Reversible membrane deformations by straight DNA origami filaments.

Henri G Franquelim,Hendrik Dietz,Petra Schwille

doi:10.1039/d0sm00150c

Abstract

Membrane-active cytoskeletal elements, such as FtsZ, septin or actin, form filamentous polymers able to induce and stabilize curvature on cellular membranes. In order to emulate the characteristic dynamic self-assembly properties of cytoskeletal subunits in vitro, biomimetic synthetic scaffolds were here developed using DNA origami. In contrast to our earlier work with pre-curved scaffolds, we specifically assessed the potential of origami mimicking straight filaments, such as actin and microtubules, by origami presenting cholesteryl anchors for membrane binding and additional blunt end stacking interactions for controllable polymerization into linear filaments. By assessing the interaction of our DNA nanostructures with model membranes using fluorescence microscopy, we show that filaments can be formed, upon increasing MgCl2 in solution, for structures displaying blunt ends; and can subsequently depolymerize, by decreasing the concentration of MgCl2. Distinctive spike-like membrane protrusions were generated on giant unilamellar vesicles at high membrane-bound filament densities, and the presence of such deformations was reversible and shown to correlate with the MgCl2-triggered polymerization of DNA origami subunits into filamentous aggregates. In the end, our approach reveals the formation of membrane-bound filaments as a minimal requirement for membrane shaping by straight cytoskeletal-like objects.

Highlights

The core DNA origami design used throughout the current work (Fig. 1A and Fig. S1, Electronic supplementary information (ESI)†) was the linear nanostructure named origami L (L for Linear), previously employed in ref. 68 as mimicry for a non-curved (‘‘flat’’) BAR domain
Throughout this work, we intentionally added 12 matching blunt ends at both edges of defined helices on our DNA origami, in order to allow for stable intermolecular stacking interactions (Fig. 1B)
In order to corroborate that the MgCl2-triggered formation of DNA origami filaments on membranes is specific to our L3E design, we examined the binding of an origami L variant (Fig. S2A, ESI†) displaying 3 TEG-chol anchors and lacking blunt-ends at the edges, named origami L3, as negative control

Summary

Introduction

Cytoskeletal filaments are non-equilibrium polymers in constant turnover with monomeric subunits in solution.[15,16]Typically, these filament-forming proteins possess ATPase/GTPase activity, requiring nucleoside triphosphate (i.e. ATP or GTP) for multimerization.[17,18,19,20] The assembly and disassembly of such filaments is highly dynamic and, in addition, tightly regulated by a multiple of stabilizing and destabilizing effector proteins and motors.[21,22,23,24,25,26] many membrane remodelling functions, such as motility, cytokinesis and vesicle trafficking, rely on this controllable ability of the cytoskeleton to dynamically (de)polymerize at different timescales and cellular localizations.[25,26,27,28,29,30,31] Notwithstanding the modest to nonexisting intrinsic curvature displayed by the above-mentioned proteins, FtsZ, actin & Co. have been only described to remodel membranes in their active filamentous state, generating for 276 | Soft Matter, 2021, 17, 276--287Paper instance wrinkled and/or tubular deformations when reconstituted with membrane model systems.[9,32,33,34,35,36,37,38] How straight filaments, like actin or microtubules, are able to bend membranes remains an open question. Cytoskeletal filaments are non-equilibrium polymers in constant turnover with monomeric subunits in solution.[15,16] There, the authors modelled how the intrinsic curvature of filaments (i.e. nematic field), and their bundling interactions (i.e. intermolecular processes) may drive tubulation. One of their predictions was that narrow tubular deformations may still emerge even in the absence of intrinsic curvature, due to the establishment of nematic interactions that allow the membrane to curve perpendicular to the filament’s alignment. The authors proposed the formation of filament bundles as a general driving force for membrane remodelling of vesicles coated with filaments, irrespective of their pre-exiting curvature

Objectives

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Results

Conclusion