Association of purified thyroid lysosomes to reconstituted microtubules

Biochimica et Biophysica Acta 969 (1988) 121-130 121 Elsevier

BBA 12244

Associa t ion of puri f ied thyro id lysosomes to recons t i tu ted micro tubules

Gil les Mi th ieux , Chr i s t ine A u d e b e t and Be rna rd Rousse t

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 complexes 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 attachment 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)

122

evidence provided by pharmacological studies using microtubule-disrupting agents: colchicine, vinblastine or nocodazole which inhibit the secretory processes in many endocrine cells such as thyroid cells [17-19]. Among the very numerous secreting cells, the thyroid cell represents a peculiar and interesting model to study the structural basis of intracellular transport phenomena. Indeed, in this cell type, hormone synthesis and secretion bring into play directional flux of different secretory granules (exocytotic vesicles, endocytotic vesicles and lysosomes) involved in the maturation and the processing of the thyroid prohormone, thyroglobulin [20]. In the light of data obtained from other cell systems, we have postulated that the different granule movements in the thyroid cell could represent microtubule-mediated translo- cation processes. In a first attempt to test this hypothesis, we have tried to determine whether purified granules could bind to reconstituted microtubules in an acellular system. Taking ad- vantage of the fact that thyroid lysosomes have been obtained in a highly purified form [21] and since intracellular movements of lysosomes have been correlated with microtubule integrity in other cells [22-24], we have studied the formation of complexes between isolated thyroid lysosomes and microtubules at steady state. Experiments were carried out in iso-osmotic or slightly hyperosmotic medium to maintain lysosomes in an intact form. The physico-chemical parameters governing the association were defined using two complemen- tary approaches: a sedimentation assay and negative-staining electron microscopy. The conditions of centrifugation used for the sedimentation assay allow separation of free microtubules (which do not sediment) from microtubule-lysosome complexes (which cosediment with free lysosomes). The use of microtubules labeled with Bolton- Hunter reagent enabled quantification of the amount of microtubules associated to lysosomes under various experimental conditions.

Materials and Methods

Purification and labeling of microtubule protein. Microtubule protein was purified from pig brain using the procedure described by Shelanski et al. [25]. Two cycles of temperature-dependent poly-

merization-depolymerization were carried out in buffer A (100 mM Mes, 0.5 mM MgCI2, 1 mM EGTA, 0.1 mM GTP, pH 6.4) supplemented with 4 M glycerol and 1 mM GTP in polymerizing steps. Microtubule-associated proteins were pre- pared as the thermostable fraction of microtubule protein cycled twice according to Francon et al. [26]. Twice-cycled microtubule protein was radio- iodinated by conjugation with the 125I-Bolton- Hunter reagent according to the method of Carlier et al. [27] with slight modifications. 100 #1 of microtubule protein in buffer A (500 /*g of protein) were incubated for 60 rain at 0°C in the presence of 1 mCi of 125I-Bolton-Hunter reagent. The reaction was stopped by the addition of glycine (final concentration, 10 raM). After 10 rain at 0°C, labeled microtubule protein was mixed with 1 ml of microtubule protein solution (about 5 mg/ml) maintained at 4°C and submitted to an assembly-disassembly cycle to select labeled molecules which remained polymerization-com- petent. The final 125I-labeled microtubule protein solution with a specific radioactivity of about 0.15 I, Ci/l~g protein was stored in liquid nitrogen. More than 95% of [125I]iodine was recovered on tubulin molecules and equally distributed between the a- and fl-subunits.

Purification of thyroid lysosomes. Lysosomes were purified from pig thyroid according to the method recently described from our laboratory [21]. The final purification step corresponds to the subfractionation of a post-nuclear particulate fraction by isopycnic centrifugation on Percoll gradients. The usual method was modified using a hyper-osmotic medium (0.5 M sucrose instead of 0.25 M sucrose in 10 mM Tris-HC1, pH 7.4) from the beginning of the purification procedure. This alteration allows (a) improvement of the separation of the dense peak (lysosomes) from the lighter peak (other cell organelles) in the Percoll gradients (Fig. 1) and (b) aquisition of a better yield: 10% of total thyroid acid hydrolase activi- ties instead of 5% in the original procedure or 20 mg instead of 10 mg of lysosomal protein per 100 g of tissue, with similar enrichment (50- to 60-fold) in lysosomal marker enzymes [21].

