High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

Abstract

Annexins are abundant cytoplasmic proteins that can bind to negatively charged phospholipids in a Ca(2+)-dependent manner, and are known to play a role in the storage of Ca(2+) and membrane healing. Little is known, however, about the dynamic processes of protein-Ca(2+)-membrane assembly and disassembly. Here we show that high-speed atomic force microscopy (HS-AFM) can be used to repeatedly induce and disrupt annexin assemblies and study their structure, dynamics and interactions. Our HS-AFM set-up is adapted for such biological applications through the integration of a pumping system for buffer exchange and a pulsed laser system for uncaging caged compounds. We find that biochemically identical annexins (annexin V) display different effective Ca(2+) and membrane affinities depending on the assembly location, providing a wide Ca(2+) buffering regime while maintaining membrane stabilization. We also show that annexin is membrane-recruited and forms stable supramolecular assemblies within ∼5 s in conditions that are comparable to a membrane lesion in a cell. Molecular dynamics simulations provide atomic detail of the role played by Ca(2+) in the reversible binding of annexin to the membrane surface.

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

“…Glt Ph , which originates from a thermophilic prokaryote, is an exceptionally favorable model system, yet future research calls for the studies of human glutamate transporters that show much faster uptake kinetics; their turnover times range from milliseconds to just under a second for different subtypes. Our current state-of-the-art HS-AFM can reach scanning speed of 20 ms per frame when the imaging area is reduced, and pixel sampling and tip velocity are kept constant (27). However, if we were to acquire single scan lines of membrane-embedded transporters, we could generate height profiles changes with submillisecond time resolution.…”

Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy

“…Nanodiscs stably hold the disc-like lipid bilayer with minimal diffusion of the lipids, and provide sufficient space on both sides of the membrane when placed in a side-on orientation (Rouck et al, 2017;Bayburt et al, 2002). Additionally, since nanodiscs can stably retain one functional unit of the membrane protein embedded in the membrane, they are suitable for single-molecule dynamic observation in real time by high-speed (HS)-AFM (Kodera et al, 2010;Miyagi et al, 2016;Shibata et al, 2017;Uchihashi et al, 2011). Single-unit imaging of membrane protein-embedded nanodiscs loaded onto solid surfaces may be achieved from various orientations using HS-AFM under appropriate conditions.…”

Facile Fabrication of Hierarchically Thermoresponsive Binary Polymer Pattern for Controlled Cell Adhesion

“…However, the number of adhered hemolytic RBCs is drastically increased (Figure S8, Supporting Information; Figure b), because of the interaction between POEGMA chain and PS exposed on the out membrane of dysfunctional cells . To confirm the PS externalization induced by LPA treatment, normal RBCs, and hemolytic RBCs are stained with Annexin V, which can strongly bind to exposed PS in cell membranes . The LPA‐treated RBCs are highly positive for fluorescent annexin V compared with the untreated cells, confirming that LPA induces PS exposure (Figure S9, Supporting Information).…”