Proteomic Analysis of Exosomes from Mutant KRAS Colon Cancer Cells Identifies Intercellular Transfer of Mutant KRAS

Endocytosis and signaling.

Ras trafficking, localization and compartmentalized signalling

Although epitope tags are useful to detect intracellular proteins and follow their localization with antibodies, background and nonspecific staining often remain problematic. We describe a simple assay based on the split GFP complementation system. Proteins tagged with the 15-amino acid GFP 11 fragment are detected with a solution of the recombinant nonfluorescent complementary GFP 1-10 fragment to reconstitute a fluorescent GFP. In contrast to antibody-based staining methods, this one-step assay presents high specificity and very low background of fluorescence, thus conferring higher signal-to-noise ratios. We demonstrate that this new application of the split GFP tagging system facilitates detection of proteins displaying various subcellular localizations using flow cytometry and microscopy analysis.

One-step split GFP staining for sensitive protein detection and localization in mammalian cells.

Anterograde and retrograde transport of neutral sphingomyelinase-2 between the Golgi and the plasma membrane

H-Ras Does Not Need COP I- or COP II-dependent Vesicular Transport to Reach the Plasma Membrane

H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes

Background: With a traditional medical use for treatment of various ailments, herbal preparations of Echinacea are now popularly used to improve immune responses. One likely mode of action is that alkamides from Echinacea bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca 2+ . Here, we show that unidentified compounds from Echinacea purpurea induce cytosolic Ca 2+ elevation in non-immune-related cells, which lack CB2 receptors and that the Ca 2+ elevation is not influenced by alkamides. Methods: A non-immune human cell line, HEK293, was chosen to evaluate E. purpurea root extracts and constituents as potential regulators of intracellular Ca 2+ levels. Changes in cytosolic Ca 2+ levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor, were tested to determine the mechanism of this Ca 2+ signaling pathway. E. purpurea root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity. Results: A rapid transient increase in cytosolic Ca 2+ levels occurs when E. purpurea extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP3 receptor dependent. Echinacea-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca 2+ elevation is sustained after the Echinacea-induced Ca 2+ release from intracellular Ca 2+ stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from E. purpurea root extracts, six induce cytosolic Ca 2+ increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of Echinacea do not explain the observed Ca 2+ response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity. Conclusions: Our data indicate that as yet unidentified constituents from Echinacea stimulate an IP3 receptor and phospholipase C mediation of cytosolic Ca 2+ levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.

RESEARCH ARTICLE Open Access Echinacea-induced cytosolic Ca 2+ elevation in HEK293

Exercise Improves Resistance To Respiratory Infection And Decreases Inflammatory Mediators In Bronchoalveolar Lavage Fluid: 2333

Publisher Summary There are two ways that Ras proteins might come to be at multiple locations within a cell. Knowledge of the vehicles, regulation of departure, and speed of Ras movement are the key to understanding signaling in this scenario. Multiple studies give glimpses of both scenarios, and show that experimental manipulation of the location of introduced Ras proteins is a tactic that can alter Ras signaling. The major shift in our understanding that has come from the study of these trafficking events is that Ras proteins are not docile travelers; in response to mitogens, they can actively signal from both cell surface and internal membrane. The challenge ahead is to find ways to apply these driving lessons to endogenous Ras proteins and learn if this helps steer Ras away from oncogenic destinations. The experiments that begin to answer these questions are described in this chapter.

The Influence of Intracellular Location on Function of Ras Proteins

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