Showing papers by "Chris Janetopoulos published in 2009"
TL;DR: The kinetics of G-protein activation as a loss of FRET prior to and after cAMP addition by using total internal reflection fluorescence microscopy (TIRFM) are reported and suggest that cAR1 activation slows the membrane dissociation rate of the α2 subunit, while simultaneously promoting βγ-subunit dissociation.
Abstract: Extracellular stimuli exert their effects on eukaryotic cells via serpentine G-protein-coupled receptors and mediate a vast number of physiological responses. Activated receptors stimulate heterotrimeric G-proteins, consisting of three subunits, alpha, beta and gamma. In Dictyostelium discoideum, cAMP binds to the cAMP receptor cAR1, which is coupled to the heterotrimer containing the Galpha2 subunit. These studies provide in vivo evidence as to how receptors influence the localization of the G-protein complex prior to and after ligand binding. Previous work has shown that the state of the heterotrimer could be monitored by changes in fluorescence (or Forster) resonance energy transfer (FRET) between the alpha2- and beta-subunits of D. discoideum. We now report the kinetics of G-protein activation as a loss of FRET prior to and after cAMP addition by using total internal reflection fluorescence microscopy (TIRFM). We also performed photobleaching experiments to measure G-protein recovery times. Our data show that inactive and active G-proteins cycle between the cytosol and plasma membrane. These data suggest that cAR1 activation slows the membrane dissociation ('off') rate of the alpha2 subunit, while simultaneously promoting betagamma-subunit dissociation.
34 citations
TL;DR: As inexpensive tools for 3D visualization, mirrored pyramidal wells may prove to be invaluable aids in nanotechnology and engineering of nanomaterials.
Abstract: A research-grade optical microscope is capable of resolving fine structures in two-dimensional images. However, three-dimensional resolution, or the ability of the microscope to distinguish between objects lying above or below the focal plane from in-focus objects, is not nearly as good as in-plane resolution. In this issue of ACS Nano, McMahon et al. report the use of mirrored pyramidal wells with a conventional microscope for rapid, 3D localization and tracking of nanoparticles. Mirrors have been used in microscopy before, but recent work with MPWs is unique because it enables the rapid determination of the x-, y-, and z-position of freely diffusing nanoparticles and cellular nanostructures with unprecedented speed and spatial accuracy. As inexpensive tools for 3D visualization, mirrored pyramidal wells may prove to be invaluable aids in nanotechnology and engineering of nanomaterials.
9 citations
TL;DR: This study will lay out the methods of conducting FRAP experiments in Dictyostelium and discuss the large amount of knowledge which can be gained by adopting this as a common technique.
Abstract: Dictyostelium discoideum is a useful cell model for studying protein-protein interactions and deciphering complex signaling pathways similar to those found in mammalian systems. Many of these interactions were analyzed using classical in vitro biochemical techniques. However, with the accessibility of fluorescently tagged proteins, extensive protein networks are now being mapped out in living cells using a variety of microscopic techniques. One such technique, fluorescent recovery after photobleaching (FRAP), has been used in Dictyostelium to investigate a number of cellular processes including actin and cytoskeleton dynamics during chemotaxis and cytokinesis (J. Muscle Res. Cell Motil. 23:639-649, 2002; Biophys. J. 81:2010-2019, 2001; Mol. Biol. Cell 16:4256-4266, 2005), to follow trafficking of proteins to organelles such as the membrane, nucleus, and endoplasmic reticulum (Development 130:797-804, 2003; J. Cell Biol. 154:137-146, 2001), and to understand the role of proteins in cell adhesion during motility and division (Mol. Biol. Cell 18:4074-4084, 2007; J. Cell Sci. 120:4302-4309, 2007). FRAP is a powerful tool that should provide a vast amount of information on the mobility of a number of proteins, not only in Dictyostelium, but in many organisms. This study will lay out the methods of conducting FRAP experiments in Dictyostelium and discuss the large amount of knowledge which can be gained by adopting this as a common technique.
5 citations