Fine structure and dynamics of EB3 binding zones on microtubules in fibroblast cells.

End-binding (EB) proteins associate with the growing tips of microtubules (MTs)and modulate their dynamics directly and indirectly, by recruiting essential factors to fine-tune MTs for their many essential roles in cells. Previously EB proteins have been shown to recognize a stabilizing GTP/GDP-Pi cap at the tip of growing MTs, but information about additional EB-binding zones on MTs has been limited. In this work, we studied fluorescence intensity profiles of one of the three mammalian EB-proteins, EB3, fused with red fluorescent protein (RFP). The distribution of EB3 on MTs in mouse fibroblasts frequently deviated from single exponential decay and exhibited secondary peaks. Those secondary peaks, which we refer to as EB3-islands, were detected on 56% comets of growing MTs and were encountered once per 44 s of EB3-RFP comet growth time with about 5 s half-lifetime. The majority of EB3-islands in the vicinity of MT tips was stationary and originated from EB3 comets moving with the growing MT tips. Computational modeling of the decoration of dynamic MT tips by EB3 suggested that the EB3-islands could not be explained simply by a stochastic first-order GTP hydrolysis/phosphate release. We speculate that additional protein factors contribute to EB3 residence time on MTs in cells, likely affecting MT dynamics.

[EXPRESSION OF THE LIGHT CHAINS OF IMMUNOGLOBULINS IN NORMAL B-CELLS AND SOME B-CELL LYMPHOMAS].

The quantitative method of determining the level of expression of immunoglobulin light chains on uncompensated data was suggested and used to examine disorders in light chain expression in various B-cell tumors. The average level of expression of the lambda isotype was 4 times higher than the level of expression of kappa isotype. The level of surface and cytoplasmic expression of LC IG varied within wide limits for different people, but there was a high degree of correlation between the levels of expression of kappa and lambda isotypes LC IG as well as between expression of the surface and cytoplasmic forms of each in isotype the same individual. In the majority of B-cell non-Hodgkin's lymphomas correlation between the expression of LC IG on the surface and in the cytoplasm of the cells was diminished. Expression of LC IG in CLL was significantly reduced on the surface of the cells and to a lesser extent--in the cytoplasm. In the case of marginal zone cell lymphoma, LC IG expression level was reduced on the surface of circulating cells and to a lesser extent--in the cytoplasm. In the case of mantle cell lymphoma and DLBCL, expression level of LC IG on the cell surface and in the cytoplasm was the same as in normal B-cells. However, in some cases DLBCL, no LC IG was expressed both on the surface and in the cytoplasm.

Noncanonical structural-functional organization of nucleoli in maturing oocytes of sea urchin Paracentrotus lividus.

The dynamics of structural and functional organization of the nucleolus in the oocytes of P. lividus is described. At the late stages of oogenesis the nucleolus is composed of two main components, namely the peripheral zone (PZ) and the central zone (CZ) which are spatially separated. This two-component structure of the nucleolus is formed, at early stages of oogenesis, by stepwise segregation of the fibro-granular component and by its migration to the nucleolar periphery. Absence of morphologically distinct fibrillar centers and dense fibrillar component in nucleoli of both somatic cells and oocytes makes it possible to classify the nucleoli of P. lividus as 'noncanonical' type. Based on detailed morphological and cytochemical analysis the following molecular interpretation of nucleolar ultrastructure in oocytes of P. lividus is proposed: 1) the PZ, containing RNP-positive granules 15 nm in size, but lacking Ag-NOR proteins and BrU incorporation, can be considered a structural equivalent of the granular component of 'typical' nucleoli; 2) the CZ, which is the site of incorporation of RNA precursors, contains intranuclear DNA, RNP-fibers and accumulates Ag-NOR proteins, corresponds to both FC and DFC of 'typical' nucleoli; 3) nucleolar growth during oogenesis, leading to the 1000-fold increase of nucleolar volume, seems to be correlated with the stockpiling of nonfunctioning mature preribosomal particles which will be utilized during embryogenesis.

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules.

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.

Interphase microtubules in cultured cells: long or short?

