Biomedical subjects

E Shelden

Publications and source records attributed to E Shelden.

6 recordsLinked to original sources

PubMed1995

Three-dimensional localization of wild-type and myosin II mutant cells during morphogenesis of Dictyostelium.

Dictyostelium amoebae that lack myosin II (mhcA-) are unable to undergo morphogenesis. The cells aggregate slowly to form hemispherical mounds, but the mounds never extend a tip upward. Expression of developmentally regulated genes appears normal in the absence of morphogenesis. When mixed with an excess of wild-type cells, some mutant cells form differentiated spores; however, rescue is extremely inefficient (Knecht and Loomis, 1988). In order to assess how morphogenesis is normally accomplished and why mutants lacking myosin II cannot develop, a new method has been developed that allows individual amoebae to be localized and tracked at high resolution within the multicellular organism during development. Amoebae are labeled with a fluorescent dye at the beginning of starvation, mixed with an excess of unlabeled cells, and allowed to develop. The three-dimensional position of labeled cells in the multicellular organism is then determined using a laser scanning confocal microscope. Using this methodology, we have shown that labeled wild-type cells are randomly distributed throughout the organism and complete development normally. When labeled mhcA- mutant cells are mixed with a 20-fold excess of wild-type cells, they are non-randomly localized even at the earliest stages of development. Mutant cells in aggregation streams are found primarily at the edges of the streams and many cells never become part of the streams or are left behind as the wild-type cells complete aggregation. Those that are incorporated into the aggregate are found at the edge and base, the backs of slugs and the base of the fruiting bodies. A few mutant cells can be found in the sorus, where they presumably become spores. The segregation of mhcA- mutant cells to the outside of the wild-type aggregation streams argues that the mutant cells are unable to penetrate a mass of adhered, wild-type cells. We hypothesize that mutant cells lacking cortical integrity are unable to generate sufficient protrusive force to break the adhesion of wild-type cells to each other. This would make the mutants incapable of moving through a mass of cells (either mutant or wild type) or of changing shape when adhered to other cells. We propose that mutants lacking myosin II are unable to accomplish morphogenesis because they cannot move correctly in a three-dimensional mass of adhered cells.

Animals↗

PubMed1995

Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis.

We have used fluorescent labeling, confocal microscopy and computer-assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dictyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5 +/- 4.9 microns/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0 +/- 4.8 microns/min, twice as fast as reported previously for isolated myosin II mutant cells, and fail to display regular periodic movement. When removed from aggregation streams myosin II mutant cells move at only 2.5 +/- 2.0 microns/min, while wild-type cells under these conditions move at 5.9 +/- 4.5 microns/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular development. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the cortex of such cells is too flacid to resist forces generated during movement. The increased rate of mutant cell movement and distortion of mutant cell morphology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a multicellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. In addition, the distortion of myosin II mutant cells within wild-type aggregation streams indicates that myosin is not required for the formation of cell-cell contacts. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium.

Animals↗

PubMed1993

Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific.

Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CHO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 +/- 10.8 microns/min, 32.2 +/- 17.7 microns/min, 11.9 +/- 6.5 microns/min, and 19.7 +/- 8.1 microns/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtK1 cells. Thus, individual microtubules in PtK1 lamellae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.

Animals↗

PubMed1992

Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to-pole motion.

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.

Anaphase↗

PubMed1990

Interzonal microtubules are dynamic during spindle elongation.

The pattern and extent of microtubule assembly during spindle elongation has been examined in PtK1 cells by microinjection of biotin-tubulin and immunolocalization of biotin-tubulin-containing microtubules using antibodies to biotin. PtK1 cells were microinjected at 30 degrees C, incubated for various intervals to allow incorporation of biotin-tubulin into microtubules, then lysed, fixed and stained for biotin-tubulin and total tubulin. When mid- to late anaphase cells were examined at short times post-injection, using conventional fluorescence light microscopy, rapid incorporation of biotin-tubulin was detected throughout the interzonal region, between the separating chromosomes, and in the spindle asters. Using confocal fluorescence microscopy, the segments of biotin-labeled microtubules in the interzonal region were found to be continuous with the distal, or plus-ends, of unlabeled microtubules. When teleophase cells were examined, a marked decline in the extent of incorporation was apparent. Quantitative analysis of the total length of labeled polymer in the interzonal region of cells from mid-anaphase through telophase further reveals that the extent of incorporation was maximal during late anaphase, and decreased during telophase. The measured rate of interzonal microtubule growth remained relatively constant during this period. Our results provide direct evidence for plus-end elongation of interzonal microtubules during spindle elongation and further reveal that interzonal microtubules are highly dynamic during late anaphase spindle elongation. The implications of these results for the mechanism of anaphase B are discussed.

Anaphase↗

PubMed1989

Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase.

The dynamic behavior of kinetochore fiber microtubules has been examined in PtK1 cells during anaphase of mitosis. Cells in anaphase were injected with biotin-tubulin and, at various intervals after injection, fixed for light or electron microscopic immunolocalization of biotin-tubulin-containing microtubules. When cells in early to mid anaphase were injected with biotin-tubulin and fixed 1-2 min later, fluorescence was observed throughout the spindle, including the region of the kinetochore fibers. Electron microscopy of early to mid anaphase cells, after processing with immunogold methods, revealed both labeled and unlabeled microtubules in the kinetochore fibers; some labeled microtubules contacted the kinetochores. When late anaphase cells were injected with biotin-tubulin, and fixed a few minutes later, little fluorescence was observed in the kinetochore fibers. Electron microscopy confirmed that kinetochore fibers in late anaphase cells were refractory to tubulin incorporation. The results of these experiments demonstrate that the kinetochore fiber incorporates new microtubules during early anaphase but that this incorporation ceases in mid to late anaphase. Thus, microtubule turnover within the kinetochore fiber does not abruptly cease at the onset of anaphase and anaphase kinetochore fiber microtubules are more dynamic than previously suspected.

Anaphase↗