DEVELOPMENT OF A PLANT MEMBRANE-ON-CHIP PLATFORM FOR INTERROGATING PROTEIN-MEDIATED METAL TRANSPORT

101 pagesTransporter proteins are ubiquitous in nature and play major roles in the uptake, redistribution, and efflux of ions and small molecules required by organisms to maintain homeostasis. Disruptions to transporter function are associated with a variety of diseases and phenotypical abnormalities. In humans, this is evident in the case of diseases such as Menkes and Wilson while in plants, disruption of copper transporters has been associated with phenotypic abnormalities such as reduced growth, fruit yield, and death. The prevalence and association of transporter proteins to many diseases and disorders have highlighted their importance and made them the target of scientific investigation. Despite the strong interest in these proteins, transporter proteins remain a challenge to study. In plants, access to the plasma membrane is hampered by the presence of the cell wall and transport proteins may also be localized to internal organelles further limiting their accessibility. Membrane proteins contain hydrophobic regions complicating attempts to isolate and study them, as these non-polar moieties must be stabilized to maintain form and function. Traditional methodologies for measuring transporter proteins heavily rely on indirect in vivo assays that often require expression in non-native systems, possibly resulting in changes in protein behavior. Direct measurement modalities such as patch-clamp are mainly amenable to certain transporter proteins, such as ion channels, which display electrogenic and fast transport activity. This precludes the measurement of ion transport of many slower or electroneutral transporters such as transport proteins. To address the demand for characterizing this class of proteins, I developed a biomimetic system capable of the direct translation of transport protein function to measurable output, called, “Plant Membrane-on-Chip” platform. This platform leverages the properties of supported lipid membranes to recapitulate the native membrane environment of the transporter proteins through the inclusion of native membrane materials and retention of orientation and fluidity properties. The crucial addition of a biocompatible electronic chip enables the measurement of transporter function using traditional electrochemical characterization techniques that are label-free, sensitive, and non-destructive. For this dissertation, I demonstrate the use of a Plant Membrane-on-Chip device derived from Arabidopsis thaliana plasma membrane material in electrically measuring the function of the copper transporter protein AtCOPT1. Critically, this project highlighted how the use of traditional resistance-based analysis methodologies can be incorporated with new bio-mimics to detect the activity of a non electrogenic transporter previously thought to be unamenable to direct electrical analyses

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

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a “plasma membrane on a chip,” also known as a supported lipid bilayer. Here, we create the “plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein–protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein–protein and protein–lipid interactions in a convenient, cell-free platform

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

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a “plasma membrane on a chip,” also known as a supported lipid bilayer. Here, we create the “plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein–protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein–protein and protein–lipid interactions in a convenient, cell-free platform

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

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a “plasma membrane on a chip,” also known as a supported lipid bilayer. Here, we create the “plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein–protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein–protein and protein–lipid interactions in a convenient, cell-free platform

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

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a “plasma membrane on a chip,” also known as a supported lipid bilayer. Here, we create the “plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein–protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein–protein and protein–lipid interactions in a convenient, cell-free platform