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First published online 1 April 2003
doi: 10.1242/jcs.00423

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Spatial and temporal dynamics of budding yeast mitochondria lacking the division component Fis1p

Stefan Jakobs^1,*,, Nadia Martini^1,*, Astrid C. Schauss¹, Alexander Egner¹, Benedikt Westermann² and Stefan W. Hell¹

¹ Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
² Institut für Physiologische Chemie, Universität München, Butenandtstrasse 5, 81377 München, Germany

View larger version (123K): [in a new window]   Fig. 1. Remodelling of mitochondria of wild-type cells upon glucose repression. Wild-type cells expressing matrix targeted GFP were grown in glycerol to log phase and transferred for 45 minutes into glucose-containing medium. (A-C) Beam-scanning confocal images of representative budding cells. Maximum intensity projections of several optical planes of the fluorescence signal overlaid with a bright field transmission image are displayed. (D-I) Confocal time-lapse microscopy recording the reorganization of the wild-type mitochondrial network upon glucose repression. Selected maximum intensity projections are displayed. The underlying data-stack comprises 165 three-dimensional stacks with 4000 single optical sections. Solid arrows indicate sites of future matrix separation. Feathered arrows indicate future fusion sites. The mitochondrial network had a morphology characteristic of cells grown in glycerol at time point zero. Bar, 2 µm. The complete dataset is available in two movies (Movie 1 and Movie 2 at jcs.biologists.org/supplemental).

View larger version (99K): [in a new window]   Fig. 2. Remodelling of mitochondria of fis1 cells upon glucose repression. fis1 mutants expressing matrix-targeted GFP were grown in glycerol to log phase and transferred for 70 minutes into glucose-containing medium. (A-C) Beam-scanning confocal images of representative budding fis1 cells as in Fig. 1A-C. (D-I) Confocal time-lapse microscopy recording the reorganization of the fis1 mitochondrial network upon glucose repression. Selected maximum intensity projections are displayed. The underlying data-stack comprises 99 three-dimensional stacks with 2200 single optical sections. Solid arrows indicate sites of future matrix separation. Feathered arrows indicate future fusion sites. At time point zero, the mitochondrial compartment resembled a large fenestrated net. Bars, 2 µm (A-C) and 1 µm (D-I). The complete dataset is available in two movies (Movie 3 and Movie 4 at jcs.biologist.org/supplemental).

View larger version (118K): [in a new window]   Fig. 3. Fission of the outer membrane in fis1 cells. (A-C) fis1 mutants expressing matrix-targeted DsRed and outer-membrane-targeted OM45-GFP were grown in PMGal to mid-log phase. After embedding in 1% agarose, live cells were imaged by beam-scanning confocal microscopy. (A) OM45-GFP, (B) DsRed in matrix and (C) bright field image. Displayed are single optical sections. Note the separation of the matrix without fission of the outer membrane (arrows). Insets: Maximum intensity projections of the separation site. (D-J) fis1 mutants expressing inner-membrane-targeted DsRed and outer-membrane-targeted OM45-GFP were grown in PMGal to mid-log phase. Displayed are maximum intensity projections. D,G,I: OM45-GFP; E,H,J: DsRed in inner membrane; F: bright field image. Time-lapse images (D-J) were taken every 2 minutes. The arrows point to a remodelling event involving complete tubule fission. Note the colocalization of the inner and outer membrane labels. Bars, 2 µm.

View larger version (25K): [in a new window]   Fig. 4. Frequency of matrix separation and fusion events during remodelling upon glucose repression. (A) All detectable separation and fusion events of the GFP labelled matrix in a single wild-type cell and a single fis1 cell were counted over a time frame of 105 minutes. Only events in the mother cell were recorded. At time point zero, the mitochondrial morphologies were of a typical glycerol type. At the end of the analysis both networks were clearly simplified. (B,C) Separation and fusion frequencies at different time points following change of carbon source. (B) Wild-type cells and (C) fis1 cells. The number of matrix separations and fusions was counted at three 15 minute time frames, namely before recognizable simplification (period 1), after final separation of mother and daughter mitochondrial compartments (period 3) and in between (period 2). In the case of fis1, period 3 was defined as the point of time when no further size reduction of the fenestrated net could be observed. Only events in the mother cells were recorded. Control: (B) Wild-type cell grown to log phase in YPD and transferred to fresh YPD medium.

View larger version (24K): [in a new window]   Fig. 5. Mitochondrial remodelling of dnm1 mutants upon glucose repression. Selected maximum intensity projections are displayed. The experimental set-up was similar as for Fig. 2D-I. Solid arrows indicate future matrix separation sites. Feathered arrows indicate matrix fusion sites. The underlying data-stack comprises 40 three-dimensional stacks. Stacks were recorded every 60 seconds for 40 minutes. Bar, 1 µm. The complete dataset is available as Movie 5 at jcs.biologists.org/supplemental.

View larger version (28K): [in a new window]   Fig. 6. Schematic representation of mitochondrial simplification upon glucose repression in wild-type and fis1 cells. Numbers indicate characteristic time points within the ongoing remodelling process. At time point zero, the mitochondrial networks exhibit morphologies typical for cells grown in medium containing glycerol. (A) In wild-type cells, the daughter cell is invaded by a single mitochondrial tubule shortly after budding. Subsequently the tubule attaches to the cell cortex within the emerging bud (arrow). During subsequent growth the mitochondrion remains attached at this site while the remaining part of the network is moving freely within the daughter cell. During budding, the connection between the mitochondrial compartments of mother and daughter cell is frequently disrupted. Concomitant with the transport of mitochondrial membranes into the daughter cell the network undergoes constantly fission and fusion. Most fusion events occur between two mitochondrial tips. It is conceivable that mitochondrial interactions with cytoskeletal elements are important for these processes (Boldogh et al., 2001). (B) The mitochondria of fis1 cells grown on glycerol are frequently large fenestrated nets located at one side of the cell cortex. In fis1 mitochondria simplification appears not to be directly correlated to budding. Upon glucose repression, single meshes of the fenestrated network are opened by fission. These fission events lead to an ongoing size reduction of the fenestrated network. Membrane fusions extend the (mostly one or two) tubules emerging from the net. The long tubules are frequently separated from the fenestrated net by fission. They subsequently refuse to the same or to a different site. Upon budding one of these single tubules penetrates into the emerging daughter cell. As observed in the wildtype, the tubule attaches with one site at the cortex of the daughter cell (arrow). The rest of the tubule moves freely within the bud. Simplifying a large fenestrated net to an almost tubular structure most probably requires several cell cycles.

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