bluuebunny/crossref_metadata_2025_split huggingface dataset:
Misdirected vimentin messenger RNA alters cell morphology and motility
Authors: Morris Eric J. , Evason Kim , Wiand Cheryl , L’Ecuyer Thomas J. , Fulton Alice B. 
Published: April 2021

ABSTRACT
Localized messenger RNAs were first observed as embryonic determinants that altered development when mislocalized. In recent years localized mRNAs have been found for several cytoskeletal proteins, including actin, vimentin and several microtubule associated proteins. We sought to determine whether redirecting mRNA for a cytoskeletal protein to an inappropriate address would alter cellular phenotypes. To do so we generated vimentin mRNAs with a myc epitope tag and the β-actin 3′ untranslated region (3′ UTR) as a localization signal. When misdirected vimentin mRNAs are expressed in either fibroblasts or SW13 cells, cells develop numerous, extremely long processes; these cells also move more slowly to enter a wound of the monolayer. In situ hybridization revealed that the misdirected mRNA was often localized in the processes, in contrast to endogenous vimentin mRNA. The processes usually contained actin distal to the transgenic vimentin and microtubules proximal to it. SW13 cells lacking vimentin produced fewer and shorter processes, suggesting a dominant negative effect that involves recruitment of endogenous vimentin. Control experiments that transfected in constructs expressing tagged, correctly localized vimentin, or β-galactosidase that localized through the β-actin 3′ UTR, indicate that neither the shape nor the motility changes are solely due to the level of vimentin expression in the cell. This is direct evidence that the site of expression for at least one cytoskeletal mRNA alters the phenotype of the cell in which it is expressed. Messenger RNA localization is proving to be as essential for the normal maintenance of somatic cell phenotypes as embryonic determinants are for embryogenesis.
A role for TGFβ1 in osteoclast differentiation and survival
Authors: Fuller K. , Lean J. M. , Bayley K. E. , Wani M. R. , Chambers T. J. 
Published: April 2021

ABSTRACT
Recently, tumour necrosis factorrelated activationinduced cytokine (TRANCE) was shown to be necessary for osteoclast formation. We now report that TGFβ, a cytokine enriched in bone matrix, is also required. TGFβ not only powerfully synergized with TRANCE for induction of osteoclastlike cells (OCL) from bone marrow precursors and monocytes, but OCL formation was abolished by recombinant soluble TGFβ receptor II (TGFβsRII). Preincubation in TGFβ was as effective as simultaneous incubation with TRANCE. TGFβ-preincubation enhanced OCL formation at least partly by preventing the development of resistance to OCL-induction that otherwise occurs when precursors are incubated in M-CSF. OCL formed in TRANCE also showed more rapid apoptosis than OCL in TRANCE plus TGFβ. Like TGFβ, incubation on bone matrix prolonged and enhanced the sensitivity of precursors to OCL-induction by TRANCE, and this was reversed by TGFβsRII. Taken together, this data is compelling evidence for a model in which TGFβ in matrix or released from bonelining or other cells maintains and enhances the osteoclastforming potential of precursors as they migrate towards sites of cellbound TRANCE. Thus, the specific circumstances necessary for osteoclast formation and survival are TRANCE expression on osteoblastic cells and TGFβ in bone.
A constitutive region is responsible for nuclear targeting of 4.1R: modulation by alternative sequences results in differential intracellular localization
Authors: Luque Carlos M. , Correas Isabel 
Published: April 2021

ABSTRACT
Red blood cell protein 4.1, 4.1R, is an extreme variation on the theme of isoform multiplicity. The diverse 4.1R isoforms, mainly generated by alternative premRNA splicing, are localized at different intracellular sites, including the nucleus. To characterize nonerythroid 4.1 proteins lacking the most upstream translation initiation site, analyze their intracellular localization and define specific domains involved in differential intracellular targeting of 4.1R, we cloned 4.1 cDNAs lacking that translation initiation site.
Seven different 4.1R cDNAs were isolated. Four of these encoded 4.1R proteins localized predominantly to the nucleus and the other three localized to the cytoplasm. Three of the nuclear 4.1R isoforms did not contain the nuclear localization signal previously identified in the alternative exon 16. A comparative analysis of the exon composition of the naturally occurring 4.1R cDNAs cloned and of appropriate composite cDNA constructs, with the subcellular distribution of their respective products, demonstrated that a region encoded by constitutive exons, which is therefore common to all 4.1R isoforms and has been termed ‘core region’, had the capacity of localizing to the nucleus. This region was able to confer nuclear targeting to a cytosolic reporter. In protein 4.1R isoforms, the nuclear targeting of the core region is modulated by the expression of alternative exons. Thus, exon 5-encoded sequences eclipsed nuclear entry of the core region, resulting in 4.1R isoforms that predominantly distributed to the cytoplasm. Exon 5 was also able to confer cytoplasmic localization to a nuclear reporter. In protein 4.1R isoforms, when exons 5 and 16 were both expressed the nuclear targeting effect of exon 16 was dominant to the inhibitory effect observed by the expression of exon 5, yielding proteins that predominantly localized to the nucleus. Taken together, these results indicate that all 4.1R molecules contain a conserved region that is sufficient to target the protein to the nucleus, but that specific exonencoded sequences modulate this capacity by acting in a hierarchical order.
Role of the ribosome in sequence-specific regulation of membrane targeting and translocation of P-glycoprotein signal-anchor transmembrane segments
Authors: Zhang Jian-Ting , Han Ernest , Liu Yang 
Published: April 2021

