The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Evaluation of Tyrosine Kinase Inhibitor Combinations for Glioblastoma Therapy

<div><p>Glioblastoma multiforme (GBM) is the most common intracranial cancer but despite recent advances in therapy the overall survival remains about 20 months. Whole genome exon sequencing studies implicate mutations in the receptor tyrosine kinase pathways (RTK) for driving tumor growth in over 80% of GBMs. In spite of various RTKs being mutated or altered in the majority of GBMs, clinical studies have not been able to demonstrate efficacy of molecular targeted therapies using tyrosine kinase inhibitors in GBMs. Activation of multiple downstream signaling pathways has been implicated as a possible means by which inhibition of a single RTK has been ineffective in GBM. In this study, we sought a combination of approved drugs that would inhibit <em>in vitro</em> and <em>in vivo</em> growth of GBM oncospheres. A combination consisting of gefitinib and sunitinib acted synergistically in inhibiting growth of GBM oncospheres <em>in vitro</em>. Sunitinib was the only RTK inhibitor that could induce apoptosis in GBM cells. However, the <em>in vivo</em> efficacy testing of the gefitinib and sunitinib combination in an EGFR amplified/ PTEN wild type GBM xenograft model revealed that gefitinib alone could significantly improve survival in animals whereas sunitinib did not show any survival benefit. Subsequent testing of the same drug combination in a different syngeneic glioma model that lacked EGFR amplification but was more susceptible to sunitinib <em>in vitro</em> demonstrated no survival benefit when treated with gefitinib or sunitinib or the gefitinib and sunitinib combination. Although a modest survival benefit was obtained in one of two animal models with EGFR amplification due to gefitinib alone, the addition of sunitinib, to test our best <em>in vitro</em> combination therapy, did not translate to any additional in vivo benefit. Improved targeted therapies, with drug properties favorable to intracranial tumors, are likely required to form effective drug combinations for GBM.</p> </div

Coexpression of neuronatin splice forms promotes medulloblastoma growth

Medulloblastoma (MB) is the most common pediatric brain cancer. Several important developmental pathways have been implicated in MB formation, but fewer therapeutic targets have been identified. To locate frequently overexpressed genes, we performed a comprehensive gene expression survey of MB. Our comparison of 20 primary tumors to normal cerebellum identified neuronatin (NNAT) as the most frequently overexpressed gene in our analysis. NNAT is a neural-specific developmental gene with α and β splice forms. Functional evaluation revealed that RNA interference knockdown of NNAT causes a significant decrease in proliferation. Conversely, coexpression of both splice forms in NNAT-negative MB cell lines increased proliferation, caused a significant shift from G1 to G2/M, and increased soft agar colony formation and size. When expressed individually, each NNAT splice form had much less effect on these in vitro oncogenic predictors. In an in vivo model, the coexpression of both splice forms conferred the ability of xenograft formation to human MB cells that do not normally form xenografts, whereas a control gene had no effect. Our findings suggest that the frequently observed over-expression of both NNAT splice forms in MB enhances growth in this cancer

A: Mice implanted intracranially with 020913 GBM oncosphere cells were treated with gefitinib (75 mg/kg), sunitinib (15 mg/kg) and a combination of gefitinib (75 mg/kg) and sunitinib (15 mg/kg

<p>). Gefitinib alone could significantly (p = 0.0001) improve survival in the animals compared to control animals. Sunitinib did not show any efficacy either when used alone (p = 0.13, compared to control) or when combined with gefitinib (p = 0.18, compared to gefitinib alone). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044372#pone-0044372-g005" target="_blank">Figure 5B:</a> Rats were implanted intracranially with 9L tumor 1 mm³ tumor pieces. Rats were then treated with gefitinib (50 mg/kg), sunitinib (8 mg/kg) and a combination of gefitinib (50 mg/kg) and sunitinib (8 mg/kg). None of the drugs including the combination of gefitinib and sunitinib showed any efficacy (p = 0.9).</p

Kinase inhibition after treatment with RTK inhibitors.

<p>A. Combining receptor tyrosine kinase inhibitors suppress downstream effectors of growth factor signaling. p-STAT3 was blocked only by the combination treatment, whereas p-AKT was blocked by all the drugs either as single agents or in combinations. The combinations of gefitinib and sunitinib as well as sunitinib and sorafenib were able to inhibit p-AKT, p-MAPK and p-STAT3 in both the GBM oncosphere lines.</p

Evaluation of Newly Developed Easy-Open Assistive Devices for Pneumatic Tube System Carriers for the Reduction of Work-Related Musculoskeletal Disorders

Musculoskeletal disorders may affect labor efficiency, cause disability, impair one’s work ability, and lower one’s quality of life. This consequently leads to a larger expenditure of medical resources. We aimed to design easy-to-open assistive devices for pneumatic tube systems to improve ergonomics and reduce musculoskeletal complaints of workers. We followed a design control process, including designs of motors, gears, sensors, and V-shaped connecting rods. Efficacy was evaluated by examining risks based on job strain index, user satisfaction, and musculoskeletal complaints of operators before and after the system’s implementation on a Nordic musculoskeletal questionnaire. We designed three assistive devices: two semiautomatic and one automatic. Each semiautomatic device costs about 300 US dollars and required space of 10×18×38 cm3. The automatic device costs about 3000 US dollars and required space of 28×38×50 cm3. The job strain index score decreased from 36 (very high risk) to 3 (low risk) with the semiautomatic devices and to 0 with the automatic device. Musculoskeletal complaints in the neck and upper limbs were reduced, with a significantly higher satisfaction rate for female operators. Our novel design of an automatic cap opening device for a pneumatic tube system was effective in improving ergonomics and reducing musculoskeletal complaints