Fluorescence interference from the control incubation was generally negligible

ages were also processed with Image-J. Mitotic Kinases Regulate F-Actin Dynamics Results Identification of a ring-like F-actin structure GW 501516 web during mitosis It is generally believed that mitotic spindle governs chromosome partitioning and segregation in eukaryotes. However, several lines of recent evidence suggest the role of F-actin-based structure in spindle assembly and positioning. To validate if there are any cytoplasmic F-actin structures in mitotic cells, we carried out immunofluorescence analyses on F-actin and spindle of mitotic cells. To this end, asynchronized HeLa cells were fixed and stained with Rhodamine-Phalloidin and anti-tubulin antibody to visualize F-actin and microtubule, respectively. As shown in Fig. 1A, F-actin staining is mainly distributed on the cortex and retraction fibers through mitosis. Careful examination revealed that the F-actin staining is organized around the spindle poles in prophase cells. As the cell goes through metaphase, F-actin staining becomes apparent around the entire spindle. In the anaphase A cell, the F-actin staining appears more recognizable. Once cells enter into anaphase B, F-actin staining becomes concentrated to contractile ring. During the course of telophase and cytokinesis, the intensity of the F-actin staining in cytoplasm is as low as its intensity in prophase. The cytoplasmic F-actin is a large collection of bundled actin filaments. It is practically a continuous structure stretching from spindle to cell cortex as well as the astral microtubule. By convention, we defined an X-axis through the spindle poles when spindle is at the equilibrium position, and a Zaxis perpendicular to the imaging plane at the center of the cell. We chose the axis perpendicular simultaneously to X-axis and Zaxis as Y-axis. We imaged cells at different PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19656604 layers with an interval of 0.2 mm. The cytoplasmic F-actin changes only slightly along the Z-axis no matter how much the spindle is rotated. We found that the relative intensity of the cytoplasmic F-actin in different regions varies slightly compared with the Z = 0 plane. Thus we concluded the distribution of cytoplasmic F-actin is isotropic. Compared to the distribution of astral microtubule, the cytoplasmic F-actin structure distributes around the spindle continuously. To examine this ring-like structure in metaphase arrested cells, an aliquot of HeLa cells was synchronized by a double-Thymidine treatment followed by release into MG132-containing DMEM to prevent anaphase onset. As predicted, treatment of Lat B perturbed the ring-like F-actin structure. These cells treated with other drugs except for Lat B exhibit a distinct actin filament ring. Unlike previous publication, F-actin bundling exists during mitosis in normal HeLa cells with considerable intensity. Comparing with the revolving network, the cytoplasmic Factin we found around the mitotic spindle is relatively more relaxed and smooth. The kymograph of the real-time images of metaphase arrested cells indicates that the ring-like F-actin structure is relatively less dynamic and cytoplasmic actin filaments have a random motile pattern. Thus, we reasoned that a ring-like cytoplasmic F-actin structure is present around the mitotic spindle in normal HeLa cells during mitosis and maintains in metaphase arrested cells. This ring-like F-actin staining was mainly observed in metaphase or anaphase of asynchronized cells, and it was observed in all MG132-synchronized cells. Modeling of spindle positioning and