Group Members: Phil Oh, Emily Holland
We have expertise in isolating specific proteins, proteins complexes, membrane microdomains, cell organelles, and cells directly from tissue. Even in highly vascularized tissue, the endothelium represents only a very small percentage of the cells present in the whole tissue. The endothelial cell plasma membrane exposed directly to the blood is a small fraction of the total membrane in the cell, and caveolae are an even smaller fraction. Because endothelial cells are such minor components of the tissue, global proteomic analysis of whole tissue is unlikely to yield much useful information about the endothelial cell proteome. This facility performs the critical subcellular fractionation of the tissue to unveil the proteins concentrated on the endothelial cell surface that otherwise are beyond the dynamic range of detection.
The Tissue Subcellular Fractionation facility currently consists of an L8-80M and an OptimaxE Ultracentrifuge, a Glas-Col variable speed tissue homogenizer, a magnetic isolator, a multi-channel animal perfusion apparatus, and multiple gel electrophoresis apparatus. This facility also has full access to the complete PRISM lab space, which includes cold rooms, radioactive labeling preparation and analysis rooms, tissue culture suites, communal instrument rooms, scintillation counters, refrigerators, chemistry hoods ultracentrifuges and key rotors, tissue culture incubators ultra-low freezers, a MilliQ water purification system, and general-use computers.
Classic techniques for isolating plasma membranes from tissues yield a membrane fraction that does indeed contain endothelial cell plasma membranes, but only as a small percentage of the total membrane isolated. This makes detection, identification, and purification of endothelial cell surface proteins very difficult and comparisons of endothelium between tissues nearly impossible. To specifically isolate the luminal surface of endothelial cell in vivo, we perfuse the vasculature with a colloidal solution of silica-based nanoparticles.These nanoparticles coat the surface of cells exposed to the blood, forming a stable adherent pellicle that increases the plasma membrane's density, adhering so strongly to the luminal membrane that after tissue homogenization, large sheets of nanoparticle-coated endothelial cell plasma membrane with their attached caveolae are readily isolated from other cellular membranes and debris by centrifugation through a high density medium. Subcellular fractionation can be readily applied to cells in culture allowing comparisons between in vitro and in vivo protein expression. Silica nanoparticles are too large to enter the caveolar invaginations; therefore, caveolae can be separated from the plasma membrane either mechanically by homogenization or more physiologically by induced budding and then isolated to homogeneity by flotation in a sucrose gradient. This technique has been successful in subfractionating the luminal endothelial cell membrane and caveolae away from other plasmalemmal microdomains, including cytoskeletal elements or lipid rafts. Electron microscopy and immunomagnetic separation confirms a homogeneous population of morphologically distinct caveolae with diameters <90nm.
To ensure that we isolate consistently high-quality material, each lot of tissue/cell homogenate, luminal endothelial cell membranes, and caveolae is subjected to rigorous quality control testing. We require the luminal endothelial cell plasma membranes to be at least 20-fold enriched, compared to the total tissue homogenated, for at least 2 endothelial cell surface marker proteins, such as caveolin-1, 5'-nucleotidase, VE-cadherin-1, angiotensin converting enzyme (ACE) or endothelial nitric oxide synthase (eNOS). In addition, the luminal membranes must be markedly depleted by >20-fold relative to the total tissue homogenate in well-established markers of other cell types and intracellular organelles. For caveolae, we require a 20-fold enrichment for caveolin-1 and 20-fold depletion in ACE or b-actin, relative to the luminal membranes.
Over nearly two decades, we have developed and optimized these tissue subfractionation techniques, which have been used in multiple published studies. To our knowledge, no other lab can isolate these EC membranes from organs in vivo to this level of rigor, which is essential to identify tissue-specific vascular proteins. Tissue subfractionation and appropriate quality control is vital to isolating pure populations of caveolae and studying the molecular components and function of caveolae with confidence and fidelity.