Presently, the question about the length of microtubules in the interphase cell became actual, since the parameters of dynamic instability of the plus end measured in vivo do not allow one to explain the rapid turnover of the long microtubule system. The problem may be solved if one of the following suppositions is assumed: either microtubules undergo rapid depolymerization from the minus end or they are on the average much shorter than it is usually considered. To check the last hypothesis, we have reconstructed microtubules using stereophotography of electron microscopic sections. Microtubules around the cell center in cultures of epithelial cells (kidney of pig embryo (PK) and bovine trachea (FBT)) and fibroblasts (MEF, primary mouse embryo fibroblasts, and L cells), as well as at the periphery of PK cells were studied. All in all, no less than 200 microtubules were found near the centrosome in each cell culture. From 2.5 to 8% microtubules were beyond the studied volume (4.0 x 5.5 x 1.5 microm). Most of microtubules in all studied cell lines were up to 1 microm and about 1/3 of them were 0.2-0.4 microm long. The mean length of microtubules surrounding the centrosome in different cell lines differed insignificantly and equalled 0.4-0.8 microm. In this case, the microtubules attached to the centrosome were on the average slightly shorter than the free ones. Thus, almost all microtubules around the centrosome are short, and the majority of those attached to it do not reach the cell periphery. A similar reconstruction of a part of the PK cell cytoplasm (10 x 35 microm) has shown that at the periphery, the mean length of microtubules is about 1.6 microm and most of them are 0.5 to 1.5 microm long. Thus, our data confirm the recent hypothesis of Vorobjev et al. (I. A. Vorobjev, T. M. Svitkina, and G. G. Borisy, J. Cell Sci. 110:2635-2645 (1997)) that most of microtubules in the cells are not connected with the centrosomes.

Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos.

A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed. Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha. However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives. This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery.

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.

Postmitotic reconstruction of nucleoli in culture cells with UV-microbeam photoinactivated centrosome.

Ultraviolet microirradiation of one of the poles of the mitotic spindle of PK cells was performed 1 min after the onset of the anaphase. Formation of the nucleolus in the telophase and G1 period was studied by vital observation, electron microscopy and indirect immunofluorescence using antibodies against B23 protein. Sister cells with nonirradiated centrosomes and cells with partially irradiated cytoplasm were used as controls. During the first hour after the anaphase, the nuclei in both sister cells were identical and contained numerous small dense particles with granular ultrastructure. B23 protein detected in the mitotic poles and at the chromosome surface in the anaphase was dispersed in the cytoplasm in both cells in the early G1 period. Later, control cells did not display any difference from intact cells: nucleoli of a typical structure were formed, B23 protein appeared in the karyoplasm and was then accumulated in the nucleoli and disappeared from the cytoplasm and karyoplasm. Nucleoli in cells with irradiated centrosomes did not achieve the normal size and contained a significantly lower amount of granular component. B23 protein was dispersed in the karyoplasm and was not accumulated in the nucleoli. Nucleoli in cells with irradiated centrosomes contained small dense particles for at least 24 h. Telophase cells where microtubule formation had been inhibited by nocodazole formed normal nucleoli. It shows that the effects observed in cells with irradiated centrosomes are not due to the absence of the microtubule radial system. We conclude that UV microirradiation of the mitotic centrosome disturbs the postmitotic reconstruction of nucleoli probably because of the photodestruction of B23 protein accumulated in the mitotic pole.

Contribution of plus and minus end pathways to microtubule turnover.

Turnover is important for the maintenance and remodeling of the cytoskeleton during the processes of cell morphogenesis, mitosis and motility. Microtubule (MT) turnover is thought to occur by dynamic instability, growth and shortening at distal (plus) ends. Recent observation of MT release from the centrosome and depolymerization from proximal (minus) ends indicates the existence of a minus end pathway. To evaluate the relative contributions of plus and minus end pathways to turnover, we analyzed MT dynamics in a model system, the fish melanophore, a large non-motile cell with a regular radial array of long MTs. MT ends were tracked in digital fluorescence time-lapse sequences and life histories of individual MTs were analyzed using random walk theory generalized to the case of diffusion with drift. Analysis of plus end dynamics gave an apparent diffusion coefficient of D=7.5 microm2/minute. The random walk model predicts that the half-time for turnover driven solely by plus end dynamics will depend strongly on position in the cell. Based on the experimentally determined value of D, turnover of MTs near the center of a typical melanophore of radius 70 microm was calculated to require over 5 hours, a paradoxically long time. To examine MT behavior deep in the cytoplasm, we developed a novel, sequential subtraction mode of image analysis. This analysis revealed a subpopulation of MTs which shortened from their minus ends, presumably after constitutive release from the centrosome. Given the relative slowness of plus end dynamics to turn over the root of a long MT, the turnover of MTs near the cell center is determined primarily by the minus-end pathway. MTs released from the centrosome become replaced by newly nucleated ones. The relative contributions of plus and minus end pathways was estimated from the diffusion coefficient, D, for the plus end, the length distribution of MTs, t he frequency of free minus ends, and the rate of minus-end shortening. We conclude that, in large animal cells with a centrosomally focussed array of MTs, turnover occurs by a combination of plus and minus end pathways, the plus end dominating at the cell periphery and the minus end dominating near the cell center.

