Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids

Authors: STUEBLER, MARTIN - Cornell University; MANZER, ZACHARY - Cornell University; LIU, HAN-YUAN - Cornell University; MILLER, JULIA - Cornell University; RICHTER, ANNETT - Boyce Thompson Institute; KRISHNAN, SRINIVASAN - Boyce Thompson Institute; SELIVANOVITCH, EKATERINA - Cornell University; BANUNA, BARITUZIGA - Cornell University; JANDER, GEORG - Boyce Thompson Institute; REIMHULT, ERIK - University Of Natural Resources & Applied Life Sciences - Austria; ZIPFEL, WARREN - Cornell University; ROEDER, ADRIENNE - Cornell University; Pineros, Miguel; DANIEL, SUSAN - Cornell University

Submitted to: ACS Applied Materials and Interfaces
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2024
Publication Date: 4/9/2024
Citation: Stuebler, M., Manzer, Z., Liu, H., Miller, J., Richter, A., Krishnan, S., Selivanovitch, E., Banuna, B., Jander, G., Reimhult, E., Zipfel, W.R., Roeder, A., Pineros, M., Daniel, S. 2024. Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids. ACS Applied Materials and Interfaces. 16(16). https://doi.org/10.1021/acsami.3c18562.
DOI: https://doi.org/10.1021/acsami.3c18562

Interpretive Summary: Transport proteins embed within the cellar membrane mediate the transport of ions and nutrients entering and leaving the cell. Therefore, they play key roles in regulating many essential biological and environmental plant responses. Understanding processes occurring at the biomembrane cellular level requires developing new techniques that allow the recreation of the complex microenvironment occurring at these molecular scales. In this study, we report on the development of a methodology that allows for the self-assembly of native plant cell membranes derived from the surface of plant cells, along both its membrane proteins and lipid environment, .thereby creating a biomimetic plant membrane on a supporting glass surface (a “chip”). This “chip” is compatible with several microscopy, biophysical and analytical tools commonly used by scientists to understand biological phenomena occurring at this interface. We demonstrate the utility and generality of this technology by characterizing the diffusion and orientation of proteins from three different plant species using this platform. This new methodology has multiple immediate applications in studies characterizing cellular phenome, such as for example, protein-protein interactions, lipid-proteins interactions, and measurement of transport across the membrane and other similar applications where recreating and preserving the complex membrane environment is essential.

Technical Abstract: The plant cell plasma membrane is a semi-permeable barrier that serves two important functions: (1) transport of essential molecules in and out of the cell, and (2) sensory transduction of environmental stimuli and developmental signals. The plasma membrane is a two-dimensional, fluid mosaic material composed of primarily lipids and proteins. Considerable progress has been made over the past two decades to understand plant membrane proteins from a genetic perspective, but complementary advances in tools for physiological, electrophysiological, and biochemical characterization of plant membrane proteins have been limited. This work describes the development of Plant Membrane-on-a-Chip, a new cell-free platform consisting of a supported proteo-lipid membrane derived from the plant plasma membrane (PM) that captures the essential features of the membrane and is compatible with state-of-the-art microscopy and biophysical characterization tools. Three model plant species (Arabidopsis thaliana, Nicotiana benthamiana, and Zea mays) were successfully used to form planar plant membranes through the straightforward production of membrane vesicles from protoplasts. Transgenic membrane proteins expressed in the plant cells are directly incorporated with defined orientation and their surrounding lipids into supported membranes without using detergents or reconstitution processes. Supported membranes can be integrated with biosensors and other techniques for the investigation of the biophysical properties of their constituents. Our plant-based supported membranes, therefore, provide a novel tool to isolate and study membrane properties, transport phenomena, biophysical processes, and the protein-lipid interactions that drive them. We illustrate single-molecule tracking of plant membrane proteins using this platform and characterization of their diffusion behavior and orientation.