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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37EB000415-22
Application #
8642647
Study Section
Special Emphasis Panel (NSS)
Program Officer
Conroy, Richard
Project Start
2012-04-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
22
Fiscal Year
2014
Total Cost
$412,129
Indirect Cost
$145,379
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Flores, Alyssa M; Casey, Scott D; Felix, Christian M et al. (2016) Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease. FASEB J 30:1789-97
Cil, Onur; Haggie, Peter M; Phuan, Puay-Wah et al. (2016) Small-Molecule Inhibitors of Pendrin Potentiate the Diuretic Action of Furosemide. J Am Soc Nephrol 27:3706-3714
Jin, Byung-Ju; Smith, Alex J; Verkman, Alan S (2016) Spatial model of convective solute transport in brain extracellular space does not support a ""glymphatic"" mechanism. J Gen Physiol 148:489-501
Cil, Onur; Phuan, Puay-Wah; Lee, Sujin et al. (2016) CFTR activator increases intestinal fluid secretion and normalizes stool output in a mouse model of constipation. Cell Mol Gastroenterol Hepatol 2:317-327
Esteva-Font, Cristina; Jin, Byung-Ju; Lee, Sujin et al. (2016) Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1. Mol Pharmacol 89:686-93
Yao, Xiaoming; Su, Tao; Verkman, A S (2016) Clobetasol promotes remyelination in a mouse model of neuromyelitis optica. Acta Neuropathol Commun 4:42
Felix, Christian M; Levin, Marc H; Verkman, Alan S (2016) Complement-independent retinal pathology produced by intravitreal injection of neuromyelitis optica immunoglobulin G. J Neuroinflammation 13:275
Zhang, Hua; Verkman, A S (2015) Aquaporin-1 water permeability as a novel determinant of axonal regeneration in dorsal root ganglion neurons. Exp Neurol 265:152-9
Cil, Onur; Esteva-Font, Cristina; Tas, Sadik Taskin et al. (2015) Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats. Kidney Int 88:311-20
Yao, Xiaoming; Smith, Alex J; Jin, Byung-Ju et al. (2015) Aquaporin-4 regulates the velocity and frequency of cortical spreading depression in mice. Glia 63:1860-9

Showing the most recent 10 out of 151 publications