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P Wadsworth

Publications and source records attributed to P Wadsworth.

At least 19 recordsLinked to original sources
PubMed2003

Peripheral, non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.

In centrosome-containing cells, microtubules utilized in spindle formation are thought to be nucleated at the centrosome. However, spindle formation can proceed following experimental destruction of centrosomes or in cells lacking centrosomes, suggesting that non-centrosome-associated microtubules may contribute to spindle formation, at least when centrosomes are absent. Direct observation of prometaphase cells expressing GFP-alpha-tubulin shows that peripheral, non-centrosome-associated microtubules are utilized in spindle formation, even in the presence of centrosomes. Clusters of peripheral microtubules moved into the centrosomal region, demonstrating that a centrosomal microtubule array can be composed of both centrosomally nucleated and peripheral microtubules. Peripheral bundles also moved laterally into the forming spindle between the spindle poles; 3D reconstructions of fixed cells reveal interactions between peripheral and centrosome-associated microtubules. The spindle pole component NuMA and gamma-tubulin were present at the foci of peripheral microtubule clusters, indicating that microtubules moved into the spindle with minus ends leading. Photobleach- and photoactivation-marking experiments of cells expressing GFP-tubulin or a photoactivatable variant of GFP-tubulin, respectively, demonstrate that microtubule motion into the forming spindle results from transport and sliding interactions, not treadmilling. Our results directly demonstrate that non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.

Animals↗
PubMed2001

Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells.

Photoactivation of caged fluorescent tubulin was used mark the microtubule (MT) lattice and monitor MT behavior in interphase cells. A broadening of the photoactivated region occurred as MTs moved bidirectionally. MT movement was not inhibited when MT assembly was suppressed with nocodazole or Taxol; MT movement was suppressed by inhibition of myosin light chain kinase with ML7 or by a peptide inhibitor. Conversely, MT movement was increased after inhibition of cytoplasmic dynein with the antibody 70.1. In addition, the half-time for MT turnover was decreased in cells treated with ML7. These results demonstrate that myosin II and cytoplasmic dynein contribute to a balance of forces that regulates MT organization, movement, and turnover in interphase cells.

Actomyosin↗
PubMed2001

Centrosome behavior in motile HGF-treated PtK2 cells expressing GFP-gamma tubulin.

In response to locomotory cues, many motile cells have been shown to reposition their centrosome to a location in front of the nucleus, towards the direction of cell migration. We examined centrosome position in PtK(2) epithelial cells treated with hepatocyte growth factor (HGF), which stimulates motility but, unlike chemotactic agents or wounding of a monolayer, provides no directional cues. To observe centrosome movement directly, a plasmid encoding human gamma tubulin fused to the green fluorescent protein was expressed in HGF-treated cells. In cells whose movements were unconstrained by neighboring cells, we found that the position of the centrosome was not correlated with the direction of cell locomotion. Further, in cells where the direction of locomotion changed during the observation period, the centrosome did not reorient toward the new direction of locomotion. Analysis of centrosome and nuclear movement showed that motion of the centrosome often lagged behind that of the nucleus. Analysis of 249 fixed cells stained with an antibody to gamma tubulin confirmed our observations in live cells: 69% of the cells had centrosomes behind the nucleus, away from the direction of locomotion. Of these, 41% had their centrosome in the retraction tail. Confocal microscopy showed that the microtubule array in HGF treated PtK(2) cells was predominantly non-centrosomal. Because microtubules are required for efficient cellular locomotion, we propose that non-centrosomal microtubules stabilize the direction of locomotion without a requirement for reorientation of the centrosome.

Animals↗
PubMed2001

Issues of normal tissue toxicity in patient and animal studies--effect of carbogen breathing in rats after 5-fluorouracil treatment.

