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First published online 17 July 2006
doi: 10.1242/jcs.03051

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Control of the AtMAP65-1 interaction with microtubules through the cell cycle

Andrei P. Smertenko^1,*, Hsin-Yu Chang^1,*, Seiji Sonobe², Stepan I. Fenyk¹, Magdalena Weingartner³, Laci Bögre³ and Patrick J. Hussey^1,

¹ The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham, DH1 3LE, UK
² Himeji Institute of Technology, Faculty of Science, Hyogo, Japan
³ School of Biological and Biomedical Sciences, Royal Holloway, University of London, Egham, TW20 0EX, UK

View larger version (46K): [in a new window]   Fig. 1. Cell-cycle-dependent phosphorylation pattern of AtMAP65-1. (A) Cell cycle synchronisation. Graphs show the frequency of metaphase (open diamonds), anaphase (open squares) and telophase (open triangles) cells in the synchronised BY-2 cell culture. Eleven sampling points were analysed: points 0-2 were collected every two hours after aphidicolin was washed out, points 3-5 were collected every 2 hours during propyzamide treatment and points 6-10 were collected every 40 minutes after propyzamide was washed out with the exception that there was a 2-hour interval between points 9 and 10. (B) Autoradiogram (i) and Coomassie Blue-stained gel (ii) showing phosphorylation of AtMAP65-1 using the total protein extract from cells collected at sampling points 0-10 described in A (i). 5 µg of recombinant AtMAP65-1 was used in each assay. The Coomassie Blue-stained gel of the extracts used as kinase in (i) and (ii) is shown in (iii). (C) Two-dimensional SDS-PAGE immunoblots of total protein extracts from interphase cells (S-phase, sampling point 0), metaphase cells (M-phase, sampling point 4) and M-phase extract treated with phosphatase, probed with anti AtMAP65-1. The area of the membrane where AtMAP65-1 isoforms were detected is shown.

View larger version (64K): [in a new window]   Fig. 2. Several protein kinases phosphorylate AtMAP65-1. (A) Effect of protein kinase inhibitors on AtMAP65-1 phosphorylation in vitro. Recombinant AtMAP65-1 was phosphorylated using M-phase extract (sampling point 4 as in Fig. 1A) without inhibitor (Control) or in the presence of 20 mM DMAP, 5 µM K252a, 5 µM staurosporine and 100 µM olomoucine. Each lane contained an equal amount of protein as shown by the amido-black staining. (B) AtMAP65-1 is phosphorylated by microtubule associating kinases. Colloidal silver staining and autoradiogram of phosphorylation assays using M-phase extract (lane 1) and a microtubule-associated protein preparation (lane 2). (C) AtMAP65-1 is phosphorylated by CDKs and MAPKs. Cyclin-dependent kinases were pulled down using pSuc1 bound beads, anti-cdc2a and anti-cdc2b. MAP kinases were immunoprecipitated with anti-MPK4 and anti-MPK6. Kinase activity was checked using histone 1 as a substrate for cyclin-dependent kinases and using myelin basic protein (MBP) as the substrate for MAP kinases. (D) Localisation of GFP:AtMAP65-1 in control cells. (Interphase (1), metaphase (2), anaphase (3) telophase (4). (E) Localisation of GFP:AtMAP65-1 in cells treated for 10 minutes with the general protein kinase inhibitor DMAP. Interphase (1), metaphase (2), anaphase (3) and telophase (4). (F) Localisation of GFP:AtMAP65-1 in cells treated for 10 minutes with the CDK inhibitor olomoucine. Prometaphase (1), metaphase (2 and 3). (G) Three consecutive movie frames (taken 2 minutes apart) of a cell treated with the phosphatase inhibitor okadaic acid. The images were recorded after 15 minutes of treatment. Note the GFP:AtMAP65-1 signal disappears and the phragmoplast does not expand in this time. (H) Localisation of AtMAP65-1 (green signal) and DNA (blue signal) in the phragmoplast of a BY-2 cell line harbouring a non-degradable form of cyclin B1 under the control of the dexamethasone-inducible promoter before induction (1) and after 16 hours induction with dexamethasone (2).

View larger version (27K): [in a new window]   Fig. 3. Identification of phosphorylation sites in AtMAP65-1. (A) Phosphorylation of four AtMAP65-1 fragments (FR1-4) using the M-phase extract (M-phase) or with the microtubule-associated proteins preparation (MTP). Cell extract without substrate was used as a negative control (Kinase). CBB, Coomassie Brilliant Blue R-250 staining; Autorad, autoradiogram. (B) Phosphorylation of synthetic peptides (PA-PI) containing putative phosphorylation sites in Fragment 4 using the M-phase extract. The inset shows the total counts per minute (CPM) in the corresponding reaction mixtures. (C) Autoradiogram (Autorad) of the recombinant wild-type AtMAP65-1 (WT) and the mutant, AtMAP65-19D (9D) phosphorylated with M-phase or S-phase (interphase, sampling point 0) extracts. The corresponding nitrocellulose membrane was stained with amido-black (Amidoblack).

