Notes

Robust Metal-Support Interaction for 2nd Components: Software in Noble Metal/TiB2 Heterointerfaces and their Enhanced Catalytic Performance regarding Formic Acidity Dehydrogenation.
The microscopic nematode Caenorhabditis elegans (C. elegans) serves as an excellent animal model for studying membrane traffic. This is due in part to its highly advanced genetics and genomics, and a transparent body that allows the visualization of fluorescently tagged molecules in the physiologically relevant context of the intact organism. Notably, C. elegans oocytes, coelomocytes, and intestinal epithelia have been established as facile cellular models to explore nonpolarized and polarized cell membrane trafficking mechanisms. In this chapter, we describe in vivo C. elegans assays, utilizing fluorescent dyes or proteins, to examine the molecular mechanisms that control endocytosis and endocytic recycling. Tissue-specific, steady-state imaging and associated quantitative analysis allow the identification and interpretation of subcellular events in the intact animal. To better understand the kinetic characteristics of recycling tubules that mediate membrane protein recycling, we describe recently developed dynamic-imaging assays in intestinal epithelial cells. Such methods bring new clarity to the system, helping to elucidate the functional roles of recycling mediators.Cargo export from mammalian endosomal compartments often involves membrane tubules, into which soluble and membrane-bound cargos are segregated for subsequent intracellular transport. These membrane tubules are highly dynamic and their formation is mediated by a variety of endosome-associated proteins. However, little is known about how these membrane tubules are temporally or spatially regulated, so other tubule-associated proteins are likely to be discovered and analyzed. Therefore, methods to examine the biogenesis and regulation of endosome membrane tubules will prove to be valuable for cell biologists. In this chapter, we describe methods for studying this process using both cell-free, in vitro reconstitution assays, and in vivo image analysis tools.Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates degradation through the late-endosomal/lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system.Endocytosis is a fundamental process that cells use to remove receptors, extracellular material, plasma membrane proteins and lipids from the cell surface. After entry into cells, the cargo proteins are subsequently trafficked to late endosomes and lysosomes for degradation, to the Golgi complex, or to recycling endosomes for return to the plasma membrane. Small G proteins in the Rab and Arf family are present on endosomes and coordinate the trafficking of cargo proteins. Here we describe some basic experimental approaches to begin to study the endosomal trafficking of a given cell surface protein.The ADP ribosylation factor (Arf) family of small guanosine triphosphatases (GTPases) regulates vesicular transport at several locations within the cell, and is in turn regulated by guanine nucleotide exchange factors (GEFs) via a conserved catalytic domain, termed the Sec7 domain. The catalytic activity of the Sec7 domain is well characterized in the context of a few GEFs acting at the periphery of the cell. This chapter describes the techniques used to extend the biochemical analysis of activity to the much larger GEFs acting on the Arf family in the core secretory pathway, using the activity of Saccharomyces cerevisiae Sec7 on Arf1, regulating export from the trans-Golgi network, as a model. The complete methods for purification to near homogeneity of all proteins required, including several Sec7 constructs and multiple relevant small GTPases, are detailed. These are followed by methods for the quantification of the nucleotide exchange activity of Sec7 in a physiologically relevant context, including modifications required to dissect the signal integration functions of Sec7 as an effector of several other small GTPases, and methods for identifying stable Sec7-small GTPase interactions in the presence of membranes. These techniques may be extended to the analysis of similar members of the Sec7 GEF subfamily in other species and acting elsewhere in the secretory pathway.A key function of coat proteins is the sorting of protein cargoes into intracellular transport pathways. For many years, however, it has been unclear whether this role of coat proteins would apply to pathways of endocytic recycling. This issue has been clarified in recent years through the identification of multiple coat complexes acting in the recycling pathways. Leading this charge have been studies on a coat complex defined by ACAP1 (adenosine diphosphate ribosylation factor GTPase-activating proteins with Coiled-coil, Ankryin repeat and PH domains 1), which acts in the sorting of cargoes at the recycling endosome for their return to the plasma membrane. DBZ inhibitor This chapter describes the methods used to characterize this role of ACAP1.Defining the interaction of Arf GAPs with specific Arfs is important for understanding their functions in the endocytic system. Cell-based approaches have been valuable for identifying Arfs and Arf GAPs active in the endocytic compartment; however, the cell-based assays have some limitations in establishing relationships among the Arfs and ArfGAPs. Here we describe a simple in vitro assay that will provide a means for comparing Arfs as substrates and serve to complement cell-based studies.The Rab GTPases are master regulators of endosomal trafficking in eukaryotic cells. Among them, Rab8 plays an important role in tubulovesicular trafficking from the trans-Golgi network and recycling endosomes to the plasma membrane. Rab8 is activated by its guanine nucleotide exchange