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Regulation of anaphase chromosome motion and spindle microtubule structure by calcium and related agents in the stamen hair cells of Tradescantia
Abstract
The accumulating evidence supports the idea that increases in the intracellular free calcium concentration ((Ca$\sp{2+}$) $\sb{\rm i}$) regulate chromosome-to-pole motion. To directly test this hypothesis I have iontophoretically injected Ca$\sp{2+}$, Ca$\sp{2+}$ buffers (EGTA, Br$\sb2$-BAPTA) or nucleotides into Tradescantia stamen hair cells during anaphase and measured their effect on (Ca$\sp{2+}$) $\sb{\rm i}$ and on chromosome motion. The results show that: (1) elevations of the (Ca$\sp{2+}$) $\sb{\rm i}$ within a limited physiological window (0.8-1.0 $\mu$M) accelerate motion two-fold. (2) the elevation of the (Ca$\sp{2+}$) $\sb{\rm i}$ above this window (2-10 $\mu$M) or below the physiological resting level ($<$0.05 $\mu$M) decelerates or stops motion. (3) GTP$\gamma$S, and to a lesser extent GTP, enhance motion without causing a sufficient increase in (Ca$\sp{2+}$) $\sb{\rm i}$. Further studies of the role of Ca$\sp{2+}$ and related agents in regulating chromosome motion requires a visualization of spindle microtubules (MTs). To achieve this goal I have adapted the technique of fluorescent analogue cytochemistry to plant cells. In this method carboxyfluorescein (CF) labeled brain tubulin is injected into dividing cells and acts as a reporter molecule for the MT structures into which it becomes incorporated. Direct observation of MT dynamics is then carried out with confocal laser scanning microscopy (CLSM). The results show that brain tubulin incorporates into plant MTs, and is utilized in the preprophase band, the perinuclear sheath at late prophase, the kinetochore fibers (k-fibers) during prometaphase, metaphase and anaphase, the interzone spindle during anaphase, and finally the phragmoplast during late anaphase and telophase. All of these MT-containing structures and, notably, their transitions from one to another, have been observed in single live cells progressing through mitosis and cytokinesis. To explore further the mechanism by which Ca$\sp{2+}$ and GTP$\gamma$S regulate chromosome motion I have directly observed the effect of these agents on the structure of CF-tubulin labeled kinetochore MTs (kMTs) during anaphase. Quantitative analysis of the changes in the fluorescence in k-fibers/background (ratio) shows that: (1) Ca$\sp{2+}$ levels (2-10 $\mu$M) known to inhibit motion, cause extensive degradation of kMT structure (70-90% decrease in ratio). (2) Ca$\sp{2+}$ levels (0.8-1.0 $\mu$M) known to accelerate motion, however, cause only a modest level of kMT depolymerization (30-50% decrease in ratio) in which the k-fibers appear less distinct, and the overall spindle fluorescence is more diffuse. (3) Ca$\sp{2+}$ buffers, which transiently block motion, have no detectable effect on spindle structure. (4) GTP$\gamma$S, which enhances motion also has no discernible effect on spindle structure. I propose that both MT depolymerization and a MT motor are involved in the control of anaphase chromosome motion.
Subject Area
Cellular biology|Botany
Recommended Citation
Zhang, Da-Hong, "Regulation of anaphase chromosome motion and spindle microtubule structure by calcium and related agents in the stamen hair cells of Tradescantia" (1991). Doctoral Dissertations Available from Proquest. AAI9120963.
https://scholarworks.umass.edu/dissertations/AAI9120963