Hybrid microtubules from mixtures of human fibroblast homogenates and chick brain microtubules

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Experimental

HYBRID FIBROBLAST

Cell Research 124 (1979) 403-412

MICROTUBULES HOMOGENATES

FROM AND

MIXTURES

CHICK

BRAIN

OF HUMAN MICROTUBULES

Absence of High Molecular Weight Associated Proteins KRISTIN

HINDS

and DAVID SOIFERL * **

Ltnstitute for Basic Research in Mental Retardation, Staten Island, NY 10314, and Cornell University Graduate School of Medical Sciences, New York, NY 10021, USA

SUMMARY Microtubules have been assembled from a mixture of chick-brain microtubule protein and total soluble protein of cultured human fibroblasts. In this system microtubules were assembled which did not have any high molecular weight (HMW) microtubule-associated protein (MAP). Since the tibroblasts were human in origin, the hybrid microtubules were compared to microtubules assembled from human brain, which do include HMW-MAPS. To determine whether HMW-MAP is unique to brain, microtubules were assembled from mixtures of soluble proteins fromnon-neural mouse organs and chick brain microtubules. These hybrid microtubules contain similar HMWMAPS to those of the chick brain alone. The absence of HMW-MAPs from the hybrid microtubules does not appear to be due to proteolysis. SDS-gel electrophoresis of all fractions prepared during the process of assembly of the hybrid microtubules reveals that the HMW-MAPs of the mixture are sedhnented away from disassembled microtubules during the fast centrifugation. The exclusion of the HMW-MAPs from the hybrid microtubules suggests that the assembly process in these mixtures, and in tibroblasts, may be qualitatively different from that found in extracts from brain and other organs.

Microtubules are structures ubiquitous to all eukaryotic cells [ 11, Some of the fimctions ascribed to microtubules require that they exist in a form allowing rapid assembly or disassembly to meet the changing needs of the cell [2, 3, 43. It has been suggested that dynamic equilibria exist between assembled microtubules and a pool (or pools) of the subunit protein, tubulin [5,6]. In such a system, regulatory signals must exist which cause microtubules to be assembled when and where needed by the cell. The mechanisms of the assembly of tubulin into microtubules and the regulation of the assemblyprocess have been extensively studied using proteins from brain, which is rich in tubulin [7]. Because in vitro as-

sembly of brain microtubule protein is possible [8], the effect of such parameters as cation concentration [9] and nucleotides [9, 10, 11, 12, 131on this assembly has been studied. It has also become apparent that, in the brain system, a number of proteins copurify with tubulin. A role for some of these microtubule-associated proteins (MAPS) in the process of microtubule assembly has been proposed [14, 15, 161. These investigators have been interested primarily in the purification of CCand ptubulins but the MAPS have not been described. * Present address: University of Colorado Medical School, Denver, CO 80220, USA. ** To whom reprint requests should be addressed. Exp Cell Res 124 (1979)

404

Hinds and Soifer

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35’. 30 ml”

wc”B*TE AT

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s2:

DISCARD

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’ Fig. 1. Outline of preparation of hybrid IN h%DmLY WFFW FanFuRTnEn CYCLES microtubules. Further cycles generate &, l?ESuEPEND

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It has been difficult to purify microtubules by assembly from tissues otherthan brain. Although’ success has been reported at assembling microtubules from extracts of cultured cells [17, 181,it is difficult to grow the number of cells needed to provide enough microtubule protein for meaningful experiments. For these reasons, a number of reports have appeared in which brain microtubule protein was used as a carrier to allow purification by co-assembly of microtubule protein from other sources [19, 20, 211. We have used this coassembly approach in the course of studies to determine the characteristics of microtubule protein in cultured fibroblasts from patients with Chediak-Higashi syndrome. From a mixture of embryonic chick brain microtubule protein and fibroblast cell protein, we assembled microtubules which appear to incorporate protein from both sources. The microtubules formed did not retain any of the MAPS characteristic of brain microtubules.