View larger version (50K): [in a new window]   Fig. 4. Phosphorylation regulates the binding of AtMAP65-1 to microtubules. (A) Cosedimentation assays of taxol-stabilised microtubules with the recombinant wild-type AtMAP65-1 (WT) or the mutants, AtMAP65-12D (2D), AtMAP65-14D(4D), AtMAP65-17D(7D) and AtMAP65-19D(9D). The amount of AtMAP65-1 proteins in the supernatant and pellet was measured on the Coomassie-stained SDS-PAGE gels using densitometry and the percentage of the total recombinant AtMAP65-1 protein used in the reaction that was recovered in the pellet was calculated as described in the Materials and Methods. (B) Turbidimetric analysis of a 14 µM tubulin solution and a tubulin solution mixed in an equimolar ratio with AtMAP65-1 or AtMAP65-19D. (C) Localisation of GFP:AtMAP65-14D(4D), GFP:AtMAP65-17D(7D), GFP:AtMAP65-19D(9D) fusion proteins in interphase and anaphase cells. (D) The ratio between the midzone and the pole signals of GFP:AtMAP65-1 (WT), GFP:AtMAP65-14D(4D), GFP:AtMAP65-17D(7D) and GFP:AtMAP65-19D(9D) fusion proteins. Ten anaphase cells were analysed for each transgenic cell line.

View larger version (80K): [in a new window]   Fig. 5. Non-phosphorylatable AtMAP65-1 binds spindle microtubules and affects metaphase spindle organisation. (A-F) Immunolocalisation of tubulin (red), GFP:AtMAP65-1 (green, B,D,F) and GFP:AtMAP65-19A (green, A,C,E), DNA (blue) during prometaphase (A,B), metaphase (C,D) and anaphase (E,F). The wild-type protein becomes associated with microtubules only in anaphase whereas the alanine mutant binds to microtubules in prophase and metaphase. (G) Negative images of the typical microtubule pattern in metaphase spindles of cell lines expressing GFP:AtMAP65-1 or GFP:AtMAP65-19A. The cells are immunostained with an antibody to -tubulin, DM1A. Note the microtubules are excessively bundled in the GFP:AtMAP65-19A spindles. (H) Number of all microtubules (white bars) and the number of pole to pole microtubules (grey bars) visible in a 1 µm optical section in the spindles of cells expressing GFP:AtMAP65-1 (WT) or GFP:AtMAP65-19A (9A). Twenty cells were analysed for WT and 18 for 9A (P<0.05) from two different cell lineages. (I) Western blot of total cell protein extracts from the BY-2 cell lines expressing wild-type GFP:AtMAP65-1 (WT), GFP:AtMAP65-19D (9D) or GFP:AtMAP65-19A (9A) probed with anti-GFP and anti--tubulin antibody. The colloidal silver stain shows the general pattern of proteins transferred onto the nitrocellulose membrane.

View larger version (50K): [in a new window]   Fig. 6. Expression of non-phosphorylatable AtMAP65-1 delays the metaphase/anaphase transition. (A) Localisation of wild-type GFP:AtMAP65-1 (WT) and mutant GFP:AtMAP65-19A (9A) fusions during mitosis. The wild-type protein becomes associated with microtubules only in anaphase whereas the mutant binds to microtubules at every stage of mitosis. Numbers in the top right corner indicate timing of each frame. (B) Quantification of the fluorescent signal in the cell division midzone of GFP:AtMAP65-1 (closed circles) and GFP:AtMAP65-19A (open circles).

View larger version (27K): [in a new window]   Fig. 7. A model for the mechanism of AtMAP65-1 regulation. (A) Diagrams of the secondary structure prediction and charts of charge distribution for anaphase spindle elongation protein 1 (ASE1), AtMAP65-1, PRC1, Feo and spindle defective 1 (SPD1). Blue colour represents helices, red represents ß strand and violet represents coiled coil. (B) Scheme for the interaction between AtMAP65-1 and microtubules. Two microtubule-interacting sites on AtMAP65-1 are needed for normal function. One site is not affected by phosphorylation and is not coloured, the second site is normally alkaline (shown is blue). The alkaline region binds to the acidic C-terminal region of tubulin (shown in red). After phosphorylation the charge of the second microtubule-interacting site changes to acidic (shown in red). This weakens the binding of AtMAP65-1 to microtubules.