Biochimica et Biophysica Acta 969 (1988) 121-130 121 Elsevier BBA 12244 Association of purified thyroid lysosomes to reconstituted microtubules Gilles Mithieux, Christine Audebet and Bernard Rousset Institut National de la Sant~ et de la Recherche M~dicale, Unit~ 197, Facult~ de M~decine Alexis Carrel, 69372 Lyon C$dex 08 (France) (Received 9 September 1987) Key words: Thyroid: Lysosome; Microtubule; (Pig brain) We report the characteristics of the interaction between reconstituted microtubules and purified thyroid iysosomes. Microtubules were extracted from pig brain by temperature-dependent assembly-disassembly and labelled with 12Sl 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 °C and 37 o 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 com- plexes was totally inhibited in the presence of 100/~M N-ethyimaleimide; the rate of the interaction was slightly increased in the presence of dithiothreitol (25-100 ~M). 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. Introduction It is now commonly accepted that interphasic microtubules are involved in the distribution and intraceUular motion of subcellular organelles Abbreviations: MAP, microtubule-associated protein; Mes, 4- morpholineethanesulphonic acid; NEM, N-ethylmaleimide. Correspondence: G. Mithieux, INSERM U. 197, Facult6 de M~decine Alexis Carrel, rue Guillaume Paradin, 69372 Lyon C6dex 08, France. throughout the cytoplasm. It has been reported that the Golgi apparatus [1 3], mitochondria [4,5]] and lysosomes [6,7] are associated to microtubules in several types of cultured cell. Movements of subcellular vesicles have been shown to occur along microtubules in various cell types, such as squid giant axon [8-12], ameba Reticulomyxa [13,14] or keratocytes [15]. In endocrine cells, the movement of secretory granules seems also to be dependent on the at- tachment of granules to microtubules [16]. How- ever this statement is largely based on indirect 0167-4889/88/$03.50 4) 1988 Elsevier Science Publishers B.V. (Biomedical Division)