MATERIALS

AND METHODS

A microtubule assembly system, summarized in fig. 1, was used to purify fibroblast microtubule protein. Brain microtubule protein was prepared from 17-day Exp CdRes 124(1979)

embryonic chick brains by the method Shelanski et al. [22]. Pellets of assembled microtubules were frozen at -70°C and stored for not more than one month before use. On the day of the experiment, these microtubules were carried through 1f cycles of disassembly and assembly, yielding a suspension of cold-disassembled microtubule protein. The buffer used was 0.05 M MES, 0.5 mM MgCl,, 1 mM EGTA, pH 6.9 (assembly buffer). For assembly steps, the solution was diluted 1: 1 with 8 M glycerol in this buffer and made 1 mM with GTP. The human diploid ftbroblasts were established [23] and maintained [24], as previously described. Twenty-four hours after subcultivation, the growth medium (modified Eagle’s MEM with 10% fetal bovine serum, Microbiological Associates) in six 75 cm2 Falcon flasks, was replaced by medium lacking methionine (Grand Island Biological+ Twelve hours later, 2 &i/ml [35S]methionine was added, together with one-fourth the normal amount of unlabelled methionine. After 72 h of growth in this medium, the cells were harvested by trypsinization and washed twice with phosphate-buffered saline. The resultant cell pellet was resuspended in the solution of disassembled chick brain microtubule protein, freeze-thawed three times, and homogenized in a Dounce homogenizer (Bpestle). This suspension was then centrifbged at 100000 g for 60 min. The supernatant from this centrifugation was mixed 1: 1 with 8 M glycerol in assembly buffer and 2 4 cycles of assembly and disassembly were carried out. The fmal suspension of disassembled microtubules was frozen at -20°C in 8 M glycerol until further use. Chick brain alone was canied through the assembly procedure to provide a control for the effect of the cell protein on assembly. YS-labelled microtubule protein was prepared from 3-day-old rats. Sixteen hours prior to sacrifice each rat received 150 &i of [SsS]methionine by intracranial injection. The microtubules were prepared by the method of Shelanski et al. [22] and stored as frozen pellets at -70°C. After 24 cycles, this batch of YSmicrotubules had a specific radioactivity of 108cpm/ mg protein. SDS-polyacrylamide gel electrophoresis was carried out on 0.75 mm thick 5-15% gradient slab gels. The

Hybrid microtubules

from human fibroblast

and chick brain

405

Fig. 2. (a) Typical field of assembled hybrid microtubules. Negative stain. (b) Microtubule from hybrid preparation. Note typical protofilament organization

and curling of protofdaments at end (arrow). tc) Section through pellet of hybrid microtubules. (a) X18500;@) x102000; (c)29000.

discontinuous buffer system of Laemmli [25] was used, and the gels were stained with Coomassie Blue R-250. The gels were processed for fluorography [26], dried, and exposed to X-ray film. Protein samples for negative stamina were aDDlied to glow discharge-treated carbon films on coppk; grids, and stained with 1% uranyl acetate. Assembled microtubules were pelleted in &I Airfuge (Beckman Instruments), fixed in 2 % glutaraldehyde in homogenization buffer, and post-fued in 1% osmium tetroxide, dehydrated, and embedded in Spurr’s resin. Ultrathin sec-

tions were prepared and stained with uranyl acetate followed by lead citrate [27]. The mouse tissues used in these experiments were from C3H mice. Human brain microtubules were preoared as uart of a studv of neurofibrillary proteins hi collaboration with Dr Hknryk Wisniewski. Protein determinations were done by the method of Lowry [28] or Bradford [29]. Radioactivity was estimated by liquid scintillation spectrometry. Protein samples were solubilized and counted in Ready-solv GP cocktail (Beckman). Exp Cell Res I24 (1979)

Table 1. Total protein and total CPM after each step in preparation of hybrid microtubules Fraction Homogenate S, p2 s3 P4

--

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JIEU &

123456789 Fig. 3. SDS-gel electrophoresis of fractions S3 (slots l-3), P4 (slots 4-6) and Ss (slots 7-9). Chick-CHS hybrids are in slots 1, 4, 7; chick-control fibroblast hybrids are in slots 2,5,8 and chick brain microtubule protein alone are in slots 3, 6, 9. (a) Coomassie bluestained gel. Arrows indicate HMW-MAPS. (6) Fluorograph of gel in (a) demonstrating protein contributed by cells.

Chick” Chick + Jp* Chick + S@ Chick Chick + Jp Chick + Sp Chick Chick + Jp Chick + Sp Chick Chick + Jp Chick + Sp Chick Chick + Jp Chick + Sp Chick Chick + Jp Chick + Sp

Total protein (md 20.8 31.5 38.2 17 18.7 19.5 E 1o:o 6.5 6.3 6.7 4.3

a 5 X 106cpm [%]methionine crotubules as control. b Jp, Control fibroblasts. c Sp, CHS fibroblasts.

