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Characteristics of the Polar Assembly and Disassembly of Microtubules Observed in Vitro by Darkfield Light Microscopy

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Characteristics of the Polar Assembly and Disassembly of Microtubules Observed in Vitro by Darkfield Light Microscopy CHARACTERISTICS OF THE POLAR ASSEMBLY AND DISASSEMBLY OF MICROTUBULES OBSERVED IN VITRO BY DARKFIELD LIGHT MICROSCOPY KEITH SUMMERS and MARC W. KIRSCHNER Downloaded from http://rupress.org/jcb/article-pdf/83/1/205/1073956/205.pdf by guest on 23 September 2021 From the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 ABSTRACT We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy . In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity ofgrowth off cellular nucleating centers . We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end . Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site . The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca" or colchicine as measured directly by darkfield microscopy . The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell . KEY WORDS microtubules " darkfield bility, and disappearance of groups of microtu- microscopy - kinetochore " microtubule polarity bules remains, for the most part, unexplained . One aspect of this problem has been approached During the cell cycle, microtubules are involved by the identification, isolation, and characteriza- in many different functions. Often, several groups tion of cell organelles which serve as nucleating of microtubules will be present at one time, each centers for microtubule growth (32) . Microtubules with a unique time of appearance and disappear- have been polymerized in vitro from isolated basal ance in the cell (50, 15) . In addition, microtubules bodies, kinetochores in mitotic chromosomes, and of different stability have been described in several pericentriolar material (43, 28, 44, 45, 18, 19, 21) . structures even within the same cell (5, 36). The However, in all cases the nature of the polymeri- mechanism by which the cell controls spatially zation initiating substance is unknown. In some and temporally the appearance, orientation, sta- cases, an amorphous cloud of material seems to be J. CELL. BIOLOGY ©The Rockefeller University Press - 0021-9525/79/10/0205/13 $1 .00 Volume 83 October 1979 205-217 present at the nucleating sites (18, 34, 35) . bules while at the same time varying their envi- Another means by which the cell could differ- ronment . It is then possible to observe changes entiate among classes ofmicrotubules would be by occurring at either end of single microtubules . the production of more than one molecular form Experiments can therefore be performed with ho- of tubulin or the use of different accessory pro- mogeneous preparations of microtubules, whereas teins, which are involved in the polymerization of in electron microscope experiments the nucleating tubulin . The question of tubulin heterogeneity or structure must be physically different from the the role of molecular modifications of tubulin is polymerizing microtubule so that the point of very complex . Cyclic-AMP-dependent phospho- nucleation can be identified (2, 6, 31, 24, 25, 54) . rylation of tubulin and accessory proteins has been These experiments also avoid the potentially dam- demonstrated but the effect ofphosphorylation on aging procedures of fixation, staining, and drying the system remains unknown (10, 17, 37, 40) . required for electron microscopy . The ability to Similarly, enzymes capable of adding and remov- study the dynamics of the assembly of individual Downloaded from http://rupress.org/jcb/article-pdf/83/1/205/1073956/205.pdf by guest on 23 September 2021 ing a tyrosine residue specifically on the carboxyl microtubules has enabled us to develop methods terminal of the « subunit of tubulin are known but for assessing their polarity . We have applied these no effect of this unusual modification on polym- methods here to determine the polarity of micro- erization has been detected (4, 33) . Evidence to tubule growth off cellular nucleating sites . Polar- explain how accessory proteins might control mi- ity, in these cases, can be determined if it is crotubule polymerization in vivo is also generally assumed that subunits can only add at the free lacking, though there has been a correlation made end . The results obtained here complement those between their presence in the brain tissue of de- recently obtained where polarity is assessed by veloping rats and the general polymerizability of looking at the change in bulk microtubule length. the tubulin extracted from this tissue (39, 14) . In the case of polarity of kinetochore growth, our Another potential basis for spatial and