The Trypanosoma brucei cytoskeleton: ultrastructure and localization of microtubule-associated and spectrin-like proteins using quick-freeze, deep-etch, immunogold electron microscopy.
We have used a combination of quick-freezing/deep-etching and colloidal gold immunocytochemistry (i) to analyze the molecular organization of the microtubular membrane skeleton and the flagellum of Trypanosoma brucei, and (ii) to localize two defined cytoskeletal proteins within these structures. The cell body of trypanosomatids is enveloped by a membrane skeleton consisting of a tightly packed array of microtubules which are closely associated with the cell membrane. The membrane-oriented face of these microtubules is richly decorated with microtubule-associated proteins, which form intermicrotubule and microtubule-membrane linkers. In contrast, the cytoplasmic faces of the microtubules have a smooth, nondecorated appearance. A previously identified, highly repetitive microtubule-associated protein is confined to the membrane-oriented face of the microtubular array, suggesting that the function of this protein may be that of a microtubule-membrane linker. Quick-freezing has also been used to reveal the geometric organization of the paraflagellar rod structure in the flagellum, its interaction wit the cell body, and a unique series of fleur-de-lis-like molecules which link this organelle to axonemal microtubules. Immunohistochemistry using an antibody against human erythrocyte spectrin suggests that these linker structures may contain ancestral spectrin-like molecules.
Hemphill
A
A
Institut für Allgemeine Mikrobiologie, Bern, Switzerland.
Seebeck
T
T
Lawson
D
D
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure*; D000939:Epitopes / Q000187:drug effects / Q000276:immunology; D005407:Flagella / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D005557:Formaldehyde / Q000494:pharmacology; D005613:Freeze Etching; D005976:Glutaral / Q000494:pharmacology; D006046:Gold; D006801:Humans; D016253:Microscopy, Immunoelectron; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000648:ultrastructure*; D015800:Protozoan Proteins / Q000032:analysis / Q000648:ultrastructure*; D013049:Spectrin / Q000032:analysis / Q000276:immunology / Q000648:ultrastructure*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000648:ultrastructure*
D000939:Epitopes; D008869:Microtubule-Associated Proteins; D015800:Protozoan Proteins; D013049:Spectrin; D005557:Formaldehyde; D006046:Gold; D005976:Glutaral