factor, Rabin8. In order to understand the molecular mechanisms that control endosomal recycling to the plasma membrane, it is pivotal to understand how Rabin8 is regulated in cells. Recently, biochemical and cell biological studies have identified several mechanisms for Rabin8 activation, which involves the relief of the intramolecular autoinhibition of Rabin8. Here we describe biochemical methods that we have used recently to study the activation of Rabin8.For some time, it has been known that recycling endosomes (REs) are organized in a nebulous "pericentrosomal" region in interphase cells. However, the collective use of previously developed methods, including centrosome isolation, live cell imaging, and electron microscopy, suggested that there is much more going on between the centrosome and the RE than previously imagined. By exploiting these approaches, we uncovered novel roles of the centrosome in RE function and, conversely, novel roles for REs in centrosome function. We first found that REs dynamically localized to the centrosome throughout the cell cycle. More specifically, we found that REs interacted with appendages of the older centriole in interphase cells to control endosome recycling, and this interaction was governed by RE-machinery including the small GTPase Rab11. We next determined that REs carry centrosome proteins to spindle poles as part of the "centrosome maturation" process. Here we discuss the methods used and materials needed to complete these types of studies.Rab GTPases are master regulators of intracellular membrane trafficking along endocytic and exocytic pathways. In this chapter, we began to characterize the exocytic and recycling Rabs from the filamentous fungus Magnaporthe oryzae (M. oryzae) that causes the rice blast disease. Among the 11 putative Rabs identified from the M. oryzae genome database (MoRabs), MoRab1, MoRab8, and MoRab11 appear orthologs of mammalian Rab1, Rab8, and Rab11 and likely function in exocytosis and endosomal recycling. To test this contention, we cloned, expressed, and determined intracellular localization of the three MoRabs in mammalian cells, in comparison to their human counterparts (hRabs). The MoRabs were well expressed as GFP fusion proteins and colocalized with the tdTomato-labeled hRabs on exocytic and recycling organelles, as determined by immunoblot analysis and confocal fluorescence microscopy. The colocalization supports the contention that the MoRabs are indeed Rab orthologs and may play important roles in the development and pathogenicity of M. oryzae.Recycling endosomes recently have emerged as major regulators of cytokinesis and abscission steps of cell division. Rab11-endosomes in particular were shown to transport proteins to the mitotic ingression furrow and play a key role in establishing the abscission site. Rab11 GTPase functions by binding and activating various effector proteins, such as Rab11 family interacting proteins (FIPs). FIPs appear to be at the core of many Rab11 functions, with FIP3 playing a role in targeting of the Rab11-endosomes during mitosis. Here we summarize the newest finding regarding the roles and regulation of FIP3 and Rab11 complex, as well as describe the methods developed to analyze membrane and cytoskeleton dynamics during abscission step of cytokinesis.Recycling endosomes (REs) form an extensive and complex network of subcompartmentalized vesicular and tubular elements that connect with the cell surface and other endosomes in macrophages. As surveillance and defense cells of the innate immune system, macrophages are highly dependent on REs for their active and voluminous cell surface turnover and endocytic, exocytic, and recycling of membrane and cargo. Here we set out three approaches for imaging and analyzing REs in macrophages, based on the expression of fluorescently labeled RE-associated proteins and the uptake of fluorescent cargo. Subcompartments of the REs are identified by co-expression and co-localization analysis of RE associated Rab GTPases. Transferrin is a well-known cargo marker as it recycles through REs and it is compared here to other cargo, revealing how different endocytic routes intersect with REs. We show how the movement of transferrin through REs can be modeled and quantified in live cells. Finally, since phagosomes are a signature organelle for macrophages, and REs fuse with the maturing phagosome, we show imaging of REs with phagosomes using a genetically encoded pH-sensitive SNARE-based probe. Together these approaches provide multiple ways to comprehensively analyze REs and the important roles they play in these immune cells and more broadly in other cell types.Rapid activation of the innate immune system is critical for an efficient host response to invading pathogens. However, the inflammatory reaction has to be strictly controlled to minimize harmful immunopathology. A number of mediators including the cytokine interleukin-27 (IL-27) appear to be responsible for limitation and resolution of inflammation. Despite increasing knowledge of its suppressive effects on T cells, the influence on neutrophils and macrophages is poorly understood. To determine the role of IL-27 in innate immune responses we analysed the effect of IL-27 in a T cell independent model of zymosan-induced peritonitis. Early administration of recombinant IL-27 strongly reduced the number of neutrophils recruited to the peritoneal cavity after zymosan application as well as the neutrophil frequency in the blood. Simultaneously, IL-27 reduced the release of neutrophils from the bone marrow upon inflammation. Although cytokine levels were not affected by IL-27 treatment, the levels of the chemokines KC, MCP-1 and MIP-1α in the peritoneal fluid were strongly decreased.
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