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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01690

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Ste20/GCK kinase Nak1/Orb3 polarizes the actin cytoskeleton in fission yeast during the cell cycle

Klaus Leonhard^*, and Paul Nurse^*

Cancer Research UK, Cell Cycle Laboratory, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

View larger version (42K): [in a new window]   Fig. 1. Effect of the nak1/orb3 deletion on morphology, actin cytoskeleton and cytokinesis of fission-yeast cells. (A) The nak1/orb3 deletion phenotype. Diploid cells lacking one copy of nak1/orb3 were sporulated; the spores were dissected and incubated at 25°C, and microphotographs were taken 48 hours later. One example of wild-type (WT) and three of nak1/orb3 microcolonies are shown. (B,C) Phenotype of nak1/orb3 cells after loss of a complementing nak1/orb3 plasmid. A nak1/orb3 strain complemented with the plasmid pREP4X-nmt1-nak1/orb3 and a wild-type strain were grown without selection. Fixed cells were stained with rhodamine-phalloidin for actin and with DAPI to show the nuclei. Bars, 10 µm.

View larger version (36K): [in a new window]   Fig. 2. Changes in shape and F-actin distribution in nak1/orb3-167 cells. (A) Morphology of nak1/orb3-167 cells at 25°C (top) and 4 hours after the shift from 25°C to 35°C (bottom). (B) Morphological changes of nak1/orb3-167 cells up to 5 hours after temperature shift to 35°C. Exponentially growing cells were transferred from 25°C to 35°C and the proportions of rod-shaped, pear-shaped and round cells were determined for each time point. (C) F-Actin delocalization in nak1/orb3-167 cells. Wild-type (WT) and nak1/orb3-167 cells were grown at 25°C and samples taken before (0) and 80 minutes (80) and 300 minutes (300) after transfer to 35°C. The cells were fixed and stained with rhodamine-phalloidin for actin. (D) Changes of polar F-actin patches in wild-type and nak1/orb3-167 cells after a temperature shift from 25°C to 35°C. Cells were grown at 25°C and samples were taken before (time point 0) and after transfer to 35°C at time points indicated. The proportions of fixed and rhodamine/phalloidin-stained cells with polar F-actin patches was determined. (E,F) Repolarization of the actin cytoskeleton after a shift of nak1/orb3-167 cells from 35°C to 25°C. The nak1/orb3-167 cells were grown at 25°C, transferred to 35°C for 80 minutes (E, left) and shifted down to 25°C (E, right). Cells were fixed in formaldehyde and stained with rhodamine-phalloidin. (F) Samples were taken of nak1/orb3-167 cells up to 80 minutes after transfer from 35°C to 25°C (–HU, closed circles). The nak1/orb3-167 cells were blocked in 15 mM hydroxyurea for 3 hours at 25°C, then shifted to 35°C for 80 minutes and shifted back to 25°C to follow actin relocalization in blocked cells (+HU, open circles). At the time points indicated, the cells were fixed and stained with rhodamine-phalloidin. The proportions of cells with polarized F-actin patches at the cell tips. 200 cells were counted for each time point and experiments were performed three times. Bars, 10 µm.

View larger version (39K): [in a new window]   Fig. 3. Effect of nak1/orb3-167 during cytokinesis. Wild-type and nak1/orb3-167 cells were shifted to 36°C for 3 hours. (A) Cells were fixed and stained with rhodamine-phalloidin for F-actin, DAPI and CalcoFluor. Mitotic cell (red arrow), constricting actomyosin rings (white arrows), lysed cell (star); bar, 10 µm. (B) The proportions of cells displaying F-actin rings, constricting rings and incomplete septa, and complete septa were determined for wild-type and nak1/orb3-167 cells. 200 cells have been counted for wild-type and for nak1/orb3-167; the experiments were repeated three times.

View larger version (47K): [in a new window]   Fig. 4. Effect of nak1/orb3-167 on the microtubule cytoskeleton. Exponentially growing wild-type (WT) and temperature-sensitive nak1/orb3-167 cells were transferred from 25°C to 35°C. Samples were taken before and 30 minutes and 60 minutes after a shift from 25°C to 35°C, fixed and stained with an antibody against tubulin. Bars, 10 µm.

