Thesis

Laser capture microdissection and mass spectrometry: a novel method to study the proteome of tunneling nanotubes (TNTS)

Mammalian cells produce different types of cell protrusions (CPs)—like filopodia, lamellipodia, invadopdia, and tunneling nanotubes—that have been implicated in numerous important biological functions, such as cell migration, cell-cell communication, cancer-cell invasion, and the intercellular spread of pathogens. Yet, the proteomes of such structures are not well characterized due to the inability to specifically isolate individual types of protrusions for downstream proteomic analysis. In this study we present a novel method using laser capture microdissection (LCM) in conjunction with microproteomics to specifically isolate such structures and define their proteomes. We successfully isolated TNTs and other cellular projections by LCM using glutaraldehyde (GLU) and dithiobispropionimidate (DTBP) fixation. Although we found the TNTs to be more stable with GLU fixation than the DTBP fixation, the number of protein hits from the lysates of cells fixed with DTBP fixation was found to be significantly higher than GLU fixation. Mass spectrometry (MS) analysis of cells fixed with GLU showed that, using LCM, we were able to isolate 113 proteins specific to TNTs. We found several proteins from cytoskeleton, filopodia, vesicles and mitochondria that have implications in TNT functionality. Thus we demonstrate that LCM/MS system is an innovative combination to isolate proteins specific to such small structures.

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