Quantitative assay of lysosome-microtubule complexes. Microtubule protein was mixed with 1251- labeled microtubule protein in buffer A contain-

123

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Fig. 1. Purification of lysosomes by isopycnic centrifugation on Percoll gradient. The crude post-nuclear particulate fraction (26000x g for 20 min) was homogenized in 10 mM Tris-HCl, 0.5 M sucrose (pH 7.4). 1 ml (7 mg of protein) was mixed with 23 ml of 10 mM Tris-HC1, 0.5 M sucrose, 30% Percoll (pH 7.4) and centrifuged at 60000x g for 30 rain in a Beckman 50.2 Ti rotor. Fractions of 1 ml were collected from top to bottom and assayed for protein (o) and acid phosphatase (e) as the lysosome marker enzyme and NADH-cytochrome-c reductase (D) as endoplasmic reticulum marker enzyme. 1 unit of enzyme activity corresponds to 1 /~mol of substrate transformed per rain. The lysosomes (fractions corresponding to 'L') were washed three times in 10 mM Tris-HCl, 0.5 M sucrose (pH 7.4) by centrifugation at 26000x g for 20 rain to remove Percoll. 'P' indicates the particulate fraction (mitochondria, micro-

somes... ) depleted in lysosomes.

ing 0.5 M sucrose. After 30 min at 4 ° C , the mixture was centrifuged at 50 000 x g for 20 min to remove undepolymerized material. 125I-labeled microtubules were then assembled during 20 min of incubat ion at 3 7 ° C in the presence of 1 m M GTP. Microtubules at steady state were sheared by five passages through a 25 gauge needle of a syringe and kept for 5 min at the temperature selected for the interaction experiment (usually 25 o C).

Interact ion experiments were carried out in a final volume of 200/~1, in buffer A containing 0.5 M sucrose (buffer B). The lysosome suspension was equilibrated at the selected temperature for 2 - 3 rain and mixed with the microtubule solution. At the end of incubat ion period, the mixture was layered on a 0.75 M sucrose cushion in buffer A (100/~1, placed in an Eppendor f cone) and centrifuged at 10000 x g for 5 min. The supernatant and the cushion were removed and the pellet was counted for radioactivity in a Packard scintillation

gamma counter. The amount of microtubules recovered in the pellet was calculated from the radioactivi ty measurements and the specific radioactivity of the microtubule protein solution.

Measurement of assembled microtubule protein. The extent of polymerizat ion (propor t ion of tubulin in microtubules) was determined under the various experimental conditions. The incubat ion mixture was centrifuged at 150 000 × g for 60 min at 25 ° C. The supernatant (free tubulin) and the pellet (microtubules) were assayed for radioactivity and protein.

Electron microscopy. For negative-staining studies, 50 #1 samples Were placed on 400 mesh carbonized Formvar-coated grids for 5 min. After rinsing with buffer B, preparat ions were succes- sively fixed using 1% glutaraldehyde in buffer B for 30 min and negatively stained for 5 min with 4% uranyl acetate in methanol. Grids were rinsed with distilled water, air dried and examined in a Jeol 1200 EX electron microscope (Centre de Mi- croscopie Electronique et Pathologie Ultrastruct- urale, Facult6 de Mrdecine Alexis Carrel, Lyon).

Other methods. Analysis of protein was per- formed in 9% acrylamide slab gels in the presence of SDS, as previously described [28]. Gels were dried and autoradiographed using X-ray films. Protein was assayed according to Lowry et al. [29] with bovine serum albumin as standard. Acid phosphatase was assayed with p-ni t rophenylphos- phate as substrate [30]. N A D H - c y t o c h r o m e - c reductase was assayed with cy tochrome c as substrate [31].

