Cell Motility and the Cytoskeleton 17:174-186 (1990)

Microtubule-Associated Proteins From Antarctic Fishes H. William Detrich Ill, Bonnie W. Neighbors, Roger D. Sloboda, and Robley C. Williams, Jr. Department of Biology, Northeastern University, Boston, Massachusetts (H,W.D., B. W.N.): Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire (R.D.S.); Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee (R.C.W.) Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N . coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 4 10,000-430,000, 220,000 -280,000, 140,000 155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5°C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPS. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5"C, and proteins corresponding to MAPs l and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33"C, but the microtubules depolymerized at 0°C. We conclude that MAPS are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization. Key words: MAPs, cold-stable microtubules, microtubule assembly

INTRODUCTION

Fishes native to Antarctic waters live at habitat and body temperatures in the range - 1.8"Cto 2°C.Their cytoplasmic microtubules, in contrast to those of mammals, are stable at these temperatures. Tubulin purified from the brains of several species of these fishes is capable of forming microtubules at temperatures as low as - 1.8"Cin the virtual absence of microtubule-associated proteins (MAPs) [Williams et al., 1985;Detrich et al., 19891.The critical concentration for assembly of Antarctic fish tubulin at - 1.8"Cwas found to be approxi-

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mately the same as that for assembly of bovine tubulin at 37°C when both proteins were examined in the same buffer in the absence of MAPs [Williams et al., 198.51. Received July 18, 1990; accepted July 19, 1990. Address reprint requests to H . William Detrich, Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 021 15. Bonnie Neighbors is now at Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309.

MAPs From Antarctic Fishes

Thus, some of the “cold-stability’’ of these fish microtubules is a property of their tubulins, which have unique a-chains [Detrich and Overton, 1986, 1988; Detrich et al., 1987bl. Nevertheless, one may ask whether these poikilotherms also possess MAPS that make additional contributions to the energetics of microtubule formation at their low body temperatures. MAPS were originally defined as those proteins that copurified stoichiometrically with tubulin when microtubule protein was prepared from brain tissue by cycles of assembly and disassembly in vitro [Sloboda et al., 19751. Many MAPs, including the well-characterized high molecular weight MAPS 1 and 2 (apparent molecular weight derived by gel electrophoresis [M,,] = 270,000350,000) and the tau proteins (M,, = 55,000-62,000) of warm-blooded vertebrates, share the following properties (reviewed by Purich and Kristofferson [1984], Vallee et al. [1984], Olmsted [1986], and Matus [1988]): 1) they promote the assembly of microtubules when added to solutions of pure tubulin; 2) they decorate the surfaces of microtubules assembled in vitro; 3) they are found in association with microtubules in vivo; and 4) they remain soluble upon exposure to 100°C (for thermostability of MAP 1, see Vallee [1985]). Recently, MAPS that must function at lower body temperatures have been identified in the cells and tissues of fishes. A protein of approximately 300,000 daltons that is present in the erythrophores of the squirrelfish reacts with antibodies to mammalian MAP 2 and can be localized to the cytoskeletons of these cells [Stearns and Binder, 19871. MAPs of both high (Mra > 250,000) and low molecular weight have been isolated from brain tissues of the dogfish [Langford et al., 19861 and of the carp [Maccioni and Mellado, 19811. Stromberg et al. [1989] identified three high molecular weight MAPs, including a protein that shares antigenic determinants with mammalian MAP 2, in a cold-labile microtubule fraction prepared from brains of the cold-adapted Atlantic cod, but the role of these proteins in microtubule assembly was not evaluated. Although it is clear that the fishes possess MAPs, little is known about the functional and structural properties of these proteins. MAPS have recently been isolated biochemically from the brains of the cold-adapted Antarctic fishes [Williams and Detrich, 1986al. Given the well-documented polymerization-enhancing and microtubule-stabilizing effects of mammalian MAPS [Sloboda et al., 1976; Cleveland et al., 1977; Murphy et al., 1977a; Sloboda and Rosenbaum, 1979; Matus, 19881, one might anticipate similar effects in these cold-adapted fishes. Additional and specific cold-stabilizing effects, such as those reported for the STOP (stable tubule only polypeptide) proteins [Job et al., 1983; Pirollet et al., 1983; Margolis et al., 19861, might also be present. In this

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paper we examine the effects of Antarctic fish MAPs on the polymerization and depolymerization of fish tubulin, We show that these proteins reduce the critical concentration necessary for microtubule assembly and speed its occurrence, but do not block disassembly. When added to bovine tubulin, fish MAPS also stimulate polymerization but convey no special cold-stability on the resulting microtubules. Preliminary reports of some of this work have appeared [Detrich, 1983; Williams and Detrich, 1986b; Detrich et al., 1987aI.

