The amounts of liposome-bound Drp1 and free Drp1 were analyzed by SDS-PAGE followed by immunoblotting. These data clearly show that increasing concentrations of doxorubicin led to displacement of Drp1 from the CL-containing liposomes, and this effect was confirmed by SPR experiments which also showed that doxorubicin efficiently competes for Drp1 binding to liposomes. Next, we analyzed the interaction of Drp1 with lipid monolayers using a Langmuir balance. Lipid monolayers composed of 80PC/ 20PE, 28PC/20PE/52PG, 28PC/20PE/52PS or 54PC/20PE/ 26CL were prepared with constant Tubulin Acetylation Inducer surface area and an initial pressure of 28 mNNm21. Recombinant Drp1 was added to the subphase, and the resulting increase in monolayer surface pressure was monitored in real-time. As shown in Figure 2D, the surface pressure of the monolayers containing PS, PG or CL started to rise immediately after injection of Drp1, while a pronounced lag time was observed in the case of the electrically-neutral PC/PE monolayers. These results indicate that Drp1 association with lipid monolayers depends at least in part on an electrostatic component. It is also notable that Drp1 causes a much larger surface pressure increase in CL-containing monolayers as compared to PS- or PG-containing monolayers even though the net electrical charge is the same in all cases. This suggests that other properties of CL may also contribute to the interaction with Drp1. To obtain a quantitative measure of the ability of Drp1 to penetrate into lipid monolayers, critical surface pressure values were determined. In these experiments, the increase in surface pressure upon Drp1 addition was measured as a function of the initial surface pressure. The data are fitted to a straight line, and the x-intercepts correspond to the monolayer critical surface pressure. The results in Fig 2D show that the penetrating potency of Drp1 was highest for CL-containing monolayers and lowest for PC/PE monolayers, while PG- and PScontaining monolayers displayed intermediate pc values. To sum up, results obtained using three independent lipidinteraction assays all concur in showing that Drp1 interacts preferentially with membranes containing CL compared to membranes containing other anionic lipids. In dynamins, the PH domain drives the binding of the protein to phosphoinositide-containing membranes, mediates the clustering of the phosphoinositides, and coordinates the regulation of GTPase activity through its membrane association. The dynamin-related protein, Drp1 does not contain a PH domain and the role of its B insert on protein activity is unclear. Recently, it has been shown that B insert of the yeast protein Dnm1 contains a short motif required for association with the mitochondrial adaptor protein Mdv1. However, this motif is strictly conserved only among fungi, and moreover, Mdv1 orthologues have not been identified in flies, worms or vertebrates. B insert is predicted to be unstructured and it has also been proposed, that similar to the PH domain, it could constitute a putative membrane interaction site.