Carboxyfluorescein-labeled brain tubulin has been microinjected into stamen hair cells of Tradescantia, and its distribution during mitosis and cytokinesis was examined using confocal laser scanning fluorescence microscopy. The results show that brain tubulin incorporates into plant microtubules and is utilized throughout mitosis and cytokinesis. Microtubule structures that incorporate brain tubulin include the preprophase band, the perinuclear sheath at late prophase, the kinetochore fibers during prometaphase, metaphase, and anaphase, the interzone spindle during anaphase, and finally the phragmoplast during late anaphase and telophase. All of these microtubule-containing structures and, notably, their transitions from one to another have been observed in single live cells progressing through mitosis and cytokinesis.

Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled contractile ring. Microtubules are a spindle component that is essential for the induction of cytokinesis. This induction could use central spindle and/or astral microtubules to stimulate cortical contraction around the spindle equator (equatorial stimulation). Alternatively, or in addition, induction could rely on astral microtubules to relax the polar cortex (polar relaxation). To investigate the relationship between microtubules, cortical stiffness, and contractile ring assembly, we used different configurations of microtubules to manipulate the distribution of actin in living silkworm spermatocytes. Mechanically repositioned, noninterdigitating microtubules can induce redistribution of actin at any region of the cortex by locally excluding cortical actin filaments. This cortical flow of actin promotes regional relaxation while increasing tension elsewhere (normally at the equatorial cortex). In contrast, repositioned interdigitating microtubule bundles use a novel mechanism to induce local stimulation of contractility anywhere within the cortex; at the antiparallel plus ends of central spindle microtubules, actin aggregates are rapidly assembled de novo and transported laterally to the equatorial cortex. Relaxation depends on microtubule dynamics but not on RhoA activity, whereas stimulation depends on RhoA activity but is largely independent of microtubule dynamics. We conclude that polar relaxation and equatorial stimulation mechanisms redundantly supply actin for contractile ring assembly, thus increasing the fidelity of cleavage.

The development and dynamics of the phragmoplast cytoskeleton have been analyzed in living stamen hair cells of Tradescanria. Microtubules and actin microfilaments have been identified by microinjecting either carboxyfluorescein labeled brain tubulin or rhodamine phalloidin. Examination with the confocal laser scanning microscope has permitted sequential imaging of the fluorescent cytoskeletal elements in single living cells progressing through division. Phragmoplast microtubules initially emerge through the lateral coalescence of preexisting interzone microtubules. As cytokinesis progresses, these tightly clustered microtubules shorten in length and expand centrifugally toward the cell periphery. By contrast, the phragmoplast microfilaments appear to arise de novo in late anaphase in close association with the proximal surfaces of the reconstituting daughter nuclei. The microfilaments are oriented parallel to the microtubules but conspicuously do not occupy the equatorial region where microtubules interdigitate and where the cell plate vesicles aggregate and fuse. As development proceeds the microfilaments shorten in length and expand in girth, similar to microtubules, although they remain excluded from the cell plate region. In terminal phases of cell plate formation, microtubules degrade first in the central regions of the phragmoplast and later toward the edges, whereas microfilaments break down more uniformly throughout the phragmoplast. 0 1993 Wiley-Liss, Inc.

Structural constituents of the spindle apparatus essential for cleavage induction remain undefined. Findings from various cell types using different approaches suggest the importance of all structural constituents, including asters, the central spindle, and chromosomes. In this study, we systematically dissected the role of each constituent in cleavage induction in grasshopper spermatocytes and narrowed the essential one down to bundled microtubules. Using micromanipulation, we produced “cells” containing only asters, a truncated central spindle lacking both asters and chromosomes, or microtubules alone. We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present. Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in “metaphase,” disassembling in “anaphase,” and bundling into arrays in “telophase.” Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress. We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.