With this project we propose to analyze small membrane protrusions know as tunneling nanotubes (TNTs) by exploring the limits of laser capture microdissection (LCM) and microproteomic techniques. Our research is focused on the characterization of TNTs, a novel mechanism for functional connectivity between cells, in the spreading of viruses, misfolded protein aggregates that lead to neurodegenerative diseases, and cancer. TNTs have been found in numerous cell types, allowing the transport of cytosolic and membrane-bound molecules, organelles and the spreading of pathogens. In vitro, these structures are heterogeneous and numerous disparities have emerged both in their structures and functions. Similar structures also exist in vivo and in tissue explants. Unfortunately, little s currently known about the basic mechanism of TNT formation, its structural components, transport mechanism or signaling pathways. Traditional proteomic techniques tantalizingly offer the potential to help elucidate many of these unanswered questions. But researchers studying TNTs have often faced two significant obstacles in using traditional techniques. The first is the substantial amounts of cells that are required for downstream MS analysis (usually on the order of 10,000 cells). The second is the high degree of sample homogeneity necessary to obtain significant results. In fact, the difficultly of working with TNTs is the transient nature of these structures. Cells do not form connections at all times, in all cells. Since researchers in the past have found no help in overcoming these obstacles, research on TNTs has been reduced to """"""""fishing"""""""" for potentially significant proteins, slowing progress in the field. Fortunately, recent studies have demonstrated that TNTs can be induced by protein over-expression or under stress conditions, and have offered hope in overcoming the transient nature of TNTs. We identified a protein, Myo10, that increases the relative percentage of cells connected by TNTs by over 50% compared to control cells. This reproducible manner of TNT induction finally offers us a tool to elucidate the proteins necessary for TNT function and formation. Here, we plan to use this protein to induce TNT formation and then further enrich sample populations using LCM. We will analyze these enriched samples using microproteomic techniques such as MALDI-MS which should permit the detection and comparison of protein expression differences between experimental conditions. Also, unlike traditional proteomic technologies, that require 10-50 ?g of protein, microproteomics requires only 1-2 ?g of protein for analysis. Overall, while this project will be technically challenging and will require patience and troubleshooting to identify the best conditions for the proposed analyses, we believe that there is a great opportunity to combine these new techniques and open up the field. Furthermore, a better characterization of both formation and function of TNTs will provide important insights and lead to novel targets to improve neuroprotection against prion or other proteopathies as well as against HIV-1 infection and cancer.
With this project we propose to analyze small, subcellular membrane protrusions know as tunneling nanotubes by exploring the limits of laser capture microdissection (LCM) and microproteomic analysis. Tunneling nanotubes are a novel mechanism for functional connectivity between cells involved in the spreading of viruses (e.g., HIV), misfolded protein aggregates that lead to neurodegenerative diseases (e.g., Prions and Alzheimer's diseases), and cancer. A better characterization of both formation and the signals necessary to induce these tubular structures will provide important insights and lead to novel targets to improve neuroprotection against prion or other proteopathies as well as against HIV-1 infection and cancer.