ABSTRACT
It is thought that the topology of a polytopic protein is generated by sequential translocation and membrane integration of independent signal-anchor and stop-transfer sequences. Two well-characterized cell-free systems (rabbit reticulocyte lysate and wheat germ extract) have been widely used to study the biogenesis of secretory and membrane proteins, but different results have been observed with proteins expressed in these two different systems. For example, different topologies of P-glycoprotein (Pgp) were observed in the two systems and the cause was thought to be the source of ribosomes. To understand how the ribosome is involved in dictating membrane translocation and orientation of polytopic proteins, individual signal-anchor sequences of Pgp were dissected and examined for their membrane targeting and translocation in a combined system of wheat germ ribosomes (WGR) and rabbit reticulocyte lysate (RRL). Addition of wheat germ ribosomes to the rabbit reticulocyte lysate translation system can enhance, reduce, or have no effect on the membrane targeting and translocation of individual Pgp signal-anchor sequences, and these effects appear to be determined by the amino acid residues flanking each signal-anchor. Ribosomes regulate the membrane targeting and translocation of Pgp signal-anchors in a polytopic form differently from the same signal-anchors in isolation. Furthermore, we demonstrated that ribosomes regulate the membrane targeting and translocation of each signal-anchor cotranslationally and that this activity of ribosomes is associated with the 60S subunit. Based on this and previous studies, we propose a mechanism by which ribosomes dynamically dictate the membrane targeting and translocation of nascent polytopic membrane proteins.
Syk-dependent phosphorylation of microtubules in activated B-lymphocytes
Authors: Faruki Shamsa , Geahlen Robert L. , Asai David J. 
Published: April 2021

ABSTRACT
Syk is a protein-tyrosine kinase that is essential for B-lymphocyte development and B-cell signaling. Syk phosphorylates tubulin on tyrosine both in vitro and in intact lymphocytes. Here we show that α-tubulin present within the cytoskeletal microtubule network was phosphorylated in a Syk-dependent manner following the activation of B-cells by engagement of the B-cell antigen receptor or by treatment with the phosphotyrosine phosphatase inhibitor, pervanadate. Immunofluorescence staining of microtubule cytoskeletons and western blotting studies with antibodies to phosphotyrosine confirmed the phosphorylation of polymerized tubulin in Syk-expressing, but not Syk-deficient, cells. At low concentrations of pervanadate, centrosomes appeared to be preferentially tyrosine-phosphorylated. Tubulin phosphorylated to a high stoichiometry on tyrosine assembled into microtubules in vitro, and preassembled microtubules were also phosphorylated by Syk kinase in vitro. Thus, Syk has the capacity to interact with microtubule networks within the B-lymphocyte and catalyzes the phosphorylation of the α-tubulin subunit. Syk-dependent phosphorylation of microtubules may affect the ability of the microtubule cytoskeleton to serve as a platform upon which signaling complexes are assembled.
Identification and molecular-genetic characterization of a LAMP/CD68-like protein from Caenorhabditis elegans
Authors: Kostich Mitch , Fire Andrew , Fambrough Douglas M. 
Published: April 2021