::“6 3.1 3.3 3.4

Total cpm (X 10-6)

20 102 79 0.25 8.4 6.7 0.19 4.6 3.4 0.009 2.4 1.8 0.005 1.1 1.2

added to chick brain mi-

major fibroblast protein isolated by coassembly also migrates on SDS polyacrylamide gel electrophoresis with the characteristics of a!- and P-tubulin. In the absence of the brain carrier protein, no microtubules RESULTS Microtubules were formed under standard are formed from ftbroblast extract under assembly conditions from a mixture of assembly conditions, In these experiments, the preponderance chick brain microtuble and soluble protein from cultured human skin tibroblasts (fig. of the protein in the assembly mixture was 1). Fig. 2 shows the morphology of the contributed by the chick brain microtubule structures formed, which is typical of in protein. In a typical experiment 6.0~10’ vitro assembled microtubules. There were cells were labeled with [35S]methionine and few, if any aberrant forms observed in sec- coassembled with 20.8 mg (5.2 mg/ml) brain tions or by negative staining. The protein in microtubule protein. The cell protein was the pellet obtained after thermal-induced approx. 12% of the soluble protein in which microtubule assembly consisted primarily microtubule assembly was induced. The of cr- and P-tubulin, as seen in the Coomas- microtubules formed consisted of 98% sie blue-stained gels in fig. 3 a. The fluoro- chick protein, but also included [35S]methiogram shown in fig. 3 b demonstrates that the nine-labelled protein contributed by the Exp Cell Res 124 (1979)

Hybrid microtubules from human fibroblast and chick brain

407

*

Fig. 4. (a) SDS-gel eiectrophoresis of chick b&n microtubules (slot 1) and hybrid microtubules(siti 2). (6) Fluorogram of hybrid microtubules @lot 1) and radioactive microtubules from rar brain (slot 2). ACWWS, HMW-MAPS.

@roblasts. The recovery of total and labelled protein at each preparative step is shown in table 1. The surprising finding in these experiments ws that the hybrid microtubules lacked the MAPS characteristic of brain microtubules. The most prominent MAPS in the carrier microtubules as seen in fig. 4a (slot I) are high molecular weight (HMW) proteins (see also [Iis) and [143). These proteins are not found in the coassembled microtubules as seen in slot 2 of fig. 4~. (see also fi. 3). In fig. 4f>, the proteins contributed by the cells (slot I) are compared with radioactive microtubules prepared from the brains of young rats (slot 2). In this fluorogram HMW-MAPS are major constituents of the rat brain microtubules. No proteins are present in this region of slot 1. The most

Fig. 5. .SDS-gelelectrophoresisof hybrids to which additional chick brain microtubule protein has been added. Slots I, 3 and 5 are CHS-chick hybrids; s&s 2,4,6. control fibroblast-chick hybrids. Ps(slots I-2). These hybrids were put through another cycle to give ST(slots 3 and 4) and Ps(slots 5 and 6).

prominent fibroblast MAP has an apparent molecular weight of about ~50000 D. Two faint high molecular weight bands are just detectable which migrate significantly ahead of the HMW-MAR Tn general, MAPS were not prominent components of the radioactive fibroblast protein, with ~1and &tubulins accounting for more than 90% of the total radioactivity of the hybrid microtubuIes . Another possible reason for the absence of the HMW-MAPS is proteolysjs. To examine this possibility and to further characterize the role of fibroblast protein in the hybrid microtubules, additional chick brain microtubule protein was added to the 2$ times cycled hybrids, and three more cycles of assembly and disassembly were carried out. As can be seen in fig. S, the microtubules formed &Y&Q the Ia cycles had no HMW-MAPS, and in fact, had virtually no EXJI Cell Res I24 (1979)

408 Hinds and Soifer . c. .,&

Fig. 6. SDS-ael electronhoresis of fractions S, (slots 1 and 2) and P, (slots 38nd 4) from chick bra& (slots 1 and 3) and hybrid,(slots 2 and 4) preparations. Note that HMW-MAPs (arrow) remain in S, of the hybrids (slot 2) while being sedimented with the assembled chick microtubules of Pz (slot 3).

