Research on Cell-free Mitochondria in Drosophila Hemolymph

I served as an undergraduate research assistant in a project focused on the identification and characterization of cell-free mitochondria in Drosophila adult hemolymph.

During the study, I observed small vesicle-like structures in the adult Drosophila melanogaster (hereinafter Drosophila) hemolymph. Moreover, single-cell RNA sequencing of adult Drosophila revealed that a significant majority of these cells contained more than 50% mitochondrial components. My primary role was to investigate and provide an in-depth characterization of these vesicle-like objects containing mitochondrial material.

First, mitochondrial DNA (mtDNA) PCR was performed, with and without DNase treatment, to determine whether these vesicles contained mtDNA. As depicted in the figure above, the results clearly demonstrated the presence of mtDNA within the vesicles.

Second, a western blot was performed to verify the origin of these vesicles from hemocytes. I collected hemocytes of HmlΔ>EGFP/+ Drosophila, separated the cells and vesicles and conducted western blotting targeting EGFP. The results confirmed their hemocyte origin, as evidenced by the detection of EGFP within the vesicles.

The hypothesis under consideration was that these vesicles are exosomes secreted from hemocytes via exocytosis. To validate this hypothesis, I conducted confocal microscopy imaging of hemocytes from Drosophila lines HLT>EGFP; Rab27-RNAi, HLT>EGFP;Rab35-RNAi and HLT>EGFP; + (control). Contrary to expectations, the results showed no significant differences between Rab knockdown and wild-type Drosophila.

Next, I sought to obtain a more detailed examination of these vesicles using a transmission electron microscope. Through this advanced imaging technique, I was able to confirm that the vesicles in HmlΔ>EGFP/+ hemolymph exhibited a structure highly reminiscent of mitochondria.

Finally, to ascertain whether these vesicles merely resembled mitochondria or were, in fact, cell-free mitochondria, I stained HmlΔ>EGFP/+ hemolymph with MitoTracker. Subsequent imaging using a confocal microscope revealed a positive signal. This conclusive evidence allowed me to definitively characterize these vesicles as cell-free mitochondria.

I speculated that these cell-free mitochondria might be linked to processes such as metamorphosis, larval midgut degradation during the pre-pupal stage, or the reduction in total hemocytes in adult Drosophila. However, it is important to note that further validation is required.