Cellular interactions and tubulin detyrosination in fibroblastic and epithelial cells

In mammalian cells most microtubules are enriched in tyrosinated alpha-tubulin (tyr-tubulin). Other subclasses of microtubules are present in variable amounts and some are enriched in detyrosinated alpha-tubulin (glu-tubulin). We examined the effect of cell-cell interactions on the level of glu-tubulin in microtubules. This was studied by quantitative immunofluorescence using antibodies against tyr- and glu-tubulin. We found that in cells which have established cell-cell contacts, the ratio of glu-/tyr-tubulin is higher than in isolated cells. We also examined the effect of cell-cell interactions on the glu-/tyr-tubulin ratio by using the antibody blocking method of Schulze and Kirschner [42]. Microtubules containing mainly tyr-tubulin had been blocked first by a polyclonal antibody against tyr-tubulin and several layers of secondary antibodies. The unblocked microtubules were then labeled by a monoclonal antibody against alpha-tubulin. Since the coating efficiency of microtubules by the anti-tyr tubulin depends on the amount of tyr-tubulin in each microtubule, this procedure allows the visualization of microtubules enriched or depleted in tyr-tubulin in specific domains of each cell. Microtubules were more extensively blocked in subconfluent than in confluent cells and preferentially at the periphery of the cytoplasm. In cells present at the margin of an artificial wound produced in a confluent monolayer, the amount of blocked microtubules increased slowly with time (between 2 and 4 h). These results are consistent with the hypothesis that cell-cell contacts lead to increased tubulin dytyrosination both in fibroblastic and epithelial cells.     

Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules

The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti-centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole-resistant microtubules in MDCK cells contained detyrosinated alpha-tubulin (glu tubulin). On the other hand, the first microtubules to regrow after nocodazole removal contained only tyrosinated tubulin. Glu-tubulin became detectable only after 30 min of microtubule regrowth. This strongly supports the hypothesis that alpha-tubulin detyrosination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount of glu-tubulin that we found in taxol-treated cells.     

Microtubule dynamics in fish melanophores

We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X- rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport.     

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(- 8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half- life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.     

Tubulin dynamics in cultured mammalian cells

Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells.     

An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin

Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.     

Temporal and spatial pattern of differences in microtubule behaviour during Drosophila embryogenesis revealed by distribution of a tubulin isoform

Immunofluorescence staining of Drosophila embryos with a monoclonal antibody specific for acetylated alpha-tubulin has revealed that acetylated and nonacetylated alpha-tubulin isoforms have different patterns of distribution during early development. Acetylated alpha-tubulin was not detected in either interphase or mitotic spindle microtubules during the rapid early cleavage or syncytial blastoderm divisions. Acetylated alpha-tubulin was first observed as interphase lengthened at the end of syncytial blastoderm, and at cycle 14 was localized to a ring of structures clustered around the interphase nuclei. These structures probably represent a set of stable microtubules involved in nuclear elongation. Absence of detectable acetylated alpha-tubulin prior to cellular blastoderm seems to be due to rapid turnover of microtubule arrays rather than to lack of the enzyme required for modification, since acetylated alpha-tubulin appeared in early embryos when micro-tubules were stabilized by taxol treatment or anoxia. Because acetylated alpha-tubulin seems to be characteristic of stable microtubule arrays, the appearance of the antigen at cycle 14 represents a fundamental change in microtubule behaviour in the somatic cells of the embryo. Acetylated alpha-tubulin was not detected in pole cells during the blastoderm or early gastrula stages, indicating that acetylation of alpha-tubulin is not merely a consequence of cellularization. After the onset of gastrulation, interphase microtubule arrays in most cell types contain acetylated alpha-tubulin. However, cells in mitosis lack antibody staining. The resulting unstained patches reveal the stereotyped spatial pattern of cell division during gastrulation. Although the cells that give rise to the amnioserosa have acetylated alpha-tubulin in their interphase arrays at early gastrulation, by germ band elongation these large, plastic cells completely lack staining with anti-acetylated alpha-tubulin. In contrast, differentiated cell types such as neurones, which have arrays of stable axonal microtubules, stain brightly with the specific antibody. Although acetylated and nonacetylated alpha-tubulin are present in roughly equal amounts by the late stages of embryogenesis, acetylated alpha-tubulin is partitioned into the pellet during centrifugation of extracts of embryos homogenized at 4 degrees C.     