Non-invasive magnetic resonance spectroscopy (MRS) can be used in the clinic to monitor the pharmacokinetics of the chemotherapeutic drug 5-fluorouracil (5-FU) and the effects of modifiers. We report two studies of 5-FU toxicity in normal tissue--one with patients and the other an animal study. 1) 19F MRS signals from fluoronucleotides, cytotoxic anabolites of 5-FU metabolism, were observed in the livers of two patients treated with 5-FU for colorectal cancer, shown by computed tomography (CT) and ultrasound (US) to have no liver metastases. This is the first report of non-invasive monitoring of toxic 5-FU metabolites in normal human tissues. 2) In animals, carbogen-breathing enhances tumour uptake and the efficacy of 5-FU, and the method is under trial in patients. This study demonstrates that there were no significant effects of carbogen breathing on the levels of 5-FU and its metabolites in normal rat tissues, or on the histology of the tissues assessed after treatment.

Adenocarcinoma↗
PubMed2001

Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin.

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1alpha). The mitotic index and doubling time for LLCPK-1alpha were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1alpha cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1alpha and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1alpha cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

Animals↗
PubMed2000

Region-specific microtubule transport in motile cells.

Photoactivation and photobleaching of fluorescence were used to determine the mechanism by which microtubules (MTs) are remodeled in PtK2 cells during fibroblast-like motility in response to hepatocyte growth factor (HGF). The data show that MTs are transported during cell motility in an actomyosin-dependent manner, and that the direction of transport depends on the dominant force in the region examined. MTs in the leading lamella move rearward relative to the substrate, as has been reported in newt cells (Waterman-Storer, C.M., and E.D. Salmon. 1997. J. Cell Biol. 139:417-434), whereas MTs in the cell body and in the retraction tail move forward, in the direction of cell locomotion. In the transition zone between the peripheral lamella and the cell body, a subset of MTs remains stationary with respect to the substrate, whereas neighboring MTs are transported either forward, with the cell body, or rearward, with actomyosin retrograde flow. In addition to transport, the photoactivated region frequently broadens, indicating that individual marked MTs are moved either at different rates or in different directions. Mark broadening is also observed in nonmotile cells, indicating that this aspect of transport is independent of cell locomotion. Quantitative measurements of the dissipation of photoactivated fluorescence show that, compared with MTs in control nonmotile cells, MT turnover is increased twofold in the lamella of HGF-treated cells but unchanged in the retraction tail, demonstrating that microtubule turnover is regionally regulated.

Animals↗
PubMed2000

Mesenchymal proliferation with decidual-like morphology in seminal vesicles of aging mice.

Lesions characterized by spindle and epithelioid cells and nuclear progesterone receptors are described in seminal vesicles of four aging mice. The lesions of two mice also contain granular metrial gland (GMG)-like cells. The same cellular details are seen in the uterine decidual reaction and the similar urinary bladder lesion in mice, also called mesenchymal tumor. Therefore, it is hypothesized that these lesions in male accessory sex glands and the urinary bladders of aging male and female mice are of mesenchymal origin with the potential for differentiation along several pathways, leading especially to lesions with decidual-like morphology, but also to lesions which contain only spindle cells. The decidual hypothesis is further supported by the occurrence of round eosinophilic granules and focal necrosis, interpreted as a sign of regression in all these lesion types. The bilateral lesions of a fifth mouse consist of spindle cells and scar-like tissue, the latter suggesting regression, and lack epithelioid and GMG-like cells. In this case, verification of the diagnosis depends on the demonstration of progesterone receptors, absent in normal glands. Uterine decidual reactions during pregnancy are brought about by priming with progesterone/estrogen, initiation through the blastocyst, and maintenance through progesterone. Experiments by others show that priming may also occur through growth factors/growth hormone, initiation through prostaglandins, and maintenance through testosterone in mice. It is hypothesized that upon such stimulation, certain cells in male accessory sex glands and the urinary bladder, possibly derived from the Muellerian ducts or other subperitoneal tissue, appear to have the potential in mice of developing into spindle and epithelioid cells, including decidual-like cells. All published uterine decidual reactions and lesions with decidual-like morphology in other organs of mice stayed within the peritoneal coverage of their respective organ and did not metastasize despite their "anaplastic", tumor-like appearance. Thus, they should be considered non-neoplastic. It is proposed to name above lesions in male accessory sex glands and urinary bladders "mesenchymal proliferation, decidual type" or "mesenchymal proliferation, spindle-cell type", depending on their cellular characteristics.

