Transfer of miRNA from single HDL particles to cells

Until recently communication between tissues was believed to occur solely via hormone-like substances. It becomes more and more clear that cellular communication is more complex than anticipated. Recently also genetic material was found to circulate in the blood stream, the so-called Micro-RNA (miRNA), which are short single stranded RNA molecules. These miRNAs regulate many biological processes and are known to play a role in the progression of atherosclerosis. In particular, extracellular miRNAs circulate stable in the bloodstream and were recognized as novel diagnostic markers. Recently two groups showed that high-density lipoprotein (HDL) transports and delivers functional miRNAs to the recipient cell. Still to date it has not been possible to observe the cellular release mechanism of miRNAs as they occur whether within a cell or at the cell membrane at a single molecule level. We hypothesize that miRNA delivery from HDL particles follows the lipid uptake path. We aim to unravel this transfer process beginning at the cell membrane to its final destination using high-end single molecule fluorescence and force microscopy which allows to image directly the uptake pathway of miRNA via HDL particles to the target cell. To accomplish this, we will record the fluorescent signal of labeled HDL and the incorporated miRNA and phospholipids, respectively. Imaging up to three molecules of different color bound to one core particle will give access if molecules are traveling together in a complex. Therefore we can identify whether miRNA takes the same uptake pathway as the HDL particle or is released in an earlier stage at the cell membrane. The latter will be gradually investigated by delivering single miRNA via covalently linked HDL using atomic force microscopy (AFM). Here, the AFM tip is used as a nanopipette for controlled delivery of the miRNA cargo and to measure interaction forces. Simultaneously, the fluorescence signal will allow the visualization of the uptake pathway at the single molecule level. Additionally, we will convert fluorescent miRNA signals to detect its location at specific time intervals using electron microscopy. This will facilitate the localization of miRNA in cell systems at the nanometer scale. To achieve this the close collaboration between Biophysicists and Cell Biologists, namely the groups of Birgit Plochberger, FH Campus Linz, and Herbert Stangl, Medical University Vienna, are inherent. Particular input on miRNA handling will come from our international collaboration partner Fatiha Tabet, University of New South Wales. Taken together, the techniques available enable us to study the uptake of miRNA out of an HDL particle in an unprecedented manner for the first time.

High Density Lipoprotein (HDL) particles carry besides the well-known lipid cholesterol a plethora of other lipids but also a magnitude of about 100 proteins and even micro RNAs (miRNAs), messengers able to block protein synthesis, in the blood stream. HDL has been described to have a wide range of action, in diseases the protective effects of HDL diminish and adverse effects are seen, this is sub-summarized under the term HDL functionality. Thus it is very important to describe the differences in HDL particle composition in health and disease. In the current project we found an alteration of the HDL miRNA signature of uremic patients, which suffer increased burden of atherosclerosis, the cause of this dramatic increase is still under intensive research. The alterations seen might be one piece in this puzzle. In addition transfer of lipoprotein- derived lipids - generally known as the good or bad cholesterol - to cells and synthetic membrane was assessed. All lipoproteins interacted with bilayers by integration, this could be detected by a highly sophisticated method called high speed atomic force microscopy (AFM). A cantilever -a very small capillary - moves quickly over the surface analyzed, by measuring its deflection the topology of the surface can be reconstituted. When using the AFM as a nanopipette we followed the transfer of fluorescently labeled cholesterol into such bilayers, upon contact movement of these lipids in the membrane was seen. Similar results were obtained in cells, in which also artificial tethering using the biotin- streptavidin system resulted in lipid transfer from lipoproteins to the cell membrane.