Pat Wadsworth - Academia.edu (original) (raw)

Papers by Pat Wadsworth

Individual deep cells migrating by blebbing and lamellipodial activity in a fundulus heteroclitus... more Individual deep cells migrating by blebbing and lamellipodial activity in a fundulus heteroclitus (killifish) embryo. Video made available thru MarK Cooper and Zebrafish The Living Laboratory.

Individual deep cell moving by circus movements. Fundulus heteroclitus(killifish) embryo injected... more Individual deep cell moving by circus movements. Fundulus heteroclitus(killifish) embryo injected with GFP-actin plasmid at 1-cell stage, filmed during gastrulation. Image made available through Zebrafish The Living Laboratory and Mark Cooper.

Individual deep cell moving by circus movements. Fundulus heteroclitus(killifish) embryo. Images ... more Individual deep cell moving by circus movements. Fundulus heteroclitus(killifish) embryo. Images made available through Mark Cooper and Zebrafish The Living Laboratory.

This video shows the dynamics of the apical cell surface of individual cells of the enveloping la... more This video shows the dynamics of the apical cell surface of individual cells of the enveloping layer in a killifish embryo. The fluorescent actin is seen to polymerize and depolymerize in cyclic waves that extend and retract these surface protrusions. Two neighboring cells from a continuous epithelium are expressing the fluorescent GFP-actin. The expression of this inserted gene is mosaic, with some cells expressing, and others not.

This video shows the apical (outside-facing) side of epithelia cells that form confluent tissues ... more This video shows the apical (outside-facing) side of epithelia cells that form confluent tissues where each cell is tightly glued to its neighbors by circumferential junctions like bands of velcro. These cells show that the cell surface is extremely dynamic, blistering and boiling with small surface protrusions, filled with actin. This video also shows these protrusions in parts of three neighboring cells, and the circumferential actin-filled ridges at the cell margins are particularly dramatic. The actin used in these protrusions is the same pool used by deep cells for individual migration, and these enveloping (EVL) cells use the same kinds of biochemical controls for polymerization (rapid growth) and depolymerization (shrinkage) of filaments.

Journal of Cell Science, 1995

Heat-stable brain microtubule associated proteins (MAPs) and purified microtubule associated prot... more 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 t...

Journal of Cell Science, 1994

Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophas... more Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is t 1/2 = 31 +/- 6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (t 1/2 = 67 +/- seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all fou...

Journal of Cell Science, 1992

We have previously shown that changes in the intracellular free calcium concentration ([Ca2+]i) m... more We have previously shown that changes in the intracellular free calcium concentration ([Ca2+]i) modulate the rate of anaphase chromosome motion in stamen hair cells of Tradescantia. Elevations between 0.8-1.0 μM accelerate motion, while increases above 2.0 μM or decreases below resting level inhibit motion. The related signaling agent GTPγS also accelerates motion, however, by a mechanism that does not appear to involve changes in [Ca2+]i. To explore further the mechanism by which Ca2+ and GTPγS regulate chromosome motion we have analyzed the direct effect of these agents on the structure of the spindle microtubules (MTs). First, we injected carboxyfluorescein-derivatized brain tubulin and allowed it to incorporate into spindle MTs. Then, during appropriate times of anaphase, we injected Ca2+ or related agents and monitored their effect on spindle MT fluorescence using a confocal laser scanning microscope. A high level of Ca2+ (10 μM), known to inhibit motion, causes extensive degra...

BioTechniques, 1998

Previous experiments have clearly demonstrated that microtubule dynamic instability is regulated ... more 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 microtubul...

Cell Motility and the Cytoskeleton, 1988

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of ... more Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.

Cell Motility and the Cytoskeleton, 1999

Microtubules are known to be required for locomotion of mammalian cells, and recent experiments d... more 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.

Molecular Biology of the Cell, 1999

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitoti... more 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, inliving 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 man...

Journal of cell science, 2016

TPX2 is a widely conserved microtubule-associated protein that is required for mitotic spindle fo... more TPX2 is a widely conserved microtubule-associated protein that is required for mitotic spindle formation and function. Previous studies have demonstrated that TPX2 is required for the nucleation of microtubules around chromosomes; however, the molecular mechanism by which TPX2 promotes microtubule nucleation remains a mystery. In this study, we found that TPX2 acts to suppress tubulin subunit off-rates during microtubule assembly and disassembly, thus allowing for the support of unprecedentedly slow rates of plus-end microtubule growth, and also leading to a dramatically reduced microtubule shortening rate. These changes in microtubule dynamics can be explained in computational simulations by a moderate increase in tubulin-tubulin bond strength upon TPX2 association with the microtubule lattice, which in turn acts to reduce the departure rate of tubulin subunits from the microtubule ends. Thus, the direct suppression of tubulin subunit off-rates by TPX2 during microtubule growth and...

Journal of Cell Science

ABSTRACT

Biophysical Journal, 2014

Seminars in Cell & Developmental Biology, 2010

In all eukaryotic cells, molecular motor proteins play essential roles in spindle assembly and fu... more In all eukaryotic cells, molecular motor proteins play essential roles in spindle assembly and function. The homotetrameric kinesin-5 motors in particular generate outward forces that establish and maintain spindle bipolarity and contribute to microtubule flux. Cell cycle dependent phosphorylation of kinesin-5 motors regulates their localization to the mitotic spindle. Analysis of live cells further shows that kinesin-5 motors are highly dynamic in the spindle. Understanding the interactions of kinesin-5 motors with microtubules and other spindle proteins is likely to broaden the documented roles of kinesin-5 motors during cell division.

Cell Motility and the Cytoskeleton, 1993

... Dahong Zhang, Patricia Wadsworth, and Peter K. Hepler ... rescence microscopy [Baskin and Can... more ... Dahong Zhang, Patricia Wadsworth, and Peter K. Hepler ... rescence microscopy [Baskin and Cande, 1990; Lambert et al., 1991; Staiger and Lloyd, 1991; Wick, 19911 to visualize microtubules in fixed cells, as well as with those using polarized light microscopy [Inoue, 1964; Ba ...