Nck adapter proteins: functional versatility in T cells

Nck is a ubiquitously expressed adapter protein that is almost exclusively built of one SH2 domain and three SH3 domains. The two isoproteins of Nck are functionally redundant in many aspects and differ in only few amino acids that are mostly located in the linker regions between the interaction modules. Nck proteins connect receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. Thereby, Nck regulates activation-dependent processes during cell polarisation and migration and plays a crucial role in the signal transduction of a variety of receptors including for instance PDGF-, HGF-, VEGF- and Ephrin receptors. In most cases, the SH2 domain mediates binding to the phosphorylated receptor or associated phosphoproteins, while SH3 domain interactions lead to the formation of larger protein complexes. In T lymphocytes, Nck plays a pivotal role in the T cell receptor (TCR)-induced reorganisation of the actin cytoskeleton and the formation of the immunological synapse. However, in this context, two different mechanisms and adapter complexes are discussed. In the first scenario, dependent on an activation-induced conformational change in the CD3ε subunits, a direct binding of Nck to components of the TCR/CD3 complex was shown. In the second scenario, Nck is recruited to the TCR complex via phosphorylated Slp76, another central constituent of the membrane proximal activation complex. Over the past years, a large number of putative Nck interactors have been identified in different cellular systems that point to diverse additional functions of the adapter protein, e.g. in the control of gene expression and proliferation

Perfusion-guided sonopermeation of neuroblastoma: a novel strategy for monitoring and predicting liposomal doxorubicin uptake

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however in vivo imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with microbubble ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation in vivo are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors. Methods: To monitor the therapeutic efficacy of sonopermeation in vivo, we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted ex vivo along with associated vascular changes. Results: In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment in vivo. Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid onset of doxorubicin-induced apoptosis of NB cells but without damage to blood vessels. Conclusion: Our results suggest that significant L-DOX uptake can occur by increasing tumor vascular permeability with microbubble sonopermeation without otherwise damaging the vasculature, as confirmed by in vivo qCEUS imaging and ex vivo analysis. The use of qCEUS imaging to monitor sonopermeation efficiency and predict drug uptake could potentially provide real-time feedback to clinicians for determining treatment efficacy in tumors, leading to better and more efficient personalized therapies. Finally, we demonstrate how the IGDD strategy outlined in this study could be implemented in human patients using a single case study

Rho GTPase function in flies: insights from a developmental and organismal perspective.

Morphogenesis is a key event in the development of a multicellular organism and is reliant on coordinated transcriptional and signal transduction events. To establish the segmented body plan that underlies much of metazoan development, individual cells and groups of cells must respond to exogenous signals with complex movements and shape changes. One class of proteins that plays a pivotal role in the interpretation of extracellular cues into cellular behavior is the Rho family of small GTPases. These molecular switches are essential components of a growing number of signaling pathways, many of which regulate actin cytoskeletal remodeling. Much of our understanding of Rho biology has come from work done in cell culture. More recently, the fruit fly Drosophila melanogaster has emerged as an excellent genetic system for the study of these proteins in a developmental and organismal context. Studies in flies have greatly enhanced our understanding of pathways involving Rho GTPases and their roles in development

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

Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function

Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion

Building Parallel Time-Constrained Hla Federates: A Case Study With The Parsec Parallel Simulation Language

Based on the DIS result, the HLA framework has been defined to achieve interoperability of independent simulators. Concurrently, and for the most part, independently to that effort, the parallel and distributed simulation (PADS) community has devoted much effort to define synchronization protocols for the correct execution of parallel simulation as-fast-as possible. Building parallel time-constrained federates within an HLA framework is not an easy task. We identify the potential difficulties: one or several federates, when and how to advance the federate's time, how to handle RTI notifications etc, and present our experiences with adding HLA features into the parsec parallel simulation language. 1 INTRODUCTION Simulation is becoming a common way to perform the evaluation and the study of a large variety of systems such as large scale computer networks or battlefield applications. As more powerful machines are available for this task, the level of complexity one wants to put in the m..