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.

Blue light inhibits mitosis in tissue culture cells.

Irradiation of the mitotic (prophase and prometaphase) tissue culture PK (pig kidney embryo) cells using mercury arc lamp and band-pass filters postponed or inhibited anaphase onset. The biological responses observed after irradiation were: (i) normal cell division, (ii) delay in metaphase and then normal anaphase and incomplete cytokinesis, (iii) exit into interphase without separation of chromosomes, (i.v.) complete mitotic blockage. Cell sensitivity to the light at wavelengths from 423 and 488 nm was nearly the same; to the near UV light (wavelength 360 nm) it was 5-10 times more; to the green light (wavelength >500 nm) it was at least 10 times less. To elucidate the possible mechanism of the action of blue light we measured cell adsorption and examined cell autofluorescence. Autofluorescence of cytoplasmic granules was exited at wavelengths of 450-490 nm, but not at >500 nm. In mitotic cells fluorescent granules accumulated around the spindle. We suppose blue light irradiation induces formation of the free radicals and/or peroxide, and thus perturb the checkpoint system responsible for anaphase onset.

Mutational analysis of human prothymosin alpha reveals a bipartite nuclear localization signal.

Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin alpha-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin alpha fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin alpha through an unknown mechanism, without influencing the nuclear import of the protein.

Quantitative analysis of the movements of cytoplasmic granules in polarized fibroblasts.

Movements of cytoplasmic organelles were analyzed in Vero fibroblasts. In the cells polarized at the edge of an experimental wound, cytoplasmic granules moved randomly (Brownian motions) and by separate jumps (saltatory movements). The displacement of granules by the Brownian motions exceeded by more than an order of magnitude that of the mitochondria similar by weight. Lipid droplets moved predominantly by saltations, whereas mitochondria and lysosomes moved much less often. In a front part of the polarized cells, the main directions of saltatory movements were from the nucleus to the leading edge of a cell and back, whereas the tangential movements (across the long axis of a cell) were less than 1%. 90% of saltatory movements occurred in the area starting 10-12 microm from the nucleus and ending 10-12 microm from the leading edge of a cell. The average rate of saltatory movements of the granules (2.38 microm/s) was identical in both directions. The average length of the track was 7.49 microm; the maximum track length reached 30 microm. An increase in the granule diameter from 0.3 to 1.4 microm resulted in a minor (statistically insignificant) decrease in the average rate of the movements. The average rate of saltatory movements of mitochondria was 1.00 microm/s, and the average track length was 6.04 microm. Therefore, mitochondria, in contrast to lipid droplets, are rigidly fixed in the cytoplasm, and the force holding mitochondria is equal to the force produced by the microtubule-associated motors. Taking into account the characteristic of the centrifugal saltations, we suggest that they are mediated by an unusual dynein.

Stereoscopic analysis of microtubule pattern around the centrosome in interphase PK cells after treatment with taxol and nocodazole.

