Exosomes released from breast cancer carcinomas stimulate cell movement

For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis

In the fluorescent spotlight: Global and local conformational changes of small catalytic RNAs

RNA is a ubiquitous biopolymer that performs a multitude of essential cellular functions involving the maintenance, transfer, and processing of genetic information. RNA is unique in that it can carry both genetic information and catalytic function. Its secondary structure domains, which fold stably and independently, assemble hierarchically into modular tertiary structures. Studies of these folding events are key to understanding how catalytic RNAs (ribozymes) are able to position reaction components for site-specific chemistry. We have made use of fluorescence techniques to monitor the rates and free energies of folding of the small hairpin and hepatitis delta virus (HDV) ribozymes, found in satellite RNAs of plant and the human hepatitis B viruses, respectively. In particular, fluorescence resonance energy transfer (FRET) has been employed to monitor global conformational changes, and 2-aminopurine fluorescence quenching to probe for local structural rearrangements. In this review we illuminate what we have learned about the reaction pathways of the hairpin and HDV ribozymes, and how our results have complemented other biochemical and biophysical investigations. The structural transitions observed in these two small catalytic RNAs are likely to be found in many other biological RNAs, and the described fluorescence techniques promise to be broadly applicable. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 224–241, 2002Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34325/1/10144_ftp.pd

2 Terbium(III) Footprinting as a Probe of RNA Structure and Metal Binding Sites

Introduction Cations play a pivotal role in RNA structure and function. A functional RNA tertiary structure is stabilized by metal ions that neutralize and, in the case of multivalent ions, bridge the negatively charged phosphoribose backbon

Cargo sorting to lysosome-related organelles regulates siRNA-mediated gene silencing

The activity of siRNAs in gene silencing is regulated by protein complexes that sort cargo to lysosome-related organelles

Conformational changes and metal -ion binding in the hepatitis delta virus ribozyme.

The hepatitis delta virus (HDV) ribozyme is among the class of small ribozymes that catalyze a self-cleavage reaction critical to the replication of a small RNA genome. In the present work, several fluorescence techniques were tested to investigate the catalytic strategies applied by the HDV ribozyme. Fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence were employed to examine the role that global and local conformational changes on the reaction pathway of the HDV ribozyme have in catalysis. Using fluorescence resonance energy transfer on a synthetic trans-cleaving form of the ribozyme, we were able to directly observe substrate binding and dissociation. Steady-state and time-resolved FRET experiments in solution and in non-denaturing gels revealed that the substrate (precursor) complex is slightly more compact than the free ribozyme, yet becomes significantly extended upon cleavage and product complex formation. By modifying the solvent exposed nucleotide G76 of the trefoil turn of the synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we directly monitored local conformational changes in the catalytic core that accompany catalysis. Additionally, the lanthanide metal ion terbium(III) was used to footprint the precursor and product solution structures of the cis-acting antigenomic HDV ribozyme. Subtle, yet significant differences between the terbium(III) footprinting patterns of the precursor and product forms of the antigenomic HDV ribozyme are consistent with differences in conformation. In addition, UV melting profiles provided evidence for a less tight tertiary structure in the precursor. In both the precursor and product, high-affinity terbium(III) binding sites were observed in joining sequence J4/2 and loop L3, which are key structural components forming the catalytic core of the HDV ribozyme, as well as in several single-stranded regions such as J1/2 and the L4 tetraloop. Sensitized luminescence spectroscopy confirmed that there are at least two affinity classes of Tb3+ binding sites. These experiments show that the precursor and product forms of the HDV ribozyme are structurally distinct and have different metal ion affinities. The presented biochemical and biophysical studies provide important new insights into the relationship between structure and function of the HDV ribozyme.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124686/2/3150214.pd

