ABSTRACT
Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 µm(3) without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang.
Subject(s)
Imaging, Three-Dimensional/instrumentation , Mitochondria/ultrastructure , Molecular Imaging/instrumentation , Myoblasts/ultrastructure , Animals , Carbocyanines , Cell Line , Fluorescent Dyes , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Imaging, Three-Dimensional/methods , Mice , Microscopy, Fluorescence/methods , Mitochondria/physiology , Molecular Imaging/methods , Myoblasts/physiology , Vimentin/chemistry , Vimentin/metabolismABSTRACT
Sensitivity of mammalian cells to the bacterial toxin aerolysin is due to the presence at their surface of glycosylphosphatidyl inositol (GPI)-anchored proteins which act as receptors. Using a panel of mutants that are affected in the GPI biosynthetic pathway and Trypanosoma brucei variant surface glycoproteins, we show that addition of an ethanolamine phosphate residue on the first mannose of the glycan core does not affect binding. In contrast, the addition of a side chain of up to four galactose residues at position 3 of this same mannose leads to an increase in binding. However, protein free GPIs, which accumulate in mutant cells deficient in the transamidase that transfers the protein to the pre-formed GPI-anchor, were unable to bind the toxin indicating a requirement for the polypeptide moiety, the nature and size of which seem of little importance although two exceptions have been identified.
Subject(s)
Bacterial Toxins/metabolism , Glycoproteins/metabolism , Glycosylphosphatidylinositols/metabolism , Hemolysin Proteins/metabolism , Receptors, Cell Surface/metabolism , Animals , CHO Cells , Cricetinae , HeLa Cells , Humans , K562 Cells , Lectins , Mannose/metabolism , Pore Forming Cytotoxic Proteins , Protein Binding , Variant Surface Glycoproteins, Trypanosoma/metabolismABSTRACT
Protein toxins are soluble molecules secreted by pathogenic bacteria which act at the plasma membrane or in the cytoplasm of target cells. They must therefore interact with a membrane at some point, either to modify its permeability properties or to reach the cytoplasm. As a consequence, toxins have the built-in capacity to adopt two generally incompatible states: water-soluble and transmembrane. Irrespective of their origin or function, the membrane interacting domain of most protein toxins seems to have adopted one out of two structural strategies to be able to undergo this metamorphosis. In the first group of toxins the membrane interacting domain has the structural characteristics of most known membrane proteins, i.e. it contains hydrophobic and amphipathic alpha-helices long enough to span a membrane. To render this 'membrane protein' water-soluble during the initial part of its life the hydrophobic helices are sheltered from the solvent by a barrel of amphipathic helices. In the second group of toxins the opposite strategy is adopted. The toxin is an intrinsically soluble protein and is composed mainly of beta-structure. These toxins manage to become membrane proteins by oligomerizing in order to combine amphipathic beta-sheet to generate sufficient hydrophobicity for membrane insertion to occur. Toxins from this latter group are thought to perforate the lipid bilayer as a beta-barrel such as has been described for bacterial porins, and has recently been shown for staphylococcal alpha-toxin. The two groups of toxins will be described in detail through the presentation of examples. Particular attention will be given to the beta-structure toxins, since four new structures have been solved over the past year: the staphyloccocal alpha-toxin channel, the anthrax protective antigen protoxin, the anthrax protective antigen-soluble heptamer and the CytB protoxin. Structural similarities with mammalian proteins implicated in the immune response and apoptosis will be discussed. Peptide toxins will not be covered in this review.
