ABSTRACT
Distinct types of vesicles are formed in eukaryotic cells that conduct a variable set of functions depending on their origin. One subtype designated circulating microvesicles (MVs) provides a novel form of intercellular communication and recent work suggested the release and uptake of morphogens in vesicles by Drosophila cells. In this study, we have examined cells of the hemocyte-like cell lines Kc167 and S2 and identified secreted vesicles in the culture supernatant. The vesicles were isolated and found to have characteristics comparable to exosomes and plasma membrane MVs released by mammalian cells. In wingless-transfected cells, the full-length protein was detected in the vesicle isolates. Proteomics analyses of the vesicles identified 269 proteins that include various orthologs of marker proteins and proteins with putative functions in vesicle formation and release. Analogous to their mammalian counterparts, the subcellular origin of the vesicular constituents of both cell lines is dominated by membrane-associated and cytosolic proteins with functions that are consistent with their localization in MVs. The analyses revealed a significant overlap of the Kc167 and S2 vesicle proteomes and confirmed a close correlation with non-mammalian and mammalian exosomes.
Subject(s)
Drosophila/chemistry , Exosomes/chemistry , Insect Proteins/analysis , Proteome/analysis , Animals , Blotting, Western , Cell Line , Centrifugation, Density Gradient , Exosomes/metabolism , Insect Proteins/chemistry , Proteome/chemistry , Proteomics , UbiquitinABSTRACT
The set-up presented in this article is intended for the selection of peptides which serve as specific binders to suitable materials. Additionally, the interaction of such binders with material surfaces can be characterized. Using this approach, a subset of peptides which adhere to the mineral TiO(2) was generated by means of a cell surface display library. The peptides are constrained by a thioredoxin scaffold. Selection of proteins was carried out on a silicium wafer sputtered with TiO(2) in anatase conformation. To verify binders and to analyze the binding kinetics of the diluted suspension of the purified proteins, the chip-based S-sens K5 surface acoustic wave sensor system was used. The surface of the sensor chips was also TiO(2), resembling the material of the Si wafer selection target retaining the peptides. Several peptides were identified. The respective binding behavior differed. The data derived from real-time interaction analysis were evaluated to select for strong and specific binders. For one of these peptides, the binding kinetics was analyzed. On- and off-rate binding constants were extracted from the fitted curves. With the resulting association rate constant k(on) and the dissociation constant k(off), the affinity of the peptide for the TiO(2) surface was calculated, represented by the equilibrium dissociation constant K(D)=81 nM.
Subject(s)
Acoustics , Aptamers, Peptide/chemistry , Titanium/chemistry , Amino Acid Sequence , Aptamers, Peptide/genetics , Base Sequence , Biosensing Techniques , Cloning, Molecular , DNA Primers , Escherichia coli/genetics , Genetic Vectors , Molecular Sequence Data , Surface Properties , Thioredoxins/chemistry , Thioredoxins/geneticsABSTRACT
We proof that nanomaterials can be successfully marked with relatively small amounts of purified bacteriophages acting as a molecular bar code label. Bacteriophages are DNA protected by a proteinous hull. The DNA fraction of the bacteriophages particle offers a nearly unlimited potential to encode information into the label. The information included in the molecular label can be read using PCR driven amplification. We show, how bacteriophage particles are easily applied to label a nanoscaled bulk material e.g., multi walled carbon nanotubes.
