Highlights d Upon demyelination, Trem2 -/microglia fail to upregulate lipid metabolism genes d Trem2 -/microglia accumulate cholesteryl ester derived from myelin cholesterol d Accumulation is rescued by ACAT1 inhibitor and LXR agonist d Apoe -/glia defective in cholesterol transport also accumulate cholesteryl ester

Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5 -coupled conjugate and the other the 3 -coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.DNA machine ͉ synthetic biology F usion of lipid membranes plays a central role in many biological processes. For example, in the case of synaptic transmission, the reaction is tightly regulated by a number of proteins of which the SNAREs [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors] have been shown to play a crucial role (1-3). In vitro studies have demonstrated the sufficiency of SNAREs to mediate fusion when reconstituted into a number of model systems, including freestanding lipid vesicles (4-7), supported lipid bilayers (8-10), and tethered vesicles (11). Generally these studies suggest that SNAREs are able to bring two membranes into close apposition by forming a trans complex in a docking reaction which is highly energetically favorable (12). This is illustrated in Fig. 1A, which is based on the x-ray structures of the soluble domains of SNARE proteins in complex (13). Several steps along the mechanism to achieve membrane fusion following this basic docking step have been proposed, and some of these are illustrated in Fig. 2, which also serves to illustrate assays commonly used to assess their role. In this step-wise mechanism, the bilayers first dock, then adopt an intermediate, hemifused state in which the outer leaflets have merged, but the inner leaflets and contents remain distinct. A transition into full fusion then involves mixing of both leaflets and content exchange.Despite substantial research on the role of SNARE proteins in the fusion process, many questions remain regarding the physical mechanism. Studies using simpler model systems can provide insight into the fundamental mechanisms of the rearrangement of bilayers and lead to a better understanding of the biological process (14, 15). We have prepared a series of DNA-lipid conjugates in which DNA o...

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A general method for synthesizing 5Ј-and 3Ј-coupled DNA-lipid conjugates has been developed and employed in DNA-mediated vesicle fusion. Vesicles presenting complementary DNA fuse, resulting in both outer and inner leaflet mixing as well as content mixing. Fusion is maximized using 5Ј-and 3Ј-coupled DNA on opposite vesicle partners, rather than only 5Ј-coupled DNA, showing the importance of DNA orientation to the process. Lipid and content mixing assays show a dependence of fusion kinetics on the sequence and average number of DNA per vesicle. Vesicles without DNA or presenting noncomplementary sequences also appear to undergo some degree of lipid mixing or exchange, but no content mixing. Total lipid mixing appears to occur more efficiently than inner leaflet mixing and content mixing, and this may be explained by the observed nonspecific lipid mixing and/or the rise of a hemifused intermediate. The ability to control DNA sequence and the relative experimental simplicity of this system make it highly attractive to probe fundamental questions of membrane fusion using both ensemble and single vesicle assays.

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