Results

Biochemical evidence for the formation of microtubule-lysosome complexes

In order to s tudy the association of purified thyroid lysosomes to in vitro reassembled 12sI- labeled microtubules, we first tried to define centrifugation condit ions allowing sedimentat ion of lysosomes, but not of microtubules, with the idea that putative lysosome-microtubule complexes could sediment in the same manner as do free lysosomes. Such centrifugation condit ions would represent a procedure for the separation of free from lysosome-bound microtubules and, therefore, a direct assay of the amount of lyso-

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some-microtubule complexes by simple radioactivity measurements. We have found that a centrifugation at 10000 × g for 5 min through a 0.75 M sucrose cushion fulfilled the above-mentioned re- quirements. Under this condition: (1) more than 95% of lysosomes were found in the pellet as judged by acid hydrolase activity measurements (in the supernatant plus cushion fraction and in the pellet after osmotic lysis); (2) microtubules did not sediment, 90% of 125I radioactivity or microtubule protein was present in the supernatant. We then studied the sedimentation behaviour of lysosomes and microtubules after coincubation at 25 o C. Lysosomes incubated with 1251-labeled microtubules were quantitatively recovered in the pellet. Interestingly, a large proportion of the radioactivity was found in the pellet together with lysosomes (Fig. 2). In order to determine whether

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Fig. 2. Evidence for the formation of complexes between lysosomes and 1251-labeled microtubules. Four incubation conditions were tested: lysosomes alone (LYS), ]251-1abeled microtubules alone (MTs), lysosomes plus 125I-labeled microtubules ( L Y S + M T s ) and lysosomes plus unpolymerized 1251-labeled microtubule protein (LYS+MTP) . The lysosome concentration was 0.55 mg pro te in /ml . The concentration of 1251-labeled microtubule protein (polymerized or not) was 1.85 m g / m l (spec. radioact.: 108 cpm/ktg ). Experiments with unpolymerized microtubule protein were carried out with microtubule protein preincubated with 10 # M colchicine for 45 rain at 4 ° C . At the end of the incubation period (30 min at 25°C) , mixtures were layered on 100 /*1 of 0.75 M sucrose and centrifuged at 10000 × g for 5 min. Acid phosphatase activity (panel A) and ~2Sl-radioactivity (panel B) were determined in pellets (hatched columns) and supernatants (open columns). Columns and vertical bars represent the mean___ S.D. of dupli-

cate incubations from a representative experiment.

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the radioactivity which cosediments with lysosomes corresponds to 12SI-labeled microtubules or to unpolymerized 125I-labeled tubulin (always in equilibrium with microtubules in suspension) associated to lysosomes, we incubated lysosomes with either 125I-labeled microtubules or the same amount of unpolymerized 125I-labeled tubulin preincubated with 10 /~M colchicine. The results of Fig. 2 clearly show that the radioactivity which sedimented with lysosomes was in the form of microtubules.

Time course of microtubule-lysosome complex formation: dependence upon microtubule and lysosome concentrations

Data presented in Figs. 3 and 4 show that the formation of microtubule-lysosome complexes ex- hibited three distinct phases: (1) a lag period, the duration of which depends on the lysosome concentration (Fig. 3), (2) a phase of interaction during which the amount of complex increased rather linearly and (3) a period of plateau, the level of which varies with the microtubule concentration (Fig. 4). The plateau value was constant for 30-60 rain.

In the presence of a fixed amount of microtubules the increase of the lysosome concentration led to a reduction of both the lag period and the time required to reach the plateau value. The amount of complex formed was the same whatever the lysosome concentration (Fig. 3). Therefore, the concentration of lysosomes only affects the rate of the microtubule-lysosome complex formation. The reciprocal of the time required to obtain the half- maximal interaction was a linear function of the lysosome concentration (panel B of Fig. 3). On the contrary, using a fixed amount of lysosomes, the increase of the concentration of microtubules altered neither the lag period nor the time required to reach the plateau of the interaction, but caused a concentration-dependent increase in the amount of lysosome-bound microtubules (Fig. 4). Note- worthy is that the association of microtubules to a fixed amount of lysosomes was a saturable pro- cess. Under the conditions of Fig. 4, the amount of lysosome-bound microtubules increased linearly between l and 3 m g /m l microtubule protein, then levelled off. When the starting microtubule protein concentration was lower than 3 mg/ml,