MATERIALS AND METHODS Materials

PiperazineNNI‘-bis[2-ethanesulfonicacid] (PIPES), dithioerythritol (DTE), dithiothreitol (DTT) ,ethylene glycol bis(P-aminoethyl ether)-N,N,N’,”-tetraacetic acid (EGTA), GTP (type 11-S), and p-tosyl-L-arginine methyl ester hydrochloride (TAME) were obtained from Sigma Chemical Co. DEAE-Sephacel was purchased from Pharmacia, Inc. Acrylamide (>99.9%), N,N,-methylene-bisacrylamide, and Bio-Gel P-6DG were products of BioRad Laboratories, and urea (enzyme grade) was supplied by Bethesda Research Laboratories. Araldite 502, glutaraldehyde, osmium tetroxide, sodium cacodylate, and uranyl acetate were supplied by Ted Pella, Inc. Tannic acid was obtained from Mallinckrodt. Taxol (provided by Dr. Matthew Suffness of the Natural Products Branch, Division of Cancer Treatment, National Cancer Institute) was prepared as a 10 mM stock in dimethyl sulfoxide and was stored at -70°C. Other chemicals were reagent grade. Collection of Fishes

Specimens of Notothenia coriiceps neglecta, N. gibberijirons, Chaenocephalus aceratus, and a species of the genus Chionodraco were collected by bottom trawling from the RIV Polar Duke near Low and Brabant Islands in the Palmer Archipelago. They were transported alive to Palmer Station, Antarctica, where they were maintained in seawater aquaria at 1-2°C. Preparation of Microtubule Proteins From Antarctic Fishes

Tubulins were purified from the brain tissues of C. aceratus or N. gibberijirons by a method that employed ion-exchange chromatography on DEAE-Sephacel followed by microtubule assembly [Detrich and Overton, 1986; Himes and Detrich, 19891. The DEAE/assemblypurified tubulins were stored at - 70°C as microtubule pellets. These preparations contained approximately 98% tubulin [Detrich and Overton, 19861.

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MAPs from Antarctic fishes were prepared by a modification [Detrich and Overton, 19861 of the taxoldependent, microtubule-affinity protocol of Vallee [1982, 19861. All operations were performed at 0-2°C. Briefly, brain tissue (10-30 g) was homogenized in 2 ml/g of PME buffer (0.1 M PIPES-NaOH, 1 mM MgSO,, 1 mM EGTA, pH 6.9 at 20°C) through the use of a Tekmar Tissumizer (30-60 s , speed 50, SDT-80EN generator). (For some preparations, leupeptin and TAME were added to PME buffer to final concentrations of 10 pg/ml.) The homogenate was centrifuged at 30,OOOg for 20 min, and the supernatant was recovered and subjected to a second centrifugation [ 165,OOOg at rmax (45,000 rpm), Beckman type 50 rotor, 75 min]. The resulting high-speed supernatant (HSS) was brought to 1 mM in GTP and to 10-30 FM in taxol. (In a given preparation, a fixed concentration of taxol was used at this and subsequent steps .) Following incubation for 60 min at 0°C to allow microtubules to form, the HSS was layered over cushions of 5% (w/v) sucrose in PME + 1 10-30 p M taxol(2-4 ml of HSS per 2-ml mM GTP cushion), and microtubules were collected by centrifugation (32,OOOg at rmax, type 50 rotor, 30 min). The microtubule pellets were washed twice by resuspension in PME/GTP/taxol buffer (0.25 X original HSS volume) followed by centrifugation (32,00Og, 30 min). Finally, the twice-washed pellet was resuspended in PME + 1 mM GTP 10-30 KM taxol (0.1-0.2 X initial HSS volume), and NaCl was added to a concentration of 0.36 M. Following a 15-min incubation at O"C, the solution was again centrifuged (32,00Og, 30 min). The MAPcontaining supernatant was recovered and frozen at -70°C for storage. Alternatively, solutions of MAPs were frozen dropwise in liquid nitrogen as described [Detrich and Williams, 1978; Williams and Lee, 19821 and stored at -70°C. Prior to use, samples were thawed and then equilibrated with the appropriate buffer by gel filtration on a column of Bio-Gel P-6DG. In most cases, the desalted MAP samples were centrifuged (15 ,OOOg, 20 min, 0°C) to remove denatured or aggregated protein, and the supernatant was recovered for experimentation. Microtubule proteins from the brain tissues of Antarctic fishes were also prepared by a modification of the calcium- and EGTA-dependent assembly/disassembly protocol of Webb and Wilson [ 19801. Brain tissues were homogenized in 1 ml/g of cold (0°C) PMT buffer (0.1 M PIPES-NaOH, 1 mM MgSO,, 2 mM TAME, pH 6.9 at 20°C) by use of the Tekmar Tissumizer (20 s , speed 50, SDT-80EN generator). CaCl, was added to the homogenate to a final concentration of 10 mM, and the homogenate was centrifuged (50,OOOg at rmax,Beckman type 50 rotor, 15 min, 0°C). The supernatant was removed and centrifuged again (135,OOOg at rmax,type 50 rotor, 30 min, 0°C) to produce an HSS. To initiate the first assem-

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bly/disassembly cycle, GTP and EGTA were added to the HSS to final concentrations of 1 mM and 11 mM, respectively, and the solution was warmed to 15°C for 60 min. Microtubules were collected by centrifugation (50,OOOgat rmax,type 50 rotor, 30 min, 15"C), the polymer was resuspended in PMT buffer (50% of original HSS volume), and CaC1, was added to the solution to a final concentration of 10 mM. Finally, the solution was incubated at 0°C for 30 min, and then centrifuged (135,OOOg at rmax,type 50 rotor, 45 min, 0°C) to yield once-cycled microtubule protein. The second cycle of assembly and disassembly was performed exactly as described for the first cycle. Pellets of microtubule proteins were stored at -70°C. Finally, microtubule proteins were prepared from the brain tissues of Antarctic fishes by one or two cycles of temperature-dependent assembly and disassembly [cf. Williams et al., 19851. Microtubule Proteins From Bovine Brain

