Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Abstract

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 alpha-type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.

The Type IIArabidopsisFormin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division

“…Coordination of microtubules and microfilaments in the cortical cytoplasm plays a key role during directional expansion of cells as well as during positioning of the plane of cell division. Microtubules and microfilaments cooperate during cell division to coordinate the mitotic and cytokinetic apparatus (Panteris and Galatis, 2005;Yasuda et al, 2005;Collings, 2008; Petrá š ek and Schwarzerová , 2009). However, little information is available at the protein level to understand the mechanisms by which microtubules and microfilaments interact.…”

“…In support of this idea, in metaphase I and II, microfilaments codistribute with the spindle microtubules. During the second division, microfilaments intimately connect with the radiating microtubules and guide outgrowth of the phragmoplast (Schmit and Lambert, 1987;Traas et al, 1989;Staiger and Cande, 1991;Preuss et al, 2004;Yasuda et al, 2005). Drug-based experiments have shown that fragmentation of microfilaments leads to the disappearance of spindles and phragmoplasts.…”

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane‐fly spermatocytes

“…Actin/myosin involvement in mitosis has been deduced from studies that actin, myosin and titin are present in a variety of spindles [e.g., Table I of Forer et al, 2003;Robinson and Snyder, 2005;Yasuda et al, 2005;Fabian and Forer, 2007;Fabian et al, 2007b] and, also in a variety of cells, from studies of the effects of actin inhibitors, of myosin inhibitors or of genetic alterations to myosin [review in Table II of ; also Forer, 2005, 2007;Woolner et al, 2008]. In crane-fly spermatocytes in particular, the cells used for the experiments we describe herein, anaphase chromosome movement is altered by actin inhibitors cytochalasin D and latrunculin B, both of which cause depolymerization of actin filaments, and is altered by myosin inhibitors BDM and Y27632, both of which block myosin activity [Forer and Pickett-Heaps, 1998;Silverman-Gavrila and Forer, 2001;Fabian and Forer, 2005;Fabian et al, 2007a].…”

TcCYS4, a cystatin from cocoa, reduces necrosis triggered by MpNEP2 in tobacco plants

“…Three experiments were performed; in each, 1 of the biological replicas (independent transformants) included 5 experimental replicates. The constitutively expressed b-actin gene (ID: AB158612, Yasuda et al, 2005) was used for normalization.…”