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Fig. 3. Effect of the lysosome concentration on the time course of microtubule- lysosome interaction. Panel A, t2Sl-labeled microtubules (440 ~g of microtubule protein, 116 cpm/~tg tubulin) were incubated for 0 -60 min at 2 5 ° C in the presence of lysosomes: 0.12 m g / m l (I) , 0.24 m g / m l (o ) or 0.55 m g / m l (0) in a total volume of 200 ~1. At the end of the incubation period, mixtures were layered on sucrose cushions and centrifuged at 10000 x g for 5 min, the radioactivity of the pellets was counted and transformed in absolute amounts of microtubules. Each point and vertical bar represents the mean and S.D. of duplicate incubations. Panel B, reciprocal plot of the half-maximal interaction time as a function of the lysosomal protein concentration. Values of half-maximal interaction time were determined as indicated in panel A (dotted lines relative to the curve with open symbols). The half-maximal interaction time was only calculated for the curves showing an actual plateau (lysosome concentration higher than

0.15-0.16 mg/ml ) . Symbols (O, ©, I , - ) correspond to four different preparations of lysosomes.

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Fig. 4. Effect of microtubule concentration on the formation of microtubule-lysosome complexes. Panel A, time course of complex formation, Lysosomes (0.3 mg pro te in /ml) were incubated in the presence of 12SI-labeled microtubules: 1.4 m g / m l (©); 2.2 m g / m l (o); 4.3 m g / m l (I) ; the specific radioactivity was 136 c p m / # g tubulin. After the various times of incubation, the amount of microtubules bound to lysosomes was determined as described in the legend of Fig. 3. Each point and vertical bar represents the mean and S.D. of duplicate incubations. Panel B, effect of microtubule protein concentration on the amount of microtubules bound to lysosomes. Conditions are the same as those for panel A. The microtubule protein concentration ranged from 0.8 to 6.3 m g / m l .

Values were taken at the max imum of the interaction (plateau value).

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ly sosome-bound mic ro tubu les represented 70-80% of the total a m o u n t of mic ro tubu les present .

Effect of lysosomes on microtubule stability and composition

Using a mic ro tubu le pro te in concen t ra t ion of 1 - 3 m g / m l , 60 70% was assembled into microtubules in the absence of lysosomes. Upon ad- d i t ion of lysosomes, there was only a slight reduc- t ion in the p ropo r t i on of assembled mic ro tubu le prote in . When the lysosome concen t ra t ion was equal to or lower than 0.6 m g / m l , 50-55% of mic ro tubu le p ro te in was in the form of micro tubules at the p la teau (the s teady state of the in teraction).

The incuba t ion of micro tubules with lysosomes for up to 60 min at 2 5 ° C d id not cause any pro teo ly t ic c leavage of tubul in (Fig. 5). This is a p p a r e n t cons ider ing ei ther the Coomass ie blue-

s ta ined gels or the d i s t r ibu t ion of 125I-labeled c ompone n t s af ter au to rad iography . In contrast , there was a rap id degrada t ion of the high molecular weight mic ro tubu le -assoc ia ted proteins. The d i sappea rance of M A P occurred in less than 2 min, at a t ime at which the associa t ion of microtubules to lysosomes was not observed. The pro- teolyt ic cleavage of these pro te ins was not unex- pected, bear ing in mind their high suscept ibi l i ty to proteinases . The d i sappea rance of M A P could ex- p la in the slight decrease in the p ropor t ion of assembled tubul in ment ioned above. To test whether degrada t ion produc ts of M A P could be involved in the lysosome micro tubule interact ion, pur i f ied M A P (the the rmos tab le fract ion of purified mic ro tubu le prote in) have been added to mi- c r o t u b u l e / l y s o s o m e mixtures. Exogenous M A P nei ther affect the rate of the in terac t ion nor al ter the amoun t of mic ro tubu les which interacts with lysosomes (result not shown). These da ta indicate

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Fig. 5. Analysis of the microtubule protein composition by polyacrylamide gel electrophoresis and autoradiography. Effect of the incubation in the presence of lysosomes. Microtubule protein preassembled for 30 min at 37 o C (lane 1) and further incubated for 60 min at 25 °C in the absence of lysosomes (lane 2). Microtubule protein recovered in the 10000 x g supernatant after 2 (lane 3), 20 (lane 4) or 60 rain (lane 5) of incubation in the presence of lysosomes. Lane 6, 10000x g pellet (containing microtubule-lysosome complexes) after 60 rain of incubation. The material of lane 5 plus that of lane 6 corresponds to the total incubation mixture. Lanes 1' to 6' are the corresponding autoradiograms. The composition of the microtubule/lysosome mixtures was microtubule protein (2 mg/ml, 100 cpm//tg of tubulin) and lysosomes (0.6 mg/ml). 40 ~tg (4000 cpm) of microtubule protein was analyzed in each lane. Note that 125I is essentially located on tubulin and is equally distributed between the a- and /3-subunits, and that MAP are

proteolyzed in less than 2 min of co-incubation with lysosomes.

that MAP (MAP2 and ~- protein) or MAP frag- ments did not mediate the association of lysosomes to microtubules.