Microtubule proteins from bovine brain tissue (generously provided by Dr. Leslie Wilson and Herbert P. Miller, University of California, Santa Barbara, and by Dr. George S . Bloom, University of Texas Southwestern Medical Center) were prepared by cycles of temperature-dependent assembly and disassembly either by a modification [Farrell and Wilson, 19841 of the method of Asnes and Wilson [1979] or by the protocol of Vallee and Borisy [1978]. Bovine tubulin was separated from the MAPs by chromatography of the microtubule proteins on DEAE Sephacel [cf. Vallee and Borisy, 1978; Murphy et al., 1977bl. The tubulin-containing fractions were pooled, frozen as aliquots at -70°C or dropwise in liquid nitrogen, and stored at -70°C; MAP-containing fractions were treated similarly. Before use, samples of tubulins or MAPs were thawed, desalted by passage over columns of buffer-equilibrated P-6DG (see next section for buffers), and, in most cases, centrifuged (15,OOOg, 20 min, 0°C) as described above. Polymerization Assay

Microtubule assembly was monitored turbidimetrically [Gaskin et al., 1974; Detrich et al., 19851 at 350 nm through the use of recording spectrophotometers (Perkin-Elmer Lambda 4B or Hitachi Model 100-60) equipped with thermostatted cuvette holders. Sample temperatures (measured within the cuvettes by means of an Omega Model 199 RTD thermometer) were controlled by circulating, constant-temperature baths attached to the cell holders. The assembly of microtubules was initiated by three different methods: 1) addition of GTP to solutions of microtubule proteins lacking the nucleotide; 2) addition of EGTA to solutions of microtubule proteins containing calcium ion (followed in some

MAPs From Antarctic Fishes

cases by warming); or 3) warming of solutions of microtubule proteins containing GTP. Two buffer systems were used in the polymerization experiments. PB was 0.1 M PIPES-NaOH, 2 mM MgSO,, 1 mM EGTA, and 1 mM DTE adjusted to pH 6.9 (20°C). PB, was 0.1 M PIPES-NaOH, 2 mM MgSO,, 2 mM TAME, 1 mM DTT, and 8 mM CaCl, adjusted to pH 6.9 (20°C). Additions (e.g., GTP and/or EGTA) to these buffer systems are described in the appropriate figure legends. Electron Microscopy Samples of microtubules were prepared for negative-stain electron microscopy essentially as described by Williams et al. [ 19851, with the exception that grids were not exposed to a glow discharge prior to sample application. Pellets of microtubules were processed for thinsection electron microscopy by the glutaraldehyde-tannic acid fixation protocol of Pierson et al. [ 19781, Following fixation, the pellets were postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate (pH 7.2) for 60 min, dehydrated through an acetone series, and embedded in Araldite. Grids and embedded pellets were prepared at Palmer Station and brought back to the United States for further processing and observation. Thin sections were stained with methanolic uranyl acetate followed by lead citrate [Pierson et al., 1978; Kim et al., 19791. Samples were examined by means of Philips EM 300 or JEOL Model 180 electron microscopes operated at 60-80 kV. Dark-Field Light Microscopy

At Palmer Station, the polymers formed by tubulins and MAPS were assessed by dark-field light microscopy as described by Suprenant and Dentler [1982]. Samples were observed by use of a Zeiss Standard 16 light microscope equipped with a 40 X planachromat objective lens (0.65 N.A.), an oil-immersion ultracondenser (1.2/1.4 N.A.), and a 75-watt Xenon burner. Specimen temperature was regulated by a water-jacketed microscope stage connected to a circulating constant temperature bath. Temperatures were measured directly from the microscope slides by means of a YSI thermocouple thermometer equipped with a surface temperature probe. Other Methods

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Protein concentration was assayed by the method of Bradford [1976] with bovine serum albumin as the standard. Sodium dodecyl sulfate (SDS) polyacrylamide gradient gel electrophoresis was performed on slab gels by the method of Laemmli [ 19701. The gels contained linear gradients of acrylamide [4-16% (wiv)] and urea (1-8 M) [Kim et al., 1979; Detrich and Overton, 19861 or of

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acrylamide [4-15% (wiv)] and glycerol [0-25% (v/v)]. Gels were stained with Coomassie Brilliant Blue R-250 [Detrich and Wilson, 1983; Detrich and Overton, 19861 or with silver [Wray et al., 19811. RESULTS Preparation and Characterization of MAPs

