In vivo efficacy and ultrastructural effects of mitomycin C against experimental alveolar hydatid disease

The in vivo efficacy and ultrastructural effects of mitomycin C were determined against alveolar hydatid disease in experimentally infected animals and compared to mebendazole treatment. Mitomycin C inhibited the mean cyst mass of treated versus control animals by 84.1% which was statistically significant at the alpha = 0.01 level. Mebendazole given daily inhibited the mean cyst mass by 80.1%, while mebendazole administration on the same treatment schedule as that used for mitomycin C inhibited the mean cyst mass by 70.4%. Ultrastructurally, mitomycin C was not observed to affect the tegumental microtriches (microvilli) or the microtubular system. However, an increase in the number and accumulation of round to oval electrondense vesicles was observed within the subtegument. These inclusion bodies became vacuolated, subsequently degenerated, and formed myelin-like figures. Mitomycin C, like mebendazole, was only cystistatic in its effects on the cyst stage of Echinococcus multilocularis as evidenced by the growth of treated cyst material following inoculation into helminth-free animals.     

Influence of the antimicrotubule agent,mebendazole, on the secretory activity of intestinal cells ofAscaridia galli

Summary The anthelmintic compound mebendazole caused the disappearance of microtubules in the intestinal cells ofAscaridia galli. Electron microscopy revealed that soon after the microtubules disappeared there was an accumulation of secretory vesicles near the golgi areas. subsequently many of these vesicles aggregated forming dense large vesicles near the terminal web of the intestinal cells. This provides further evidence for the involvement of microtubules in the secretion of products from eukaryotic cells. It seems likely that inhibition of microtubule directed secretory functions in various cell types is an important function in the anthelmintic activity of the benzimidazole carbamates.     

Ultrastructure of the intestinal cells of Aspiculuris tetraptera after in vivo treatment of mice with mebendazole and thiabendazole

Ultrastructural changes in the intestinal cells of female Aspiculuris tetraptera following in vivo treatment with mebendazole or thiabendazole are described. Major changes included a marked reduction in the luminal microbial flora and the appearance of large numbers of autophagic vesicles in the cytoplasm of the intestinal cells. Some mebendazole-treated nematodes were more severely affected, with a complete loss of cellular integrity and a collapse of the intestinal lumen. The possible role of the intestinal cell microtubules in inducing these anthelmintic effects is discussed.     

Cytoskeletal features of the syncytial epidermis of the tapeworm Hymenolepis diminuta

A hallmark feature of parasitic platyhelminths is a cytoarchitecturally unusual syncytial epidermis composed of a peripheral layer of continuous cytoplasm (the ectocytoplasm) connected to underlying nucleated cell bodies by small cytoplasmic bridges. The helminth epidermis, or tegument, plays important roles in protection and nutrient acquisition; cestodes, in fact, completely lack a gastrointestinal tract and absorb all nutritive material through the tegument. Perhaps not surprisingly, the cestode tegument bears certain resemblances to the mucosal epithelium of the vertebrate small intestine, including the possession of a microvillous brush border upon the surface of the ectocytoplasm. In contrast to the intestinal epithelial cell, however, very little is known concerning the nature and organization of the cytoskeleton within the helminth epidermis. Therefore, a number of different microscopical preparative techniques were used to examine the tegument of the tapeworm Hymenolepis diminuta for the presence and distribution of microfilaments, intermediate filaments, and microtubules. It was found that both actin-containing microfilaments and intermediate-sized filaments are present but are restricted to specific locations along the plasmalemmae of the ectocytoplasm. In contrast, microtubules are found throughout the tegument, and are concentrated in the supranuclear regions of the perikarya and in the cytoplasmic bridges interconnecting the perikarya and ectocytoplasm. Unlike brush borders of most other epithelia, the cestode epidermal brush border lacks a filamentous terminal web and is instead associated with microtubules. A network of fine filaments, 5-8 nm in diameter but distinct from actin-containing microfilaments, runs throughout the ectocytoplasm and appears to interlink tegumental vesicles. These fine filaments may represent the primary "skeletal" system responsible for maintaining the structure of the tegumental cytoplasm.     

