Light-induced translocation of cyclic-GMP phosphodiesterase on rod disc membranes in rat retina

PURPOSE. Cyclic GMP phosphodiesterase (PDE) is the light-regulated effector enzyme of vertebrate rods. Upon photo-activation of rhodopsin followed by activation of transducin/GTP, PDE rapidly hydrolyzes 3′, 5′-cyclic GMP (cGMP) to 5′-GMP, which results in closure of cGMP-dependent ion channels and generation of a nerve signal. In the rod photoreceptors, PDE is entirely localized within the rod outer segment (ROS), a specialized compartment consisting of thousands of disc stacks. This study investigated the effects of light on the subcellular localization of PDE in ROS. METHODS. Adult rats were either dark- or light-adapted for various durations before eyes were isolated and processed for transmission electron microscopy. Immunogold electron microscopy was performed with antibodies against PDE. Lateral displacement of PDE on ROS disc membrane was analyzed from electron micrographs. PDE enzymatic activities were measured with thin layer chromatography. RESULTS. Light exposure induced translocation of PDE away from the edges of the dark-adapted disc membranes adjacent to the ROS plasma membrane. In dark-adapted ROS, a substantial portion (19±2%) of total PDE was localized near the edges of the disc membranes. Within 1 min of light exposure in the presence of GTP, over half of such PDE molecules (10±1% of total PDE) had moved away from the edges of the discs toward disc center. This light induced translocation of PDE was GTP dependent, as the effect was abolished when hydrolysis-resistant GTPγS was used in place of GTP. The percentage of PDE found near the disc edge corresponds to the fraction of PDE activity relative to maximal PDE activity revealed by limited trypsin proteolysis. CONCLUSIONS. These results suggest that light and GTP modulates lateral displacement of PDE, which might contribute to light-induced reduction of rod photoreceptor sensitivity.National Eye Institute (R01 EY14057); Juvenile Diabetes Research Foundation International fellowship (10–2008–603

Biomimetic autoseparation of leukocytes from whole blood in a microfluidic device

Leukocytes comprise less than 1% of all blood cells. Enrichment of their number, starting from a sample of whole blood, is the required first step of many clinical and basic research assays. We created a microfluidic device that takes advantage of the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, to separate leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels designed to enhance lateral migration of leukocytes and their subsequent extraction from the erythrocyte-depleted region near the sidewalls. A single pass through the device produces a 34-fold enrichment of the leukocyte-to-erythrocyte ratio. It operates on microliter samples of whole blood, provides positive, continuous flow selection of leukocytes, and requires neither preliminary labeling of cells nor input of energy (except for a small pressure gradient to support the flow of blood). This effortless, efficient, and inexpensive technology can be used as a lab-on-a-chip component for initial whole blood sample preparation. Its integration into microanalytical devices that require leukocyte enrichment will enable accelerated transition of these devices into the field for point-of-care clinical testing