Abstract
Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present. Here, we show that the IM domain (also called I-BAR domain) from the protein ABBA, forms semi-flexible nanotubes protruding into Giant Unilamellar lipid Vesicles (GUVs). By simultaneous quantification of tube intensity and tubular shape we find both the diameter and stiffness of the nanotubes. I-BAR decorated tubes were quantified to have a diameter of ~50 nm and exhibit no stiffening relative to protein free tubes of the same diameter. At high protein density the tubes are immobile whereas at lower density the tubes diffuse freely on the surface of the GUV. Bleaching experiments of the fluorescently tagged I-BAR confirmed that the mobility of the tubes correlates with the mobility of the I-BAR on the GUV membrane. Finally, at low density of I-BAR the protein upconcentrates within tubes protruding into the GUVs. This implies that I-BAR exhibits strong preference for negatively curved membranes.
Highlights
Membrane shaping constantly takes place in cellular processes such as endocytosis[1], cell division[2] and in tubular protrusions like filopodia[3,4,5] and Tunneling NanoTubes (TNT’s)[6]
A number of different BAR domain proteins have been studied with respect to mechanical effects on membranes[7] and on curvature sensing and curvature induction[11], but remarkably little information exists regarding the mechanical effect of I-BAR domains on membranes and how the domains are recruited to certain membrane regions
Possible stiffening effects have so far not been studied with I-BAR proteins despite its presence in stiff cellular membrane tubes where the stiffening is often attributed to stiff polymers like F-actin inside the tube[18]
Summary
Membrane shaping constantly takes place in cellular processes such as endocytosis[1], cell division[2] and in tubular protrusions like filopodia[3,4,5] and Tunneling NanoTubes (TNT’s)[6]. Recently the I-BAR domain from IRSp53 was found to be highly sensitive to negative membrane curvatures[12] and another investigation has shown that IRSp53 induces shape instability in membranes in a tension dependent manner[13] by a similar mechanism as was found previously with N-BAR domains[14,15] Physical characteristics, such as tube width, have been investigated by cryo-electron microscopy (cryo-EM) in fixed samples using multilamellar vesicles tubulated by different types of I-BAR domains. The width of tubules formed by BAR domains binding to multilamellar lipid vesicles have been quantified by using electron microscopy[8,16,19] Another approach is to employ optical trapping together with micropipette aspiration to pull tubes, with specific curvatures, out of GUVs which has allowed investigation of curvature sensing by various BAR domain proteins[11,20]. Addition of I-BAR to the outside results in formation of inwards pointing tubes which cannot readily be held fixed by optical tweezers and a different approach is needed to study such tubes
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