Using DNA origami to study nanoscale organization of plasma membranes.

Plasma membrane (PM) lipids and proteins are organized into nanoscale regions called nanodomains, which regulate essential cellular processes by controlling local membrane organization. Despite advances in super-resolution microscopy and single particle tracking, the small size and temporal instability of nanodomains make them difficult to study in living cells. To overcome these challenges, we built fluorescent DNA origami probes that insert into the PM via lipid anchors displayed on the cell. The number and spatial distribution of anchors between the origami and the cell surface were precisely defined by the origami, enabling nanometer-scale sampling of the cell surface. Inserting these DNA origami particles into the membrane with lipid anchors allowed them to passively diffuse across the membrane, and we tracked their movement using single particle tracking to survey the PM landscape. By varying the number and spatial arrangement of lipid anchors connecting the DNA origami to the cell surface, we showed that stable immobilization of DNA origami particles requires simultaneous interactions with multiple nanodomains. Disruption of the actin cytoskeleton reduced immobilization, confirming its role in supporting nanodomain stability. Moreover, transient mechanical stretching of cells led to reversible increases in DNA origami mobility, indicating that mechanical force can reversibly regulate PM nanodomain organization. Altogether, we present a novel membrane-integrated DNA origami approach that provides mechanistic insights into PM nanodomain architecture and dynamics in living cells. Significance: Plasma membrane (PM) nanodomains regulate essential cellular processes including signaling, trafficking, and mechanotransduction. However, their small size and dynamic nature make them challenging to study in living cells. We developed a DNA origami-based probe that inserts into the membrane via lipid anchors spaced at defined nanometric distances, enabling inference of PM organization through analysis of probe diffusion. Live-cell tracking revealed that PM nanodomains are densely packed, smaller than 20 nm, and sensitive to actin disruption and mechanical stretching. This tool provides a powerful strategy for mapping membrane architecture at the nanoscale, offering insights into how cells dynamically regulate PM nanodomain organization in response to biochemical and mechanical signals.