Aging↗
PubMed1999

Regional regulation of microtubule dynamics in polarized, motile cells.

Microtubules are known to be required for locomotion of mammalian cells, and recent experiments demonstrate that suppression of microtubule dynamic turnover reduces the rate of cell motility and induces wandering of growth cones [Liao et al., 1995: J Cell Sci. 108:3473-3483; Tanaka et al., 1995: J Cell Biol. 128:139-155]. To determine how microtubule dynamic instability behavior contributes to directed cell locomotion, the behavior of individual microtubules has been directly observed and quantified at leading and lateral edges of hepatocyte growth factor-treated motile cells. Microtubules extended into newly formed protrusions at the leading edge; these "pioneer" microtubules [Waterman-Storer and Salmon, 1997: J Cell Biol. 139:417-434] showed persistent growth when compared with microtubules in non-leading, lateral edges. The percentage of total observation time spent in the growth phase was 68.2% at the leading edge compared with 32.0% in non-leading edges, and net microtubule elongation was observed in lamellipodia at the leading edge. The frequency of catastrophe transitions was threefold greater and the average number of transitions/microtubule/min was twofold greater in non-leading edges, as compared with the leading edge. These observations demonstrate that pioneer microtubules that enter newly formed lamellipodia at the leading edge of motile cells are characterized by persistent growth excursions, and directly demonstrate that the frequency of catastrophe transitions can be regionally regulated in polarized motile cells. The data indicate that region specific differences in the organization and dynamics of actin filaments may regulate microtubule dynamic instability behavior in vivo.

Actins↗
PubMed1999

Taxol suppresses dynamics of individual microtubules in living human tumor cells.

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30-100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

Adenocarcinoma↗
PubMed1998

Similarities between the uterine decidual reaction and the "mesenchymal lesion" of the urinary bladder in aging mice.

The histopathologic characteristics of the decidual reaction in the uterus of aging mice and the "mesenchymal lesion/tumor" in the urinary bladder of aging mice are compared and found to be very similar. Both lesions consist of spindle and epithelioid cells, may contain round eosinophilic granules and possess nuclear progesterone receptors and cytoplasmic desmin. The decidual reaction derives from endometrial stromal cells, while the "mesenchymal lesion" apparently develops from mesenchymal cells near the trigone area, carrying or developing progesterone receptors. If the hypothesis is accepted that in aging mice the uterine decidual reaction and the "mesenchymal lesion" in the urinary bladder represent an equivalent type of tissue reaction, then it follows that the typical "mesenchymal lesion" is not a tumor and could be called more specifically "decidual-like reaction".

Aging↗
PubMed1998

Functional assays to identify and characterize regulators of microtubule behavior.

Previous experiments have clearly demonstrated that microtubule dynamic instability is regulated in living cells, but the molecular mechanisms that are responsible for this regulation are not well understood. We describe two rapid, functional assays that can be used to screen cell extracts for regulators of microtubule dynamic instability behavior. In both assays, highly purified tubulin is used to assemble microtubules from Tetrahymena axonemes. In the immunofluorescence assay, microtubules are visualized by fixation and staining with anti-tubulin antibodies. Alternatively, microtubule assembly has been visualized by the addition of rhodamine-labeled tubulin to axonemes, followed by low-light-level fluorescence microscopy. In either case, polymerization is quantified by measuring polymer length, total polymer and the number of microtubules per axoneme. In these assays, addition of brain microtubule-associated proteins (MAPs) results in a 2-fold-3-fold increase in average microtubule length, and addition of vinblastine results in a 50%-75% decrease in average microtubule length. The number of microtubules per axoneme was significantly increased by the addition of MAPs and significantly decreased by the addition of vinblastine. These functional assays can detect molecules that stimulate or suppress net microtubule assembly and provide a useful initial screen to isolate regulators of microtubule dynamic behavior.

Animals↗
PubMed1997

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.

Animals↗
PubMed1997

Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells.

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

Centrosome↗
PubMed1996

Stimulation of microtubule dynamic turnover in living cells treated with okadaic acid.

