Mitochondrial transformation: time for concerted action.

the mitochondrial genome is essential for the expression of several crucial proteins. Defects in mitochondrial DNa (mtDNa) are known to cause a wide variety of clinical disorders, and mutations have been implicated in the ageing pro­ cess. Our detailed understanding of the mechanisms that govern mammalian mito­ chondrial gene expression, however, is still surprisingly sketchy, particularly in com­ parison with our successes in un ravelling gene expression in prokaryotes and in the nucleus or cytosol of many eukaryotes. given the relatively small size of the mito­ chondrial genome, why is this the case? the answer, in large part, is due to our inability to manipulate the mitochondrial genome or to interrogate the role in vivo of various cis­acting sequences in mtDNa replication, transcription and messenger rNa expression. Mitochondrial transfection is com­ plicated for several reasons. First, the mitochondrion is a dynamic organelle sur­ rounded by two membranes, the innermost of which is impermeable to large hydro­ philic polyanions such as DNa or rNa. thus, any successful nucleic­acid­mediated trans formation of mitochondria requires that the molecule crosses the plasma membrane, targets the mitochondrion and is imported across two membranes into the matrix, in which it can be expressed. Second, assuming the molecule is able to access the matrix, DNa would need to be stably recombined into an endogenous copy of mtDNa, or maintained indepen­ dently. this is complicated because the level of in organellar recombination in some species, including mammals, is believed to be low in most tissues. this is compounded by our limited understanding of the crucial elements for DNa replication and faithful transmission. this means that the entire genome needs to be used as a potential vec­ tor for replication, and even then we are unsure whether an incoming genome would associate with the soluble and membrane­ bound factors necessary to promote mtDNa transmission. Finally, any transfecting mole­ cule would need to carry either a select able marker or express a foreign element that could be identified, such as a mitochondri­ ally translated fluorescent protein. as many hundreds or thousands of copies of mtDNa are present in any mammalian cell, selec­ tion would have to be highly efficient, as it is unlikely that methods of transfection would introduce many copies of foreign DNa. there have, nevertheless, been many attempts to transfect the mitochondrion, with varying degrees of success and repro­ ducibility. the field has been punctuated by many claims of success, yet no method has been accepted or repeated by in dependent research groups. thus, with the excep­ tion of biolistic­mediated transformation of yeast mitochondria, we are still frus­ tratingly devoid of robust methods in nearly all species. Khan & Bennett (2004) suggested an intriguing approach to the transfection of mammalian mitochondria, termed ‘pro­ tofection’. this methodology has been continually updated and is notable for a history of impressive claims (for example, Keeney et al, 2009), including in vivo mito­ chondrial transfection in live rats in 2004 (http://www.sens.org/files/conferences/ sens2/talks/Smigrodzki.mp3). although the details of the methods were hard to come by, several more recent papers have detailed the agent vector used for mtDNa protofection. the inventors have tagged the well­characterized mtDNa­binding protein tFaM with a virally based amino­terminal protein transduction domain immediately upstream from a mitochondrial­targeting sequence. theoretically, this allows the pre­packaged mtDNa–fusion protein com­ plex to cross the plasma and mitochondrial membranes. constructs with an N­terminal viral­transduction domain upstream from a mitochondrial pre sequence have also been used to import extra cellular fusion proteins into mitochondria (for example, rapoport et al, 2011). However, the way in which a construct relying on an ionic inter­ action between DNa and binding protein is able to facilitate mitochondrial import of DNa through three membranes remains unclear. although the data are intriguing, the methodology has only been applied by one research group so far. until experi­ ments using protofection can be reliably reproduced by other researchers, it must remain a promising yet unfulfilled method, particularly given its long gestation period. another approach has been to use nanocarriers, including DQasomes and MitO­porter. DQasomes—derived from the compound dequalinium—are cationic, self­assembling vesicles that target the mito­ chondrion. these mole cules can condense with DNa and have been shown to localize to mitochondria, in which their DNa cargo is released (Weissig et al, 2001). the prob­ lem of how DNa could be delivered across the mitochondrial membranes has been addressed by the production of another liposome­based carrier, MitO­porter, which enters cells by macro pinocytosis and mediates mitochondrial membrane fusion (for example, yasuzaki et al, 2010). import into the mitochondrial matrix requires DNa transfer through both the inner and outer membranes. although MitO­porter has yet to be shown to deliver large DNa molecules, it seems to be a promising Mitochondrial transformation: time for concerted action