Regulation of anaphase chromosome motion in Tradescantia stamen hair cells by calcium and related signaling agents

Abstract

Several lines of evidence support the idea that increases in the intracellular free calcium concentration ([Ca2+]i) regulate chromosome motion. To directly test this we have iontophoretically injected Ca2+ or related signaling agents into Tradescantia stamen hair cells during anaphase and measured their effect on chromosome motion and on the Ca2+ levels. Ca2+ at (+)1 nA for 10 s (∼1 μM) causes a transient (20 s) twofold increase in the rate of chromosome motion, while at higher levels it slows or completely stops motion. Ca2+ buffers, EGTA, and 5,5′-dibromo1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′- tetraacetic acid, which transiently suppress the ion level, also momentarily stop motion. Injection of K+, Cl-, or Mg2+, as controls, have no effect on motion. The injection of GTPγS, and to a lesser extent GTP, enhances motion similarly to a low level of Ca2+. However, inositol 1,4,5-trisphosphate, ATPγS, ATP, and GDPβS have no effect. Measurement of the [Ca2+]i with indo-1 reveals that the direct injections of Ca2+ produce the expected increases. GTPγS, on the other hand, causes only a small [Ca2+]i rise, which by itself is insufficient to increase the rate of chromosome motion. Further studies reveal that any negative ion injection, presumably through hyperpolarization of the membrane potential, generates a similar small pulse of Ca2+, yet these agents have no effect on motion. Two major conclusions from these studies are as follows, (a) Increased [Ca2+]i can enhance the rate of motion, if administered in a narrow physiological window around 1 μm; concentrations above 1 μm or below the physiological resting level will slow or stop chromosomes, (b) GTPγS enhances motion by a mechanism that does not cause a sustained uniform rise of [Ca2+]i in the spindle; this effect may be mediated through very localized [Ca2+]i changes or Ca2+-independent effectors.