Microtubules which interacted with lysosomes were still depolymerizable upon cold treatment. When microtubule / lysosome mixtures taken at the plateau of the interaction (after 30 min at 25°C) were cooled for 5 min at 4°C, less than 10% of microtubule protein was recovered in either the 10000 x g pellet (which indicates that microtubule-lysosome complexes were no longer present) or in the 100 000 x g pellet (which indicates that microtubules were depolymerized). Similar results were obtained when microtubule-lysosome complexes (formed at 25 ° C) were first pelletted at

127

10000 X g then resuspended in cold medium and recentrifuged at 10000 x g or 100000 x g.

Morphological characterization of microtubule-lysosome complexes

Free microtubules, free lysosomes and micro-

tubule- lysosome complexes were studied by elec- t ron microscopy on negatively s tained samples. Microtubules were rather short due to the shearing

procedure used to avoid or decrease artefactual en t rapp ing of lysosomes within microtubule net- works. Lysosomes appeared as spherical particles with a diameter ranging from 100 to 400 nm.

After 30 min of incuba t ion of microtubules in the

presence of lysosomes, two main types of micro-

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tubule-lysosome complex were observed (panel C of Fig. 6): (a) complexes between one microtubule and one lysosome and (b) complexes formed by several microtubules attached to a lysosome. Some more complex pattern corresponding to clusters of several lysosomes and many microtubules were occasionally found. It must be noted that at the plateau of the interaction, there were no free lysosomes. When microtubules were mixed with lysosomes and immediately layered on the grids, lysosomes in close contact with microtubules were only rarely observed. Therefore, microtubule- lysosome complexes observed after 30 rain of incubation at 25 °C did not correspond to artefactual structures obtained by the overlaying on the grids of lysosomes on microtubules or vice versa.

Modulation of the interaction of microtubules with [ysosomes

The rate of microtubule-lysosome complex formation increased with temperature. When the temperature was increased from 25 to 37 ° C, the plateau of the interaction was reached more rapidly. Temperatures below 25 ° C were not tested,

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Fig. 7. Effect of temperature on the rate of microtubule-lysosome complex formation. 1251-labeled microtubules (1.9 nag microtubule protein/ml, 130 c p m / # g tubulin) were incubated in the presence of lysosomes: 0.19 mg/ml (e) or 0.55 mg/ml (c~) for 30 min at 25, 30 or 37°C. Values of half-maximal interaction times were obtained from the time-dependent inter-

action curves as reported in panel A of Fig. 3.

since the assembly state of microtubules could have been altered. The effect of temperature is reported in Fig. 7. There was a linear relationship between the reciprocal of the time required to obtain the half-maximal interaction and the temperature of the incubation medium. This is shown for two concentrations of lysosomes, the concentration of microtubule protein being constant. For a fixed composition of the lysosome/ microtubule mixture, the amount of complex formed was independent of temperature.

The interaction between microtubules and lysosomes was altered by the addition of N-ethylmaleimide into the incubation mixture. The thiol reagent decreased both the rate and the extent of the reaction (Table I). At a concentration of 100 /~M, N-ethylmaleimide completely blocked the formation of microtubule-lysosome complexes. This effect was not related to microtubule disassembly. Indeed, we have checked that, when added on assembled microtubules, NEM did not induce any decrease in the amount of polymerized tubulin. On the contrary, it was observed that NEM prevented the slight lysosome-induced depolymerization effect. The proportion of microtubule protein which remained in the polymerized

TABLE I

EFFECT OF THIOL REAGENTS ON MICROTUBULE- LYSOSOME INTERACTION

Time courses of microtubule-lysosome interaction were determined under the conditions of standard assay, with effectors present since the beginning of the incubation (lysosomes, 0.3 mg/ml ; 125I-microtubules, 2.4 mg microtubule protein/ml, 110 c p m / # g of tubulin). Values of the half-maximal interaction time and the plateau value of microtubules bound to lysosomes were obtained as described in Figs. 3 and 4.