Taxol-dependent isolation of MAPs. Lanes B-J of Figure 1 show protein fractions obtained from brain tissue of the Antarctic cod, N . coriiceps neglecru, at each stage of the taxol-dependent MAP preparation. The microtubules (lane H) contained tubulin and a complement of MAPs. Sedimentation of the presumptive MAPs from the high-speed supernatant was not observed in the absence of taxol, nor could these proteins be pelleted following incubation of an HSS depleted of tubulin (by prior chromatography of the HSS on DEAE-Sephacel) with taxol (10 p M final concentration, 60 min, 0°C) (data not shown). Thus, these proteins were associated specifically with the taxol-induced microtubules. Nearly quantitative displacement of the MAPS from the taxolstabilized microtubules was obtained when the resuspended microtubule pellet (lane H) was exposed to elevated concentrations of NaCl (compare lanes I and J). Prominent in this final MAP preparation were proteins of apparent molecular weight ca. 430,000, 230,000270,000, 89,000, 45,000-50,000, and 32,000-34,000. Many of the MAPs present in the taxol preparation (e.g., the protein with M,, = 430,000) co-migrated electrophoretically with nontubulin proteins observed in oncecycled microtubules (lane K) obtained from N . coriiceps neglectu by a temperature-dependent assembly protocol [cf. Williams et al., 19851. Some or all of these proteins may represent functional homologs of the high molecular weight MAPs 1 and 2, the tau proteins, and the low molecular weight MAPs (Mra = 30,000-35,000) found in microtubule protein isolated from the brains of warmblooded vertebrates. The yield of microtubule protein obtained by taxol-dependent microtubule assembly, 1 mg/g of brain tissue for each fish species, was identical with that reported by Vallee [ 19821 for calf brain cerebral cortex. Of the total microtubule protein, -15% was recovered in the MAP fraction. Figure 2 shows MAP preparations from the brains of two Antarctic cods ( N . gibberifrons and N . coriiceps neglectu) and from an ice fish (Chionodraco sp.). The MAPs from these fishes displayed similarities in general electrophoretic pattern. For example, there are clusters of proteins in the molecular weight ranges 4 10,000430,000,220,000-270,000, 140,000 -1 55,000,85,00095,000, 40,000-45,000, and 32,000-34,000 (lanes DG). However, small interspecific differences in the MAPS are also apparent. For example, the major high

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Fig. 1. Taxol-dependent purification of microtubule proteins (tubulin and MAPs) from brain tissues of the Antarctic cod, N . coriiceps neglectu. Samples from each step of the purification were electrophoresed in the presence of SDS on a urea-polyacrylamide gradient gel (see “Materials and Methods”). Lane A: High molecular weight standards, including myosin (205,000), P-galactosidase (1 16,000), phosphorylase b (97,400), bovine serum albumin (66,000), ovalbumin (45,000), and carbonic anhydrase (29,000). Lanes B,C: High-speed pellet and high-speed supernatant (HSS), respectively. Lanes D,E: First supernatant and first pellet, respectively, after centrifugation of the HSS through the sucrose cushion. Lanes F,G: Supernatants from the first (F) and second (G) centrifugal washes of pellet E in PME buffer containing 1 mM GTP and 30 pM taxol. Lane H: Pellet after second wash. Lanes 1,J: MAP-containing supernatant and tubulin-

containing pellet, respectively, obtained by exposure of sample H to elevated ionic strength (PME buffer containing 1 mM GTP, 30 pM taxol, and 0.36 M NaCl) followed by centrifugal resolution. Lane K: Once-cycled microtubule protein obtained from N . coriiceps neglecta brain by the temperature-dependent assembly procedure [Williams et al., 19851. Lane L: Three-times-cycled microtubule protein from bovine brain. Lane M: Low molecular weight standards, including bovine serum albumin, ovalbumin, trypsinogen (24,000), P-lactoglobulin (18,400), and lysozyme (14,300). Lanes E-J were loaded with six times the volume of sample used for lanes B-D. The molecular weights of the standards (in thousands) and the positions of the tubulin chains (Tb) and of bovine MAP 2 (Mra = 275,000) are indicated on the vertical axes.

molecular weight MAP from N . coriiceps neglecta (Mra 430,000) migrated more slowly than did the comparable proteins (Mra = 415,000) from N . gibberifrons, from the Chionodraco sp., and from Chaenocephalus aceratus (not shown in Fig. 2). Nevertheless, the similarity of these preparations suggests that many of the MAPs from the different species may be structurally or functionally related, or both. MAPs obtained by cycles of microtubule assembly and disassembly. To verify further that the proteins obtained by the taxol-dependent method were microtubule-associated, microtubule proteins were also prepared by a modification of the assembly/disassembly protocol of Webb and Wilson [ 19801. MAP preparations obtained from C . aceratus brain by the taxol-dependent procedure or by two cycles of calcium- and EGTA-dependent microtubule assembly and disassembly were comparable in protein composition (data not shown), but the yield of

MAPs obtained by the cycle method was somewhat smaller (-50% that obtained by the taxol protocol). The similarity of the MAP preparations obtained by the two independent procedures provides further support for the specific association of these proteins with microtubules . Morphology of MAP-containing microtubules. Figure 3 presents thin-section electron micrographs of microtubules prepared from brain tissue of N . gibberifrom by taxol-dependent assembly. Numerous thin filamentous projections decorate the surfaces of all microtubules in this preparation (Fig. 3a). The longest projections observed measured -25 nm. Following release of the MAPs by exposure to elevated ionic strength, however, the microtubules collected by centrifugation were found to be smooth-walled (Fig. 3b). Microtubules assembled from DEAE-purified Antarctic fish tubulins were also found to be smooth-walled. The projections on the taxol-stabilized microtubules thus appear

