SUMMARY
mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggest metabolite profiling can help assess the quality and function of mtDNA modified cells.
BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three “injection type” type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown.ResultsKnown T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection.ConclusionsT3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation.
We demonstrate direct nuclear delivery of DNA into live mammalian cells using the photothermal nanoblade. Pulsed lasertriggered cavitation bubbles on a titanium-coated micropipette tip punctured both cellular plasma and nuclear membranes, which was followed by pressure-controlled delivery of DNA into the nucleus. High-level and efficient plasmid expression in different cell types with maintained cell viability was achieved.
We demonstrated direct nuclear delivery of macromolecules into live mammalian cells using the photothermal nanoblade. Pulsed laser triggered cavitation bubbles on a titanium thin film coated micropipette tip was utilized to puncture both the cell plasma and nuclear membranes followed by pressure controlled delivery of cargo into the nucleus. High plasmid expression (79-100%) in different cell types with maintained cell viability was achieved.
Despite a wealth of publications about the role of alpha-synuclein in Parkinson's disease, very little has been reported about this protein's physiological function in health. This current study established an important function for native a-synuclein in the regulation of ATP synthase activity. Life cell imaging and mitochondrial respiration analysis revealed that deficiency in synuclein (a,b,g-synuclein knockout mouse) leads to mitochondrial uncoupling characterized by increased mitochondrial respiration (V2; 12658.1% of control) and reduced mitochondrial membrane potential (8553% of control). Furthermore, a reduced efficiency of the ATP synthase (7454.2% of control) and lower ATP levels (8751.3%) were detected in synuclein deficient cells. Application of exogenous, native a-synuclein rescued the mitochondrial membrane potential (by 50%) and improved ATP synthase efficiency (by 113%). To provide direct evidence that a-synuclein acts on the ATP synthase, ATP synthase from brain mitochondria were immunocaptured and exposure to unfolded a-synuclein was able to directly increase its activity. These data suggest that asynuclein is a physiological modulator of mitochondrial bioenergetics through its ability to alter ATP synthase efficiency.
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