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Calmodulin-containing substructures of the centrosomal matrix released by microtubule perturbation

Nicoleta Moisoi, Muriel Erent*, Sheena Whyte, Stephen Martin and Peter M. Bayley{ddagger}

Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
* Present address: Department of Biophysics, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany



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Fig. 1. Spectroscopic properties of CaM-EGFP prove that the fusion protein preserves the CaM characteristics with respect to calcium and peptide binding. (A) Absorption spectra of CaM-EGFP: Ca2+-free and with saturating Ca2+; inset: CaM-EGFP absorbance (489 nm) with Ca2+ titration. Different Ca2+ saturation levels do not affect the EGFP characteristic absorption spectra (with maximum at 489 nm); at 280 nm there is the same change in the single Tyr absorbance as for wtCaM. (B) Ca2+ titration of CaM-EGFP: 5,5'-Br2BAPTA absorbance at 263 nm; CaM-EGFP competes with Br2BAPTA showing no change of the average Ca2+ affinity of the fusion protein compared with wtCaM. (C) WFF peptide fluorescence titration of Ca2+- saturated CaM-EGFP ({lambda}ex=290 nm, {lambda}em=330 nm) indicates a 1:1 stoichiometry and dissociation constant similar to that of wtCaM.

 


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Fig. 2. Calmodulin distribution in the mitotic spindle in control cells and in mild treatment with nocodazole. (A) Control metaphase HeLa (projection of deconvolved sections). (B) Stage I: MDCK (nocodazole 0.01 µg/ml, 2 hours; one section deconvolved). (C) Stage II: MDCK (nocodazole 0.01 µg/ml, 2 hours; projection of deconvolved section; adjacent panel shows only the CaM-EGFP (green) of the same cell). In the control, as in the first two stages of the mitotic spindle perturbation with depolymerising drug, CaM-EGFP is located at polar level in a ring-like pattern, and in the mitotic spindle along microtubules from pole to kinetochore with a decreasing intensity (green, CaM-EGFP; red, {alpha}-tubulin; blue, chromosomes). Bar, 10 µm.

 


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Fig. 4. CaM-EGFP redistribution in relation to kinetochores and microtubules in MDCK. (A) Metaphase MDCK control (no drug). (B) Star-like structures in nocodazole (0.1 µg/ml, 4 hours)-treated MDCK. The control cell shows the calmodulin (green) concentrated at the spindle pole in a ring-like shape, the microtubules (red; overlapping green and red seen as yellow) and the kinetochores (white, false colour) at the metaphase plate (blue). The star-like structure in stage III of mitotic spindle disruption seen with four colours gives the detailed arrangement of the four elements (B, detail shown in inset i): calmodulin as a core with radiating microtubules and the corresponding kinetochores attached to chromosomes. (C) CaM-EGFP redistribution relative to kinetochores induced with nocodazole in stage IV of spindle disruption. The kinetochores (red) are situated in immediate proximity to the CaM-EGFP (green) accumulation and chromosomes (blue). Bar, 10 µm.

 


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Fig. 3. Effect of nocodazole (6 hours, 0.1 µg/ml) on CaM-EGFP localisation in MDCK cells. (A) Stage III of microtubule disruption; (i) star-like structure; (ii) centrosomal remnant; (a1) centrosomal remnant; (a2) star-like structure in control experiments with cold treatment (4°C, 15 minutes to 2 hours). The star-like substructure shows the CaM-EGFP concentrated centrally with short microtubules radiating from it and the chromosomal material present at the outermost region. The centrosomal remnants show an alternating pattern of tubulin and CaM-EGFP with no obvious colocalisation, seen as `ring' in projection. (B) Stage IV shows CaM location at kinetochore level in the absence of residual microtubules as seen with immunofluorescence. No centrosomal remnants could be seen (green, CaM-EGFP; red, {alpha}-tubulin; blue, chromosomes; projection of deconvolved series). Bar, 10 µm.