View larger version (22K): [in a new window]   Fig. 5. F-Actin delocalization in nak1/orb3-167 cells blocked at different stages of the cell cycle. Wild-type (WT), nak1/orb3-167, nak1/orb3-167 cdc2-M26 and nak1/orb3-167 cdc25-22 cells were grown at 25°C and shifted to 35°C for 2 hours. The nak1/orb3-167 culture was split in half before the temperature shift. One half was directly shifted to 35°C (orb3-167 –HU) and the other half was incubated with 15 mM hydroxyurea for 4 hours at 25°C before the shift up (orb3-167 +HU). Cells were fixed and stained with rhodamine-phalloidin, and the proportion of cells with polar F-actin patches at the tips was determined. 200 cells were counted for each mutant; experiments were performed at least twice.

View larger version (27K): [in a new window]   Fig. 6. Localization of Nak1/Orb3. (A) Localization of Nak1/Orb3-GFP in vivo. Living cells expressing Nak1/Orb3 tagged with GFP under the endogenous promoter. Arrows point at dots at the nuclei that might correspond to the spindle pole body. (B) Colocalization of Nak1/Orb3-GFP and Sad1-DsRed in live cells. (C) Colocalization of Nak1/Orb3-GFP and actin. Cells expressing GFP-tagged Nak1/Orb3 were fixed and double stained with antibodies against actin (red) and GFP (green). Overlay of both channels is shown in yellow. Bars, 10 µm.

View larger version (29K): [in a new window]   Fig. 7. Nak1/Orb3 protein levels in synchronized Nak1/Orb3-Myc cdc25-22 cells. The cells were blocked in G2 phase at 36.5°C for 3.5 hours and synchronously released into mitosis by cooling the culture to 25°C. Samples were taken every 20 minutes. (A) Septation index. The proportion of cells displaying a septum was determined for each time point. (B,C) Nak1/Orb3-Myc protein levels. Denatured protein extracts were prepared and equal amounts for each time point were separated by SDS-PAGE. The proteins were detected with monoclonal antibody 9E10 against the Myc tag. (B) Separation on a 4-12% acrylamide gradient gel. (C) Separation on a 8% acrylamide/0.1% bisacrylamide gel (left). Phosphorylated (Nak1/Orb3-P) and unphosphorylated (Nak1/Orb3) forms of Nak1/Orb3 are visible. The extract of the 40 minutes time point was either incubated with -phosphatase (+) or buffer only (–) (right).

View larger version (38K): [in a new window]   Fig. 8. Protein-kinase activity of Nak1/Orb3 in vitro. (A) Nak1/Orb3-dependent kinase activity throughout the cell cycle. Samples were taken from cultures synchronized by cdc25-22 block-and-release at the time points indicated and kinase assays were performed with casein as a substrate. Synchronous cdc25-22 cells expressing untagged Nak1/Orb3 were used as a control. Protein levels of precipitated Nak1/Orb3-Myc were checked by western blotting and immunodetection with anti-Myc antibodies (data not shown). (B) Kinase activity of Nak1/Orb3-Myc samples (closed circles) and of untagged control samples (open circles) was quantified using a phosphor imager. Septation index (closed triangles). (C) Temperature-sensitive kinase activity of Nak1/Orb3-167/Myc in vitro. Kinase assays were performed at 25°C and 37°C, with samples taken from asynchronous cultures of wild-type cells (WT) expressing untagged Nak1/Orb3, tagged wild-type Nak1/Orb3 or tagged mutant Nak1/Orb3-167. Protein levels of precipitated (prec.) Nak1/Orb3 and of total Nak1/Orb3 and -tubulin in the extracts were checked by western blotting and immunodetection.

View larger version (38K): [in a new window]   Fig. 9. The roles of the actin and the microtubule cytoskeletons, and Nak1/Orb3 kinase activity in localizing the protein. (A-C) The effects of F-actin and microtubule depolymerization on the localization of Nak1/Orb3-GFP. Cells were incubated with 10 µM latrunculin A for 20 minutes (+ Lat A), with 25 µg ml–1 of the microtubule-depolymerizing agent carbendazim (+ MBC) or with their solvent dimethylsulfoxide as a control (+ DMSO). Arrows show patches of Nak1/Orb3-GFP. Samples with and without MBC were fixed in methanol and stained with anti-tubulin antibodies to control for microtubule polymerization (data not shown). (D) Effects of nak1/orb3-167 mutation on Nak1/Orb3-GFP localization. Nak1/Orb3-GFP cells grown at 25°C and cells shifted to 36°C for 60 minutes were fixed in formaldehyde and stained with anti-GFP antibodies. Bars, 10 µm.

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