Lipid-Composition-Mediated Forces Can Stabilize Tubular Assemblies of I-BAR Proteins

Abstract

Collective action by inverse-Bin/Amphiphysin/Rvs (I-BAR) domains drive micron-scale membrane remodeling. The macroscopic curvature sensing and generation behavior of I-BAR domains is well characterized, and computational models have suggested various mechanisms on simplified membrane systems, but there remain missing connections between the complex environment of the cell and the models proposed thus far. Here, we show a connection between the role of protein curvature and lipid clustering in the relaxation of large membrane deformations. When we include phosphatidylinositol 4,5-bisphosphate-like lipids that preferentially interact with the charged ends of an I-BAR domain, we find clustering of phosphatidylinositol 4,5-bisphosphate-like lipids that induce a directional membrane-mediated interaction between membrane-bound I-BAR domains. Lipid clusters mediate I-BAR domain interactions and cause I-BAR domain aggregates that would not arise through membrane fluctuation-based or curvature-based interactions. Inside of membrane protrusions, lipid cluster-mediated interaction draws long side-by-side aggregates together, resulting in more cylindrical protrusions as opposed to bulbous, irregularly shaped protrusions.

Figure 1

Membrane presentation (A) and three protein representations first with curved I-BAR domain with uniform binding surface (orange) (B, upper), low-curvature I-BAR domain with uniform binding surface ((B) center), and curved I-BAR domain with negatively charged lipid binding site (yellow) (B, lower), highlighting the transverse (solid) and longitudinal (dashed) curvatures. Schematic of two angles (C) used to characterize protein aggregates is shown: the angle formed by vectors describing the long dimension of two neighboring proteins (Prot-Prot) and the angle formed by a vector describing the long dimension of a protein and the center of mass between two neighboring proteins (Prot-CoM). To see this figure in color, go online.

Figure 2

Characterization of aggregation behavior for separate membrane-protein systems: (A) I-BAR domains with uniform binding surface and various protein radii and membrane-protein interaction strengths and (B) I-BAR domains with a nonuniform membrane-binding surface and various PIP₂-like surface coverages and I-BAR domain to PIP₂-like membrane beads interaction strengths. The snapshots on the right are outlined in a color corresponding to markers in plots (A) and (B), demonstrating the various aggregation behavior: no aggregation (black lines), end-to-end aggregate (yellow triangles), intermediate aggregates (red squares), and side-by-side aggregate (purple circles). The snapshots show I-BAR domains in red, PIP₂-like membrane bead in blue, and generic membrane bead in gray. To see this figure in color, go online.

Figure 3

Snapshots of protrusions oriented in the z axis with initial radii of 15- (A) and 21-nm (B) radii stabilized by I-BAR domains of 15-, 21-, and 50-nm (left to right) radii. A significant portion of the membrane has been omitted to clearly visualize the protrusion aggregates. Gaussian curvature and number density as position along the length of the protrusion (C) for the corresponding protrusion and I-BAR domain radii are shown. To see this figure in color, go online.

Figure 4

Snapshots of protrusions stabilized oriented in the z axis by 15-nm I-BAR domains with and without PIP₂-like membrane bead (A, left and right, respectively) with surrounding half of the membrane and flat sheet region omitted for clarity, the reduced representation of membrane (B, negative and positive Gaussian curvature shown in red and blue, respectively), and Gaussian curvature and number density as position along the length of the protrusion with ^∗p < 0.01 (Student’s t-test) (C). To see this figure in color, go online.