Structure of kinetochore fibers: Microtubule continuity and inter-microtubule bridges

To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 m sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and in cells recovering from Colcemid arrest were similar in two respects: the number of microtubules per kinetochore (mean 14 and 12, respectively) and the nearest neighbor intermicrotubule distance (mean90 nm). In Colcemid recovered cells, over 90% of the microtubules in kinetochore fibers were attached to the kinetochore (i.e. kinetochore microtubules) and extended most or all of the distance to the pole. Few free microtubules were present in the kinetochore fibers; most non-kinetochore microtubles terminated in the pole. Since kinetochores in this Colcemid-recovered system have been demonstrated to nucleate microtubules (Witt et al., 1980), it seems likely that most if not all of these kinetochore microtubules originated at the kinetochore. Some of the reconstructed kinetochore fibers were attached to chromosomes with bipolar orientation, suggesting that kinetochore microtubules need not interact with many polar microtubules for orientation to occur. In Colcemid recovered cells lysed to reduce cytoplasmic background, microtubules in kinetochore fibers were preferentially preserved. The parallel and near-hexagonal order typical of microtubules in kinetochore fibers was maintained, as was the number of kinetochore microtubules (mean, 13). The intermicrotubule distance was slightly reduced in lysed cells (mean, 60 nm). Crossbridges about 5 nm wide and 30–40 nm long were visible in kinetochore fibers of lysed cells. Such crossbridges probably contribute to the stabilization and parallel order of microtubules in kinetochore fibers, and may have a functional role as well.     

Giardia lamblia: ultrastructural study of the in vitro effect of benzimidazoles.

Axenically grown Giardia lamblia trophozoites treated with low concentrations of the benzimidazole carbamates albendazole and mebendazole detach from glass culture tubes and lose viability. Scanning electron microscopic observations revealed that these drugs produce grotesque modifications of the cell shape of the parasite and disassembly of the adhesive disc. Transmission electron microscopy showed several stages of the fragmentation of adhesive discs with dispersion of microtubules and microribbons in the cytoplasm. Flagella appeared undamaged. In drug-treated trophozoites electron-dense precipitates were selectively deposited on microtubules and microribbons. The results indicate that the antigiardial effect of benzimidazoles is the result of binding to microtubules and subsequent alterations of the cytoskeleton. The electron microscopic observations also suggest that the drugs may bind to microribbon components of the adhesive disc, possibly giardin proteins.     

Giardia lamblia: Ultrastructural Study of the In Vitro Effect of Benzimidazoles

ABSTRACT Axenically grown Giardia lamblia trophozoites treated with low concentrations of the benzimidazole carbamates albendazole and mebendazole detach from glass culture tubes and lose viability. Scanning electron microscopic observations revealed that these drugs produce grotesque modifications of the cell shape of the parasite and disassembly of the adhesive disc. Transmission electron microscopy showed several stages of the fragmentation of adhesive discs with dispersion of microtubules and microribbons in the cytoplasm. Flagella appeared undamaged. In drug-treated trophozoites electron-dense precipitates were selectively deposited on microtubules and microribbons. The results indicate that the antigiardial effect of benzimidazoles is the result of binding to microtubules and subsequent alterations of the cytoskeleton. The electron microscopic observations also suggest that the drugs may bind to microribbon components of the adhesive disc, possibly giardin proteins.     

The Dynamics of Microtubules in Cultured Cells

The behavior of microtubules in cultured cells in a cooled matrix after the microinjection of fluorescent tubulin was studied using a frame recording with a digital camcorder. In the cell lamella, the positive ends of individual microtubules extend and shorten at random. The histograms of rate distribution have an almost normal distribution with a mode close to 0. The maximum rate of lengthening and shortening reaches 30 and 50 m/min, respectively. The positive ends of microtubules in PtK cells were in an equilibrium state, while in murine embryonic fibroblasts and Vero cells, they were usually displaced to the cell edge. Free microtubules were present in the cells of all three cultures. In the epithelial cells, they were numerous and relatively stable, while in the fibroblasts, they occurred rarely and were depolymerized at the proximal end. Free microtubules in PtK cells appeared mostly due to spontaneous assembly in the cytoplasm (not in the relationship with the preexisting microtubules) and, more rarely, due to breakage of long microtubules. Separation of microtubules from the centrosome is a very rare event. Unlike positive ends that were characterized by dynamic instability, negative ends were stable and were sometimes depolymerized. When long microtubules were broken, new negative ends were formed that were, as a rule, stable, while in the lamella of fibroblasts (in murine embryonic fibroblasts and Vero cells), new negative ends were immediately depolymerized: free microtubules existed in these cells no more than 1–2 min. A diffusion model has been proposed where the behavior of microtubule ends is considered as unidimensional diffusion. The coefficient of diffusion of positive ends in the epithelial cells is several times less than in the fibroblasts, thus suggesting a higher rate of tubulin metabolism in the fibroblasts as compared to the epithelium. The results obtained indicate that for the exchange of long microtubules, the dynamic instability is not sufficient. In the fibroblasts, their exchange takes place mostly at the expense of depolymerization of the liberating negative ends, which agrees with the previously proposed conveyer hypothesis of microtubule assembly on the centrosome.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