Probing RNA Structure and Metal‐Binding Sites Using Terbium(III) Footprinting

The function of an RNA molecule is determined by its overall secondary and tertiary structure. The tertiary structure is facilitated and stabilized by the interaction with metal ions. The current chapter offers a detailed protocol on the use of the lanthanide metal ion terbium(III) as a powerful probe of RNA structure and metal‐binding properties. When incubating RNA with low (micromolar) concentrations of terbium(III), specific backbone scission by partially deprotonated aqueous terbium(III) complexes can be used to detect high‐affinity metal‐binding sites, while incubation with high (millimolar) terbium(III) concentrations cleaves the RNA backbone preferentially at structurally accessible regions, providing a footprint of the RNA secondary and tertiary structure.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153092/1/cpnc0608.pd

Local conformational changes in the catalytic core of the trans-acting hepatitis delta virus ribozyme accompany catalysis. Biochemistry

ABSTRACT: The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the doublerolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C 75 ) with a pK a close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5′-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G 76 of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP 76 is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP 76 becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C 75 becomes positioned for reaction chemistry only along the trajectory from precursor to product. The hepatitis delta virus ribozyme is among a class of small endonucleolytic RNAs that catalyze a reversible selfcleavage reaction necessary for the replication and propagation of their satellite RNA genomes. Specifically, the hepatitis delta virus ribozyme is a unique RNA motif found in the human hepatitis delta virus (HDV) 1 (1). HDV is a satellite of the hepatitis B virus (HBV); coinfection of HDV and HBV results in intensification of the disease symptoms associated with the hepatitis B virus (2). The small RNA genome of HDV replicates through a double-rolling circle mechanism, whereby multimeric units of genomic and antigenomic RNA strands are produced, followed by self-cleavage and ligation into circular monomers (1, 3). Self-cleavage activity in the genomic and antigenomic RNAs resides within continuous 85-nucleotide sequences that both form a nearly identical secondary structure consisting of a nested double pseudoknot (4, 5). The genomic and antigenomic forms of the HDV ribozyme catalyze self-cleavage by a transesterification reaction, which requires deprotonation of the adjacent 2′-OH group and its nucleophilic attack on the scissile phosphate, resulting in formation of 2′,3′-cyclic phosphate and 5′-OH termini (5). The reaction mechanism of the HDV ribozyme has been extensively studied. The crystal structure of the self-cleaved genomic ribozyme reveals that the base cytosine 75 (C 75 ) is situated in the active site cleft and, thus, in the proximity of the 5′-OH leaving group Several biochemical and mutagenesis studies support the idea that C 75 in the genomic ribozyme and the corresponding RC 76 (R used to distinguish antigenomic numbering) in the antigenomic ribozyme are involved in catalysis (7-10). The pH dependence of self-cleavage (or cis cleavage) by the HDV ribozyme reveals a macroscopic apparent pK a that approaches neutrality. In a widely accepted model, this pK a reflects the ionization equilibrium of N3 in C 75 which therefore is strongly shifted in the folded ribozyme compared to that in the free base (pK a ≈ 4.2). A decrease in this pK a for selfcleavage of an antigenomic ribozyme with an RC 76 A mutation was observed, consistent with A substituting for C in this position to act as a general base catalyst (8). However, the pH profile of the genomic ribozyme in the presence of 1 M NaCl and 1-100 mM EDTA favors a model where C 75 acts as a general acid during catalysi

Endocytosis of exosomes in breast cancer cells.

<p>(A) Time-course curve of exosome uptake (endocytosis) by determining fluorescent intensity of TAMRA-labeled exosomes from “donor” cancer cells at specific times. Inset shows a MCF-7 “recipient” cells incubated with TAMRA-labeled MCF-7 “donor” exosomes at 8 hours. Errors were calculated from fluorescence intensity of cells (n = 50), at each time point, normalized to the intensity at the final time point (24 hours). (B) Colocalization of TAMRA-labeled exosomes added to MCF-7 cells transiently transfected with LAMP1-GFP. Scale bar is 10μm.</p