fixed cells. To establish its localization in live cells, we followed the intracellular fate of annexin 1 molecules fused to the Green Fluorescent Protein (GFP). We show that annexin 1-GFP associates with distinct, transferrin receptor-positive membrane structures in living HeLa cells. A GFP chimera containing the Ca2+/phospholipid-binding protein core of annexin 1 also shows a punctate intracellular distribution, although the structures labeled here do not resemble early but, at least in part, late endosomes. In contrast, the cores of annexins 2 and 4 fused to GFP exhibit a cytoplasmic or a different punctate distribution, respectively, indicating that the highly homologous annexin core domains carry distinct membrane specificities within live cells. By inactivating the three high-affinity Ca2+ binding sites in annexin 1 we also show that endosomal membrane binding of the protein in live HeLa cells depends on the integrity of these Ca2+ binding sites. More detailed analysis identifies a single Ca2+ site in the second annexin repeat that is crucially involved in establishing the membrane association. These results reveal for the first time that intracellular membrane binding of an annexin in living cells requires Ca2+ and is mediated in part through an annexin core domain that is capable of establishing specific interactions.
Fast transport of neurofilament protein along microtubules in squid axoplasm
Authors: Prahlad Veena , Helfand Brian T. , Langford George M. , Vale Ron D. , Goldman Robert D. 
Published: April 2021

ABSTRACT
Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 μm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 μm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal proteins, throughout neurons.
Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells
Authors: Yan Li , Moses Marsha A. , Huang Sui , Ingber Donald E. 
Published: April 2021

ABSTRACT
The growth and regression of capillary blood vessels during angiogenesis is greatly influenced by changes in the activity of matrix metalloproteinases (MMPs), which selectively degrade extracellular matrix (ECM) and thereby modulate capillary endothelial cell shape, growth and viability. However, changes in cell-ECM binding and cell spreading have also been reported to alter MMP secretion and activation. Studies were carried out to determine whether changes in integrin binding or cell shape feed back to alter MMP-2 processing in human capillary endothelial (HCE) cells. Catalytic processing of proMMP-2 to active MMP-2 progressively decreased when HCE cells were cultured on dishes coated with increasing densities of fibronectin (FN), which promote both integrin binding and cell spreading. Conversely, the highest levels of active MMP-2 were detected in round cells cultured on low FN. When measured 24 hours after plating, this increase in active MMP-2 was accompanied by a concomitant rise in mRNA and protein levels for the membrane-type 1 MMP (MT1-MMP), which catalyzes the cleavage of proMMP-2. To determine whether proMMP-2 processing was controlled directly by integrin binding or indirectly by associated changes in cell shape, round cells on low FN were allowed to bind to microbeads (4.5 μm diameter) coated with a synthetic RGD peptide or FN; these induce local integrin receptor clustering without altering cell shape. ProMMP-2 activation was significantly decreased within minutes after bead binding in these round cells, prior to any detectable changes in expression of MT1-MMP, whereas binding of beads coated with control ligands for other transmembrane receptors had no effect. This inhibitory effect was mimicked by microbeads coated with activating antibodies against αVβ3 and β1 integrins, suggesting a direct role for these cell-surface ECM receptors in modulating proMMP-2 activation. Similar inhibition of proMMP-2 processing by integrin binding, independent of cell spreading, was demonstrated in cells that were cultured on small, microfabricated adhesive islands that prevented cell spreading while presenting a high FN density directly beneath the cell. Interestingly, when spread cells were induced to round up from within by disrupting their actin cytoskeleton using cytochalasin D, proMMP-2 processing did not change at early times; however, increases in MT1-MMP mRNA levels and MMP-2 activation could be detected by 18 hours. Taken together, these results suggest the existence of two phases of MMP-2 regulation in HCE cells when they adhere to ECM: (1) a quick response, in which integrin clustering alone is sufficient to rapidly inhibit processing of proMMP-2 and (2) a slower response, in which subsequent cell spreading and changes in the actin cytoskeleton feed back to decrease expression of MT1-MMP mRNA and, thereby, further suppress cellular proteolytic activity.
The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1
Authors: Bryan Janine T. , Morasso Maria I. 
Published: April 2021

ABSTRACT
The murine Dlx3 protein is a putative transcriptional activator that has been implicated during development and differentiation of epithelial tissue. Dlx3 contains a homeodomain and mutational analysis has revealed two regions, one N-terminal and one C-terminal to the homeodomain, that act as transcriptional activators in a yeast one-hybrid assay. In addition to transactivation, data are presented to demonstrate specific DNA binding and an association between Dlx3 and the Msx1 protein in vitro. Immunohistochemical analysis confirmed coexpression of Dlx3 and Msx1 proteins in the differentiated layers of murine epidermal tissues.
Transcription factor function requires nuclear localization. In this study, the intracellular localization of the green fluorescent protein fused to Dlx3 was examined in keratinocytes induced to differentiate by calcium and is shown to localize to the nucleus. A bipartite nuclear localization signal (NLS) was identified by mutational analysis and shown to be sufficient for nuclear localization. This was demonstrated by insertion of the Dlx3 bipartite NLS sequence into a cytoplasmic fusion protein, GFP-keratin 14, which functionally redirected GFP-keratin 14 expression to the nucleus. Further analysis of Dlx3 NLS mutants revealed that the Dlx3 NLS sequences are required for specific DNA binding, transactivation potential and interactions with the Msx1 protein.
SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands
Authors: Niemeyer Barbara A. , Schwarz Thomas L. 
Published: April 2021