temporal experiments support the recent analysis by Borisy differentiation among groups of microtubules (7) using extent ofassembly, as judged by electron within a cell comes from the polar nature of microscopy, to analyze the rate of microtubule microtubules themselves . Microtubules are intrin- polymerization . sically polar as indicated by the subunit lattice structure seen by X-ray diffraction and electron MATERIALS AND METHODS microscopy (20, 3, 12, 9) . The intrinsic polarity is Microtubule protein was prepared from hog brain tissue also indicated by the fact that tubulin polymerizes as described previously (5l) . The protein was stored at to a different extent off the proximal and distal -20°C in 8 M glycerol . Before each experiment, a sample ends of flagellar microtubules (2, 6, 31, 24, 25, 54) . was diluted into an equal volume of polymerization If cells were able to initiate microtubule polymer- buffer (0.1 M MES (2-[N-morpholino]ethane sulfonic ization from nucleation centers with a specific acid), pH 6.4, 2 mM EGTA, l mM mercaptoethanol, 0 .5 polarity and if they also possessed a mechanism mM MgC12, 0.1 mM EDTA, 1 mM GTP), polymerized for differentiating between such polar microtu- at 37°C, and the microtubules were collected by centri- fuging at 75,000 g for 30 min. The pellet of microtubules bules, they would effective control system have an was then resuspended in polymerization buffer and put for discrimination between two groups of micro- on ice. After allowing 20 min for depolymerization, the tubules . sample was centrifuged again at 75,000 g for 30 min to To investigate the role of polarity in microtubule remove insoluble aggregates. polymerization and depolymerization under a va- Chromosomes were isolated from mitotic CHO cells riety of conditions, we have observed the growth by an adaptation of the methods of Gould and Borisy of individual microtubules by darkfteld light mi- (18) . CHO cells were grown to near confluence in 100- croscopy . This technique has been used previously mm culture flasks in F-10 medium . Mitotic cells were to study ATP-induced sliding of doublet micro- shaken loose from their culture bottle and collected by centrifuging at 500 g for 5 min . The pellet of cells was tubules of flagellar axonemes (46) and the move- gently rinsed with distilled water and then suspended in bacterial . ment of flagella (26) Observations on 0.1 ml of distilled water . After l min, 0.1 ml of 0.1 M reconstituted neural microtubules have also been Mes pH 6.4, 0.5 mM MgCl,, 0 .5% Triton X-100 was reported (25, 54, 47) . By using darkfteld light added and the cells were lysed by forcing the suspension microscopy it is possible, in principle, to make through a blunt syringe needle held against the bottom continuous observations on individual microtu- of the tube. In most cases, 0.4 ml of microtubule protein 2W THE JOURNAL OF CELL BIOLOGY " VOLUME 83, 1979 was added immediately and the sample was incubated from monomers and oligomers of tubulin are dif- at 37°C for 5 min . These chromosome preparations were ficult to visualize by darkfield microscopy . The used immediately for experiments since the chromosome polymeric components in the initial steps scatter morphology tended to deteriorate after a few hours at light less intensely than do microtubules. Since experiments, chromosomes room temperature . In a few they also move rapidly around by Brownian mo- mM CaC1 2 and I M were preserved by adding 0.5 tion, it has not been possible to photograph the hexylene glycol to the lysing media . Such chromosomes nucleation process in real time . These components were stable at 0°C for several hours . In addition, we have been photographed by darkfield microscopy obtained chromosomes from colchicine-arrested HeLa cells from U. K . Laemmli (Princeton University) (1) . and electron microscopy after fixation with glutar- Axonemes were prepared from cilia detached from aldehyde (25, 47) and suggest that the flexible Tetrahymena by exposing a concentrated suspension of intermediates could represent protofilament the cells to I mM dibucaine . The membranes were sheets, which have been reported to be present removed by suspending the cilia in 0.1% Triton, 0 .1 M during the assembly reaction both in vitro and in Downloaded from http://rupress.org/jcb/article-pdf/83/1/205/1073956/205.pdf by guest on 23 September 2021 Mes, pH 6 .4, 0.5 mM MgCl2- vivo (23, 11, 8, 41, 49) . Observations were made with a Zeiss Axiomat light It is possible, however, to photograph clearly N. Y.) with a 200-watt microscope (Carl Zeiss, Inc ., the elongation of microtubules, which takes place mercury arc light source fitted with infrared filters and in the later phase ofpolymerization .
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