Carmichael’s Concise Review Coming Events
2011 Microscopy Conference MC 2011 August 28–September 11, 2011 Kiel, Germany
www.mc2011.de
Multinational Congress on Microscopy September 4–9, 2011 Urbino, Italy
www.mcm2011urbino.it
ICXOM21 September 5–8, 2011 Campinas, Brazil
icxom21.lnls.br
EMAG 2011 September 6–9, 2011 Birmingham, UK
www.emag-iop.org
National Society for Histotechnology September 16–21, 2011 Cincinnati, OH
www.nsh.org
FEMMS 2011 September 18–23, 2011 Sonoma County, CA
www.femms2011.llnl.gov
CIASEM 2011 September 25–30, 2011 Mérida, Mexico
www.ciasem.com
Neuroscience 2011 November 12–16, 2011 Washington, DC
www.sfn.org
MRS Fall Meeting 2011 November 26–October 2, 2011 Boston, MA
www.mrs.org
American Society for Cell Biology December 3–7, 2011 Denver Convention Center, CO
www.ascb.org/meetings
2012 Microscopy & Microanalysis 2012 July 29–August 2, 2012 Phoenix, AZ
2013 Microscopy & Microanalysis 2013 August 4–8, 2013 Indianapolis, IN
2014 Microscopy & Microanalysis 2014 August 3–7, 2014 Hartford, CT
More Meetings and Courses Check the complete calendar near the back of this magazine and in the MSA journal Microscopy and Microanalysis.
8
Better Protein Localization Stephen W. Carmichael1 * and Philip Oshel2 1 Mayo Clinic, Rochester, MN 55905 2 Central Michigan University, Mt. Pleasant, MI 48859
*
carmichael.stephen@
mayo.edu Localizing specifi c proteins within cells, tissues, and organisms has been a goal of
microscopists for generations. In the early 1990s, a breakthrough was made when a molecule originally derived from a jellyfi sh was introduced as a probe for fl uorescence microscopy. T is molecule is green fl uorescent protein (GFP), and it has become well known for its usefulness in localizing proteins at the level of the light microscope. It is also well known that electron microscopy (EM) off ers far superior spatial resolution over light microscopy, but the application of probes to localize specifi c proteins has required antibodies conjugated with colloidal metals (such as gold). Delivery of antibodies into the cell commonly requires detergents to permeabilize the cell membrane, which compromises the ultrastructural detail. Another breakthrough was recently published on-line by Xiaokun Shu, Varda Lev-Ram, T omas Deerinck, Yingchuan Qi, Ericka Ramko, Michael Davidson, Yishi Jin, Mark Ellisman, and Roger Tsien [1]: they have developed a method similar to using GFP for light microscopy, but for specifi cally tagging proteins at the EM level. T e essential molecule for this technique is miniSOG (mini singlet oxygen
generator); this is a small (106 amino acids), genetically encodable protein that does not need exogenous cofactors to fl uoresce and generate 1O2 (singlet oxygen) when exposed to blue light. Shu et al. began with the concept that the domain (LOV, for light, oxygen, and voltage) of phototropin that binds fl avin mononucleotide could be converted into a molecule that generates 1O2. T is was accomplished by mutagenesis of the phototropin LOV domain in Arabidopsis thaliana. T ey then manipulated the resulting molecule by shuffl ing the DNA to produce miniSOG, which resulted in an increased fl uorescent brightness. T is miniSOG was then fused with the target protein, which allows for effi cient labeling that can be visualized by fl uorescent microscopy, in the same manner as GFP-target fusion proteins. However, the real advantage is that the illumination of miniSOG generates suffi cient 1O2 to locally catalyze the polymerization of diaminobenzidine (DAB) into an osmiophilic reaction product that can be visualized by EM. Furthermore, this can be done on specimens fi xed by conventional techniques that yield images of detailed ultrastructure. Shu et al. demonstrated correct localization of well-understood proteins (α-actinin,
histone 2B, part of cytochrome c, and connexin 43 [Figures 1A and 1B]) tagged with miniSOG in cultured cells. T ey also localized cytochrome c in a multicellular organism
Figure 1: A and B show light microscopic and EM localization, respectively, of Cx43, and C and D show mitochondrial-targeted miniSOG. E shows EM localization of SynCAM2.
doi:10.1017/S1551929511000861
www.microscopy-today.com • 2011 September
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