In the interphase PK cells, more than 85% of microtubules radiating from the centrosome were not longer than 1.5 microns. A half of microtubules had their proximal ends free. After nocodazole treatment (20 microM), the number of microtubules attached to the centrosome decreased by 20% after 10 min of treatment, remained the same after 20 min of treatment, and increased after 60 min of nocodazole treatment slightly above the control level. After 5 and 60 min of treatment, the number of attached microtubules with the length over 0.7 micron increased twice as compared to the control level. During the first 20 min of nocodazole treatment, the immunofluorescent staining of cells with antibodies to gamma-tubulin was the same as in the control cells. The number of free microtubules decreased fourfold during the first 5 min, then it decreased slowly (for 20 min) and remained at the same level after 60 min. After 10 min of taxol (12 microM) treatment, the number of attached and free microtubules increased more than two times, whereas the number of attached microtubules with the length over 0.7 micron increased more than tenfold. After 15 min of treatment, the number of attached microtubules was slightly higher, and the number of free microtubules was half of the control level. After 20-60 min of treatment, the number of microtubules of all types decreased. Thus, upon the nocodazole treatment, the microtubules attached to the centrosome were more resistant to depolymerization: however, these microtubules were the most reactive to taxol treatment. The data obtained suggest that (a) in PK cells, the centrosome-attached microtubules occupy not all of the existing templates; (b) during prolonged treatment with inhibitors, the centrosome performed the compensatory reaction-the inhibition of microtubule assembly results in the decrease in the number of active templates on the centrosome; the inhibition of microtubule depolymerization results in the inactivation of hitherto active reserve templates. The microtubules formed on the centrosome within the first minutes disengage from it and then leave the chromosomal region.

Cytoplasmic assembly of microtubules in cultured cells.

The origin of non-centrosomal microtubules was investigated in a variety of animal cells in culture by means of time-lapse digital fluorescence microscopy. A previous study (Keating et al. (1997) Proc. Nat. Acad. Sci. USA 94, 5078-5083) demonstrated a pathway for formation of non-centrosomal microtubules by release from the centrosome. Here we show a parallel pathway not dependent upon the centrosome. Correlative immunostaining with anti-tubulin antibodies and electron microscopy established that apparent free microtubules observed in vivo were not growing ends of long stable microtubules. Free microtubules appeared spontaneously in the cytoplasm and occasionally by breakage of long microtubules. Estimates of the frequencies of free microtubule formation suggest that it can be a relatively common rather than exceptional event in PtK1 cells and may represent a significant source of non-centrosomal microtubules. The observation of free microtubules permitted analysis of the microtubule minus end. Unlike the plus end which showed dynamic instability, the minus end was stable or depolymerized. Breakage of long microtubules generated nascent plus and minus ends; the nascent minus end was generally stable while the plus end was always dynamic. The stability of microtubule minus ends in vivo apparently provides the necessary condition for free microtubule formation in the cytoplasm. Parameters of the dynamic instability of plus ends of free microtubules were similar to those for the distal ends of long microtubules, indicating that the free microtubules were not exceptional in their dynamic behavior. Random walk analysis of microtubule end dynamics gave apparent diffusion coefficients for free and long microtubules which permitted an estimate of turnover half-times. The results support the concept that, in PtK1 cells, a pathway other than plus end dynamics is needed to account for the rapidity of microtubule turnover.

Role of the centrosome in mitosis: UV micro-irradiation study.

Ultraviolet micro-irradiation (UV-MI) of the PK (pig kidney embryo) cell centrosome (lambda max = 280 nm, spot diameter 1.6 mm, exposure time 5-15 s) at metaphase and anaphase resulted in functional damage of the centrosome. After UV-MI of the centrosome at early metaphase, chromosomes quickly (in 1-3 min) moved away from the irradiated pole and then encircled the non-irradiated pole. Within 10 min after UV-MI the spindle disassembled and chromosomes remained unseparated. The minimal dose inducing this effect in 90% of cells was accumulated in 5 s. After the same UV-MI at late metaphase, chromosomes shifted towards the non-irradiated pole; however, anaphase started and chromosome motion towards the non-irradiated pole continued normally. UV-MI of the centrosome at early anaphase for 5-15 s slowed down and then stopped chromosome motion towards the irradiated pole. This was a result of rapid (within 2-3 min) disorganization of the half-spindle. Chromosomes continued to move towards the opposite pole normally, while cytokinesis was significantly retarded. No visible lesion was revealed by electron microscopy after 5 s UV-MI, while 15 s irradiation resulted in the truncation of the microtubule bundles 1.5-2 microns from the centrosome. We concluded that UV-MI inactivates the centrosome and induces disaggregation of microtubule initiation sites. The critical point (checkpoint) in mitosis up to which this damage induces mitotic arrest is mid-metaphase.