Antigenic localities in the tissues of Metagonimus yokogawai observed by immunogoldlabeling method]

In order to determine the antigenic localization in the tissues of the adult Metagonimus yokogawai, immunogoldlabeling method was applied using serum immunoglobulins(IgG) of cats which were infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex(particle size: 12 nm). It was observed by electron microscopy at each tissue of the worm. The gold particles were observed on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were also labeled on the lumen of bladder and egg shell. The above findings showed that antigenic materials in the tissue of adult worms were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Origin of kinetochore microtubules in Chinese hamster ovary cells

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 m thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 g/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. — In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubles were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.     

Fertilization-induced changes in the microtubular architecture of the maize egg cell and zygote—an immunocytochemical approach adapted to single cells

By using single cell micromanipulation techniques, we developed an immunocytochemical procedure to examine subcellular protein localization in isolated and cultured cells. Localization of microtubules was examined in isolated single egg cells and developing zygotes of maize with anti--tubulin antibodies. In egg cells, a few cortical microtubules were detected but well organized microtubules were rarely observed. In contrast, distinct cortical microtubules and strands of cytoplasmic microtubules radiating from the nucleus to the cell periphery were observed in developing zygotes. Solely cortical microtubules were observed in zygotes up to 7 h after in vitro fertilization. After this time, radiating microtubules additionally appeared, and persisted during zygote development. These results indicate early and pronounced fertilization-induced changes in microtubular organization in the fertilized egg cell of maize.     

Distribution of microtubules and microfilaments in exocrine (Ventral prostatic epithelial cells and pancreatic exocrine cells) and endocrine cells (Cells of the adenohypophysis and islets of Langerhans)

Summary We investigated the distribution of microtubules and microfilaments in some exocrine and endocrine cells in rats. Microtubules were stained by applying an immunofluorescent technique using antibodies against -tubulin, while microfilaments were stained with rhodamine-phalloidin, which binds selectively to polymerized actin filaments. In the cytoplasm of some exocrine cells (pancreatic acinar cells and ventral prostatic epithelial cells), the microtubules were distributed longitudinally from the apical region to the basal region, but no microtubules were found in the nuclear region. In exocrine cells, most of the microfilaments were localized beneath the apical plasma membrane. In some endocrine cells (those of the adenohypophysis and the islets of Langerhans), the microtubules exhibited a radial or reticular distribution in the cytoplasm, and intense fluorescence was observed in the perinuclear region. The immunofluorescence produced by the antibodies against -tubulin was more intense in endocrine cells than in exocrine cells. The microfilaments observed in the endocrine cells studied were homogeneously distributed beneath the plasma membrane. Dot-like rhodamine-phalloidin staining was often observed in the cytoplasm of both the exocrine and endocrine cells. The present study clearly demonstrated marked differences in the distribution of cytoskeletal elements in exocrine and endocrine cells, and these may reflect differences in the secretory direction of such cells as well as in epithelial-cell polarity.     

Relationship between the Golgi complex and microtubules enriched in detyrosinated or acetylated α-tubulin: studies on cells recovering from nocodazole and cells in the terminal phase of cytokinesis

Double immunofluorescence microscopy was used to study the relationship between the Golgi complex and microtubules enriched in posttranslationally modified tubulins in cultured mouse L929 fibroblasts. In interphase cells, the elements of the Golgi complex were grouped around the microtubule-organizing center. From here, tyrosinated microtubules extended to the periphery of the cells, whereas the distribution of detyrosinated and acetylated microtubules largely overlapped with that of the Golgi complex. Treatment of cells with 10 M nocodazole led to the disruption of all microtubules and dispersion of the Golgi elements. Following withdrawal of the drug, tyrosinated microtubules reformed first, followed by acetylated and then detyrosinated microtubules. In parallel, the Golgi elements moved back toward the juxtanuclear region and reestablished a close spatial relationship first with the acetylated and later also with the detyrosinated microtubules. Long-term recovery in the presence of 0.15 or 0.3 M nocodazole allowed partial reformation of tyrosinated and acetylated microtubules, whereas no or only a few detyrosinated microtubules were detected. At the same time, the Golgi elements were grouped closer together around or on one side of the nucleus in close relation to acetylated microtubules. In synchronized cells released from a mitotic block, a radiating array of tyrosinated microtubules was first formed, followed by acetylated and detyrosinated microtubules. The Golgi elements initially came together in a few groups and thereafter took an overall morphology similar to that in interphase cells. During this reunification, they showed a close spatial relationship to acetylated microtubules, whereas detyrosinated microtubules appeared only later. Microtubules enriched in acetylated and/or detyrosinated tubulin thus appear to take part in establishing and maintaining the organization of the Golgi elements within an interconnected supraorganellar system. Whether the acetylation and detyrosination of tubulin are directly involved in this process or merely represent two modifications within this subpopulation of microtubules remains unknown.On leave of absence from the Department of Histology and Embryology, Institute of Biostructure, Medical School, Warsaw, Poland     

