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Microinjected Fluorescent Phalloidin in Vivo Reveals the F-Actin Dynamics and Assembly in Higher Plant Mitotic Cells

Anne-Catherine Schmit and Anne-Marie Lambert’ Laboratoire de Biologie Cellulaire Végétale, Universite Louis Pasteur, Centre National de Ia Recherche Scientifique, Unité Associée 1182, lnstitut de Botanique, 28, rue Goethe, F-67083 Strasbourg Cedex, France Endosperm mitotic cellsmicroinjected with fluorescent phalloidin enabled us to follow the in vivo dynamics of the F-actin cytoskeleton. The fluorescent probe immediately bound to plant microfilaments. First, we investigated the active rearrangement of F-actin during chromosome migration, which appeared to be slowed down in the presence of phalloidin. These findings were compared with the actin patterns observed inmitotic cells fixed at different stages. Our second aim was to determine the origin of the actin filaments that appear at the equator during anaphase-telophase transition. It is not clear whether this F-actin is newly assembled at the end of mitosis and could control plant cytokinesis or whether it corresponds to a passive redistribution of broken polymers in response to microtubule dynamics. Wemicroinjected the same cells twice, first in metaphase with rhodamine-phalloidin and then in late anaphase with fluorescein isothiocyanate-phalloidin. This technique enabled us to visualize two F-actin populations that are not co-localized, suggesting that actin is newly assembled during cell plate development.
These in vivo data shed new light on the role of actin in plant mitosis and cytokinesis.INTRODUCTION

A large body of evidence obtained during the last few years that reveals entirely new features of the plant actin cytoskeleton suggests that plant microfilaments may play an important role in specific properties of the higher plant cells (for review, see Lloyd, 1989). To date, these data have been obtained after labeling of fixed and permeabilized cells, either by indirectimmunofluorescence or with fluorescent phalloidin (Seagull, Falconer, and Weerdenburg, 1987; Schmit and Lambert, 1987; Lloyd and Traas, 1988), as a specific probe for F-actin (Wieland, 1977; Wulf et al., 1979). The identification of an actin network in the cytoplasm of endosperm cells (Schmit and Lambert, 1985, 1987; Mole-Bajer, Bajer, and Inoué, 1988), as in various cell types (for reviews, see Staigerand Schliwa, 1987; Lloyd, 1989), showed that F-actin is, like tubulin, one of the major components of the plant cytoskeleton. Its role, however, is hardly understood. A much-debated question is to define the potential activity of actin during plant mitosis and cytokinesis (Forer, 1985, 1988; Schmit and Lambert, 1987, 1988; MolbBajer et al., 1988). In most cultured animal ‘cells, mitotic induction ischaracterized by the disassembly of actin stress fibers (for review, see Aubin, 1981). On the contrary, in higher plant cells it has been found that the interphase cytoplasmic actin network is rearranged within the cell cortex at the onset
of mitosis and remains as a permanent cage around the microtubular mitotic spindle, as seen after permeabilization, in endosperm (Schmit and Lambert, 1987)and in other higher plant cells (Traas et al., 1987; Lloyd and Traas, 1988). Microfilaments were also detected inside the spindle (Forer, 1985), but their potential activity is still unknown. Indeed, cytochalasins (Schmit and Lambert, 1988) and phalloidin may affect chromosome velocity but do not stop chromosome migration. Besides these characteristics and the unexpected behavior of the corticalactin during the process of mitosis, a particular accumulation of actin filaments is detected during strategic events that control cytokinesis, suggesting that actin may regulate plant cytokinesis. Two steps are of particular interest. (1) At the onset of mitosis, actin filaments were detected within the preprophase band that is believed to predetermine the future cell division plane (Palevitz,...