proteins coassembling which had a molecular weight greater than that of tubulin. The most prominent MAPS seen in these later cycles had molecular weights of 34ooo-45ooo. When the fractions are examined that are normally discarded during the process of preparation of hybrid microtubules, the fate of the HMW-MAPS becomes apparent (fig. 6). The Sz fraction of chick brain is formed almost entirely of tubulin. The pellet of assembled chick microtubules (P2) includes the HMW protein, as is usually the case. When microtubules are assembled from the cell-chick mixture, the HMW Exp CellRes 124(1979)

123456 Fig. 7. SDS-gei electrophoresis of fractions obtained during cycling of human microtubule proteins. Slots l-6 are S,, P2, S,, Pa, S5, and POrespectively. Arrows, positions of HMW-MAPS.

stays in S, and is discarded. The intact microtubules of Pz of the mixture do not include the HMW-MAPS. High molecular weight MAPS could be a property of brain microtubules, and not characteristic of microtubules in other tissues. Alternatively, human microtubules might not contain MAPS. Experiments were performed to examine these possibilities of species-specific MAPS or tissue-specific MAPS. In the first, microtubules were puritied by assembly from post-mortem human brain. As shown in fig. 7 human brain microtubules contain protein in the region of the high molecular weight MAPS seen in the

Hybrid microtubules

from humanfibroblast

and chick brain

409

with Chediak-Higashi syndrome (CHS) would be different from normal cells. It was found that, indeed, coassembly of labeled fibroblast protein with chick brain microtubular protein did occur. The CHS cells behaved like the normal cells in the coassembly system, as will be discussed in a separate report on microtubule biology in CHS cells [39]. That microtubule assembly actually occurred in the mixture of fibroblast homogenate and chick brain microtubules was demonstrated by two criteria. (1) Electron microscopic examination of negativestained or thin-sectioned pelleted fractions showed structures with the appearance of microtubules following incubation under assembly conditions. There were no apparent differences between these microFig. 8. SDS-gel electrophoresis of microtubules pre- tubules and those seen in brain preparapared from homogenates of mouse testis or kidney in chick brain microtubule protein. Samples are as tions. In particular, there were none of the follows: slots 1, 4, 7, chick alone; 2, 5, 8, chick plus atypical forms reported by others [30] when testis; 3, 6, 9, chick plus kidney. Slots l-3 are Pa, 4-6 phosphocellulose-purified tubulin is assemare S,, 7-9 are Pa. bled in the absence of MAPS. (2) Analysis by SDS-gel electrophoresis of each fraction during repeated cycles of disassembly and brain microtubule preparations from other assembly showed LY-and /3-tubulins to be species. To examine tissue specificity, the primary peptides present. It is not poschick brain microtubule protein was assemsible to say whether the microtubules bled in the presence of protein from mouse formed under the conditions described are kidney or testis. As seen in fig. 8 the micro- true hybrids, with brain and cell protein tubules formed in these circumstances contributing to the same tubule, or whether showed the high molecular weight characa small number of microtubules, derived teristic of assembled chick brain micro- exclusively from the fibroblasts, are prestubules. ent. The fact that cell protein does not form microtubules in the absence of carrier DISCUSSION protein suggests that coassembly may proThe initial purpose of the experiments de- duce hybrid microtubules. Spiegelman et al. scribed in this paper was to determine [21] also concluded that their CHOXbrain whether microtubule protein from cultured microtubules were probably hybrids. In examining the characteristics of the human fibroblasts would coassemble with brain microtubule protein, and if it did, coassembled microtubules, the most interwhether the coassembly characteristics of esting finding was the lack of HMW-MAPS protein from cells derived from patients in the putative hybrids. This lack of HMWExp Cell Res 124 (1979)