ABSTRACT
Fusion of vesicles with target membranes is dependent on the interaction of target (t) and vesicle (v) SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) proteins located on opposing membranes. For fusion at the plasma membrane, the t-SNARE SNAP-25 is essential. In Drosophila, the only known SNAP-25 isoform is specific to neuronal axons and synapses and additional t-SNAREs must exist that mediate both non-synaptic fusion in neurons and constitutive and regulated fusion in other cells. Here we report the identification and characterization of SNAP-24, a closely related Drosophila SNAP-25 homologue, that is expressed throughout development. The spatial distribution of SNAP-24 in the nervous system is punctate and, unlike SNAP-25, is not concentrated in synaptic regions. In vitro studies, however, show that SNAP-24 can form core complexes with syntaxin and both synaptic and non-synaptic v-SNAREs. High levels of SNAP-24 are found in larval salivary glands, where SNAP-24 localizes mainly to granule membranes rather than the plasma membrane. During glue secretion, the massive exocytotic event of these glands, SNAP-24 containing granules fuse with one another and the apical membrane, suggesting that glue secretion utilizes compound exocytosis and that SNAP-24 mediates secretion.
Defective organellar membrane protein trafficking in Ap3b1-deficient cells
Authors: Yang Wei , Li Chaoying , Ward Diane M. , Kaplan Jerry , Mansour Suzanne L. 
Published: April 2021

ABSTRACT
AP-3 is a heterotetrameric protein complex involved in intracellular vesicle transport. Molecular analyses show that Ap3b1, which encodes the AP-3 β3A subunit, is altered in pearl mice. To provide genetic evidence that mutation of Ap3b1 is responsible for the pearl phenotype and to determine the null phenotype, the Ap3b1 gene was disrupted by homologous recombination. Mice homozygous for the resulting allele, Ap3b1LN, or compound heterozygotes with pearl, displayed phenotypes similar to those of pearl mice, confirming that Ap3b1 is the causal gene for pearl. Moreover, pearl is likely to be a hypomorph as the Ap3b1LN homozygotes had a lighter coat color and accumulated fewer of the μ3 and δ3 subunits of AP-3 than did pearl mice. Finally, immunofluorescence analysis of fibroblasts and melanocytes cultured from Ap3b1LN homozygotes revealed that the lysosomal membrane proteins Lamp I and Lamp II and the melanosomal membrane protein tyrosinase were mislocalized. In particular, the Lamp proteins were clustered on the cell surface. These findings strengthen the evidence for an alternate pathway via the plasma membrane for cargo normally transported to organelles by AP-3.
Analysis of gene expression by microarrays: cell biologist’s gold mine or minefield?
Authors: Schulze Almut , Downward Julian 
Published: April 2021

ABSTRACT
The development of DNA microarrays to study simultaneously the level of mRNA expressed from thousands of genes offers great promise to cell biologists. Microarrays can be used to gain detailed information about transcriptional changes involved in a specific pathway, potentially leading to the identification of novel components of the signalling system. They can also be used to obtain a fingerprint of the transcriptional status of the cell under a given condition, which may be useful for characterising the pathways used in response to novel stimuli. The use of microarrays will generate huge amounts of expression data, contributing to the transformation of biology from a data-poor to a data-rich science. Whether this leads to real advances in the understanding of cell biological problems will depend on the development of methodologies, both in experimental biology and in bioinformatics, that allow meaningful knowledge to be extracted from all this information.
LIF transduces contradictory signals on capillary outgrowth through induction of Stat3 and P41/43MAP kinase
Authors: Paradis Hélène , Gendron Robert L. 
Published: April 2021

ABSTRACT
The signaling pathways regulating blood vessel growth and development are not well understood. In the present report, an in vitro model was used to identify signaling pathways regulating capillary formation in embryonic endothelial cells. Basic fibroblast growth factor (bFGF) plus leukemia inhibitory factor (LIF) optimally stimulate the formation of capillary-like structures of the embryonic endothelial cell line IEM. LIF stimulation of IEM cells leads to activation of the Stat3 as well as the P41/43mitogen-activated protein kinase (P41/43MAPK) cascade, while bFGF does not activate Stat3 but does induce the P41/43MAPK cascade. Inhibition of Stat3 DNA-binding activity by expression of a dominant inhibitory Stat3 mutant increases the capillary outgrowth of the IEM cells induced by LIF. Increased Stat3 activity by overexpression of the wild-type Stat3 greatly reduced capillary outgrowth. In contrast, inhibition of the P41/43MAPK cascade using a MEK-1 inhibitor dramatically inhibits the LIF-induced capillary outgrowth. Moreover, the increased formation of capillary-like structures of the IEM cells mediated by Stat3 inhibition does not overcome the requirement for activation of the P41/43MAPK pathway for capillary outgrowth. Stat3 activity correlates with the LIF-induced expression of the negative feedback regulators of the Janus (JAK) family of tyrosine kinases, SOCS-1 and SOCS-3. These results provide evidence that Stat3 acts as a negative regulator of capillary outgrowth, possibly by increasing SOCS-1 or SOCS-3 expression. The contradictory signals stimulated by LIF could be necessary to control the intensity of the response leading to capillary outgrowth in vivo.