Confocal and video imaging of cytoskeleton dynamics in the leech zygote

Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors.     

Acetylated alpha-tubulin in microtubules during mouse fertilization and early development

alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.     

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.     

Organization of microtubules in stabilized meristematic plant cells revealed by a rat monoclonal antibody reacting only with the tyrosinated form of alpha-tubulin

A rat monoclonal antibody against yeast tubulin (clone YL 1/2; Kilmartin et al., 1982) that reacts specifically with mammalian alpha-tubulin carrying a carboxyterminal tyrosine residue (Wehland et al., 1983) was used to localize microtubules in plant cells derived from onion root apices (Allium cepa L.). YL 1/2 reacted with all types of microtubular arrays known to occur in higher plant meristematic cells such as interphase cortical microtubules, pre-prophase bands, the mitotic spindle and phragmoplast microtubules. The specific labeling of microtubules in isolated cells from onion root tips by YL 1/2 indicates that plant cells like animal cells contain tubulin tyrosine ligase, the enzyme which posttranslationally modifies alpha-tubulin. This enzyme could be involved in the dynamic regulation of microtubular arrays in all eukaryotic cells.     

Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells

Immunofluorescence with specific peptide antibodies has previously established that tyrosinated (Tyr) and detyrosinated (Glu) tubulin, the two species generated by posttranslational modification of the COOH-terminus of alpha-tubulin, are present in distinct, but overlapping, subsets of microtubules in cultured cells (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789). Similar results were observed by light microscopic immunogold staining in the two cell types used in this study, CV1 and PtK2 cells: most microtubules were stained with the Tyr antibody, whereas only a few were stained with the Glu antibody. We have examined immunogold-stained preparations by electron microscopy to extend these results. In general, electron microscopic localization confirmed results obtained at the light microscopic level: the majority of the microtubules in CV1 and PtK2 cells were nearly continuously labeled with the Tyr antibody, whereas only a few were heavily labeled with the Glu antibody. However, in contrast to the light microscopic staining, we found that all microtubules of interphase and mitotic CV1 and PtK2 cells contained detectable Tyr and Glu immunoreactivity at the electron microscopic level. No specific localization of either species was observed in microtubules near particular organelles (e.g., mitochondria or intermediate filaments). Quantification of the relative levels of Glu and Tyr immunoreactivity in individual interphase and metaphase microtubules showed that all classes of spindle microtubules (i.e., kinetochore, polar, and astral) contained nearly the same level of Glu immunoreactivity; this level of Glu immunoreactivity was lower than that found in all interphase microtubules. Most interphase microtubules had low levels of Glu immunoreactivity, whereas a few had relatively high levels; the latter corresponded to morphologically sinuous microtubules. Quantification of the relative levels of Tyr and Glu immunoreactivity in segments along individual microtubules suggested that the level of Tyr (or Glu) tubulin in a given microtubule was uniform along its length. Understanding how microtubules with different levels of Tyr and Glu tubulin arise will be important for understanding the role of tyrosination/detyrosination in microtubule function. Additionally, the coexistence of microtubules with different levels of the two species may have important implications for microtubule dynamics in vivo.     

Assembly and turnover of detyrosinated tubulin in vivo

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.     

A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.     

Acetylated and detyrosinated alpha-tubulins are co-localized in stable microtubules in rat meningeal fibroblasts

We have examined the distribution of acetylated alpha-tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meninges. Meningeal fibroblasts showed heterogeneous staining patterns with a monoclonal antibody against acetylated alpha-tubulin ranging from staining of primary cilia or microtubule-organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti-alpha-tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double-labeling experiments using an antibody against acetylated alpha-tubulin (6-11B-1) and antibodies against either tyrosinated or detyrosinated alpha-tubulin, it was found that acetylated alpha-tubulin and tyrosinated alpha-tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated alpha-tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated alpha-tubulin and was cold stable, and the other contained tyrosinated alpha-tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of alpha-tubulin are involved in the specification of stable microtubules.     

