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First published online 12 September 2006
doi: 10.1242/jcs.03166

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Dynamin-related proteins Vps1p and Dnm1p control peroxisome abundance in Saccharomyces cerevisiae

Kasinath Kuravi^1,*, Shirisha Nagotu^1,*, Arjen M. Krikken¹, Klaas Sjollema¹, Markus Deckers², Ralf Erdmann², Marten Veenhuis¹ and Ida J. van der Klei^1,

¹ Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, NL-9750 AA Haren, The Netherlands
² Institut für Physiologische Chemie, Abt. Systembiochemie, Ruhr-Universität Bochum, 44801 Bochum, Germany

View larger version (18K): [in a new window]   Fig. 1. Vps1p, Dnm1p and Fis1p play a role in regulating peroxisome numbers in S. cerevisiae. Quantitative data on peroxisome abundance were obtained by fluorescence microscopy of GFP-SKL producing S. cerevisiae strains cultured on glucose (A,C,E,G,I) or in the presence of oleate (B,D,F,H,J). The number of fluorescent spots per cell was counted from randomly taken fluorescence microscope images. For each sample, fluorescent spots were counted in 300 non-budding cells taken from two independent cultures (150 cells per culture). The frequency distributions of cells with different numbers of fluorescent spots are shown. Error bars represent the s.e.m.

View larger version (68K): [in a new window]   Fig. 2. Peroxisome and mitochondrial morphology in oleate-induced cells. (A) Representative fluorescence images of GFP-SKL-producing, oleate-induced cells of WT and the various mutant strains. In dnm1 and vps1 cells the peroxisome number is reduced. In vps1 and dnm1 vps1 cells large peroxisomes are present, which are often constricted. (B) Examples of the mitochondrial morphology in oleate-induced cells of the same strains. Mitochondria were stained using MitoTracker Orange. In WT and vps1 cells a branched mitochondrial network is evident. In dnm1, fis1 and dnm1 vps1 cells generally one long tubular structure is present that contains fewer branches than in WT cells and remains near the cell cortex. (C) A GFP-Ant1p-producing dnm1 vps1 cell induced on oleate medium. GFP-Ant1p is localized to peroxisomal membranes. As in the dnm1 vps1 cells producing GFP-SKL, generally a single, large peroxisome is observed per cell. Bars, 5 µm.

View larger version (19K): [in a new window]   Fig. 3. Deletion of VPS1, DNM1 or FIS1 does not affect peroxisome positioning. Quantitative data on peroxisome positioning in budding cells were obtained by fluorescence microscopy of GFP-SKL-producing S. cerevisiae strains cultured on glucose (A,C,E,G,I) or in the presence of oleate (B,D,F,H,J). The number of fluorescent spots in each cell region (1-4) was counted from randomly taken fluorescence microscope images. Region 1 is the part of the mother cell opposite to the bud, region 2 is the central region in the mother cell, region 3 represents the region in the mother cell near the bud neck and region 4 is the developing bud (see A). For each sample, fluorescent spots were counted in 300 cells taken from two independent cultures (150 cells per culture). The frequency distributions of cell regions with different numbers of fluorescent spots are shown. Error bars represent the s.e.m.

View larger version (85K): [in a new window]   Fig. 4. Peroxisome dynamics in S. cerevisiae WT and mutant strains. Selected images (a-e) taken from time-lapse videos of WT (lane A), vps1 (lane B) and dnm1 vps1 cells (lane C), grown in the presence of oleate. The corresponding Movies 1-3 are shown in the supplementary material. In each panel the fate of two cells that resulted from budding is depicted. The data show that in WT cells (A) peroxisomes developed in buds at the early stage of development. In vps1 cells (B) low numbers of peroxisomes are present. Developing buds may be administrated by peroxisomes in two different ways. In the cell on the right-hand side, a small peroxisome had initially migrated into the bud, followed by a second organelle that arose from the larger organelle located in the neck region. In the cell on the left-hand side, a single organelle is initially observed that migrates to the neck, elongates and subsequently divides at a late stage of budding. A similar elongation effect is observed during the second budding of the cells on the right-hand side, confirming that administration of peroxisomes to developing buds may occur by two different mechanisms. In cells of the dnm1 vps1 double mutant (C) only one mode of segregation is observed. In these cells the single peroxisome structure migrates to the neck between the mother cell and the bud, elongates into the bud and is separated at a very late stage of bud development, probably during cytokinesis. Bars, 5 µm.

View larger version (46K): [in a new window]   Fig. 5. 3D model of a peroxisome in dnm1 vps1 cell. Characteristic 3D image of a peroxisome in a budding dnm1 vps1 cell, grown in the presence of oleate. The image is reconstituted based on a Z-stack of 22 CLSM images at 0.05 µm intervals. The figure demonstrates that in these cells typically one elongated organelle is present that protrudes into the bud (see also supplementary material Movie 4). Characteristically, the part of the organelle located in the mother cell is enlarged.

View larger version (107K): [in a new window]   Fig. 6. Dnm1-GFP co-localizes with mitochondria and peroxisomes. (A) The location of Dnm1-GFP to mitochondria visualized by MitoTracker Orange. Note that all GFP spots, except for one (upper right), co-localize with mitochondria. Cells were grown on glucose media. (B) A section through a cell in which two peroxisomes are observed, labelled with DsRed-SKL. One of these shows co-localization with Dnm1-GFP (arrow), the remaining Dnm1-GFP spots are considered to localize to mitochondria (compare with A). The images represent a selected single section through a Z-stack, prepared by CLSM (see supplementary material Movie 5). Cells were grown on glucose. The inset shows a magnification of the organelle indicated by the arrow. (C) Location of Dnm1-GFP is shown in glucose-grown fis1 cells. In these cells the number of Dnm1-GFP spots is strongly reduced (compare with A,B). These spots do not co-localize with peroxisomes, which are labeled with DsRed-SKL. (D) Location of GFP-Fis1p. Most of the GFP is present on large structures, which represent mitochondria. A minor portion of the GFP-Fis1p fluorescence is present in small spots, which co-localize with the peroxisomal marker protein DsRed-SKL. Bars, 5 µm.