1,720,959 research outputs found
Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells
No full textThe lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy
Intracellular targeting of tail-anchored proteins
No full textTail-anchored (TA) proteins are integral membrane proteins that carry out important and diverse functions and that are targeted to their destination by unique post-translational pathways. Although the main pathway for targeting to the endoplasmic reticulum (ER) of TA proteins is represented by the TRC40/Get3 pathway, many TA proteins, i.e., those targeted in vivo to the mitochondrial outer membrane (MOM) and some ER targeted ones, can insert in vitro into pure phospholipid bilayers without assistance from any chaperone. The mechanism of precise in vivo targeting of these TA proteins is unclear. Cytochrome b5 is a spontaneously inserted TA protein, of which two forms are known, targeting the ER (b5-ER) or the MOM (b5-RR). The recombinant proteins microinjected into cultured cells are faithfully targeted, indicating that the targeting information is present in the protein and not in the mRNA. Using digitonin semi-permeabilized cells and in the presence of rabbit reticulocyte lysate as the source of cytosol, we have obtained faithful targeting for both forms of cytochrome b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. We tested also the effects of energy depletion of the RRL and we observed an effect on b5-ER, confirmed by the reduction of ER localization. Thus, energy-dependent chaperones are required for b5-ER’s avoidance of the MOM and its specific targeting to the ER. Taking this into consideration, we investigated the role of the principal TA protein target TRC40 system on b5-ER targeting, by using two different approaches in our system: the coiled-coil domain of WRB (ER membrane receptor of TRC40), which act as a decoy receptor, and WRB silencing. These experiments demonstrated that TCR40 plays only a modest role in the post-translational delivery of b5-ER to the ER membrane, suggesting the contribution of redundant pathways to b5 biogenesis
Mechanism of precise intracellular targeting of spontaneously inserting tail-anchored proteins
No full textTail-anchored (TA) proteins are membrane proteins that are targeted to their destination by unique post-translational pathways. Cytochrome b5 is a spontaneously inserted TA protein, of which two forms are known: one targeting the ER (b5-ER) and other targeting the MOM (b5-RR). Microinjection of the recombinant proteins into cells results in precise targeting of each of the two proteins, indicating that the targeting information is present in the protein. Using digitonin semi-permeabilized cells and in the presence of rabbit reticulocyte lysate (RRL), we have obtained faithful targeting for both forms of cytochrome b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. Attempting to find the chaperone responsible for the ER targeting, we observed that both TCR40 and Snd2 pathways play only a modest role, suggesting the contribution of redundant pathways to the biogenesis of b5. Recently, we found that Eyerestatin I, a p97 inhibitor, strongly inhibits the glycosylation of b5-ER. The effect seems to be specific for spontaneously inserted tail-anchored proteins, but the exact mechanism is still under investigation
Mechanism of precise intracellular targeting of tail-anchored proteins
No full textTail-anchored (TA) proteins are a subclass of type II integral membrane proteins that carry out important and diverse functions within cells. Many TA proteins, i.e., those targeted in vivo to the mitochondrial outer membrane (MOM) and a certain proportion of endoplasmic reticulum (ER) targeted ones, can insert into pure phospholipid bilayers without assistance from any chaperone. Cytochrome b5 is a spontaneously inserted TA protein, of which two forms are known: one targeting the ER (b5-ER) and other targeting the MOM (b5-RR). Microinjection of the recombinant proteins results in faithful targeting of each of the two proteins, indicating that the targeting information is present in the protein and not in the mRNA. Now, using digitonin semi-permeabilized cells and in presence of rabbit reticulocyte lysate (RRL) as the source of cytosol, we have obtained faithful targeting for both forms of cytochrome b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. We tested also the effects of energy depletion of the RRL and we observed an effect on b5ER, confirmed by the reduction of endoplasmic reticulum colocalization. Thus, energy-dependent chaperones are required for b5ER’s avoidance of the MOM and its specific targeting to the ER. Taking this into consideration, we have started now testing the role of the energy-dependent TRC40 system on b5ER targeting, by adding the coiled-coil domain of WRB (ER membrane receptor of TRC40). So far, our results tell us that WRBcc causes a minor shift in the insertion from ER to MOM, suggesting a modest role of this chaperone in the post-translational delivery of b5-ER to the ER membrane. We are presently investigating the role of other factors in b5 targeting, including chaperones of the Hsp70 family. Although this study is being carried out on model proteins b5-ER and b5-RR, it could be relevant for other physiologically crucial proteins, such as those of the Bcl2 family. Thus, one could speculate that under different conditions, or in different tissues, the availability of different targeting factors could result in different subcellular distribution of these proteins, with different effects on cell physiology
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Nicotine-Modulated Subunit Stoichiometry Affects Stability and Trafficking of α3β4 Nicotinic Receptor
No full textHeteromeric nAChRs are pentameric cation channels, composed of combinations of two or three α and three or two β subunits, which play key physiological roles in the central and peripheral nervous systems. The prototypical agonist nicotine acts intracellularly to upregulate many nAChR subtypes, a phenomenon that is thought to contribute to the nicotine dependence of cigarette smokers. The α3β4 subtype has recently been genetically linked to nicotine dependence and lung cancer; however, the mode of action of nicotine on this receptor subtype has been incompletely investigated. Here, using transfected mammalian cells as model system, we characterized the response of the human α3β4 receptor subtype to nicotine and the mechanism of action of the drug. Nicotine, when present at 1 mm concentration, elicited a ∼5-fold increase of cell surface α3β4 and showed a more modest upregulatory effect also at concentrations as low as 10 μM. Upregulation was obtained if nicotine was present during, but not after, pentamer assembly and was caused by increased stability and trafficking of receptors assembled in the presence of the drug. Experimental determinations as well as computational studies of subunit stoichiometry showed that nicotine favors assembly of pentamers with (α3)2(β4)3 stoichiometry; these are less prone than (α3)3(β4)2 receptors to proteasomal degradation and, because of the presence in the β subunit of an endoplasmic reticulum export motif, more efficiently transported to the plasma membrane. Our findings uncover a novel mechanism of nicotine-induced α3β4 nAChR upregulation that may be relevant also for other nAChR subtypes
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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