Endosomal signalling via exosome surface TGF beta-1

Extracellular vesicles such as exosomes convey biological messages between cells, either by surface-to-surface interaction or by shuttling of bioactive molecules to a recipient cell's cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor beta-1 (TGF beta-1) on their surfaces. The latent form of TGF beta-1 is associated with the exosomes via heparinase-II and pH-sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes-associated TGF beta-1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free-TGF beta-1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD-dependent pathways. Our results show that mast cell-derived exosomes are decorated with latent TGF beta-1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.11Ysciescopu

Detecting the dynamics of single biomolecules

This thesis is concerned with the detection of single biomolecules. Through detection of individual molecules it can be clarified whether a behavior which has been observed in a population is representative for all molecules. For detection confocal fluorescence microscopy was used, in particular Fluorescence Correlation Spectroscopy (FCS). Complexes of a 217 base-pairs DNA and the fluorescent molecule TMR were immobilized at low concentration on glass coverslips. Through an automatic search single complexes were positioned into the open laser excitation volume (I [my]m). The detected fluctuating fluorescence intensity of the single molecules was interpreted as association / dissociation of TMR to the nucleotide guanosine. Within the limiting survival time of the fluorophores, the fluctuation rates of the DNA-TMR molecules were found to be heterogeneously distributed. From an analysis of non-fluctuating DNA-TMR complexes an exponential distribution of survival time and number of detected photons was found. Enzymatic activity of horseradish peroxidase (HRP) and of ribonuclease T1 (RNase T1) was analyzed by FCS. For RNase T1, enzyme (~ fM conc.) mediated release of surface immobilized DNA was demonstrated. In the case of HRP, the enzymes were immobilized. The analyzed enzymatic cycle involved formation of a fluorescent enzyme-product complex. It was found that the rate with which the enzyme-product complex was formed was widely distributed. An FCS-setup for ultraviolet excitation and emission was built. With UVexcitation the fluorescence of certain nucleotides and aminoacids can be used. In particular biomolecular dynamics in which such fluorophores are involved could be analyzed. The modified nucleotide 2-aminopurine (2-AP) was used as fluorophore. The diffusion and triplet state dynamics of 2-AP were analyzed. The dynamics of voltage gated ion channels in cell membranes were measured. The voltage- sensing segment S4 of the Shaker potassium channel was labeled with TMR. A combination of the patch-clamp technique and FCS was used. In an allopen or all-closed measurement, correlation between ion current through the channels and fluorescence change generated by S4's conformational rearrangement was observed. This indicates that cross-correlation of the same signals during spontaneous millisecond opening and closing should be detectable

Inverse-fluorescence correlation spectroscopy: more information and less labeling

Inverse-Fluorescence Correlation Spectroscopy(iFCS) is a recently developed modification of standardFCS that allows analysis of particles and biomoleculeswithout labeling. The particles generate no signal; insteadthe signal is generated by a surrounding medium. Particlesdiffusing through the FCS-detection volume displace afraction of the surrounding medium, causing transient dipsin the detected signal. These give information about themobility and concentration of the analyzed particles. Alsolabeled particles can be analyzed, whereby their signal iscross-correlated with that from the surrounding medium(iFCCS). This can give information about the volume of thelabeled particles, or alternatively about the size of thedetection volume. Also the interaction of unlabeledparticles with small, labeled ligands can be analyzed withiFCCS. This allows using cross-correlation as a sensitiveindication of binding, even though only one binding-partneris labeled. This review describes the principles of iFCS andiFCCS and measurements of microspheres dissolved in asurrounding medium containing alexa 488. We also discusspractical considerations, and future possibilities foranalyses of biomolecules.QC 2012021

On Death Numbers and Survival Times of Single Dye Molecules

QC 20160104</p

Studying Individual Events in Biology

Studying the properties of individual events and molecules offers a host of advantages over taking only macroscopic measurements of populations. Here we review such advantages, as well as some pitfalls, focusing on examples from biological imaging. Examples include single proteins, their interactions in cells, organelles, and their interactions both with each other and with parts of the cell. Additionally, we discuss constraints that limit the study of single events, along with the criteria that must be fulfilled to determine whether single molecules or events are being detected.QC 2012020