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Fig. 2 . MAPs isolated from the brain tissues of three species of Antarctic fishes by the taxol-dependent protocol. Samples were examined by electrophoresis on an SDS urea-polyacrylamide gradient gel. Lane A: High molecular weight standards (see Fig. 1 for identification). Lanes B,C: Once-cycled microtubules from N . gibberifrons and from N . coriiceps neglecfu, respectively, obtained by the temperature-dependent assembly method [Williams et al., 19851. Lanes D,E: MAPs from N . coriiceps neglecfu (two preparations). Lane F: MAPS from N . gibberifrons. Lane G: MAPs from a Chionodruco sp. Proteins in samples D-G were recovered in the supernatants obtained by exposure of taxol-stabilized microtubules to elevated ionic strength (PME buffer containing 1 mM GTP, 30 p M taxol, and 0.36 M NaCI) followed by centrifugation. The molecular weights of the standards (in thousands) and the positions of the tubulin chains (Tb) and of bovine MAP 2 (sample not shown; M,, = 275,000) are indicated on the vertical axes.

to correspond to one or more of the proteins identified biochemically as MAPs (Figs. 1, 2). MAP-Mediated Microtubule Assembly

Formation of microtubules by the MAPs and tubulins of Antarctic fishes. Figure 4 shows the polymerization of DEAE-purified N . gibberifrons brain tubulin in the presence of taxol-purified MAPS from C. aceratus at o ~ c Assembly . was initiated by the addition Of GTP to Of the proteins lacking the otide. Both the initial rate and the final extent of assembly increased with increasing concentrations of the MAPS (curves 3-7). Similar results were obtained when from c. aceratus solutions containing tubulin and or from N . gibberifrons at 2-5°C were released from calcium-mediated assembly inhibition by addition of excess EGTA. Numerous filamentous elements were observed when the products of the assembly reactions (Fig.

Fig. 3. Electron micrographs of N . gibberifrons microtubules. Microtubules prepared from N . gibberifrons brain tissue by the taxol-dependent procedure were examined by thin-section electron microscopy. Specimens were prepared both before (a) and after (b) exposure of the microtubules to elevated ionic strength. a: MAP-containing microtubules prepared by taxol-dependent assembly. Examples of filaments projecting from their surfaces are indicated by the arrowheads. b: Salt-extracted microtubules. Note the absence of projections. The bar in each panel represents 0.1 p m .

4, curves 3-7) were examined by dark-field light microscopy at O"C, and the filaments corresponded to microtubules when examined by negative-stain electron

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Fig. 4. MAP-induced assembly of N . gibberifrons brain tubulin at 0°C. Polymerization of DEAEiassembly-purified tubulin (0.67 mgiml in PB, buffer) in the presence of increasing concentrations of C. acerafus MAPs was monitored at 0°C by turbidimetry Assembly was initiated at zero time by adding GTP (1 mM) to solutions of the proteins lacking the nucleotide. Sample 1: N . gibberifrons tubulin alone, 0.67 mgiml. Sample 2: C. aceratus MAPs alone, 0.17 mgiml. Samples 3-7: Tubulin (0.67 mgiml) containing MAPs at final concentrations of 0.07, 0.12, 0.18, 0.24, and 0.45 mgiml, respectively. The bar in the upper left-hand corner indicates the approximate extent of experimental uncertainty in these results.

microscopy. MAP-induced microtubules depolymerized upon addition of CaC1, to 10 mM or of podophyllotoxin to 50 pM. A sample of tubulin alone (at the same subcritical concentration used for the MAPitubulin reconstitution experiments) did not polymerize (curve l ) , nor did the MAPs alone develop appreciable turbidity (curve 2). Clearly, some component or components of the MAP preparations increase both the rate and the extent of assembly of microtubules from Antarctic fish tubulin at physiological temperatures. The incorporation of MAPs into microtubule polymer during hlAP-induced assembly at low temperature was examined by sedimentation and electrophoresis. Figure 5 presents the results of an experiment with proteins from C. aceratus. Analysis of the Coomassiestained gel revealed that small quantities of at least six proteins with apparent molecular weights between 60,000 and 300,000 co-sedimented with the microtubules (Fig. 5a, lane 5 ) . These proteins did not sediment from a sample containing MAPs alone (Fig. 5a, compare lanes 2 and 3) and therefore must have been specifically attached to microtubules. The six proteins, as well as others present in smaller amounts, are clearly visible in the silver-stained gel (Fig. 5b, lane 5 ) . In the absence of the MAP fraction, tubulin did not sediment appreciably (Fig. 5a, compare lanes 6 and 7). Therefore, the cosedimenting C. aceratus proteins appear to be bona fide

