Morphogenesis and the Control of Microtubule Dynamics in Cells

M. Kirschner a nd E. Schulze 0 0 Department of Biochemistry and Biophysics, University of California at San Francisco , San Francisco, CA 94143-0448 , USA Microtubules show unusual dynamic properties at steady state in vitro. While overall the polymer mass remains stable, individual polymers in the population are either growing or shrinking. T his phenomenon called dynamic instability is best explained by the known coupling of polymerization to G T P hydrolysis, and the hypothesis that the stability or instability of the whole polymer is determined by whether G T P or GDP is bound to the terminal subunit. Similar unusual dynamics have now also been found in vivo. By visualizing new subunit assembly after injection of tubulin modified with biotin into living fibroblast cells, we can visualize new growth on individual microtubules with antibody to biotin. Microtubules grow in vivo at about 4/im m in_1 and after rapid and precessive depolymerization old microtubules are replaced by new growth from the centrosome. Some microtubules turn over much more slowly and these stable microtubules have a different spatial distribution from the majority of dynamic ones. The existence of both stable and dynamic microtubules in the same cell suggests a model for morphogenesis of the microtubule cytoskeleton. The rapid turnover of microtubules in the cell provides a complex population upon which selective factors can act. Stability can be generated at the end of the polymer and affects the entire microtubule. This model of selective stabilization at the microtubule ends is discussed in terms of recent experiments on the establishment of kinetochore-pole microtubules during mitosis. - T he allure of cell biology, to those willing to endure complex and often inelegant experimentation, has been the chance to understand the spatial organization of living things. In the past biologists have emphasized the chemical and molecular biological genesis of structure as the key to understanding this organization. But for all we have learned about metabolism and the molecular structure of proteins and nucleic acids, we are still unable to answer the basic question of how a cell organizes itself. The cytoskeleton is one aspect of cellular organization that has recently become amenable to investigation at the molecular level. In particular the microtubule cytoskeleton poses real morphogenetic questions and has been valuable as a model for the study of the self-assembly of large-scale intracellular structures. T h e arrangement of microtubules in the cloned SK N SH retinoblastoma cells (Bluestein, 1978) serves as an example of how genetically identical cells express different cellular morphology and microtubule distributions (see Fig. 1). These same cells, however, will all produce functional mitotic spindles, and even respond in a similar manner to retinoic acid (a morphogenetic signal), by extending similar neurite processes (see Fig. 2). How can we explain the variability of structure in genetically identical cells in a nearly identical environment? How can we then explain Fig. 1. Undifferentiated retinoblastoma cells stained with antibody to tubulin. Note the heterogeneity of both morphology and the distribution of microtubules between cells. Bar, 10 /im. Prepared according to the methods of Schulze & Kirschner (1986). Fig. 2. Retinoblastoma cells exposed for 2 days to 30 jUgml 1 retinoic acid and stained with antibody to tubulin as stated in legend to Fig. 1. Bar, 10 fim. the similar responses these cells make to extracellular signals such as retinoic acid or to internal cell cycle signals that will produce in each cell a functionally equivalent response? Although we will be concerned in this review mainly with the assembly and maintenance of microtubules in the cell, we must remember that this is only one aspect of cell organization and, as we shall see, poses many general questions about cellular morphogenesis. In order to study the organization of the microtubule network, it is first necessary to understand the mechanism of assembly of the microtubule polymer. It was difficult initially to study the molecular mechanism of microtubule assembly in vivo, except in the special case of mitosis, and much investigation, therefore, has been done on microtubule assembly and dynamics in vitro. These studies have addressed the question ofwhetherthe microtubule polymer is inan equilibrium state, a steady state, ameta-stablestate, or is simply a series ofrapidly changing kinetic inter mediates. Any simple equilibrium process such as the addition of a subunit to the end of a linear polymer is governed by equation (1): m (1 ; This equation, though simple in form ties the fraction of monomer in polymer to the off-and-on rate of subunit assembly. T he dissociation constant K diss represents the free monomer concentration in equilibrium with the polymer (Oosawa & Asakura, 1975). If tubulin subunits in the cell are used efficiently to make polymer, then K diss will be low. T he on rate, k on, is ultimately limited by the diffusion of subunits to the end of the polymer. Measurements of subunit assembly in vitro suggest that the measured value is close to the theoretical limit of diffusion. T he off rate ko{{ is in fact what is measured, when microtubules depolymerize if the free monomer pool is lowered either by dilution or by complexing the monomer with drugs such as colchicine or nocodazole. Given the physical limit on k on it is clear from equation (1) that the extent of polymerization and k an are inversely related and that polymers at true equilibrium within the cell can choose either efficient use of tubulin subunits (low/Qiss) or rapid dynamics (high ka{() but not both. As we shall see, the unusual physical chemical properties of tubulin allow the cell to circumvent the physical limitations posed by equation (1). Evidence that rates of exchange in vivo of the monomer of tubulin with the polymer were very rapid was previously found only for mitotic cells (Inoue, 1981; Saxton et al. 1984), but recently it has been demonstrated for interphase cells. T ubulin modified with fluorescein has been injected into cells and the rates of turnover have been measured by allowing the tubulin to exchange with the total monomer and polymer pool and then measuring the re-equilibration after photobleaching (Salmon et al. 1984; Saxton et al. 1984). These experiments confirmed the rapid microtubule turnover inferred previously in mitotic cells from polarization microscopy studies and extended these results to interphase cells where the dynamics were found to be slower but the microtubules are much longer. T he question raised by the fluorescent photobleaching studies w as: how could microtubules turnover so quickly and yet maintain appreciable polymer mass under the constraints imposed by equation (1)? U N U S U A L P R O P E R T I E S OF M I C R O T U B U L E S I N VITRO An explanation for the puzzling in vivo properti (...truncated)