Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection.

Abstract

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. lnjection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability t o act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive t o microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells. INTRODUCTIONPlant cells contain extensive arrays of actin filaments, which appear to participate in a variety of cellular functions, including cytoplasmic streaming (Salitz and Schmitz, 1989;Kuroda, 1990; Emons et al., 1991 ;Williamson, 1993; Staiger et al., 1994), nuclear positioning (Katsuta and Shibaoka, 1988;Lloyd, 1989;Cho and Wick, 1990;Staiger and Lloyd, 1991), and cell division (Schmit and Lambert, 1990;Lambert et al., 1991;Wick, 1991;Cleary et al., 1992;Hepler et al., 1993;Staehelin and Hepler, 1996). Of particular pertinence to our study is their participation in cell plate formation and alignment during cytokinesis. Microscopic investigations indicate that actin filaments are a major structural component of the cytokinetic apparatus. Actin filaments are distributed throughout the phragmoplast and aligned parallel to the palisade of microtubules (Kakimoto and Shibaoka, 1987; Schmit and Lambert, 1990;Zhang et al., 1993;Staehelin and Hepler, 1996). Although the microtubules of the phragmoplast initially arise through the coalescence of preexist-' Both authors contributed equally to this work. * Current address: Plant Cell Biology Group, University of Nijmegen, Toernooiveld, NL-6525 ED Nijmegen, The Netherlands.To whom correspondence should be addressed. E-mail annemie. emons@algem.pcm.wau.nl; fax 31 -31 7-485005.ing spindle-interzone elements, the actin filaments seem to originate de novo as two opposing groups, each initially associated with the surface of the re-forming daughter nuclei (Zhang et al., 1993;Valster and Hepler, 1997).Although it is clear that actin filaments are associated with the phragmoplast, it is much less clear how they participate in cell plate formation. One p...

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

“…Alternatively, cells can be microinjected with fluorescently labelled phalloidine. This method is successfully applied to study AFs role in plant cells (Cleary et al 1992;Valster et al 1997).…”

“…Actin filaments have been observed in association with the PPB and phragmoplast of dividing cells as well as with the cell cortex and throughout the cytoplasm of interphase cells (Collings and Emons 1999), in spindles during division (e.g. Van Lammeren et al 1989) and in the phragmoplast (Valster et al 1997). To gain insight in the structural organisation and the function of the microtubular and actin cytoskeleton, BY-2 suspension cells are frequently used because they have a short cell cycle and the events of the cell cycle progression from the M phase to G 1 phase can be well monitored microscopically (Nagata et al 1992;Hasezawa et al 1997;Kumagai et al 2001;Stoeckel and Takeda 2002).…”

Utilization of specific primers in legume allergens based polymorphism screening

“…Profilins are small 14-to 17-kDa cytosolic proteins expressed in all eukaryotic cells [10]. The major role of profilin in plant cells is the rapid reorganization of microfilaments during processes like cytokinesis, cytoplasmic streaming, cell elongation, and growth of pollen tubes and root hairs [11][12]. Plant profilin was discovered by Valenta et al [13] as an allergen source in birch pollen.…”

Visualization and analysis of actin cytoskeleton organization in plants

“…MFs carry the cytoplasmic strands and are responsible for cyclosis (Valster et al, 1997;Ketelaar et al, 2002). In the stele, root hairs and trichomes, actin microfilaments are assembled into thick bundles oriented along the cell's long axis.…”

“…Vilins belong to the gelsolin family of actin-interacting proteins and facilitate actin bundling into thick bundles (Klahre et al, 2000). Profilins are small (12-15 kDa) proteins that prevent the spontaneous polymerization of actin (Valster et al, 1997). Actin-depolymerizing factors (ADF) are also small (15-20 kDa) proteins that are active in complex with actin-interacting protein 1, severe MFs and further facilitate actin depolymerisation by making free ends in MFs .…”