ABSTRACT
Lysosome associated membrane proteins (LAMPs) constitute a family of vertebrate proteins located predominantly in lysosomes, with lesser amounts present in endosomes and at the cell surface. Macrosialin/CD68s are similar to LAMPs in their subcellular distribution and amino acid sequence and presumed structure across the carboxyl terminal two thirds of their length. The functions of LAMPs and CD68s are not known. In the present study, a bioinformatics approach was used to identify a Caenorhabditis elegans protein (LMP-1) with sequence and presumed structural similarity to LAMPs and CD68s. LMP-1 appears to be the only membrane protein in C. elegans that carries a GYXXΦ vertebrate lysosomal targeting sequence at its C terminus (where Φ is a large, hydrophobic residue). LMP-1 was found to be present from early embryonic stages through adulthood and to be predominantly localized at the periphery of a population of large, membrane-bound organelles, called granules, that are seen throughout the early embryo but in later stages are restricted to the cells of the intestine. Analysis of an LMP-1 deficient C. elegans mutant revealed that LMP-1 is not required for viability under laboratory conditions, but the absence of LMP-1 leads to an alteration in intestinal granule populations, with apparent loss of one type of granule.
Different properties of two isoforms of annexin XIII in MDCK cells
Authors: Lecat Sandra , Verkade Paul , Thiele Christoph , Fiedler Klaus , Simons Kai , Lafont Frank 
Published: April 2021

ABSTRACT
Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.
Sustained elevation in inositol 1,4,5-trisphosphate results in inhibition of phosphatidylinositol transfer protein activity and chronic depletion of the agonist-sensitive phosphoinositide pool
Authors: Speed Caroline J. , Mitchell Christina A. 
Published: April 2021

ABSTRACT
The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in a signal-terminating reaction. We have utilised cell lines that stably underexpress the 43 kDa 5-phosphatase, as a model system to investigate whether Ins(1,4,5)P3 can control the rate of its own formation by regulating the resupply of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). A sustained 2.6-fold elevation in the basal concentration of Ins(1,4,5)P3, in cell lines underexpressing the 43 kDa 5-phosphatase, correlated with a 32% reduction in the total cellular mass of PtdIns(4,5)P2. The depletion in cellular PtdIns(4,5)P2 was confined to a Triton-insoluble cell compartment, enriched in caveolin. In resting cells with elevated Ins(1,4,5)P3 concentrations resulting from underexpression of the 43 kDa 5-phosphatase, phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns(4)P) were depleted by 50% and PtdIns(4,5)P2 by 61% in the caveolin-enriched Tritoninsoluble compartment. Agonist stimulation resulted in the rapid turnover of phosphoinositides in the caveolinenriched Triton-insoluble fraction of vector-transfected cells, but not in cells with high basal Ins(1,4,5)P3 concentrations. Depletion of phosphoinositides from the caveolin-enriched Triton-insoluble pool in cells underexpressing the 43 kDa 5-phosphatase did not result from activation of phospholipase C isoenzymes, or inhibition of PtdIns 4-kinase or PtdIns(4)P 5-kinase activities. Significant inhibition of phosphatidylinositol transfer protein (PITP) activity (up to 70%) was observed in cells with elevated basal Ins(1,4,5)P3 concentrations; however, no reduction in PITPα protein expression was detected. These studies indicate that chronic elevation in cellular Ins(1,4,5)P3 concentrations decreases the PITP-mediated resupply of phosphoinositides in the caveolinenriched agonist-sensitive pool.
Inhibiting proteasome activity causes overreplication of DNA and blocks entry into mitosis in sea urchin embryos
Authors: Kawahara Hiroyuki , Philipova Rada , Yokosawa Hideyoshi , Patel Rajnikant , Tanaka Keiji , Whitaker Michael 
Published: April 2021

ABSTRACT
The proteasome has been shown to be involved in exit from mitosis by bringing about destruction of mitotic cyclins. Here, we present evidence that the proteasome is also required for proper completion of S phase and for entry into mitosis in the sea urchin embryonic cleavage cycle. A series of structurally related peptide-aldehydes prevent nuclear envelope breakdown in their order of inhibitory efficacies against the proteasome. Their efficacies in blocking exit from S phase and exit from mitosis correlate well, indicating that the proteasome is involved at both these steps. Mitotic histone HI kinase activation and tyrosine dephosphorylation of p34cdc2 kinase are blocked by inhibition of the proteasome, indicating that the proteasome plays an important role in the pathway that leads to embryonic p34cdc2 kinase activation. Arrested embryos continued to incorporate [3H]thymidine and characteristically developed large nuclei. Pre-mitotic arrest can be overcome by treatment with caffeine, a manoeuvre that is known to override the DNA replication checkpoint. These data demonstrate that the proteasome is involved in the control of termination of S phase and consequently in the initiation of M phase of the first embryonic cell cycle.
Differential distribution and defence involvement of antimicrobial peptides in mussel
Authors: Mitta Guillaume , Vandenbulcke Franck , Noël Thierry , Romestand Bernard , Beauvillain Jean Claude , Salzet Michel , Roch Philippe 
Published: April 2021

