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 degradation of spindle MT structure. An intermediate level (2 μM), which slows but does not stop movement, produces a distinct decay of fluorescence. A level of Ca²⁺ (0.8-1 μM) known to accelerate motion, however, generates only a small change in which the kinetochore fibers appear less distinct, and the overall spindle fluorescence is more diffuse. The Ca²⁺ buffer EGTA, which transiently blocks motion, has no detectable effect on spindle structure. GTPyS, which enhances motion also has no discernible effect on spindle structure. The results with Ca²⁺ support the idea that the ion facilitates anaphase motion through depolymerization of kinetochore MTs. However, if the depolymerization is extensive then inhibition of motion ensues. GTPγS, on the other hand, accelerates motion without inducing a detectable effect on spindle MT structure.

• Received July 1, 1991.
• Accepted January 31, 1992.

• © 1992 by Company of Biologists