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First published online 29 March 2005
doi: 10.1242/jcs.02305

Journal of Cell Science 118, 1705-1714 (2005)
Published by The Company of Biologists 2005
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The nuclear rim protein Amo1 is required for proper microtubule cytoskeleton organisation in fission yeast

Mercedes Pardo1,*,{ddagger} and Paul Nurse2

1 Cell Cycle Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK
2 The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA



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  		Fig. 1. A role for Amo1 in microtubule organisation and polarity. (A) Overexpression of amo1 leads to bundling of microtubules on one side of the cell. (B) Differential interference contrast microscopy images of wild-type cells (wt) and amo1{Delta} cells from plates. (C) Tubulin immunofluorescence of wild-type and amo1{Delta} cells. (D) MBC sensitivity spot assay. Cells from indicated strains were grown to mid-exponential phase, and tenfold serial dilutions spotted on plates containing the indicated amount of MBC. (E) Calcofluor White staining of septated wild-type and amo1{Delta} cells. (F) Growth patterns of pairs of cells after cell division were monitored. The two patterns observed are shown, with percentages of total cells scored. Bar, 10 µm.

 


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  		Fig. 2. Localisation of Amo1 to the nuclear surface. (A) Projection of deconvolved focal sections of live interphase and mitotic cells containing Amo1-GFP. Arrowheads indicate threads of nuclear membrane between separating nuclei at anaphase. (B) Single focal section showing that Amo1-GFP (green) localises to the nuclear periphery surrounding chromosomal DNA (blue) and nucleolus. (C) Four consecutive deconvolved focal sections showing Amo1-myc (green) and Sad1 (red) detected by immunofluorescence. (D) Amo1-GFP localisation in control (DMSO) and MBC-treated cells. E. Deconvolved projection showing live MBC-treated cells expressing GFP-tubulin (green) and Amo1-mRFP1 (red). Bar, 10 µm.

 


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  		Fig. 5. Amo1{Delta} cells are not defective in NPC organisation. (A) Alignment of S. pombe Amo1 and S. cerevisiae Nup42 sequences, showing conservation of some FG repeats (in red). Protein alignment was carried out with CLUSTALW. (B) Immunofluorescence of cells expressing Amo1-myc with anti-myc (green) and anti-Nup189 (red) antibodies. (C) NLS-LacI-GFP localisation in live wild-type and amo1{Delta} cells. (D) Nup189 immunofluorescence in wild-type and amo1{Delta} cells. Bar, 10 µm.

 


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  		Fig. 3. Microtubular defects in amo1{Delta} cells. (A) Tubulin immunofluorescence of cold and MBC treated cells at time 0 (t0) and 1 minute (1') after allowing microtubule repolymerisation. (B,C) Time-lapse series of GFP-tubulin in live wild-type (B) and amo1{Delta} cells (C) showing uncoordinated growth and shrinkage in amo1{Delta} cells. Numbers indicate time in seconds. Arrowheads in B indicate polymerising microtubules (white) and depolymerising microtubules (yellow) in the same bundle. White arrowheads in C indicate polymerising microtubules and yellow arrowheads depolymerising microtubules in bundles with more than one microtubule touching the cell tip at the same time. (D) Tea1 (green) and tubulin (red) immunofluorescence in wild-type and amo1{Delta} cells. Arrowhead depicts abnormal accumulation of Tea1. Bar, 10 µm.

 


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  		Fig. 4. Microtubule loading is not defective in amo1{Delta} cells. Localisation of Klp5-GFP on interphase microtubules, Tea2-GFP on microtubules and at cell ends, Tip1-GFP on microtubules and at cell ends, Alp4-GFP at the SPB and on microtubule satellites, and Mod20-GFP at MTOCs and microtubule satellites in live wild-type and amo1{Delta} cells. Bar, 10 µm.

 

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© The Company of Biologists Ltd 2005