This MERIT extension remains focused on the development and application of novel fluorescence methods to measure solute mobility and interactions in live cells and tissues.
The aims of the research are a direct extension of ongoing work to understand the determinants of extracellular space (ECS) diffusion and volume (Aim 1), plasma membrane protein diffusion (Aim 2) and protein-protein interactions (Aim 3).
In Aim 1, novel optical methods will be applied to study regulated ECS diffusion, volume and ionic homeostasis in brain and tumor. We will apply microfiberoptic methods, polarization correlation microscopy, and K'^-sensing fluorescent indicators to study ECS regulation in brain and tumor, addressing questions regarding the microviscosity of the extracellular matrix, the dynamic changes in ECS volume and [K*] during neural signal transduction, and the influence of cellular crowding on macromolecule diffusion in the ECS.
In Aim 2, single-molecule fiuorescence methods will be applied to investigate the determinants of membrane protein translational and rotational diffusion. Building on quantum dot-single particle tracking (SPT) studies of aquaporin-4 (AQP4) water channels, we will use multi-color SPT and quantum rod polarization correlation microscopy to investigate the determinants of AQP4 translational and rotation diffusion in live cells, and to study AQP diffusion in lamellipodia of migrating cells and the determinants of AQP4 supramolecular assembly.
In Aim 3, single-molecule fluorescence methods will be applied to investigate protein-protein interactions of membrane water and ion transporters. We have implemented methodology to quantify protein-protein interactions in live cells, including multicolor SPT, two-color FCS, and super-resolution microscopy. Building on our recent studies of AQP water channels and CFTR Cl"""""""" channels, we will investigate the size and dynamics of AQP4 supramolecular assembly, and the cytoskeletal and other interacting proteins of AQP4 and CFTR. These data will provide molecular-level information about the mechanisms and biological consequences of AQP4 and CFTR protein-protein interactions. In addition, the proposed studies will establish novel and widely applicable methodology to measure protein dynamics and interactions in cell plasma membrane and in the extracellular space.
Our research involves the development and application of novel microscopy methods to study protein dynamics and interactions in live cells. The research addresses basic mechanistic questions about the extracellular space in brain and tumor, and how water and ion channels are regulated and assembled at cell membranes.
|Smith, Alex J; Verkman, Alan S (2018) The ""glymphatic"" mechanism for solute clearance in Alzheimer's disease: game changer or unproven speculation? FASEB J 32:543-551|
|Duan, Tianjiao; Smith, Alex J; Verkman, Alan S (2018) Complement-dependent bystander injury to neurons in AQP4-IgG seropositive neuromyelitis optica. J Neuroinflammation 15:294|
|Lee, Sujin; Cil, Onur; Diez-Cecilia, Elena et al. (2018) Nanomolar-Potency 1,2,4-Triazoloquinoxaline Inhibitors of the Kidney Urea Transporter UT-A1. J Med Chem 61:3209-3217|
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|Phuan, Puay-Wah; Veit, Guido; Tan, Joseph-Anthony et al. (2018) ?F508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter. SLAS Discov 23:823-831|
|Verkman, Alan S; Yao, Xiaoming; Smith, Alex J (2018) The evolving mystery of why skeletal muscle is spared in seropositive neuromyelitis optica. J Cell Mol Med 22:2039-2040|
|Jin, Byung-Ju; Verkman, A S (2017) Microfluidic platform for rapid measurement of transepithelial water transport. Lab Chip 17:887-895|
|Felix, Christian M; Lee, Sujin; Levin, Marc H et al. (2017) Pro-Secretory Activity and Pharmacology in Rabbits of an Aminophenyl-1,3,5-Triazine CFTR Activator for Dry Eye Disorders. Invest Ophthalmol Vis Sci 58:4506-4513|
|Haggie, Peter M; Phuan, Puay-Wah; Tan, Joseph-Anthony et al. (2017) Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product. J Biol Chem 292:771-785|
|Yao, Xiaoming; Verkman, Alan S (2017) Complement regulator CD59 prevents peripheral organ injury in rats made seropositive for neuromyelitis optica immunoglobulin G. Acta Neuropathol Commun 5:57|
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