1,720,979 research outputs found
Studying the effects of semaphorins on oligodendrocyte lineage cells
No full textOligodendrocytes are the myelinating cells of the central nervous system. The role of oligodendrocytes in health and disease has been considerably enhanced by the development of methods to isolate and culture oligodendrocytes from central nervous system tissue. The cellular and molecular mechanisms involved in oligodendrocyte differentiation can be identified by challenging oligodendrocyte progenitors cells (OPCs) by altering their extracellular environment and intrinsic differentiation pathways. To address these issues, it is imperative to develop an in vitro protocol where pure OPCs are isolated and cultured in the presence of inhibitory developmental and differentiation cues like Semaphorin 3A. In this chapter, we describe methods to isolate and culture OPCs from neonatal rat brain tissue and further characterise their differentiation into oligodendrocytes. The described protocol is relatively simple in comparison to existing protocols and can be used to study the effect of lesion-associated inhibitors like Semaphorin 3A on oligodendrocyte differentiation
Characterizing the Molecular Mechanisms of Axon Guidance: Activation and Regulation of the Axon Guidance Receptor Plexin A
No full textNeuronal connectivity is precisely determined by axonal pathfinding during development. The navigating axons detect attractive and repulsive environmental cues by axon guidance receptors. However, the biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are the largest family of axon guidance cues and utilize Plexin receptors to exert repulsive effects on axon extension. The intracellular region of Plexins contains a Ras GTPase activating protein (GAP) domain, which is necessary for repulsive guidance effects. Previous studies suggest that activation of Plexin RasGAP requires interactions with both Semaphorin at the extracellular region and a Rho-family GTPase at the Rho family GTPase-binding domain (RBD). Interestingly, Semaphorin repulsion can be rapidly "turned-off" by other distinct cues and signaling cascades. However, the molecular mechanisms to activate or modulate Plexin RasGAP remain unclear. First, to further understand how the Plexin RasGAP is activated, I collaborated with the Zhang lab, and following determination of the crystal structure of the intracellular region of Plexin, I examined the roles of residues interfacing with the RasGAP domain using functional mutagenesis in the Drosophila model system. Our results demonstrate that Plexin exhibits an auto-inhibited conformation, and suggest that interaction among the previously uncharacterized juxtamembrane segment, the RBD, and the RasGAP domain is critical for Plexin RasGAP activation. Second, to better understand how Semaphorin/Plexin signaling is modulated, I characterized the results of a large-scale screen to look for proteins interacting with the cytoplasmic portion of Plexin and identified the phosphoserine binding protein 14-3-3epsilon as a specific Plexin-interacting protein. My results reveal that 14-3-3epsilon is specifically required for axon guidance during development. Moreover, Protein kinase A is found to phosphorylate Plexin in the RasGAP domain and mediates the 14-3-3epsilon interaction. Plexin-14-3-3epsilon interactions prevent Plexin from interacting with its Ras-family GTPase substrate, which effectively switches Plexin-mediated axonal repulsion to Integrin-mediated adhesion. These findings uncover both a new molecular integration point between important axon guidance signaling pathways and a biochemical logic by which this guidance information is coalesced to steer the growing axon. Therefore, these new observations on activating and silencing specific signals that are repulsive to axon growth also illuminate new approaches to neutralize axonal growth inhibition and encourage axon regeneration
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Actin filaments—A target for redox regulation
Get PDFActin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through noncovalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates—the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL—and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. © 2016 Wiley Periodicals, Inc.Fil: Wilson Rodriguez, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Chile; Chile. Center for Geroscience; ChileFil: Terman, Jonathan R.. The University of Texas Southwestern; Estados UnidosFil: González Billault, Christian. Universidad de Chile; Chile. Center for Geroscience; Chile. The Buck Institute for Research on Aging; Estados UnidosFil: Ahmed, Giasuddin. The University of Texas Southwestern; Estados Unido
Variations on the Author
Get PDF“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
Get PDFWe 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
Characterizing New Molecules and Mechanisms of Semaphorin/Plexin/MICAL Signaling
No full textThe mechanisms that regulate the cellular behaviors including morphology, motility, navigation, and connectivity that are critical for normal human health are still incompletely understood. These types of behaviors are regulated through the ability of guidance cues that are present outside of cells to exert precise effects on the cell’s internal actin cytoskeleton. To help characterize an underlying logic for these cellular changes and proper cellular function I have been characterizing how one of the largest families of extracellular guidance cues, members of the Semaphorin family of proteins, induces actin cytoskeletal changes. Interestingly, Semaphorins have been found to inhibit the movement of cells and their membranous processes but the molecules linking them to these specific behaviors have remained poorly understood. Therefore, during my graduate work, I began further characterizing a new family of proteins, the MICALs that were identified as cytoplasmic binding partners of the Semaphorin