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.     

Exposure of lumenal microtubule sites after mild fixation

High-resolution analysis of tubulin structure and docking the structure of tubulin dimer into a map of microtubules led to a prediction that sites for tubulin acetylation are in the interior of microtubules. This is somehow difficult to reconcile with their susceptibility to proteases and acetylation in assembled microtubules. To assess the availability of acetylated alpha-tubulin for antibodies, immunofluorescence on detergent-extracted cells, on cells fixed under various conditions and in microinjected cells was performed with monoclonal antibodies of known epitope locations. The presented data indicate that acetylated alpha:Lys40 is not exposed on unfixed microtubules but that this region of lumenal microtubule surface becomes easily exposed under mild fixation conditions.     

Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum

We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporates into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-alpha-tubulin incorporates into microtubules at a lower efficiency than does the endogenous alpha-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP-alpha-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-alpha-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics.     

Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells

Subsets of microtubules enriched in posttranslationally detyrosinated (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779) or acetylated (Piperno, G., M. Le Dizet, and X. Chang. 1987. J. Cell Biol. 104:298), alpha tubulin have previously been described in interphase cultured cells. In this study an immunofluorescence comparison of these minor populations of microtubules revealed that, in African green monkey kidney epithelial cells (TC-7 line), the population of microtubules enriched in detyrosinated tubulin was virtually coincident with the population enriched in acetylated alpha tubulin. In some cell types, however, such as human HeLa or marsupial PtK-2 cells, only one posttranslationally modified form of tubulin, i.e., acetylated or detyrosinated, respectively, was detectable in microtubules. In TC-7 cells, although both modifications were present, dissimilar patterns and kinetics of reappearance of microtubules enriched in detyrosinated and acetylated tubulin were observed after recovery of cells from microtubule-depolymerizing treatments or from mitosis. Thus, a minor population of microtubules exists in cultured cells that contains an elevated level of tubulin modified in either one or two ways. While these two modifications occur primarily on the same subset of microtubules, they differ in their patterns of formation in vivo.     

Translocation and clustering of endosomes and lysosomes depends on microtubules

Indirect immunofluorescence labeling of normal rat kidney (NRK) cells with antibodies recognizing a lysosomal glycoprotein (LGP 120; Lewis, V., S.A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) reveals that lysosomes accumulate in the region around the microtubule-organizing center (MTOC). This clustering of lysosomes depends on microtubules. When the interphase microtubules are depolymerized by treatment of the cells with nocodazole or during mitosis, the lysosomes disperse throughout the cytoplasm. Lysosomes recluster rapidly (within 30-60 min) in the region of the centrosomes either upon removal of the drug, or, in telophase, when repolymerization of interphase microtubules has occurred. During this translocation process the lysosomes can be found aligned along centrosomal microtubules. Endosomes and lysosomes can be visualized by incubating living cells with acridine orange. We have analyzed the movement of these labeled endocytic organelles in vivo by video-enhanced fluorescence microscopy. Translocation of endosomes and lysosomes occurs along linear tracks (up to 10 microns long) by discontinuous saltations (with velocities of up to 2.5 microns/s). Organelles move bidirectionally with respect to the MTOC. This movement ceases when microtubules are depolymerized by treatment of the cells with nocodazole. After nocodazole washout and microtubule repolymerization, the translocation and reclustering of fluorescent organelles predominantly occurs in a unidirectional manner towards the area of the MTOC. Organelle movement remains unaffected when cells are treated with cytochalasin D, or when the collapse of intermediate filaments is induced by microinjected monoclonal antivimentin antibodies. It can be concluded that translocation of endosomes and lysosomes occurs along microtubules and is independent of the intermediate filament and microfilament networks.