title: Metallothionein as a clonable high-density marker for cryo-electron microscopy.
abstract: Cryo-electron microscopy is expanding its scope from macromolecules towards much larger and more complex cellular specimens such as organelles, cells and entire tissues. While isolated macromolecular specimens are typically composed of only very few different components that may be recognized by their shape, size or state of polymerization, cellular specimens combine large numbers of proteinaceous structures as well as nucleic acids and lipid arrays. Consequently, an unambiguous identification of these structures within the context of a whole cell may create a very difficult challenge. On plastic-embedded specimens, or Tokuyasu sections (Tokuyasu, 1980), epitopes that are exposed at the surface can be tagged by antibodies. However, vitrified sections have to be kept at strict cryo-conditions (below -140 °C) and therefore do not allow any post-sectioning treatment of the specimens other than data acquisition in the microscope. Hence, the labels have to be placed into the specimen before freezing. Here we report on the application of a small metal-clustering protein, metallothionein (MTH), as a clonable label capable of clustering metal atoms into a high-density particle with high spatial resolution. We tested MTH as a label for kinesin-decorated microtubules (MTs) as well as the building blocks of desmin intermediate filaments (IFs).
authors: Dept. of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, CO 80309-0347, USA.
authors: Cédric
authors: C
authors: Bouchet-Marquis
authors: Maria
authors: M
authors: Pagratis
authors: Robert
authors: R
authors: Kirmse
authors: Andreas
authors: A
authors: Hoenger
mesh_terms: D003001:Cloning, Molecular; D020285:Cryoelectron Microscopy; D003893:Desmin / Q000235:genetics / Q000648:ultrastructure; D005786:Gene Expression Regulation; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007382:Intermediate Filaments / Q000235:genetics / Q000648:ultrastructure; D008668:Metallothionein / Q000235:genetics / Q000648:ultrastructure*; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D015388:Organelles / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D013048:Specimen Handling / Q000379:methods
chemical_list: D003893:Desmin; D008668:Metallothionein