MAPs that promote the formation of microtubules at low temperatures. The MAPitubulin stoichiometry of the reconstituted microtubules (Fig. 5 ) was smaller than that observed for the original microtubule preparation (e.g., Fig. l ) , and some of the MAPs (e.g., the protein of highest apparent molecular weight, 415,000) did not cosediment appreciably with the microtubules under the assembly conditions employed. Proteolysis of the MAPs appears not to be responsible for this result because purified MAP preparations (in PB, containing 10 mM EGTA and 1 mM GTP) incubated either in the presence or in the absence of tubulin at temperatures between 0 and 10°C for periods up to 10 h showed no discernable change in electrophoretic pattern. Rather, the reduced and differential binding of MAPs by the reconstituted microtubules may result from denaturation of some of the proteins during preparation (perhaps as a consequence of freezing for storage, thawing for experimentation, or both). To assess the effect of freezing and/or thawing on MAP activity, microtubules were polymerized at 10°C from a solution containing tubulin and freshly prepared (i.e., never frozen) MAPs from N . gibberifrons. Again only a subset of the added MAPs cosedimented at low stoichiometry with the polymer under these conditions, but some of the largest MAP (Mra = 415,000) was incorporated into microtubules (data not shown). Thus, preparative variables may contribute in part to the selectivity and low stoichiometry of MAP incorporation observed in the co-polymerization experiments. Other factors may also be involved (see “Discussion’’). Formation of microtubules by heterologous combinations of tubulins and MAPs. To determine the relative contributions of tubulins and MAPs to the formation of cold-stable microtubules, we have investigated the influence of heterologous MAPs on the assembly of purified brain tubulins from the cow and from Antarctic fishes. As shown in Figure 6, addition of bovine MAPs to subcritical concentrations of Antarctic fish tubulin (curves 3, 4) stimulated the formation of microtubules (verified by dark-field light microscopy) at low temperatures (2°C). Further increases in temperature (to 5 and 10°C; see arrows) produced additional microtubule assembly, whereas cooling a sample from 10 to 2°C (curve 3, see arrow at t = 450 min) caused the microtubules to depolymerize to yield an amount of polymer equivalent to that initially attained following release from calciummediated inhibition of assembly. Pure fish tubulin at the same concentration (curve 2), in contrast, began to polymerize only at 10°C. Therefore, bovine MAPs, like the MAPs of Antarctic fishes, promote the reversible assembly of microtubules from Antarctic fish tubulins at temperatures near 0°C. When the polymer (sample 4) was

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the three samples were centrifuged at 25,000g for 20 min at 5°C. The supernatants were recovered, the pellets were resuspended to the original sample volume, and equal volumes were electrophoresed in the presence of SDS on a glycerol-polyacrylamide gradient gel. Lane 1: High molecular weight standards as in Figure 1. Lanes 2-7: Supernatants (2,4,6) and pellets (3,5,7) from the samples containing MAPs alone (2,3), tubulin plus MAPs (4,5), and tubulin alone (6,7). The gel was stained with Coomassie Blue R-250. b: Samples 1-5 from panel a restained with silver. The molecular weights of the standards (in thousands) and the positions of the top of the gel, of the tubulin chains (Tb),and of the dye front (DF) are indicated on the vertical axes.

collected by centrifugation (25,00Og, 20 min, 1O"C), bovine proteins corresponding to MAP 2A, MAP 2B, a MAP 1 species, and several proteins with lower molecular weights (possibly including some tau proteins) cosedimented specifically with the microtubules under these conditions. As was true for the fish-MAP/fish-tubulin system (Fig. 3, the proportion of bovine MAPs that co-sedimented with the polymer was smaller (not shown) than that commonly observed in mammalian microtubule assembly systems [Asnes and Wilson, 1979; Kim et a]., 19861. Addition of Antarctic fish MAPs to bovine tubulin (final concentrations of 0.06 and 0.56 mg/ml, respectively, in PB, plus 1 mM GTP) enhanced the rate and the extent of microtubule assembly at 33°C. The assembly was cold-reversible, demonstrating that the fish MAPs do not function in the capacity of the STOP proteins of

mammalian brain [Job et al., 1983; Pirollet et al., 1983; Margolis et al., 19861. Furthermore, the fish MAPs did not promote the polymerization of bovine tubulin at an intermediate temperature of 20°C. Thus, MAPs from the cold-adapted fishes and from the warm-blooded mammal appear to function similarly at both low and high temperatures. The MAPs from Antarctic fishes do not possess special capacities to prevent cold-induced microtubule disassembly or to promote microtubule assembly at low temperatures. Heat treatment of Antarctic fish MAPs. Several mammalian MAPs, including MAP 2 and tau, remain soluble and retain their assembly-promoting activity following exposure to 100°C [Weingarten et al., 1975; Fellous et al., 1977; Herzog and Weber, 1978; Kim et al., 1979; Vallee, 198.51. To determine whether Antarctic fish MAPs share these properties, a solution of C. acer-

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Fig. 6. Assembly of N . gibberijrons tubulin induced by MAPs from bovine brain. Polymerization at 2°C was initiated at zero time by addition of EGTA (10 mM) to solutions of the proteins in PB, buffer containing 1 mM GTP. Subsequent temperature jumps to 5°C and 10°C were initiated at the times indicated by the arrows. Sample 1: Bovine MAPs alone, 0.39 mgiml. Sample 2: N . gibberijrons tubulin alone, 1.1 mgiml. Samples 3, 4:Tubulin (1.1 mgiml) containing bovine MAPs at final concentrations of 0.20 and 0.39 mgiml, respectively. At 395 rnin podophyllotoxin (PLN) was added to sample 2 to a final concentration of 50 )*M. At 445 min, samples 1, 2 , and 4 were recovered for sedimentation analysis (see “Results”), and at 450 min the temperature of sample 3 was lowered to 2°C.

atus MAPs was incubated at 100°C for 10 rnin and cen-

trifuged. The “heat-stable’’ supernatant fraction contained 7 1% of the total protein. Electrophoretic analysis showed that the overall protein compositions of heattreated and control (unheated) supernatants were similar. As shown in Figure 7, though, heat treatment abolished the assembly-stimulating activity of the MAPs. Thus, the Antarctic fish MAPs remain in solution upon heat treatment, but, unlike the MAP 2 and tau proteins of mammals, they lose much of their polymerization-enhancing activity. DISCUSSION Evidence for Association of These Proteins With Microtubules

