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Defining the Oligomerization Domains of Tau Using a Split-Luciferase Strategy
Get PDFMicrotubules (MTs) are highly dynamic components of the cell cytoskeleton that are necessary for many functions, including cell division, cellular locomotion, and intracellular transport. An essential mechanistic feature of MT physiology is dynamic instability, which is characterized by the frequent polymerization and depolymerization of tubulin subunits at MT ends. This dynamicity is critical to MT function and is regulated by MT-associated proteins (MAPs), which interact with tubulin dimers and/or the MTs themselves. Tau is a prominent neuronal MAP that stabilizes MTs by promoting growth events, stability, and by suppressing shortening events. On the other hand, dysregulation and mutation of tau are associated with pathogenesis in various neurodegenerative diseases, such as Alzheimer’s disease (AD), frontotemporal dementia with Parkinsonism-17 (FTDP-17), and progressive supranuclear palsy (PSP). Taken together, it is critical to understand both normal tau physiology as well as how altered tau function leads to disease pathogenesis.
Previous research has suggested that tau is able to dimerize or oligomerize via its N-terminal projection domain as part of its normal function. One currently proposed model, based on in vitro data, is that two tau molecules form an “electrostatic zipper” in which the N-termini of the two molecules associate in an antiparallel fashion, with the C-termini containing the MT-binding region of each tau molecule extending away from one another. If correct, this model could explain many features of tau action. We investigated this hypothesis in mammalian cells using a split-luciferase strategy in order to (i) test the above stated model for tau oligomerization in cells and (ii) identify and map regions of the protein that are capable of tau-tau oligomerization.
We found that constructs containing the N-terminus of tau produce significantly higher luciferase signals indicative of oligomerization compared to constructs containing the C-terminus. More specifically, the construct containing amino acids 1-120 produces the strongest luciferase signal, consistent with our proposed model that the N-terminus of tau is responsible, at least in part, for its oligomerization activity. Interestingly, C-terminal regions of tau are also capable of promoting tau oligomerization. Taken together, our data suggest that both the N- and C- termini of tau are each sufficient to promote tau oligomerization in mammalian cells
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Mechanistic Insights into the Regulation of Microtubule Assembly and Dynamic Instability by Tau and MMAE
Get PDFMicrotubules (MTs) are dynamic cytoskeletal polymers that are essential for many cellular processes, including cell division, maintenance of cellular shape, intracellular transport, and cell signaling. Their dynamic, switch-like behavior, known as MT dynamic instability, arises from the conformational changes associated with GTP hydrolysis by β-tubulin, but exactly how the tubulin conformational cycle contributes to the overall MT dynamic state remains unclear. Proper regulation of MT dynamicity is critical across almost all cell types, but their necessity for proper chromosome alignment and division during mitosis make them an especially effective target for anti-cancer drugs targeting rapidly dividing tumor cells. Despite the broad use of these MT-targeting agents (MTAs) as chemotherapeutics, we often lack understanding of the mechanisms of action underlying the global changes they enact on MT dynamics. We investigated the binding of MTA Monomethyl auristatin E (MMAE), as a free drug or as an antibody-drug conjugate (ADC), to MTs and free tubulin subunits, and characterized its effects upon MT dynamics and MT morphology as well as cell proliferation, cell cycle regulation, and the generation of mitotic spindle abnormalities in cultured human cells. In combination with comparisons made to other MTAs, our data provide further insights into the molecular mechanisms underlying normal MMAE action as well as those governing MMAE ADC-induced peripheral neuropathy. In cells, MT-associated proteins (MAPs) help to regulate MT dynamics. Tau is a neuronal MAP that regulates the critical growing and shortening behaviors of neuronal MTs, and its normal activity is essential for neuronal development and maintenance. Accordingly, aberrant tau action is tightly associated with Alzheimer's disease and is genetically linked to several additional neurodegenerative diseases known as tauopathies. Indeed, one often suggested model for pathological tau action in Alzheimer's and related, dementia-causing tauopathies is the destabilization of axonal MTs, leading to aberrant axonal transport and neuronal cell death. Although tau's most well-characterized activity is its promotion of net MT growth and stability, the precise mechanistic details governing its regulation of MT dynamics remain unclear. We used the slowly-hydrolyzable GTP analog, guanylyl-(α,β)-methylene-diphosphonate (GMPCPP), to examine the structural effects of tau at MT ends that may otherwise be too transient to observe. We found that co-incubation of GMPCPP tubulin and tau resulted in the formation of extended, multiprotofilament-wide tubulin spirals emanating i) from the ends of pre-assembled MTs at 25 °C, ii) from free tubulin heterodimers at 4 °C, and iii) from free tubulin heterodimers at 34 °C. While 3R and 4R tau isoforms promoted MT assembly intermediates similarly, 4R tau stabilized disassembly spiral intermediates much more effectively than 3R tau, consistent with 4R tau's more effective suppression of MT shortening events. Importantly, all of these spiral structures were also observed, albeit at much lower frequencies, in the absence of tau, and have also been observed in previous studies of both GTP and GMPCPP tubulin, consistent with the notion that that they are bona fide intermediates in the MT assembly/disassembly processes. Finally, three tau proteins harboring mutations that cause neurodegeneration and dementia were differentially compromised in their abilities to stabilize intermediate structures. Taken together, we propose that tau promotes the formation/stabilization of intermediate states in MT assembly and disassembly by promoting both longitudinal and lateral tubulin-tubulin contacts. We hypothesize that these activities represent fundamental aspects of tau action that normally occur at the GTP-rich ends of GTP/GDP MTs and that may be compromised in neurodegeneration-causing tau variants
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
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
Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules
No full textMicrotubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from alpha/beta-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry (D. J. Needleman et al., Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16099). Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes (I. Koltover et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 14046). The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results which show that, unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (e. g. Ca2+, Sr2+, Ba2+). Instead, many divalent cations pre-empt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca2+ depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the known stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg2+, Mn2+, Co2+, and Zn2+. GdCl3 (3+) is found to be extremely efficient at MT depolymerization requiring ion concentrations of about 1 mM, while oligolysine (2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents either disrupting lateral interactions between PFs (which are strengthened for taxol containing beta-tubulin), or interfering with taxol's ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tubulin dimers in a PF)
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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