ABSTRACT
In previous papers, we characterised 3 types of 4-kDa, cysteine-rich, cationic antimicrobial peptides: MGDs (for Mytilus galloprovincialis defensins), mytilins and myticins, which are abundant in the mussel hemocytes. In the present work, we revealed a differential distribution of the cells expressing the different genes. In addition, using confocal and electron microscopy, we confirmed that defensins and mytilins were partially located in different sub-types of circulating hemocytes although the peptides can be located in the same cell, and even in the same granule. We also demonstrated that mytilins exert their microbicidal effect within the cells through the process of phagosome-mytilin granule fusion leading to the co-location of ingested bacteria and mytilins.
The COOH-terminal nuclear localization sequence of interferon γ regulates STAT1α nuclear translocation at an intracellular site
Authors: Subramaniam Prem S. , Larkin Joseph , Mujtaba Mustafa G. , Walter Mark R. , Johnson Howard M. 
Published: April 2021

ABSTRACT
We have recently shown that the nuclear localization of IFNγ is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFNγ, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1α activated by IFNγ. Treatment of IFNγ with antibodies to the C- terminal region (95-133) containing the NLS blocked the induction of STAT1α nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1α in IFNα treated cells. A deletion mutant of human IFNγ, IFNγ(1-123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFNγ receptor complex on cells with a Kd similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFNγ(1-123) to initiate the nuclear translocation of STAT1α, which is required for the biological activities of IFNγ following binding to the IFNγ receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1α nuclear localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFNγ with a Kd approx. 3×10−8 M−1 has been described by previous studies on the intracellular cytoplasmic domain of the IFNγ receptor α-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C- terminal NLS domain of IFNγ into the cytoplasm of cells before treatment of cells with IFNγ. These intracellular antibodies specifically blocked the nuclear translocation of STAT1α following the subsequent treatment of these cells extracellularly with IFNγ. These data show that the NLS domain of IFNγ interacts at an intracellular site to regulate STAT1α nuclear import. A C-terminal peptide of murine IFNγ, IFNγ(95-133), that contains the NLS motif, induced nuclear translocation of STAT1α when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1α nuclear translocation. In cells activated with IFNγ, IFNγ was found to as part of a complex that contained STAT1α and the importin-α analog Npi-1, which mediates STAT1α nuclear import. The tyrosine phosphorylation of STAT1α, the formation of the complex IFNγ/Npi-1/STAT1α complex and the subsequent nuclear translocation of STAT1α were all found to be dependent on the presence of the IFNγ NLS. Thus, the NLS of IFNγ functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1α.
Actin disruption alters the localization of tau in the growth cones of cerebellar granule neurons
Authors: Zmuda Jonathan F. , Rivas Rodolfo J. 
Published: April 2021

ABSTRACT
Cultured cerebellar granule neurons initially extend a single axon, followed by the extension of a second axon to attain a bipolar morphology. Differentiation culminates with the extension of several short dendrites from the cell body. In the present study, we determined the location of the dephosphorylated form of the microtubule-associated protein tau (dtau) within the growth cones of newly forming axons and examined whether this localization was influenced by the actin cytoskeleton. Following elongation of the initial axon at 2-3 days in vitro, dtau immunoreactivity was present along the entire length of the axon, becoming most intense just proximal to the growth cone. Dtau labeling dropped off dramatically along the microtubules of the growth cone and was undetectable along the most distal tips of these microtubules. As the initial axon continued to elongate at 3-4 days in vitro, the actin-rich growth cone peripheral domain characteristically underwent a dramatic reduction in size. Dtau immunoreactivity extended all the way to the most distal tips of the microtubules in the growth cones of these cells. Cytochalasin D and latrunculin A mimicked the effects of this characteristic reduction in growth cone size with regard to dtau localization in the growth cone. Depolymerization of filamentous actin caused the collapse of the peripheral domain and allowed dtau to bind all the way to the most distal tips of microtubules in the axon. Upon removal of the drugs, the peripheral domain of the growth cone rapidly re-formed and dtau was once again excluded from the most distal regions of growth cone microtubules. These findings suggest a novel role for actin in determining the localization of the microtubule- associated protein tau within the growth cones of neurons.
Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells
Authors: Xu Jiliu , Ziemnicka Dorota , Merz George S. , Kotula Leszek 
Published: April 2021

ABSTRACT
Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 μm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid α-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.