410

Hinds and Soifer

MAPS is underscored by the fact that chick brain microtubules assembled in the same experiment contained HMW-MAPS as prominent components of the total microtubule protein. Prolonged exposure of the fluorograph failed to reveal any cell protein having a molecular weight similar to the HMW-MAPS. The most prominent nontubulin band seen in the fibroblast protein had a molecular weight of approx. 150000. Extensive evidence has been presented by a number of investigators which shows that MAPS have a role both in the initiation of the assembly of tubulin into microtubules and in the rate of elongation of microtubules [ 14, 15, 161.It has been reported that in the absence of MAPS, tubulin can be induced to assemble only at very high protein concentrations or under non-physiologic conditions such as high magnesium [31] or with DMSO [32]. Which of the MAPS are necessary for assembly has been the subject of some discussion. The HMW-MAPS are the primary protein found in most microtubule preparations; these have been implicated in assembly by some investigators [14, 15-J. It has also been suggested that a group of proteins of 52-70000 D, and known collectively as tau, are necessary for assembly [ 161, and of these a 67 000 D protein contains the major portion of the activity [33]. All these assembly-stimulating MAPS have been described in microtubules purified by assembly from brains of a number of species. In a few reports of microtubules from cultured cells, the role of MAPS has been less clearly defined. It has been reported that C6, a glial cell line, contains HMWMAPS [34]. Assembly of microtubules from 3T3 cells and from Simian virus-transformed 3T3 cells has been reported, but MAPS were not discussed [17]. Nagle et al. [ 181 have reported assembly of microtuExp Cell Res 124 (1979)

bules from several non-human cell lines. In all the cell lines they examined, MAPS were present but did not include HMW protein. These authors concluded that 49000 D protein which co-purified in a quantitative fashion with tubulin might be required for assembly. Weatherbee et al. [35] and Bulinsky & Borisy [36] have recently succeeded in assembling microtubules from HeLa cells. These lack HMW-MAPS, but include proteins of lower molecular weight as components of the microtubules. Klein et al. [37] have reported, however, that when extracts of mouse embryo tibroblasts are used to induce assembly of phosphocellulose purified brain tubulin there is a fibroblast protein in the assembled microtubules which co-migrates with HMW-MAPS. There are several possible explanations for the absence of HMW-MAPS from the hybrids. One possibility is that fibroblast proteases have degraded the HMW-MAPS. The experiments with the mouse tissue homogenates provide one piece of evidence suggesting that proteolysis cannot explain the absence of HMW-MAPS, since the kidney in particular is rich in proteases. A different kind of evidence comes from the finding that the addition of fresh chick brain microtubule protein to the 2+ times cycled fibroblast x brain microtubules does not result in the presence of HMW-MAPS. If proteolysis accounted for the initial lack of HMW-MAPS, their continued absence after addition of fresh brain material would mean that fibroblast-derived protease was copurifying with the microtubule protein. In addition, undegraded HMW-MAPS is found in the supematant of the first assembly cycle. The hybrid microtubules may lack HMW-MAPS because a tibroblast protein competes for the same binding site on the tubulin molecule. This would not seem to be the explanation since there is such a

Hybrid microtubules

from human fibroblast

large excess of brain protein present in the assembly system. The continued absence of HMW-MAPS after the addition of more chick brain microtubule protein to the cycled hybrid microtubules would appear to exclude this possibility. Experiments described here were also designed to test the possibility that HMWMAPS are species- or organ-specific and might be replaced in the hybrid microtubules either by specific human MAPS or by MAPS of non-neuronal origin. To determine whether human microtubules might, in general, have different MAPS, human brain microtubules were isolated by cycles of assembly and were found to have protein in the molecular weight range of the HMWMAPS. The human HMW-MAPS have the same electrophoretic mobility as other mammalian HMW-MAPS. These differ somewhat from the chick brain protein [40]. Ikeda & Steiner [38] have found HMWMAPS in preparations of microtubules from another human tissue, platelets. To assess the effect of cell homogenates of organs other than brain, microtubules were assembled from a mixture of chick brain microtubules protein and homogenates of mouse kidney or testis. The microtubules assembled under these conditions retained the HMW-MAPS characteristic of chick brain microtubules. In the studies reported here, no protein of a higher molecular weight than a-tubulin contributed significantly to the hybrid microtubules after 3 cycles of assembly and disassembly. It would appear that MAPS play a much less significant role than in any physiologic system previously reported. The finding that the hybrid microtubules not only do not include HMW, but are able to exclude it when added, suggests that the mechanism of assembly of the hybrids is different from that seen in neuro-

and chick brain

411

tubules. The relationship of these in vitro studies to the assembly occurring in vivo can only be speculated upon. We are grateful to Dr B. S. Danes for support which she has provided. We thank MS K. Mack for her assistance, K. H. was supported through the predoctotal training program in Genetics at Cornell Medical

College;NIH no. 5 ~01GMO191s-0s.

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Received February 28, 1979 Revised version received July 2, 1979 Accepted July 10, 1979