The group of proteins whose isolation from Antarctic fish brain is described above can be identified as MAPs on the basis of three kinds of evidence obtained in vitro: they co-sediment with microtubules both in the presence of taxol (Figs. 1, 2) and in its absence, they stimulate assembly of microtubules at physiological temperatures when added to pure tubulin (Figs. 4, 5 , 7), and they give rise to filamentous projections on the surfaces of microtubules (Fig. 3). Furthermore, the similarity of the MAP preparations obtained by two independent pro-

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TIME (min) Fig. 7. Effect of heat treatment on the assembly-promoting activity of MAPs from an Antarctic fish. A solution of MAPs from C. aceratus (0.49 mgiml in PB,) was heated to 100°C for 10 rnin and then centrifuged (30,00Og, 10 min, 0°C). The supernatant (0.35 mgiml remaining) was recovered as the “heat-stable’’ fraction, and the pellet was discarded. An unheated control solution of MAPs was treated otherwise identically. Polymerization of C. aceratus tubulin (in PB, buffer plus 1 mM GTP plus 10 mM EGTA) in the presence of “heatstable” or control MAPs was initiated at zero time by warming the solutions from 5 to 10°C. Tb Heat-treated MAPs: 0.70 mgiml tubulin plus 0.24 mgiml MAPs; Tb + Control MAPs: 0.70 mgiml tubulin plus 0.33 mgiml MAPs.

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cedures, taxol-dependent assembly and cycles of EGTAand calcium-dependent assembly/disassembly, further supports the specific association of these proteins with microtubules, Because evidence of intracellular localization is lacking, we cannot exclude the possibility that some have different functions and locations in vivo and only become adventitiously associated with microtubules after cellular disruption. Previous studies [reviewed by Vallee et al., 1984; Olmsted, 1986; Matus, 1988; see also Hirokawa et al., 1985; Shiomura and Hirokawa, 19871 have shown, however, that proteins with the properties listed above most often prove to be associated with microtubules in vivo. For the purpose of functional study, we refer to proteins of the isolated group as MAPs. Functional and Structural Relationships of Antarctic Fish MAPs to the MAPs of Other Organisms

Stimulation by the fish MAPS of microtubule assembly from fish tubulin follows the same qualitative pattern at 0°C as is seen at 37°C in the classic mammalian case [Sloboda et al., 1976; Murphy et al., 1977al. Both speed and final extent of assembly are enhanced, and the degree of enhancement increases with the concentration of added MAPs. Assembly is reversed by the addition of podophyllotoxin or calcium. It therefore seems likely that the Antarctic fish MAPs enhance microtubule as-

MAPs From Antarctic Fishes

sembly through the same mechanisms that operate for mammalian MAPs: promotion of microtubule nucleation and enhancement of microtubule elongation by stabilization of formed polymer [Murphy et al., 1977al. This conclusion is reinforced by the demonstration that the MAPs are interchangeable with respect to their assembly-enhancing function: bovine MAPs stimulate polymerization of fish tubulin, and fish MAPs stimulate polymerization of bovine tubulin. This interchangeability also suggests that the proteins may have similar binding domains and may occupy similar sites on the microtubule lattice. The similarity appears to extend to cold reversibility of assembly, because bovine microtubules induced to form at 33°C by the addition of fish MAPs depolymerize upon cooling to 0°C. In addition to their microtubule-binding domains, the MAPs of poikilotherms and homeotherms may share structural features in other regions. The general similarity of the apparent molecular weights of the Antarctic fish MAPs to those of the major mammalian and avian MAPs argues for more extensive interspecific homology of some of the proteins. For example, the fish MAP preparations contain groups of proteins similar in size to the high molecular weight MAPs 1 and 2 (Mra = 270,000-350,000) [Sloboda et al., 1975; Murphy et al., 1977a,b] and the tau proteins (Mr, = 55,000-62,000) [Cleveland et al., 19771 of mammals and birds. Interestingly, the C . aceratus MAP of greatest apparent molecular weight (Mra = 415,000) cross-reacted (G.S. Bloom, B.W. Neighbors, and H.W. Detrich 111, unpublished results) with a monoclonal antibody that recognizes a phosphorylated epitope present on MAP lB, MAP l A , and two neurofilament proteins from mammals [Luca et al., 19861. Finally, the two low molecular weight fish MAPs (Mra = 32,000-34,000) may be related to the low molecular weight mammalian MAPs [Berkowitz et al., 19771 that have been identified as light chains of the native MAP I complex [Vallee and Davis, 19831. However, the heat-lability of the assembly-promoting activity of MAPs from C. aceratus differentiates them from some mammalian brain MAPs [Weingarten et al., 1975; Fellow et al., 1977; Herzog and Weber, 1978; Kim et al., 19791. Although it is possible that the loss of assembly-promoting activity resulted from precipitation of the active protein, it is more likely that the MAPs remained soluble but no longer active. The MAP fraction from each of the Antarctic fishes contains a protein of unusually high apparent molecular weight (Mra 415,000-430,000, depending on the species). To date, MAPs of comparable size have not been found in the brain tissues of mammals or of birds. However, Stromberg et al. [1989] have described an apparent MAP of similar molecular weight (M,, = 400,000) that is present in a once-cycled, cold-labile brain microtubule ^I

