Association of purified thyroid lysosomes to reconstituted microtubules.
We report the characteristics of the interaction between reconstituted microtubules and purified thyroid lysosomes. Microtubules were extracted from pig brain by temperature-dependent assembly-disassembly and labelled with 125I by conjugation with the Bolton-Hunter reagent. Thyroid lysosomes were purified from pig thyroid by isopycnic centrifugation on Percoll gradients. The formation of microtubule-lysosome complexes has been studied by electron microscopy, using negative staining, and by differential centrifugation. The association of lysosomes to microtubules is time- and temperature-dependent (between 25 degrees C and 37 degrees C). The rate of microtubule-lysosome complex formation is related to the concentration of lysosomes. The higher the lysosome concentration is, the higher also is the rate of the interaction. Changes in microtubule concentration merely alter the amount of complex formed; there is a linear relationship between the amount of complexes and the microtubule concentration. However, lysosomes seem to possess a limited number of 'microtubule-binding sites', since a saturation of the complex formation can be obtained at high microtubule concentration. Two main types of complex have been observed by electron microscopy on negatively stained samples; simple complexes composed of a lysosome in close contact with a microtubule and complexes formed by a lysosome surrounded by several microtubules. The formation of microtubule-lysosome complexes was totally inhibited in the presence of 100 microM N-ethylmaleimide; the rate of the interaction was slightly increased in the presence of dithiothreitol (25-100 microM). The interaction we describe here in an acellular system might be relevant to the association of lysosomes to microtubules observed in intact cells (Collot, M., Louvard D. and Singer S.J. (1984) Proc. Natl. Acad. Sci. USA 81, 788-792) and will constitute a useful model to study the regulation mechanisms of microtubule-vesicle interaction.
Mithieux
G
G
Institut National de la Santé et de la Recherche Médicale, Unité 197, Faculté de Médecine Alexis Carrel, Lyon, France.
Audebet
C
C
Rousset
B
B
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D007457:Iodine Radioisotopes; D007700:Kinetics; D008247:Lysosomes / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008868:Microtubule Proteins / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000648:ultrastructure*; D008970:Molecular Weight; D013552:Swine; D013961:Thyroid Gland / Q000648:ultrastructure*
D007457:Iodine Radioisotopes; D008868:Microtubule Proteins