bottom-up-vitro-methods-to-assay-ultrastructural-organization

Bottom-Up <em>In Vitro</em> Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic ...

Sorbonne Université

We report that user-defined DNA nanostructures, such as two-dimensional (2D) origamis and nanogrids, undergo a rapid higher-order folding transition, referred to as supra-folding, into three-dimensional (3D) compact structures (origamis) or well-defined μm-long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer-by-layer deposition of polyelectrolytes. Once supra-folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA-polyelectrolyte complexation. Orthogonal to DNA base-pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site-specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami-tethered proteins.

Reversible Supra-Folding of User-Programmed Functional DNA Nanostructures on Fuzzy Cationic Substrates.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.

Koyomi Nakazawa

Laboratoire Physico Chimie Curie,
Institut Curie,
PSL Research University,
Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University

Sorbonne Université

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Koyomi Nakazawa

Laboratoire Physico Chimie Curie,Institut Curie,PSL Research University,Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University

Sorbonne Université

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Laboratoire Physico Chimie Curie,
Institut Curie,
PSL Research University,
Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University