Half a century of biochemical and biophysical experiments has provided attractive models that may explain the diverse functions of microtubules within cells and organisms. However, the notion of functionally distinct microtubule types has not been explored with similar intensity, mostly because mechanisms for generating divergent microtubule species were not yet known. Cells generate distinct microtubule subtypes through expression of different tubulin isotypes and through post-translational modifications, such as detyrosination and further cleavage to Δ2-tubulin, acetylation, polyglutamylation and polyglycylation. The recent discovery of enzymes responsible for many tubulin post-translational modifications has enabled functional studies demonstrating that these post-translational modifications may regulate microtubule functions through an amazing range of mechanisms.

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Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.

(8), however, have hypothesized that a dynamic array of MTs is a source of potential morphogenetic determinants; by the selective stabilization of individual MTs, a MT-based cellular asymmetry could be generated.Considerable evidence exists, both from drug studies (9-11) and from the direct measurement of MT turnover (6,12,13), for a small population of MTs in cultured fibroblasts that is more stable than the bulk of MTs. These stable MTs frequently exhibit a sinuous morphology and are typically comprised of tubulin that has been post-translationally modified (10-13). Such stable MTs are prime candidates for a specialized cytoskeletal role-e.g., in organelle transport or in the maintenance of cellular polarity. However, reported distributions of stable MTs seem at odds with such a role; stable MTs appear randomly located and often do not extend to the cell periphery (9)(10)(11)(12)(13)(14)32).In this study we examine the distribution of stable MTs in mouse 3T3 fibroblasts after applying a specific directional signal to the cells. We (Costar, Cambridge, MA). The medium was removed, and the wounded monolayer was washed once before incubating the cells in fresh medium. Cells were treated with the MT-depolymerizing drug, nocodazole (Aldrich) as described (15), including the extraction step to remove monomeric tubulin before fixation.

Abstract. We previously demonstrated (Ookata et al., 1992(Ookata et al., , 1993 that the p34~¢2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34 ~dc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anticyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34~d~Vcyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34~cVcyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34 ~c2, cyclin B, and the COOHterminal domain of MAP4, Phu, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34 ~d~2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.p ROGaESSION through M-phase of the cell cycle is controlled by M-phase promoting factor (MPF) i, which consists of a complex of p34 cd~2 and cyclin B (for

Turnover MATERIALS AND METHODSCells and Tissue Culture. African green monkey kidney epithelial cells (TC-7) were cultured in Dulbecco's modified Eagle's medium containing 10% (vol/vol) fetal bovine serum as described (24).Preparation of Hapten-Labeled Tubulin. Microtubule protein was isolated under conditions favoring tyrosination as described (33,34). Tubulin was separated from microtubuleassociated proteins by DEAE-Sephadex chromatography (35) and was then hapten-labeled with either dichlorotriazinylaminofluorescein (DTAF) (4) or N-hydroxysuccinimidyl biotin (36). The hapten-labeled material, after two cycles of temperature-dependent assembly and disassembly using 5% (vol/vol) dimethyl sulfoxide (37), was injected at a concentration of --4 mg/ml. [Glu] tTo whom correspondence and reprint requests should be addressed. 9040The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

This review discusses the possible role of alpha-tubulin detyrosination, a reversible post-translational modification that occurs at the protein's C-terminus, in cellular morphogenesis. Higher eukaryotic cells possess a cyclic post-translational mechanism by which dynamic microtubules are differentiated from their more stable counterparts; a tubulin-specific carboxypeptidase detyrosinates tubulin protomers within microtubules, while the reverse reaction, tyrosination, is performed on the soluble protomer by a second tubulin-specific enzyme, tubulin tyrosine ligase. In general, the turnover of microtubules in undifferentiated, proliferating cells is so rapid that the microtubules accumulate very little detyrosinated tubulin; that is, they are enriched in tyrosinated tubulin. However, an early event common to at least three well-studied morphogenetic events--myogenesis, neuritogenesis, and directed cell motility--is the elaboration of a polarized array of stable microtubules that become enriched in detyrosinated tubulin. The formation of this specialized array of microtubules in specific locations in cells undergoing morphogenesis suggests that it plays an important role in generating cellular asymmetries.

Abstract. Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat ~ muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating ~ myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Giu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (<1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of I~ cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.