Dynamic and Stable Populations of Microtubules in Cells Eric Schulze and Marc Kirschner Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California 94143-0448 Abstract. Using a new immunocytochemical tech- with a half-time of 10 min. A separate population of nique, we have visualized the spatial arrangement of microtubules can be detected, using the above tech- those microtubules in cells that are stable to bio- niques, that are stable to exchange for 1 h or more; tin-tubulin incorporation after microinjection. Cells these have a characteristic pericentrosomal spatial ar- fixed at various periods of time after injection were rangement as compared to the majority of dynamic exposed to antibody to biotinylated tubulin and several microtubules. Unlike the dynamic microtubules, most layers of secondary antibodies; these layers prevented of the stable microtubules are nongrowing. The aver- reaction of biotin-containing microtubules with anti- age BSC-1 cell contains *700 microtubules: ~500 tubulin antibodies. The microtubules that had not in- growing at 4 l~m min -I, 100 shrinking at m20 ~tm corporated biotin-tubulin could then be stained with min -1, and *100 that are relatively more stable to ex- anti-tubulin and a fluorescent secondary antibody. In change. The potential significance of these stable BSC1 cells, most microtubules in the cell exchange microtubules is discussed. fundamental problem in cell biology is the asymmetric and photobleaching (Saxton et al., 1984; Soltys and Borisy, placement of components within the cell. The cyto- 1985) suggest that microtubules turnover and grow very rap- skeleton and, in particular, microtubules are thought idly in vivo, displaying a pattern of nucleation, growth, and to play an important role in cell asymmetry, since their de- rapid shrinkage, which leads to replacement of microtubules polymerization often leads to withdrawal of cell processes, one by one. Most microtubules followed this pattern of re- inhibition of cell movement, and displacement of the nu- placement and were found to have a half-life of 5-10 min. cleus and cell organelles. In many cells, microtubules have However, some microtubules were more stable and unla- an overall asymmetric distribution. Polarity can also be beled microtubule segments could still be seen at 1-6.5 h, al- achieved if the distribution is symmetric but some microtu- though all microtubules exchanged by 10 h. bules in the cell are differentiated from others. Recently, sup- In this report we examine in more detail the turnover, num- port for spatial differentiation of microtubules has come ber, and spatial distribution of stable microtubules in two cell from the study of posttranslational modifications of tubulin, lines. We demonstrate, by means of a new immunocytochem- such as detyrosination and acetylation (Gunderson et al., ical technique, that these stable microtubules are generally 1984; LeDizet and Piperno, 1986). Such modifications have continuous nongrowing polymers with a distribution distinct been shown to occur on a spatially distinct subpopulation of from that of the dynamic population of microtubules. We microtubules in the cell. also report the results of a more detailed analysis of the dy- A simple explanation for the differentiation of neighboring namic microtubule population. These data on the number microtubules is that microtubules in the cell differ in age and fraction of growing microtubules, along with our better (Kirschner and Mitchison, 1986). In the case of detyrosina- understanding of stable microtubules, allows us to model, in tion, the available evidence supports the view that older general terms, the behavior of microtubules in an interphase microtubules could rather uniformly accumulate posttrans- cell. How the various microtubule classes arise from the lational changes. This pattern of modification of neighboring presumably homogenous dynamic microtubule population, microtubules is consistent with the pattern of microtubule and their possible functional significance are also discussed. growth expected from the model of dynamic instability in vitro (Mitchison and Kirschner, 1984a, b; Horio and Ho- Materials and Methods tani, 1986). Recently, we have studied the pattern and rate of microtu- Materials bule growth in fibroblast cells by injecting cells with biotin- labeled tubulin and observing the pattern of new assembly N-Hydroxysuccinimidyl biotin was from Polysciences, Inc. (Warrington, (Schulze and Kirschner, 1986). These experiments and PA); rabbit anti-biotin antibody was from Enzo Biochem, Inc. (New York, NY). Mouse monoclonal anti-13-tubulin was a kind gift of Dr. S. H. Blose others involving incorporation of fluorescein-labeled tubulin © The Rockefeller University Press, 0021-9525/87/02/277/12 $1.00 The Journal of Cell Biology, Volume 104, February 1987 277-288 277 (Protein Databases, Inc., Huntington Station, NY). Unlabeled, and rhoda- tography were carried out as in Sehulze and Kirachner (1986). The use of mine- and fluorescein-labeled secondary antibodies were from Cappel hypersensitized technical Pan 