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First published online 12 April 2005
doi: 10.1242/jcs.02311

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End4/Sla2 is involved in establishment of a new growth zone in Schizosaccharomyces pombe

¹ Cell Cycle Lab Cancer Research UK, 44 Lincoln's Inn Field, London, WC2A 3PX, UK
² Paul-Ehrlich-Institute, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany
³ Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

View larger version (57K): [in a new window]   Fig. 1. sla2 cells have an aberrant morphology. (A) Morphological phenotype, (B) microtubules, (C) distribution of actin patches, (D) Tea1-GFP localization in wild type (upper panels) and sla2 (lower panels) grown at 25°C. Bar, 5 µm.

View larger version (36K): [in a new window]   Fig. 2. End4/Sla2 talin-like domain is required for actin organization at new growth zones. (A) Schematic organization of End4/Sla2 mutant proteins generated in these study and percentage of cells with monopolar growth in each strain. (B) Wild-type (left) and sla2talin (right) cells were stained with calcofluor (upper panel) and for actin (lower panel). (C) Western blot analysis of total extracts from wild-type, Sla2-GFP and Sla2talin-GFP strains with -GFP (top) and -tubulin (bottom) antibody. (D) Patterns of growth of sla2talin, tea1 and sla2talin tea1 cells after division.

View larger version (44K): [in a new window]   Fig. 3. End4/Sla2 talin-like domain is required for establishment of a new growth zone. (A) Schematic representation of latA pulse experiment (top) and scoring (bottom) of cells with monopolar actin distribution before the treatment (grey) and 2 hours after wash out (black). (B) Cells before (top), during (middle) and 2 hours after latA treatment (bottom): cdc10-129 (left) cdc10-129 sla2talin (right). Bar, 5 µm.

View larger version (88K): [in a new window]   Fig. 4. End4/Sla2 localization in vivo. (A) Sla2-GFP accumulation (green) at cell ends during interphase (top) and in a medial ring over the nucleus (middle), which persists until the end of mitosis (bottom). The nucleus is stained with DAPI (blue). (B) Central accumulation of Sla2-GFP is unaffected in tea1 cells (arrow). Sla2-GFP is enriched only at the growing end in tea1 and (C) in tea1(200) cells. (D) Accumulation of Sla2-GFP (bottom) and actin (top) at the growing end in cdc10-129 cells. (E) Staining of Sla2-GFP-expressing cells with -actin and (F) -Tea1 antibody. Bar, 5 µm.

View larger version (36K): [in a new window]   Fig. 5. Localization of Sla2-GFP is independent of the actin and microtubule cytoskeletons. (A) cdc10-129 cells expressing Sla2-GFP were grown at 25°C and then transferred to 36°C for 3 hours (left). After addition of DMSO or 40 µM latA or 25 µg/ml MBC, or 25 µg/ml MBC plus 40 µM latA (indicated by an arrow in the scheme) respectively, cells were released to permissive temperature (25°C) for an hour in the presence of the drug and then analysed for Sla2-GFP localization (right panels). (B) Quantification of cells with bipolar actin distribution at 36°C (black bar) and 30 minutes after shift down to 25°C (white and grey bars) in the presence of the different drugs. Bar, 5 µm.

View larger version (54K): [in a new window]   Fig. 6. Sla2-GFP localization depends on vesicular transport. (A) Sla2-GFP cells were treated with (a) ethanol and DMSO, (b) 40 µM latA (10 minutes at 25°C), (c) 100 µg/ml of brefeldin A (BfA) (10 minutes at 25°C), or (d) 100 µg/ml BfA and 40 µM latA (10 minutes at 25°C). BfA treatment delocalizes Sla2-GFP in 5 minutes. Sla2-GFP de-localization is abolished by co-treatment with latA. (B) Localization of Sla2talin-GFP (a) and calcofluor staining (b). Sla2talin-GFP cells treated with BfA at 25°C for 10 minutes (c). (C) Staining of Sla2talin-GFP-expressing cells with Rhodamine phalloidin. (D) Sla2-GFP local movement is abolished by treatment with 40 µM latA. Arrowheads point to the same GFP dot in different frames of a time-lapse recording (see Movie 1 in supplementary material; +latA frames are from Movie 2). Bar, 5 µm.

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