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The role of mitochondria in tunneling nanotube formation
Tunneling nanotubes (TNTs) have been recently discovered as an essential form of cell-cell communication in multicellular organisms. Although TNTs appear to be an important cell-cell signaling pathway, its mechanism of formation and functions are still not fully understood. A central issue is determining whether mitochondria play a vital role in the formation of TNTs. Previous studies have shown that exposure to a low concentration of ethidium bromide (EtBr) in mammalian cells caused a complete loss of mitochondrial DNA (mtDNA) leading to non-functional mitochondria (Rho0 cells); therefore, this technique will be applied to produce a HeLa Rho0 cell line to study the effects of non-functional mitochondria on TNT. Here we will determine if Rho0 cells were successfully engineered by labeling the mtDNA with Quant-it PicoGreen Reagent and labeling of the plasma membrane with WGA Rhodamine to determine the effects on TNT formation. We will also determine the expression levels of Myosin-X (Myo10), a known TNT inducer, in Rho0 cells versus control cells. Overall results revealed that mitochondria do play a role in TNT formation. As cells were in the process of becoming Rho0 cells, TNT formation increased peaking at week 4. As Rho0 cells stabilized, TNT formation significantly decreased. In addition, cells were seen to expel their mitochondria into the extracellular medium in week 6, a process known as mitochondrial extrusion. Lastly, Myo10 protein levels significantly increased during the production of a stable Rho0 cell line at week 4. The increase in Myo10 protein levels correlated with an increase in TNT formation.