Articles by Mauricio R. Galiano in JoVE
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography Sarah H. Shahmoradian1, Mauricio R. Galiano2, Chengbiao Wu3, Shurui Chen4, Matthew N. Rasband2, William C. Mobley3, Wah Chiu4 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Neuroscience, Baylor College of Medicine, 3Department of Neuroscience, University of California at San Diego, 4National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.
Other articles by Mauricio R. Galiano on PubMed
Re-examination of the Post-translational Arginylated Protein of 125-kD Initially Identified As N-STOP Neurochemical Research. Feb, 2004 | Pubmed ID: 15002739 Post-translational modification of proteins is a complex mechanism by which cells regulate protein activities. One post-translational modification is the incorporation of arginine into the NH2-terminus of proteins. It has been hypothesized that in rat brain extracts, one of the proteins modified by this reaction is the microtubule-associated protein Neuronal Stable Tubule Only Polypeptide (N-STOP). This was inferred from its electrophoretic mobility (125 kD) and because it was immunoprecipitated with a monoclonal antibody against the N-STOP. However, this hypothesis is not supported by our recent results. Herein, we found that rat N-STOP interacts with Ca(2+)-calmodulin, whereas the 125-kD [14C]-arginylated protein does not. The 125-kD [14C]-arginylated protein from rat brain is separated from the N-STOP by two-dimensional electrophoresis, and it is not recognized by a STOP monoclonal antibody. Mouse brain contains N-STOP, which migrates as a protein of 116 kD and could not be labeled by the post-translational incorporation of [14C]-arginine. The 125-kD [14C]-arginylated protein appears in wild-type as well as in STOP knock out mice. Based on these results, we conclude that the 125-kD arginylated protein is different from N-STOP.
Post-translational Arginylation of Calreticulin: a New Isospecies of Calreticulin Component of Stress Granules The Journal of Biological Chemistry. Mar, 2007 | Pubmed ID: 17197444 Post-translational arginylation consists of the covalent union of an arginine residue to a Glu, Asp, or Cys amino acid at the N-terminal position of proteins. This reaction is catalyzed by the enzyme arginyl-tRNA protein transferase. Using mass spectrometry, we have recently demonstrated in vitro the post-translational incorporation of arginine into the calcium-binding protein calreticulin (CRT). To further study arginylated CRT we raised an antibody against the peptide (RDPAIYFK) that contains an arginine followed by the first 7 N-terminal amino acids of mature rat CRT. This antibody specifically recognizes CRT obtained from rat soluble fraction that was arginylated in vitro and also recognizes endogenous arginylated CRT from NIH 3T3 cells in culture, indicating that CRT arginylation takes place in living cells. Using this antibody we found that arginylation of CRT is Ca2+-regulated. In vitro and in NIH 3T3 cells in culture, the level of arginylated CRT increased with the addition of a Ca2+ chelator to the medium, whereas a decreased arginine incorporation into CRT was found in the presence of Ca2+. The arginylated CRT was observed in the cytosol, in contrast to the non-arginylated CRT that is in the endoplasmic reticulum. Under stress conditions, arginylated CRT was found associated to stress granules. These results suggest that CRT arginylation occurs in the cytosolic pool of mature CRT (defined by an Asp acid N-terminal) that is probably retrotranslocated from the endoplasmic reticulum.
A Distal Axonal Cytoskeleton Forms an Intra-axonal Boundary That Controls Axon Initial Segment Assembly Cell. May, 2012 | Pubmed ID: 22632975 AnkyrinG (ankG) is highly enriched in neurons at axon initial segments (AISs) where it clusters Na(+) and K(+) channels and maintains neuronal polarity. How ankG becomes concentrated at the AIS is unknown. Here, we show that as neurons break symmetry, they assemble a distal axonal submembranous cytoskeleton, comprised of ankyrinB (ankB), αII-spectrin, and βII-spectrin, that defines a boundary limiting ankG to the proximal axon. Experimentally moving this boundary altered the length of ankG staining in the proximal axon, whereas disruption of the boundary through silencing of ankB, αII-spectrin, or βII-spectrin expression blocked AIS assembly and permitted ankG to redistribute throughout the distal axon. In support of an essential role for the distal cytoskeleton in ankG clustering, we also found that αII and βII-spectrin-deficient mice had disrupted AIS. Thus, the distal axonal cytoskeleton functions as an intra-axonal boundary restricting ankG to the AIS.