A Tail-Anchored Myotonic Dystrophy Protein Kinase Isoform Induces Perinuclear Clustering of Mitochondria, Autophagy, and Apoptosis

Contains fulltext : 79678.pdf (publisher's version ) (Open Access)BACKGROUND: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. CONCLUSION/SIGNIFICANCE: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients

Modular actin nano-architecture enables podosome protrusion and mechanosensing

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries

Sell-side analysts' use and communication of intellectual capital information

Structural economic changes in many countries, together with unprecedented developments in the business environment, have significantly affected the value creation processes of firms and the way business is conducted. The traditional financial reporting model is inadequate as a consequence of these developments, and intellectual capital (IC) information has gained importance for investment decision making. Empirical capital markets research demonstrates the value-relevance and predictive ability of certain types of IC information. The use of IC information by capital market participants is a topic that has begun to gain attention from contemporary researchers, but for which scant empirical evidence exists. Much of the research in this area relies on the literature about the use of non-financial information (NFI), which is inadequate in its examination of certain types of IC information. Therefore, the main aim of this thesis is to examine the use and communication of IC information by sell-side analysts. Sell-side analysts are of particular interest because they are capital market intermediaries and sophisticated processors of corporate information. The reports they produce provide an opportunity to examine their use and communication of IC information. The specific objectives of this thesis are to examine: the extent and types of IC information used by sell-side analysts in initiating coverage reports produced by them; how IC information is used and communicated in these reports; and factors that may influence the use of IC information by sell-side analysts. In order to address these research objectives a content analysis of IC references in 64 initiating coverage reports written on an equivalent number of S&P/ASX 200/300 companies is performed. The content analysis identifies and measures IC references by topic, evidence (discursive, monetary, numerical, or visual), news-tenor (positive, neutral or negative) and time orientation (forward-looking, past-oriented or non-time-specific). The findings indicate that Australian sell-side analysts appreciate the importance of IC in firm valuation, and thus are not ambivalent about the use of IC information in general. However, the findings suggest that their communication of IC information is inconsistent and unsystematic, and inadequate in relation to certain types of IC. This highlights the need for undertaking work at a policy level to educate and train sell-side analysts to deal with IC information, and the development of better models and guidelines for analysing and communicating IC information. On how IC information is used, this thesis finds that sell-side analysts have varying uses of IC information. It was found that IC is predominantly communicated discursively, positively, and in a past-oriented manner; and in doing so IC is used as a tool to further the sell-side analysts’ agenda for the company analysed. Further, the results highlight that the type of investment recommendation in analyst reports impacts on the evidence, news tenor, and time-orientation of IC communicated. These findings alert future researchers to the wider role played by IC beyond its use in forecasts and valuations. Also, the findings indicate inter-sectoral differences in the use of IC information in analyst reports, highlighting the need to improve IC reporting practices of firms by including additional information on industry-specific IC value drivers. Further, it was found that sell-side analysts emphasise IC information in analyst reports for companies from high IC-intensive sectors compared to those from low IC-intensive sectors. Similarly, it was found that analyst reports on risky companies contain significantly more IC information than analyst reports on less risky companies. Contrary to expectations, the extent of IC information is not found to vary with firm size and firm profitability. Also, the results support that the extent of certain types of IC information differs between types of analysts’ investment recommendations. More generally, the findings of this thesis suggest that the corporate reporting process could be improved by including additional types of IC information and providing this information more effectively in a manner that enables users to visualise the interrelationships between resources (both tangible and intangible) and outcomes. This study calls for standards or guidelines for intellectual capital reporting (ICR) in Australia and the expansion of the role of auditing and assurance services to enhance reliability of firm provided IC information in a bid to improve the use of IC information in company analysis by sell-side analysts

Actin reorganization is altered during NAMPT inhibition in RAW 264.7 macrophages.

<p>Actin organization was assessed in wild type cells seeded on glass coverslips, treated with FK866 for 15 or 24 hours, and simultaneously stimulated overnight with 100/ml LPS. After fixation in 2% PFA, cellular actin was stained with phalloidin-Alexa568 and cells were imaged on a Zeiss LSM510 META confocal laser scanning microscope. (Bar = 10 µm).</p

The effect of FK866 on RAW 264.7 metabolism.