Centrosome and microtubules behavior in the cytoplasts.

After enucleation of PK (pig kidney embryo) cells using cytochalasin D centrioles remained approximately in 80% of cytoplasts. Some cytoplasts retained a single centriole. 14-16 h after enucleation large secondary lysosomes and lipid droplets were evident around the centrosome of many cytoplasts. In part of the cytoplasts replicating centrioles were found 16 h after enucleation. Ouabain treatment (1 mM, 30 min) of the cytoplasts resulted in the appearance of mainly perpendicular orientation of mother centrioles relative to the substrate surface. Reconstruction of microtubule pattern around the centrosome showed that a total of approximately 15 microtubules were attached in the centrosome of normal cells and twice more than that in cytoplasts. Microtubules in the cytoplasts were more resistant to nocodazole induced depolymerization. We suggest that microtubule instability is modulated by regulatory effector that is under direct nucleus control.

Centrosome behaviour and orientation of centrioles under the action of energy transfer inhibitors.

2,4-dinitrophenol, dinitrophenol together with deoxyglucose, sodium azide and ouabain didn't alter cytoplasmic microtubule (MT) network of cultured PK (pig kidney embryo) cells, meanwhile they induced an increase in the average number of pericentriolar satellites and percentage of centrioles with the primary cilium in these cells. Also all drugs studied increase number of MTs attached to and oriented towards the centrosome. Under the action of ouabain the total number of MTs around the centrosome doubled, meanwhile the number of long MTs emanating from the centrosome increased more than 15 times. Under the action of all drugs studied, except sodium azide, the number of maternal centrioles oriented perpendicularly to the substrate surface increased significantly from that in control cells.

Centrosome behavior under the action of a mitochondrial uncoupler and the effect of disruption of cytoskeleton elements on the uncoupler-induced alterations.

Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) induced in pig kidney embryo cells a loss of rhodamine 123 staining of mitochondria in 2-3 min. Within 5 min after FCCP inoculation of cells prestained with rhodamine 123, the diffuse staining of the cytoplasm was absent. FCCP did not induce changes in the cytoplasmic microtubule complex, but induced nonrandom (preferentially perpendicular to the substrate surface) orientation of maternal centrioles. Nonrandom orientation of maternal centrioles occurred 10 min after treatment and remained for 2 hr. At 30 min after introduction of the drug, FCCP treatment increased the mean number of pericentriolar satellites on maternal centrioles and the frequency of primary cilia. The percentage of centrioles perpendicular to the substrate induced by FCCP treatment was slightly increased by disruption of microtubules and slightly diminished by disruption of microfilaments. In both cases centrioles were oriented significantly differently from random (P < 0.01). These results suggest that microtubules are neither involved in the signaling pathway from plasma membrane to the centriole, nor do they anchor the centrioles perpendicular to the substrate, as proposed by Albrecht-Buehler and Bushnell (Experimental Cell Research 120, 1979).

Optical trapping for chromosome manipulation: a wavelength dependence of induced chromosome bridges.

Using a tunable titanium-sapphire laser, we have compared different wavelengths (from 700 to 840 nm) for their utility in optical trapping of chromosomes in mitotic rat kangaroo Potorous tridactylus (PtK2) cells. It was found that irradiation with a near-infrared light induces the sticking together of chromosome shoulders. The attached chromatids failed to separate, or separated with significant delay and formed a chromosome bridge during anaphase. Using this bridge (and induced c-mitosis) as a reference, we compared the action of different wavelengths (from 700 to 840 nm). Chromosomes were irradiated at metaphase and the cells were observed until the end of cytokinesis. Chromosomes were irradiated for different periods of time, using 130 mW of power at the objective focal plane. The biological responses observed after optical trapping were: (1) normal cell division, (2) formation of a temporary chromosome bridge, (3) formation of a permanent chromosome bridge, (4) complete blockage of chromosome separation (c-mitosis). The chromosomes were found to have a maximal sensitivity to 760-765 nm light and minimal sensitivity to 700 and 800-820 nm light. Cells with chromosomes irradiated for a long time, using wavelength 760-765 nm, generally were incapable of going through anaphase and remained in c-mitosis. We conclude that the optimal wavelengths for optical trapping are 700 and 800-820 nm.