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Calcium Lability of Cytoplasmic Microtubules and Its Modulation By Proc. Natl. Acad. Sci. USA Vol. 78, No. 2, pp. 1037-1041, February 1981 Cell Biology Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins (detergent extraction/calmodulin/calmodulin inhibitors/immunofluorescence microscopy) MANFRED SCHLIWA*, URSULA EUTENEUER*, JEANNETTE CHLOE BULINSKItt, AND JONATHAN G. IZANT* *Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309; and tLaboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706 Communicated by Keith R. Porter, October 31, 1980 ABSTRACT Detergent-extracted BSC-1 monkey cells have The discovery that calmodulin, a ubiquitous Ca2+-binding been used as a model system to study the Ca2+ sensitivity of in vivo protein that regulates a number of Ca2+-dependent functions polymerized microtubules under in vitro conditions. The effects (for reviews, see refs. 9 and 10), influences microtubule assem- of various experimental treatments were observed by immuno- has the fluorescence microscopy. Whereas microtubules are completely bly in vitro in a Ca2+-dependent manner (11) opened stable at Ca2+ concentrations below 1 pM, Ca2+ at greater than possibility that Ca2+ effects on microtubules are mediated by 1-4 ,uM induces microtubule disassembly that begins in the cell this protein. Immunofluorescence microscopy with antibodies periphery and proceeds towards the cell center. At concentrations against calmodulin further showed an association of this protein of up to 500 jiM, both the pattern and time course of disassembly with certain components of the mitotic spindle (12, 13), sug- are not markedly altered, suggesting that, within this concentra- gesting an important role in Ca2+-dependent functions during tion range, Ca2+ effects are catalytic rather than stoichiometric. Higher (millimolar) Ca2+ concentration results in rapid destruc- mitosis. tion of microtubules. Of other divalent cations, onl' Sr2+ has a Until it becomes possible experimentally to manipulate Ca2+ slight depolymerizing effect, whereas millim'olar BaY+, Mg2+, or concentrations inside a living cell, the hypothesis that Ca2+ and Mn2+ is ineffective. Disassembly induced by micromolar Ca2+ is calmodulin regulate microtubule assembly can be tested only inhibited by pharmacological agents known to bind to calmodulin in vitro. Here we describe the use of an in vitro system that and inhibit its function, suggesting that calmodulin mediates Ca2+ the in vivo situation. We report on the Ca2+ lability effects. Both the addition of exogenous brain microtubule-asso- resembles ciated proteins (MAPs) after lysis and the retention of endogenous of in vivo polymerized microtubules in detergent-extracted cellular MAPs normally extracted during the lysis step stabilize cells and present evidence for the involvement of calmodulin microtubules against the depolymerizing effect of micromolar mediating the Ca2+ effect. In addition, we show that the Ca2+ Ca2+. The results indicate that, in this model system, microtubules lability of these microtubules is modulated by microtubule-as- are sensitive to physiological Ca2+ concentrations and that this sociated proteins (MAPs). Part of the data have been presented sensitivity may be conferred by calmodulin associated with the at International Symposium on Microtubules and microtubules. MAPs appear to have a modulating effect on mi- the Second crotubular Ca2+ sensitivity and thus may function as a discrimi- Microtubule Inhibitors, Beerse, Belgium, 1980 (14). nating factor in cellular functions performed by calmodulin. It is hypothesized that Ca2+-stimulated microtubule disassembly de- MATERIALS AND METHODS pends on the relative amount of MAPs. Cell Culture. African green monkey kidney cells (BSC-1) were grown in Dulbecco's modified Eagle's medium supple- Several important cellular functions depend on an ordered as- mented with 10% fetal calf serum. Cells were seeded into 35- sembly and disassembly of microtubules. The factors control- mm plastic Petri dishes that contained four round glass cover- ling these processes are, however, not fully understood. Since slips. After 24-48 hr, coverslips were removed from the Petri the discovery of conditions for the reversible polymerization of dishes and used for experiments. microtubules in vitro (1) it has been suggested that the con- Detergent Extraction. Cells were washed twice for 5 sec each centration of Ca2+ might be a regulator of the assembly/di- at room temperature with a buffer containing 60 mM 1,4-pi- sassembly process. For the most part the evidence supporting perazinediethanesulfonic acid (Pipes), 25 mM N-2-hydroxy- this hypothesis is circumstantial. Kiehart and Inoue (2) microin- ethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 