Freeze-fracture Labeling Research Articles

Nanoscale analysis reveals no domain formation of glycosylphosphatidylinositol-anchored protein SAG1 in the plasma membrane of living Toxoplasma gondii.

Glycosylphosphatidylinositol (GPI)-anchored proteins typically localise to lipid rafts. GPI-anchored protein microdomains may be present in the plasma membrane; however, they have been studied using heterogeneously expressed GPI-anchored proteins, and the two-dimensional distributions of endogenous molecules in the plasma membrane are difficult to determine at the nanometre scale. Here, we used immunoelectron microscopy using a quick-freezing and freeze-fracture labelling (QF-FRL) method to examine the distribution of the endogenous GPI-anchored protein SAG1 in Toxoplasma gondii at the nanoscale. QF-FRL physically immobilised molecules in situ, minimising the possibility of artefactual perturbation. SAG1 labelling was observed in the exoplasmic, but not cytoplasmic, leaflets of T. gondii plasma membrane, whereas none was detected in any leaflet of the inner membrane complex. Point pattern analysis of SAG1 immunogold labelling revealed mostly random distribution in T. gondii plasma membrane. The present method obtains information on the molecular distribution of natively expressed GPI-anchored proteins and demonstrates that SAG1 in T. gondii does not form significant microdomains in the plasma membrane.

Freeze-fracture immunocytochemistry: fracture-label and label-fracture for the localization of membrane proteins.

Freeze-fracture is a unique investigative tool for visualization of the en face topography of individual membrane leaflets of cell membranes at high resolution under the electron microscope. The development of a system of freeze-fracture cytochemical and immunocytochemical techniques has further advanced the utility of this methodological approach for high-resolution localization of specific membrane and intracellular macromolecules in tissues and cells. The unit focuses on description, in a step-by-step manner, of the experimental procedures for two specific freeze-fracture labeling techniques, namely fracture-label and label-fracture. Users are guided in a stepwise manner, starting from the preparation of tissue or cell samples to the final retrieval and mounting of fracture-label and label-fracture specimens for examination on the electron microscope.

Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes

The sodium dodecyl sulfate (SDS)-solubilized freeze fracture replica labeling (SDS-FRL) technique has been successfully applied to a vascular plant, Vigna angularis [Kimura et al. 1999, Plant Cell 11: 2075–2085] and a bacterium, Acetobacter xylinum [Kimura et al. 2001, J. Bacteriol. 183: 5668–5674] for understanding cellulose biosynthesis. However, we do not know yet how many gold particles can be bound with antibodies that label a single rosette or linear TC. We analyzed these techniques from a theoretical background by considering the size of gold particle, primary and secondary antibody, and the rosette and linear TC. The 10 nm gold particles coated with primary and secondary antibodies result in a 30 nm diameter for the gold-conjugated antibody that binds to a rosette TC. Therefore, a rosette TC theoretically could be labeled with, at most, four gold particles and commonly with 1–3 gold particles after the topological consideration of the immunogold labeling of the rosette TC. We interpreted an actual image of freeze-fracture labeling of TCs by comparison with a scale model of the antibody-labeling to a rosette TC. The labeling of linear TCs with the gold-conjugated antibody of c-di-GMP binding protein was quite stable, and 75% of the 277 gold particles were found within 20 nm of the linear row. Labeled TCs with depressions and indistinct particle arrays and non-labeled TCs with distinct particle arrays were observed on the P-fracture face of the outer membrane, suggesting that TCs in A. xylinum are composed of two types of subunits.

Localization of c-di-GMP-binding protein with the linear terminal complexes of Acetobacter xylinum.

