Susan Hester

THE G PROTEIN-COUPLED RECEPTOR KINASE 2 IS A MICROTUBULE-ASSOCIATED PROTEIN KINASE THAT PHOSPHORYLATES TUBULIN

The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulinin vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied β2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.

Identification of Focal Viral Infections by Confocal Microscopy for Subsequent Ultrastructural Analysis

A correlative microscopy method for the ultrastructural analysis of focal viral tissue infections is presented. Using a confocal scanning laser microscope, foci of infection are identified in tissue sections prior to embedment; a variety of techniques can be employed for viral detection, including staining with standard histochemical reagents and fluorescently labeled antibodies. Areas of infection identified using confocal microscopy are excised from the tissue sections, embedded, and examined by transmission electron microscopy. Applications of this technique in both diagnostic and basic research settings are described.

Electron Tomography of Thick Sections of Insect Flight Muscle

Insect flight muscle (IFM) is a good model system within which to visualize actin-myosin interactions due to its highly ordered lattice of actin and myosin filaments. Lethocerus flight muscle is perhaps the best ordered muscle in nature. Electron tomography (ET) of Lethocerus IFM has recently resulted in a model for the weak to strong transition that incorporates large azimuthal changes in the position of the lever arm compared to that predicted from crystal structures of myosin subfragment 1 in both the nucleotide free and transition states (Wu et al. PLoS-ONE, Sept. 2010). Those studies did not visualize the S2 domain in either the raw tomogram or in subvolume averages which would clarify the crossbridge origin. Here we have used ET of IFM fibers in rigor in which the filament lattice has been swollen in low ionic strength buffer to view where S2 emerges from the thick filament backbone as a test of the weak to strong transition. Previous ET on myac layers (single filament layers containing alternating myosin and actin filaments) of these same swollen rigor fibers revealed the S2 domain with clarity. In the present work, we are examining 80 nm thick transverse and longitudinal sections of swollen rigor IFM fibers in order to visualize all of the crossbridges originating from each 14.5 nm crown on the thick filament, but especially the so-called lead bridges, which bind the thin filament within the same target zone of isometric contraction. Class averages of both thick filaments as well as myac layers are being pursued. The thick filaments show subfilaments in the backbone and many of the myac layer raw repeat subvolumes show S2. Progress on this study will be presented. Supported by NIGMS and NIAMSD.