receptor Plexin. Combining Drosophila genetics with in vitro biochemical assays, my work revealed that Mical regulates actin organization both in vivo and in vitro and is a novel actin disassembly factor. These results provide a new basis for understanding how extracellular guidance cues regulate the actin cytoskeleton. I then went on to further explore Mical-mediated actin filament (F-actin) disassembly. In one line of investigation, my work revealed that Mical plays an antagonistic role to F-actin stabilizing/bundling proteins including fascin and espin in regulating the F-actin cytoskeleton in vivo. This work also indicated that Semaphorin/Plexin/Mical activity not only directly disassembles the F-actin cytoskeleton but also triggers other actin regulatory proteins to reorganize a more complex F-actin network, resulting in increased cellular plasticity. In another line of investigation, I found that the Abl non-receptor tyrosine kinase is a new Mical-interacting protein. My functional assays revealed that Abl and Semaphorin/Plexin/Mical work together to regulate F-actin arrangements and these interactions are conserved in many different contexts including bristle cell morphology, axon guidance, and cancer cell survival and invasion. Thus, I have found that MICAL family proteins are novel controllers of the actin cytoskeleton, functioning directly on F-actin and with other actin regulatory proteins to modulate diverse cellular behaviors
Regulatory Mechanisms of Semaphorin/Plexin/Mical-Mediated F-actin Disassembly and Cellular Remodeling
Get PDFDynamic changes to the actin cytoskeleton modify the shape of cells and their membranous extensions, and underlie diverse developmental and functional events in multiple tissues including migration, navigation, and connectivity. Semaphorins, together with their Plexin receptors, are a large family of extracellular cues that trigger complex cytoskeletal rearrangements to direct these cellular phenomena, but the mechanisms regulating their effects are poorly understood. Emerging evidence identifies Mical, a conserved oxidoreductase (Redox) enzyme, as a critical component in Semaphorin/Plexin signaling through its post-translational oxidation of F-actin, which promotes actin instability and disassembly. How this Mical-mediated redox regulation of actin dynamics is locally positioned and coordinated with the activity of other actin regulatory proteins to achieve specific, targeted effects on the cytoskeleton remains unknown. Therefore, as a part of my dissertation research, I used a genetic assay to begin to address these questions and search for proteins that could alter Semaphorin/Plexin/Mical signaling effects on the cytoskeleton. In this dissertation, I present my discovery of a functional interplay between Mical and two critical new interactors - cofilin, a well-known ubiquitous F-actin regulatory protein, and Sisyphus, an unconventional class XV myosin. With regards to cofilin, my in vivo genetic/functional assays reveal that cofilin activity is required for and enhances Semaphorin/Plexin/Mical-dependent cytoskeletal rearrangements and morphological changes. Additionally, in vitro biochemical assays demonstrate that cofilin preferentially binds Mical-oxidized actin and accelerates its disassembly. Together, these findings indicate that cofilin and Mical act as a functional pair in both neuronal and non-neuronal cells to rapidly and efficiently disassemble actin filaments. Similarly, my results reveal that Sisyphus is necessary and sufficient for triggering Semaphorin/Plexin/Mical-dependent F-actin disassembly/cellular remodeling. Moreover, using in vivo functional assays, I find that Sisyphus uses its myosin motor activity and the first MyTH4 domain of its C-terminal tail region to modify the subcellular localization of Mical. In this way, Sisyphus spatially controls Mical-dependent F-actin disassembly/cellular remodeling. Therefore, both cofilin and Sisyphus function to promote Mical-mediated F-actin disassembly; thereby, they act as critical regulators of Semaphorin/Plexin/Mical-mediated effects on cytoskeletal and morphological dynamics. Thus, my findings unveil novel molecular and biochemical mechanisms that orchestrate cellular, developmental, and neural biology
Progranulin Biology: Small Molecule Enhancers of Progranulin Expression and Biochemical Analysis of Granulin Receptors
No full textFrontotemporal dementia (FTD) is the second most common presenile dementia syndrome. Mutations in the GRN gene account for about 20% of patients with familial FTD. The protein encoded by GRN, progranulin, is a secreted glycoprotein with growth factor-like and immunomodulatory activities. Human progranulin contains seven granulin domains (denoted granulins A through F) that can be individually liberated following proteolytic cleavage. It is uncertain whether the holoprotein, the granulins or both mediate the biological effects of progranulin. All pathogenic GRN mutations result in haploinsufficiency and decreased extracellular progranulin. Therefore, increasing progranulin expression from the wild-type allele or (pro)granulin receptor agonists may be therapeutic in FTD. The overall goals of the work presented here were to identify small molecule enhancers of progranulin expression and (pro)granulin receptors that can be drug targets for the treatment and prevention of GRN deficient FTD. As described here, I discovered that suberoylanilide hydroxamic acid (SAHA), an FDA-approved histone deacetylase (HDAC) inhibitor, enhances GRN expression and nearly normalizes progranulin levels in haploinsufficient primary human cells from GRN mutation carriers. I also discovered that granulin A binds three proteins in solubilized extracts of rodent brain membranes: wolframin, excitatory amino acid transporter 1 (EAAT1), and the α3 subunit of the Na+/K+ ATPase. I argue that these proteins are candidates for a putative granulin receptor
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