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.     

What moves chromosomes, microtubules or microfilaments?

An investigation of the spindle apparatus of crane-fly (Nephrotoma suturalis) spermatocytes has been undertaken using methods that permit combined light and electron microscopy of selected cells. At the ultrastructural level, spindles contain microtubules in a granular matrix. Microtubules have been classified as kinetochore microtubules (which connect to kinetochores of chromosomes) and non-kinetochore microtubules (not attached to kinetochores). Kinetochore microtubules are distributed in densely packed bundles, which are the birefringent chromosomal fibers seen in living cells. Actin filaments were not observed in spindles of unglycerinated cells or in cells fixed in glutaraldehyde containing tannic acid, which negatively stains F-actin in situ and thus can be used to aid the localization of actin filaments in non-muscle cells. The absence of actin filaments in the spindle coupled with their presence in the "contractile ring" of spermatocytes fixed during cytokinesis is evidence against the hypothesis that chromosome movements are microfilament-based. The results are compatible with the hypothesis that microtubules are involved in the mechanism of chromosome transport. The details of that mechanism remain to be clarified.     

Behavior of mitochondria, microtubules, and actin in the triangular yeastTrigonopsis variabilis

Summary Dimorphic yeastTrigonopsis variabilis is a unique species that can form either an ellipsoidal or a triangular cell depending upon nutritional conditions. This fluorescence microscopic study was intended to correlate morphological changes of mitochondria in the triangular cells with the distribution of the cytoskeleton. In addition, unique features in the behavior of the cytoskeleton were also examined during triangular cell formation. In log-phase cells stained with 4,6-diamidino-2-phenylindole, mitochondrial nucleoids appeared as a string of beads throughout the vegetative growth. The profile of mitochondria stained by 3,3-dihexyloxacarbocyanine iodide showed a network corresponding to the fluorescence images of mitochondrial nucleoids in both mother and daughter cells. Cell-cycle-dependent fragmentation of mitochondria was not discerned. As the culture reached stationary phase, a network of mitochondria gradually changed to form unique rings that were located near the angles of triangular cells. When examined by immunofluorescence microscopy with anti-tubulin antibody, microtubules were found to be well developed along the sides of cells in the cytoplasm ofT. variabilis interphase cells. Although distributions of microtubules and mitochondria are different during cell cycle as a whole, cytoplasmic microtubules frequently extended along a part of the mitochondria in budded cells, suggesting correlation of microtubules and mitochondria. Rhodamine-phalloidin staining revealed both actin patches and cables. Actin cables elongated from mother cells into the buds and showed close proximity to mitochondria, although complete overlapping of both structures was rare. Moreover, actin patches localized on the mitochondrial network at a frequency of 65%. These results suggested that actin cables and patches, as well as microtubules, participated in the distribution of mitochondria. The localization of actin patches separated towards opposite ends at a bud tip when the bud grew to medium size. The unique localization of actin patches is responsible for bi-directional growth of the bud, forming triangular cells.     

Microtubules in statocytes from roots of cress (Lepidium sativum L.)

Summary Statocytes in root caps ofLepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem cells are observable rarely, structures which can be regarded as nucleating sites for microtubules are lacking. During ageing of the root cap cells, the number of microtubules increases in combination with a decrease of microtubule length. Development of the roots on a horizontal clinostat preserves a younger developmental stage of the microtubule system regarding amount and length of the individual microtubules. Evidence for an involvement of microtubules in graviperception is low, whereas their role in orienting cellulose microfibrils cannot be ruled out. Compression of the distal network of microtubules after centrifugation of the roots indicates that microtubules in statocytes ofLepidium sativum L. roots might function in stabilizing the distal complex of endoplasmic reticulum.     

How we discovered fluorescent speckle microscopy

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ~10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual "speckled" appearance-there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.