 


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Fig. 5. CaM-EGFP redistribution in mitotic MDCKs under vinblastine treatment. Mitotic cells (A-G) stained for microtubules at different levels of spindle impairment (green, CaM-EGFP; red, {alpha}-tubulin; blue, chromosomes; projection of deconvolved sections). (A,B) (1 hour, 0.5 nM) Stages I and II with spindle shortening and CaM in a ring-like structure at spindle poles and along spindle microtubules with decreasing intensity. (C,D) (1 hour, 1 nM) Stage III with CaM at the cores of the star-like structures. (E) (1 hour, 10 nM) Stage IV shows the CaM in a punctate distribution but immunofluorescence does not identify remaining kinetochore microtubules. (F) (1 hour, 50 nM) Stage V with striking release of the calmodulin from the punctuate accumulation in an even cytoplasmic distribution. (G) (1 hour, 100 nM) Stage VI in which tubulin forms paracrystals, with a similar cytoplasmic distribution of CaM as in stage V. (H) (1 hour, 100 nM) Interphase cells with tubulin paracrystals. (I-L) Relative distribution of CaM-EGFP (green) and kinetochores (red) with chromosomes (blue) in vinblastine-treated MDCK cells (projection of deconvolved series). (I) (10 nM, 1 hour) At the arrest of mitosis in prophase in a monopolar spindle, the CaM shows the gradient distribution along microtubules. (J, stage III and K, stage IV) (0.5 nM, 1 hour) star-like structures (details shown in insets i and ii); the distance between the kinetochores and CaM core shortens progressively (K), and the CaM along the microtubule is progressively redistributed into the cytoplasm in the stage V (L; 100 nM, 1 hour). Bar, 10 µm.

 


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Fig. 6. (A-E) CaM-EGFP redistribution at the paclitaxel-induced microtubule polar structures in mitotic MDCKs (4 hours, 10 nM) (green, CaM-EGFP; red, {alpha}-tubulin; blue, chromosomes); (A) Monopolar spindle; (B) Bipolar spindle; (C) Three-polar spindle; (D) Quadri-polar spindle; (E) Multipolar spindle; Calmodulin redistributes in a ring-like shape at the paclitaxel-induced multipolar spindle without an accumulation at the kinetochores level. CaM-preserves the ring-like shape as seen in projection at every pole. Note that in the bipolar structure the CaM content at the two poles (B) is symmetrically distributed, and at the three (C) and four poles (D) its content decreases with the increasing number of poles. (F-I) Example of steps in the paclitaxel-induced multipolar structure as shown by CaM-EGFP at polar level (green, CaM-EGFP; red, kinetochore; blue, chromosomes). The CaM-containing ring-like structure divides (A,B), producing two or more poles (C,D). The kinetochores decorate chromosomes at the metaphase plate and those that were removed together with the related microtubules; projection of deconvolved series. Bar, 10 µm.

 


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Fig. 7. Schematic view of steps in calmodulin redistribution under microtubule disrupting treatments. In control metaphase cells and in the stages I/II of microtubule spindle impairment, CaM preserves a ring-like structure at the spindle pole and the intensity distribution along microtubules decreases from poles to kinetochores. Stage III shows that the chromosomal matrix has divided, the kinetochores lose the metaphase alignment, tending to group around the CaM-containing cores and there is a punctate distribution of CaM, in multiple small star-like structures, associated via short residual microtubules to kinetochores/chromosomes together with the remnants of the calmodulin-microtubule polar structure. In stage IV, CaM-EGFP is close to kinetochores that are attached to the chromosome mass and tubulin is punctate. In stages V and VI (vinblastine only) there is a striking dispersion of the CaM from its accumulation sites in the cytosol and tubulin paracrystals are formed. Immunofluorescence and live cell monitoring suggest that the main mechanism of the CaM-EGFP redistribution throughout these stages is by the splitting of the pericentriolar material that contains the targets for calmodulin by shortening of the spindle microtubules until they are progressively removed.

 


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Fig. 8. Schematic view of steps in calmodulin redistribution under microtubule stabilising treatment with paclitaxel. The CaM initially remains associated with centrosomal matrix and kinetochore-to-pole microtubules, as in control cells. At the spindle pole the CaM-EGFP appears to subdivide from its ring-like shape, along with entire fragments of the spindle, including microtubules and associated kinetochores and chromosomes. Further repetition of this process generates a multipolar spindle, with CaM seen in a ring-like structure at each of these poles, some of which contain centrioles, and the chromosomal material becomes disorganised.

 





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