A novel embedment-free immunoelectron microscopy technique reveals association of apoptosis-regulating proteins with subcellular structures.
By employing two electron microscopy techniques, postembedding double- and triple immunocytochemical gold-labelling combined with embedment-free electron microscopy (EF-EM), we have detected previously unreported nuclear and cytoplasmic complexes between different proapoptatic proteins in a human cancer cell line COLO 205 stimulated to apoptosis by nimesulide, a specific cyclooxygenase-2 inhibitor. Experiments with the use of double- and triple immunolabelling visualized the colocalization of proapoptotic proteins such as Bax with Bid, Bax with Bid and voltage-dependent anion channel protein (VDAC-1), and Bax with Bid and caspase-8, on organellar membranes and within the nucleus. Application of this technique in combination with EF-EM technique augments our knowledge on the precise identification and relationship of subcellular structures containing Bax, Bid, VDAC-1 and caspase-8.
Gajkowska
B
B
Laboratory of Cell Ultrastructure, Medical Research Centre, Polish Academy of Sciences, 5 Pawinski Street, 02 106 Warsaw, Poland.
Wojewódzka
U
U
D000230:Adenocarcinoma / Q000378:metabolism / Q000648:ultrastructure; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002462:Cell Membrane / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D022082:Cellular Structures / Q000378:metabolism / Q000648:ultrastructure*; D003110:Colonic Neoplasms / Q000378:metabolism / Q000648:ultrastructure; D051546:Cyclooxygenase 2; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008565:Membrane Proteins; D016253:Microscopy, Immunoelectron / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D009685:Nuclear Envelope / Q000648:ultrastructure; D018272:Porins / Q000378:metabolism / Q000648:ultrastructure; D011451:Prostaglandin-Endoperoxide Synthases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D013194:Staining and Labeling; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D050994:Voltage-Dependent Anion Channels; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D018272:Porins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D050994:Voltage-Dependent Anion Channels; D051028:bcl-2-Associated X Protein; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C012655:nimesulide