Effcctor Concen- Half-maximal Microtubules tration interaction bound to (t~m) time lysosomes

(min) (plateau value)

(t~g) None 19 184

N-Ethylmaleimide 10 28 165 25 38 101

100 - 0

Dithiothreitol 25 16 181 100 11 188

form at the plateau of interaction appeared higher by about 10% in the presence of 100 /LM NEM than that in the absence of NEM. Dithiothreitol added to the incubation mixture slightly increased the rate of the interaction, the same amount of complexes being formed at the plateau. It appears, therefore, that the lysosome-microtubule association is dependent upon the existence of free thiol groups.

Discussion

This study represents the first demonstration of the interaction of lysosomes with microtubules in a complete acellular system. Our data further document the observation of Collot et al. [6] in intact cells. These authors unequivocally demon- strated by immunofluorescence techniques that lysosomes are attached to microtubules. The existence of specific interaction between microtubules and different cell organelles is now well accepted [32]. Data reported in this paper give some new insight in the properties of such a binding phenomenon.

The formation of the microtubule-lysosome complexes exhibits a lag phase which could mean that some molecular component(s) involved in the interaction has to be transformed from an inactive to an active form. This step seems to be either shortened or by-passed when the lysosome concentration is increased. Once triggered, the interaction of microtubules with lysosomes occurs at a rather constant rate, which is a function of the concentration of lysosomes. Even in the presence of a high concentration of lysosomes, a fraction of microtubules (about 30%) was not capable of interacting with the vesicles. This fact agrees with the results of Suprenant and Dentler [33], who reported that a maximum of 50-60% of in vitro- assembled microtubules interacted with purified pancreatic secretory granules under the most favourable conditions. This could indicate the presence of subclasses of microtubules; if the interaction is dependent upon the presence of a microtubule-associated component, some microtubules could lack this molecular species. Alterna- tively, the existence of uncomplexed microtubules in the presence of microtubule-lysosome complexes could give evidence of a kind of equi-

129

librium between free and lysosome-bound microtubules.

Lysosomes exhibit several interacting sites as judged by electron microscope analysis, but a limited number of sites, since the curve describing the formation of complexes shows a clear saturation at a high microtubule concentration. These results suggest that lysosomes bear on their surface a subset of membrane-associated molecules which could link the vesicle to microtubules. This hypothesis is in agreement with recent reports on the axoplasmic vesicle-microtubule interaction [34, 35]. Polypeptides essential for binding and trans- location are present on the vesicle surface. Given the size of the lysosome, one might expect a microtubule to interact with several binding molecules on the lysosomal membrane. The fact that it is possible for a lysosome to interact through several bridges with a microtubule and to interact with several microtubules should favour the anchorage or the positioning of this rather large vesicle at a given place within the cells.

For some authors, MAP2 [33] or a MAP2-1ike protein [34] are believed to mediate the vesicle-microtubule interactions. For others, kinesin [11] or other polypeptides [35] seem to represent the fundamental components for the microtubule-vesicle attachment and transport [11], or the formation of stable axoplasmic vesicle-microtubule complexes [35]. High-molecular-weight microtubule-associated proteins do not seem to be involved in the microtubule-lysosome binding pro- cess, since we report here that these proteins are degraded by the proteolytic enzymes which are spontaneously released by the lysosome suspension. Furthermore, addition of MAPs (MAP2 and ~- protein) did not change the kinetics of the complex formation. It is also very likely that cleavage products of MAP (which increase upon addition of exogenous MAP) do not represent the key molecules for the interaction.

Our system of measuring the microtubule-lysosome interaction has allowed us to contribute additional information to the knowledge of the association of microtubules to cell organelles; it permits the analysis of both the rate of formation and the amount of complex formed. It will un- doubtedly be very useful in searching for regu- latory mechanisms and putative cytosolic compo-

130

nents able to modulate the association. Finally, our experimental approach could be convenient in the study of the interaction of microtubules with any other subcellular organelles.

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Association of purified thyroid lysosomes to reconstituted microtubules