Specific labeling of a single row of cellulose-synthesizing complexes (terminal complexes, TC subunits, TCs, or TC arrays) in Acetobacter xylinum by antibodies raised against a 93-kDa protein (the cyclic dignanylic acid-binding protein) has been demonstrated by using the sodium dodecyl sulfate (SDS)-freeze-fracture labeling (FRL) technique. The antibodies to the 93-kDa protein specifically recognized the TC subunits on the protoplasmic fracture (PF) face of the outer membrane in A. xylinum; however, nonlabeled TCs were also observed. Two types of TC subunits (particles or pits) are observed on the PF face of the outer membrane: (i) immunogold-labeled TCs showing a line of depressions (pits) with an indistinct particle array and (ii) nonlabeled TC subunits with a distinct single row of particle arrays. The evidence indicates that the labeling patterns differ with respect to the presence or absence of certain TC subunits remaining attached to the replica after SDS treatment. This suggests the presence of at least two TC components, one in the outer membrane and the other in the cytoplasmic membrane. If the TC component in the outer membrane is preferentially fractured and remains attached to the ectoplasmic fracture face (or outer leaflet) of the outer membrane, subsequent replica formation reveals a pit or depression with positive antibody labeling on the PF face of the outer membrane. If the TC component in the outer membrane remains with the PF face (or inner leaflet) of the outer membrane, the innermost TC component is removed during SDS treatment and labeling does not occur. SDS-FRL of TCs in A. xylinum has enabled us to provide the first topological molecular analysis of component proteins in a cellulose-synthesizing TC structure in a prokaryotic organism.

Probing of the Membrane Topology of Sarcoplasmic Reticulum Ca2+-ATPase with Sequence-specific Antibodies: EVIDENCE FOR PLASTICITY OF THE C-TERMINAL DOMAIN

The topology of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles was investigated with the aid of sequence-specific antibodies, produced against oligopeptides corresponding to sequences close to the membranous portions of the protein. The antisera in competitive enzyme-linked immunosorbent assays only reacted with intact SR vesicles to a limited extent, but most epitopic regions were exposed by low concentrations of nondenaturing detergent, octaethylene glycol dodecyl ether (C12E8) or after removal of cytosolic regions by proteinase K. In particular, these treatments exposed the loop regions in the C-terminal domain, including L7-8, the loop region located between transmembrane segments M7 and M8, with a putative intravesicular position, which had immunochemical properties very similar to those of the C terminus with a documented cytosolic exposure. In contrast to this, the reactivity of the N-terminal intravesicular loop regions L1-2 and L3-4 was only increased by C12E8 treatment but not by proteinase K proteolysis. Complexation of Ca2+-ATPase with beta,gamma-CrATP stabilized the C-terminal domain of Ca2+-ATPase against proteinase K proteolysis and reaction with most of the antisera, but immunoreactivity was maintained by the L6-7 and L7-8 loops. Immunoelectron microscopic analyses of vesicles following negative staining, thin sectioning, and the SDS-digested freeze-fracture labeling method suggested that the L7-8 epitope, in contrast to L6-7 and the C terminus, can be exposed on either the intravesicular or cytosolic side of the membrane. A preponderant intravesicular location of L7-8 in intact vesicles is suggested by the susceptibility of this region to proteolytic cleavage after disruption of the vesicular barrier with C12E8 and in symmetrically reconstituted Ca2+-ATPase proteoliposomes. In conclusion, our data suggest an adaptable membrane insertion of the C-terminal Ca2+-ATPase domain, which under some conditions permits sliding of M8 through the membrane with cytosolic exposure of L7-8, of possible functional significance in connection with Ca2+ translocation. On the technical side, our data emphasize that extreme caution is needed when using nondenaturing detergents or other treatments like EGTA at alkaline pH to open up vesicles for probing of intravesicular location with antibodies.

Freeze fracture immunocytochemistry of light-harvesting pigment complexes in a cryptophyte

Immunocytochemical techniques using colloidal gold as the marker have been used to examine the location of the two light harvesting pigment-protein complexes in cryptophyte chloroplasts. A comparison of post-embedding thin section labelling and freeze fracture labelling has been carried out onRhodomonas salina using polyclonal antibodies to a chlorophylla/c2 light-harvesting complex, phycoerythrin and the β-subunit of phycoerythrin. The effect of different fixation procedures on the intensity of labelling and ac curacy of antigen location have been examined and the effectiveness of uranyl acetate and tannic acid in improving both the preservation of thylakoid structure and labelling density of phycoerythrin has been demonstrated. Freeze fracture labelling gives better spatial res olution of the different antigens than post-embedding labelling, as well as better definition of thylakoid membranes. It confirms the location of phycoerythrin in the thylakoid lumen and the location of the chlorophylla/c2 LHC in both appressed and unappressed thylakoid membranes.

Effect of Integral Proteins on Frozen Membrane Fracture

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.