10 mM eth- jected Ca2+ into dividing sea urchin eggs and observed a local ylene glycol bis(f3-aminoethyl ether)-N,N,N',N'-tetraacetic and transient reduction of spindle fiber birefringence. Like- acid (EGTA), and 1 mM MgCl2 (extraction buffer; pH 6.9). Cells wise, ionophore-mediated Ca2' entry has been shown to re- were lysed in extraction buffer supplemented with 0.15% Triton versibly affect the cytoplasmic microtubule complex of cultured X-100 or Brij 58 for 30-120 sec. The supernatant was removed cells (3) and microtubule-containing heliozoan axopodia (4). The and the extracted cells on the coverslip were washed with ex- concentration of Ca2' required to inhibit microtubule assembly traction buffer before application of the Ca/EGTA buffer. in vitro apparently depends on the purification protocol used. Preparation of Ca/EGTA Buffers. Ca/EGTA buffers with Assembly in crude brain extracts is inhibited by near micromo- free Ca2+ in the range of 0.1-100 ,AM were made by using a lar Ca2+ (1, 5, 6), whereas the assembly of purified protein is computer program prepared by John Gilkey that took into ac- sensitive to Ca2+ concentrations in the millimolar range only count the pH of the final solution, ionic strength, and Mg2+ (7). Interestingly, the sensitivity to micromolar Ca2+ of purified concentration. The buffers were made up in 60 mM pipes/25 microtubule protein can be restored by a factor present in crude mM Hepes/1 mM MgCl2, pH 6.9. brain extracts (8). Abbreviations: MAP, microtubule-associated protein; EGTA, ethylene The publication costs of this article were defrayed in part by page charge glycol bis(3-aminoethyl ether)-N,N,N',N'-tetraacetic acid. payment. This article must therefore be hereby marked "advertise- t Present address: Dept. of Biochemistry, University of Washington, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Seattle, WA 98195. 1037 Downloaded by guest on September 30, 2021 1038 Cell Biology: Schliwa et al. Proc. NatL Acad. Sci. USA 78 (1981) r" I% A...-I- o il. t .;.Il a .64,, A .1il.. O 11 *rS .............J XXt£2 .......a .' Ai 0" 4 hi ? .". I 1. I 4":.%. .-It- ... -z v s .i ,.. dsr :av I FIG. 1. (Legend appears at the bottom of the next page.) Downloaded by guest on September 30, 2021 Cell Biology: Schliwa et al. Proc. Natd Acad. Sci. USA 78 (1981) 1039 Antisera. The tubulin antiserum was a generous gift of Keigi Table 1. Concentrations of calmodulin inhibitors required to attain Fujiwara. It was prepared according to procedures described virtually complete inhibition of microtubule depolymerization elsewhere (15). The antiserum against a Mr 210,000 assembly induced by 100 ,uM Ca2" in lysed tissue culture cells MAP isolated from HeLa cells was prepared as described (16). Inhibitor Conc.,,uM Procedures for the isolation of a monoclonal antibody against MAP2 have been outlined (17). R24571 5 Preparation of MAPs. MAPs were separated from three- Pimozide 20 times-cycled beef brain microtubules in a DEAE-Sepharose Trifluoperazine 50 column according to Vallee and Borisy (18). As judged by so- Chlorpromazine 200 dium dodecyl sulfate/polyacrylamide gel electrophoresis, the MAP fraction contained the high molecular weight MAPs and ments do disappear first before more central portions of a. mi- a number of low molecular weight proteins but very little tubu- crotubule are affected. lin (<10%). Within the range of 5-500 uM Ca2 , the pattern and time Immunofluorescence Microscopy. Cells were fixed for im- course of microtubule disassembly appears to be independent munofluorescence microscopy with 0.3% glutaraldehyde for 10 of the Ca2+ concentration, suggesting that within this concen- min in the buffer used in the step immediately preceding fix- tration range Ca2+ effects are catalytic rather than stoichio- ation. Coverslips were washed with phosphate-buffered saline metric. Only at 1 mM Ca2+ or above is microtubule breakdown and treated with NaBH4 (1 mg/ml) for 6-8 min (19). After two accelerated and more irregular; after 1 min, only a few short additional washes with phosphate-buffered saline, cells were fragments without any apparent connection to the centrosome incubated at 370C for 45 min with the respective antisera, are present (Fig. ic). Microtubules are essentially stable at Ca2+ washed thoroughly, and allowed to react for 30-45 min with concentrations below 1 MuM (Fig. ld). fluorescein-conjugated goat immunoglobulins specific for rab- The depolymerizing effect is highly specific for Ca2+. Of bit immunoglobulin. Coverslips were mounted on slides and other divalent cations tested, only Sr2' at 0.5 mM induces mi- viewed in a Leitz Orthoplan light microscope equipped with crotubule disassembly; Mg2' at 10 mM-or Ba2' and Mn2+ at epifluorescence illumination.
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