2415 film (Eastman Kodak Co., Rochester, Laboratories (CooperBiomedical Inc., Malvern, PA). Texas red-labeled rat NY) with its extremely fine grain, made possible the accurate analysis of anti-mouse antibody was from Accurate Chemical & Scientific Corp. highly magnified images. (Westbury, NY). Nocodazole was from Aldrich Chemical Co. (Milwaukee, WI). Nocodazole Experiments Tubulin Preparation SKNSH cells were injected with 10 mg/ml biotin-tubnlin, and incubated for 1 h in a 370C tissue culture incubator. The cells were then incubated in either Phosphocellulose-purified tubulin was prepared from bovine brains by a 1 I~g/ml or 10 gg/ml nocodazole 3"/0C in tissue culture media for either 2 modification of the procedure of Weingarten et al. (1975) as described in or 10 min, respectively. The cells are then immersed into a modified per- Mitchison and Kirschner (1984a, b). Protein concentrations were deter- meabilization buffer (80 mM Pipes, 5 mM EGTA, 1 mM MgC12, 0.5% mined by the method of Bradford (1976), using BSA as a standard. NP-40, 20% glycerol by volume) for45 s at room temperature. The addition of glycerol to the permeabilization buffer helps stabilize the microtubules Preparation of Biotinylated Tubulin during the slightly longer permeabilization necessary to remove the large pool of free tubulin in these nocodazole-treated cells. Glutaraldehyde Biotinylated tubulin was prepared as described in Kristofferson et al. (1986) (0.5%) is then added with gentle mixing, and the cells are fixed for 10 min and Mitchison and Kirschner (1985) with modifications for microinjection followed by the above described antibody-blocking technique. as in Schulze and Kirschner (1986). Cell Area Measurement and Average Cell Culture Microtubule Length Measurement African Green monkey kidney fibroblasts (BSC1) (a kind gift of U. Euten- Negatives of microinjected ceils were projected onto a GITCO (RockviUe, euer, University of California at Berkeley) were grown in DME sup- MD) digitizing pad interfaced to an IBM PC. The perimeter of the cell was plemented with 10% FCS (Gibco, Grand Island, NY). Human retino- traced, and area calculated using the supplied soRware. The length of grow- blastoma cells (SKNSH) (a kind gift of Manfred Schwab, University of ing microtubules was measured from negatives of three cells microinjected California at San Francisco) were grown in RPMI 1640 with 10% FCS and incubated for short times (2 min). The length of each growing microtu- (Bluestein, 1978). For microinjection studies, the cells were trypsinized off bule was approximated by measuring from a defined point in the cell center tissue culture dishes and replated onto l-in-diameter poly-D-lysine-coated to the end of a growing segment. The digitized lengths were stored on an coverslips. BSC1 cells were allowed to settle for 1 d before use; SKNSH IBM PC for subsequent statistical analysis. cells were allowed to settle for 1 d, and differentiated in 30 I~g/ml retinoic acid for '~3 d before use. Microtubule Number and Percent of Growing Microtubules at the Periphery Microinjection Microtubnle segments were counted by using 8 × 10-in enlargements of Cells on coverglasses were placed in a thermostatted microinjection cham- cells injected with biotin-tubnlin and incubated for short times (5 min). The ber at 37°C as described in Schulze and Kirschner (1986). The chamber was percent of microtubules growing at the periphery was calculated from in- then placed on a Zeiss ICM 405 inverted microscope allowing direct access jected ceils with short incubations which were prepared using either the from above. The cells were then pressure microinjected using the technique antibody-blocking technique, or standard immunofluorescence techniques. of Graessman and Graessman (1976). Microinjection needles were drawn High magnification pictures were printed of both the anti-tubulin and out to ,x,0.5-1 lan diameter tips using a micropipette puller model 720 anti-biotin staining; all unambiguous ends were recorded as either bio- equipped with patch clamp adapter 728A (David Kopf Instruments, Tu- tin-tubulin ends, or unexchanged ends, from which we calculated the frac- junga, CA). We estimate that approximately one-tenth of a cell volume was tion of microtubule ends with biotin segments. typically injected (Graessman et al., 1980). Antibody-blocking Technique Results After injection of cells with 10 mg/mi biotin-tubulin, cells were allowed to incubate at 37°C in a CO2 incubator for the desired period of time, from Visualization of Stable Microtubules 5 rain to 2 d. The cells were then washed with PBS for 2 s, then immersed We have previously described the existence of a minority in room temperature permeabilization buffer (80 mM Pipes, 5 mM EGTA, 1 mM MgCI2, 0.5% NP-40) for 20 s, after which glutaraldehyde (0.5% class of microtubules in BSC1 cells that was more stable final concentration) was added to the permeabilization buffer with gentle to exchange with free tubulin than the majority of microtu- mixing. After fixation (10 min), the cells were washed for 10 s in PBs, before bules.