<p>A, B, Glucose consumption and lactate production in the presence and absence of FK866. Cells were incubated in control or FK866 medium for a total of 24 hours. Glucose and lactate was measured in medium supernatants collected over the last 6 hours (18–24 h) of incubation. C, Intracellular ATP levels of RAW 264.7 macrophages treated with 5 nM FK866 for 3, 15, or 24 hours. D, Mitochondrial NAD(P)H-level. Cellular autofluorescence was measured after 24 hour incubation in control medium or medium with 5 nM FK866. E, After 24 hour pre-incubation with or without FK866, oxygen consumption was measured in suspensions of 1×10⁶ cells in either FK866 or control medium. Control and FK866 treated cells were analyzed in parallel on the same day. The basal oxygen consumption was measured where after oligomycin, FCCP, and rotenone was added successively in order to determine the leak respiration, maximal respiration (Max), and residual oxygen consumption (Res). Data in a-c represent means ± SEM of three experiments performed in triplicate, in d means ± SEM of three experiments, and in e means ± SEM of five experiments. (*<i>p</i><0.05, **<i>p</i><0.01; unpaired t-test).</p

LPS-stimulated spreading of RAW 264.7 macrophages is compromised by glucose deprivation.

<p>RAW 264.7 macrophages expressing Lifeact-EYFP were pre-incubated in control medium or medium containing 2.5 µM oligomycin and 25 mM glucose (A), 10 mM 2-DG and 25 mM glucose (B), 10 mM galactose and no glucose (C), or 1 mM glucose and 10 mM galactosel (D) medium for the indicated time periods. To assess spreading efficiency, cells were detached with EDTA, re-suspended, seeded in 96 well plates and allowed to adhere. Cell spreading of EYFP-positive cells was recorded over time using a BD Pathway high content microscope. The average pixel area per cell was determined at 10 minute intervals. Lines and bars represent means ± SEM of three independent experiments performed in triplicate. For every condition, representative images of cells at 0 and 200 minutes are presented in the panel on the right.</p

Proliferation and viability of RAW 264.7 cells under different metabolic conditions.

<p>Proliferation was monitored for 24 hours in the presence of LPS and expressed as the increase in total cellular protein in either control medium (25 mM glucose) or medium containing 2.5 µM oligomycin and 25 mM glucose (A), 10 mM 2-DG and 25 mM glucose (B), or 10 mM galactose and no glucose (C). Cell viability was also assessed for 24 hours under the same medium conditions in the presence of pSIVA apoptosis biosensor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096786#pone.0096786-Kim2" target="_blank">[50]</a> (D). The appearance of the fluorescent pSIVA signal was recorded in real time and the total pixel area per frame was measured using Fiji Imaging software. An increase in the pSIVA pixel area correlates linearly with increase in the amount of apoptotic cells. Data in A–C represent means ± SEM of three independent experiments performed in triplicate and in D the averages of one experiment performed in triplicate. (*p<0.05, **p<0.01, ***p<0.001, unpaired t-test).</p

NAMPT-mediated salvage synthesis of NAD⁺ controls morphofunctional changes of macrophages: Synthesis of literature evidence.

<p>NAD⁺ is required to sustain high glycolytic activity in macrophages. Metabolism of glucose yields cellular ATP which is loaded onto G-actin monomers by profilin before incorporation into an actin filament, or used to activate the Arp2/3 complex. ATP also binds to Rho GTPase Rho A, which in turn also stimulates actin polymerization (pink arrows). LPS stimulation induces the expression of the NAD⁺ synthesizing enzyme NAMPT (turquoise arrows). NAMPT-mediated NAD⁺ synthesis may regulate actin dynamics by activating Cdc42 (green arrows) or by influencing cellular metabolism and, thereby, intracellular pH and cofilin activity (purple arrows). Alternatively, NAMPT may control redox regulation of the actin cytoskeleton via mical or NADPH oxidase by regulating NADPH levels (orange arrows). GLUT, glucose transporter; NOX, NADPH oxidase; PPP, pentose phosphate pathway; ROS, reactive oxygen species; SSH-1L, slingshot-1L; TLR4, toll-like receptor 4.</p

Actin cytoskeletal structural changes induced by inhibition of glycolysis or mitochondrial OXPHOS.

<p>RAW 264.7 cells were seeded on glass coverslips, incubated in control medium or medium containing 2.5 µM oligomycin and 25 mM glucose (A–F), 10 mM 2-DG and 25 mM glucose (G–J), 10 mM galactose and no glucose (M–P), or 1 mM glucose and 10 mM galactose (Q–T) for the indicated time periods and stimulated overnight with LPS or left unstimulated. After fixation in 2% PFA, cellular actin was stained with phalloidin-Alexa568 and cells were imaged on a Zeiss LSM510 meta confocal laser scanning microscope. The number of filopodia extending radially from the cell surface (expressed as # filopodia per µM contour length; see M&M) was determined for control cells and cells treated for 24 hours with oligomycin, in the presence and absence of LPS (K). The average cell circumference was determined for cells in control medium or medium containing 10 mM galactose, or 1 mM glucose and 10 mM galactose (L). (*p<0.05, **p<0.01, ***p<0.001, unpaired t-test).</p