title: Measurement of trehalose loading of mammalian cells porated with a metal-actuated switchable pore.
abstract: Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. Among the key determinants of desiccation tolerance is the intracellular trehalose concentration, and thus quantifying the amount and rate of trehalose accumulation has now become very critical to the success of these desiccation approaches. We introduced trehalose into 3T3 fibroblasts, human keratinocytes, and rat hepatocytes using a genetically engineered mutant of the pore-forming alpha-hemolysin from Staphylococcus aureus. Manipulating the extracellular Zn(2+) concentration selectively opens and closes this pore ( approximately 2 nm) and enables controlled loading of cells with sugars. We quantified intracellular trehalose using gas chromatography-mass spectroscopy (GC-MS) to examine the trimethylsilyl derivative of intracellular trehalose. Using the GC-MS method, we demonstrate that the switchable characteristics of H5 alpha-hemolysin permit controlled loading of the high concentrations of trehalose (up to 0.5 M) necessary for engineering desiccation tolerance in mammalian cells.
authors.affiliation: The Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospitals for Children, 51 Blossom Street, Room 266, Boston, Massachusetts 02114, USA.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Acker
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Lu
authors.forename: Vernon
authors.initials: V
authors.lastname: Young
authors.forename: Stephen
authors.initials: S
authors.lastname: Cheley
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
authors.forename: Alex
authors.initials: A
authors.lastname: Fowler
authors.forename: Mehmet
authors.initials: M
authors.lastname: Toner
mesh_terms: D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D019610:Cytoprotection / Q000187:drug effects / Q000502:physiology; D003890:Desiccation / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006460:Hemolysin Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016062:Porosity / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D014199:Trehalose / Q000378:metabolism* / Q000493:pharmacokinetics; D015032:Zinc / Q000494:pharmacology*
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C021091:staphylococcal alpha-toxin; D014199:Trehalose; D015032:Zinc