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fraction from the Atlantic cod, Gadus morhua. We suggest that these large MAPs from the austral and boreal fishes may be structurally and functionally homologous. Thin projections or arms up to -25 nm in length were found to decorate Antarctic fish microtubules assembled from solutions containing both MAPs and tubulin (Fig. 3), but not microtubules assembled from pure tubulin. They are similar to projections formed by the high molecular weight MAPs of mammals. In thin section the MAP 1 complex appears to extend up to 23 nm from the microtubule wall [Vallee and Davis, 19831, and MAP 2 molecules are found to project up to 40 nm [Herzog and Weber, 1978; Kim et al., 1979; Zingsheim et al., 19791. When visualized by similar methods, microtubules decorated by tau lack detectable projections [Herzog and Weber, 1978; Zingsheim et al., 19791. Thus, we suggest that the arms of the fish microtubules are composed of one or more of the high molecular weight fish MAPs. Stability of Fish MAPs

In previous studies of cold-water fishes, a near absence of MAPs was observed in twice-cycled preparations of microtubules obtained from brain tissues of two Antarctic nototheniids, Pagothenia borchgrevinki and Dissostichus mawsoni, and of the Atlantic cod by conventional cycling procedures involving assembly at high temperature (30-37°C) and disassembly at low temperature (0°C) [Williams et al., 1985; Stromberg et al., 19861. Similarly, twice-cycled brain microtubules from N . coriiceps neglecta and from C. aceratus, prepared by the method of Williams et al. [1985], contained only small quantities of a few nontubulin proteins (H.W. Detrich 111, unpublished results). By contrast, MAPs copurified with tubulin when twice-cycled microtubules were prepared from C. aceratus brain by a protocol that employed a lower temperature (15°C) for polymerization (see “Results”). Thus, it appears likely that the MAPs were lost in the conventional preparations due to denaturation or inactivation induced by the prolonged and repeated incubations of the microtubule protein solutions at high temperatures (37°C) during the assembly steps. Sensitivity to inactivation by high temperature may be a property common to many of the MAPs of cold-adapted marine fishes [cf. Stromberg et al., 19891. The selectivity and low stoichiometry of MAP incorporation observed in microtubule assembly systems reconstituted from the piscine proteins suggests that some of the MAPs may be inactivated by preparative or experimental manipulations such as freezing, thawing, or exposure to high concentrations of NaCl (during extraction of the taxol-stabilized microtubules). An addi415,000) was tional MAP species (the largest of M,, found to bind to microtubules when the reconstitution ^I

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experiments were performed with N . gibberifvons MAPS that had never been frozen, suggesting that it is labile in the freeze-thaw process. When frozen preparations of MAPS were thawed, equilibrated with PB, by gel filtration chromatography, and then centrifuged, substantial (- 10-20933) amounts of the MAPS were recovered in the pellet, probably due to simple denaturation. The fundamental affinity of fish tubulin for MAPS may be low, however, since relatively small amounts of bovine MAPS were recovered in microtubules polymerized from fish tubulin and bovine accessory proteins.

ence Foundation, by the personnel of ITT Antarctic Services, Inc., and by the captains and crews of RIV Polar Duke. We wish to thank Dr. Leslie Wilson, Herbert P. Miller, and Dr. George S . Bloom for their gifts of microtubule proteins from bovine brain, and Dr. Matthew Suffness for his contribution of the taxol. This paper is based upon work supported by National Science Foundation grants DPP-83 17724 and DPP-8614788 (to H.W.D.), and, in part, by National Institutes of Health grant GM25638 (to R.C.W.).

Antarctic Fish MAPs Are Not End-Capping Proteins

REFERENCES

Besides their cold-labile microtubules, many mammalian cells contain a subpopulation of microtubules that resist depolymerization at temperatures near 0°C [Jones et al., 19801. At metaphase, for example, the kinetochore microtubules of the mitotic spindles of PtK, cells are cold stable, whereas the adjacent interpolar microtubules are not [Brinkley and Cartwright, 19751. One possible explanation of the cold-stability of such microtubules invokes the presence of proteins that cap the ends of the polymers. Margolis and colleagues have proposed that the cold-stability of mammalian microtubules is conferred by random, substoichiometric incorporation of one or a few nontubulin proteins, termed STOPS, into otherwise cold-labile microtubules [Job et al., 1981, 1982, 1983; Pirollet et al., 1983; Margolis et al., 19861. When exposed at the ends of microtubules, these protein caps are thought to prevent the end-wise depolymerization of the polymer that is normally effected by low temperature, by antimitotic drugs (e.g., podophyllotoxin), or by other agents [Job et al., 1981, 1982, 1983; Pirollet et al., 1983; Margolis et al., 19861. By contrast, microtubule polymerization supported by the Antarctic fish MAPS is completely reversible (see above). Thus, the MAPS of Antarctic fishes do not function as capping proteins that inhibit subunit exchange at microtubule ends. We conclude that these cold-adapted Antarctic fishes possess tubulins that polymerize well at 0°C and MAPs that act to enhance the assembly at the same temperature. Together, these proteins appear to function in a manner homologous to the mammalian tubulin-MAP system. They do not convey unusual kinetic properties, such as prevention of the ordinary reversibility of microtubule assembly.

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ACKNOWLEDGMENTS

We gratefully acknowledge the logistic support provided to this project over the past 5 years by the staff of the Division of Polar Programs of the National Sci-

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