We have examined the effects of okadaic acid, an inhibitor of protein phosphatases type 1 and 2A, on the dynamic instability behavior of individual microtubules in living cells. Addition of 1 microM okadaic acid to PtK1 epithelial cells induced ruffling of lamellar regions; after 50 min in okadaic acid, many cells were observed to round up. Confocal microscopy of okadaic acid-treated cells stained with an antibody to tubulin showed that microtubules were more densely packed near the periphery of the rounded cells, and in many cells, a reduction in the density of microtubules near the microtubule-organizing center was observed. The dynamic behavior of individual microtubules in cells previously injected with rhodamine-labeled tubulin was quantified by tracking individual microtubules from image sequences. Microtubule dynamic turnover was markedly stimulated in cells treated with 1 microM okadaic acid for 50-60 min: The average rates of both microtubule growing and shortening increased, and the average duration of pause, or attenuation, a phase in which neither growth nor shortening could be detected, was significantly decreased. Further, okadaic acid induced an approximately twofold increase in the frequency of catastrophe transitions and a threefold decrease in the frequency of rescue transitions. Dynamicity, a measure of the net gain and loss of polymer at microtubule plus ends, increased nearly threefold in okadaic acid-treated cells. These results demonstrate that microtubule turnover is stimulated in okadaic acid-treated cells and suggest that phosphorylation of molecules which interact with microtubules may result in increased microtubule dynamic turnover in vivo.

Animals↗
PubMed1996

Microtubule dynamic turnover is suppressed during polarization and stimulated in hepatocyte growth factor scattered Madin-Darby canine kidney epithelial cells.

The dynamic behavior of microtubules has been measured in non-polarized, polarized, and hepatocyte growth factor treated Madin-Darby canine kidney epithelial cells. In a nocodazole disassembly assay, microtubules in polarized cells were more resistant to depolymerization than microtubules in non-polarized cells; microtubules in scattered cells were nearly completely disassembled. Analysis of fluorescent microtubules in living cells further revealed that individual microtubules in polarized cells were kinetically stabilized and microtubules in scattered cells were highly dynamic. Individual microtubule behavior in polarized cells was characterized by a suppression of the average rate of shortening, an increase in the average duration of pause, a decrease in the frequency of catastrophe transitions, and an increase in the frequency of rescue transitions, when compared with microtubules in non-polarized cells. In contrast, microtubule behavior in epithelial cells treated with hepatocyte growth factor was characterized by increase in the average rates of microtubule growth and shortening, a decrease in the frequency of rescue transitions, and an increase in the frequency of catastrophe transitions, when compared with polarized cells. Dynamicity, a measure of the gain and loss of subunits from microtubule plus ends, was 2.7 microns/min in polarized cells and 11.1 microns/min in scattered cells. These results demonstrate that individual microtubule dynamic behavior is markedly suppressed in polarized epithelial cells. Our results further demonstrate that in addition to its previously characterized effects on cell locomotion, hepatocyte growth factor stimulates microtubule dynamic turnover in lamellar regions of living cells.

Animals↗
PubMed1995

Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells.

Animals↗
PubMed1995

Modulation of microtubule dynamic instability in vivo by brain microtubule associated proteins.

Heat-stable brain microtubule associated proteins (MAPs) and purified microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine-labeled tubulin. The dynamic instability behavior of individual microtubules was then examined using low-light-level fluorescence microscopy and quantitative microtubule tracking methods. Both MAP preparations suppressed microtubule dynamics in vivo, by reducing the average rate and extent of both growing and shortening events. The average duration of growing events was not affected. When measured as events/unit time, heat-stable MAPs and MAP-2 did not significantly alter the frequency of rescue; the frequency of catastrophe was decreased approximately two-fold by heat-stable MAPs and MAP-2. When transition frequencies were calculated as events/unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. The percentage of total time spent in the phases of growth, shrink and pause was determined. Both MAP-2 and heat-stable MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Heat-stable MAPs increased the average pause duration, decreased the average number of events per minute per microtubule and increased the probability that a paused microtubule would switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual microtubules primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the microtubule length can be detected. The results further suggest that the expression of MAPs directly contributes to cell type-specific microtubule dynamic behavior.

Animals↗