Nocodazole, vinblastine and taxol at low concentrations affect fibroblast locomotion and saltatory movements of organelles

Microtubules (MTs) are essential for the maintenance of asymmetric cell shape and motility of fibroblasts. MTs are considered to function as rails for organelle transport to the leading edge. We investigated the relationship between the motility of Vero fibroblasts and saltatory movements of particles in their lamella Fibroblasts extended their leading edges into the experimental wound at a rate of 20+/-11 microm/h. Intracellular particles in the front parts of the polarized fibroblasts moved saltatorily mainly along the long axis of the cells. MT depolymerization induced by the nocodazole at a high concentration (1.7 microM) resulted in the inhibition of both fibroblast motility and saltatory movements of the particles. Taxol (1 microM) inhibited the fibroblast locomotion but not the saltatory movements. The saltatory movement pattern was disorganized by taxol by decreasing the portion of longitudinal saltations and consequently by increasing the part of saltations perpendicular to the cell long axis. This effect may be explained by disorganization of the MT network resulting from the inhibition of dynamic instability. To further investigate the relationships between the MT dynamics instability, saltatory movements, and fibroblast locomotion, we treated fibroblasts with microtubule drugs at low concentration (nocodazole, 170 nM; vinblastine, 50 nM; and taxol, 50 nM). All these drugs induced rapid disorganization of the saltatory movements and decreased the rate of cell locomotion. Simultaneously, the amount of acetylated (stable) MTs increased. The treatment also induced reversible changes in the actin meshwork. We suggest that decrease in the fibroblast locomotion rate in the case of MT stabilization occurred because of the appearance of numerous free MTs. Saltations along free MTs are poorly organized and, as a result, the number of organelles reaching the fibroblast leading edge decreases.     

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.     

Radial-organized microtubules provide cell shape support and more effective intercellular transport then free microtubules

Microtubules take part in various cell processes, including cell polarization, migration, intercellular transport, and some others. Therefore, the spatial organization of microtubules is crucial for normal cell behavior. Fibroblasts have radial microtubule arrays that consist of microtubules that run from the centrosome. Two components compose this microtubule array, i.e., (1) minus ends attached to the centrosome microtubules with their plus ends radiating to the cell periphery and (2) free microtubules with ends not attached to the centrosome. Distinctions in the dynamic properties, intercellular organization, and structure of centrosome-attached and free microtubules allow us to assume that their cellular functions are also different. To study centrosome-attached and free microtubules functions, we used cytoplasts, i.e., nucleus-lacking cellular fragments that, under certain conditions, also lose their centrosomes. In these cytoplasts, there are only free microtubules. The shape, general morphology, and size of cytoplasts that retain their centrosomes differ only slightly from whole cells. Cytoplasts who have lost their centrosomes have an extremely thin network of microtubules located in their central region; furthermore, they lose the shape that is typical for fibroblast and become rough lamellae with protrusions. The internal architecture of the cytoplasm and organoid arrangement are also broken. Saltatory movements in cytoplasts with centrosomes are similar to those in whole cells; in cytoplasts without centrosomes, saltatory movements occur with velocities that are twofold less and by shorter distances. Saltatory movements of granules in centrosome-lacking cytoplasts took place basically in the central region of cytoplast and were less ordered than in whole cells and in cytoplasts with centrosomes. We believe that radial organized microtubules ensure the effective transport and dynamical interaction of microtubule plus ends with cellular cortical structures, which is sufficient to support the common fibroblast-like shape, whereas the disorganized free microtubules are not able to maintain the external fibroblast shape and its intercellular organization.     

Analysis of Different Methodological Approaches to Measuring Microtubule Length in the Cytoplasm of Cultured Cells

It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 ± 3.69 m. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 ± 0.72 m long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 m, averaging 3.33 ± 2.43 m; the average length of centrosomal microtubules was 1.49 ± 0.82 m. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of the free microtubules in Vero cells actually have a length of 2–5 m; i.e., they are much shorter than the cell radius (about 25 m). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.