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Comparative Study
. 2003 Dec;2(6):1315-26.
doi: 10.1128/EC.2.6.1315-1326.2003.

Two Dictyostelium orthologs of the prokaryotic cell division protein FtsZ localize to mitochondria and are required for the maintenance of normal mitochondrial morphology

Paul R Gilson  1 , Xuan-Chuan Yu, Dale Hereld, Christian Barth, Amelia Savage, Ben R Kiefel, Sui Lay, Paul R Fisher, William Margolin, Peter L Beech
Affiliations
Comparative Study

Two Dictyostelium orthologs of the prokaryotic cell division protein FtsZ localize to mitochondria and are required for the maintenance of normal mitochondrial morphology

Paul R Gilson et al. Eukaryot Cell. 2003 Dec.

Abstract

In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszA-green fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.

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Figures

FIG. 1.
FIG. 1.
Mitochondrial morphology in wild-type Dictyostelium vegetative amoebae and the Δfsz mutants. The mitochondria are dark cytoplasmic organelles in bright-field (phase-contrast) images and bright fluorescent organelles when stained with MitoTracker Red. Arrowheads show examples of intense MitoTracker Red-staining regions within the mitochondria (SMBs). See the text for descriptions and Table 1 for frequencies of cells with mitochondrial spheres, rods, or tubules. Scale bar = 5 μm.
FIG. 2.
FIG. 2.
FszA formed belts and puncta within the mitochondria of Dictyostelium. (A and B) Immunofluorescence of Dictyostelium amoebae labeled with anti-FszA (green) and MitoTracker Red (red). (A) FszA formed belt-like (arrows) or punctate (arrowheads) structures in spherical mitochondria. (B) In rod-shaped mitochondria, one or two FszA foci were detected (arrowheads), often located near the ends of the organelles. Scale bar = 5 μm. (C to E) Immunoelectron microscopy with anti-FszA indicated that FszA (arrowheads) was located within the mitochondrion but did not concentrate within the electron-dense SMB (arrow) (C). Scale bars = 1 μm. (F and G) Expression of FszA-GFP in Dictyostelium. (F) At low expression levels, FszA-GFP was targeted to the mitochondria where it mostly formed a single belt in each spherical organelle (arrowhead). (G) At high expression levels, FszA-GFP formed extended helices (arrowheads) in long, tubular mitochondria. Scale bar = 5 μm.
FIG. 3.
FIG. 3.
FszB-GFP localizes to an SMB in Dictyostelium amoebae. (A and B) FszB-GFP accumulated within mitochondria to regions called SMBs that stained brightly with MitoTracker Red. SMBs are present in spherical (A) and tubular (B) organelles. (B) Mitochondria have been outlined. Usually, a single SMB was detected near the end of tubular mitochondria (arrow), but occasionally one or two additional SMBs are also visible within an organelle (arrowheads). (A and B) Scale bars = 5 μm. (C to E) Immunoelectron microscopy with anti-GFP gold demonstrates that FszB-GFP almost exclusively localized to the electron-dense SMB and in mitochondrial tubules this structure was often near one end (E). Scale bar = 0.5 μm.
FIG. 4.
FIG. 4.
Double labeling of FszA and FszB in Dictyostelium cells. FszA (red) has been detected by immunofluorescence in cells transformed with FszB-GFP (green). (A) In more than half the spherical and rod-shaped mitochondria, one FszB punctum paired with or partially colocalized with an FszA punctum (arrowheads). (B) Tubular mitochondria usually contained a single FszB punctum and two or more FszA foci dispersed along the length of the organelle. FszA and FszB usually formed adjacent or overlapping puncta near one end of the mitochondrion (arrowheads). Scale bars = 5 μ m.
FIG. 5.
FIG. 5.
At low expression levels, FszA-GFP partially complemented the loss of FszA in ΔfszA cells. A phase-contrast, bright-field image of several closely packed and flattened ΔfszA amoebae transformed with FszA-GFP is shown, and the outlines of the cells have been traced in white. Colabeling with MitoTracker Red showed that individual amoebae contained rod (R)- or tubule (T)- shaped mitochondria. The level of FszA-GFP expression, ascertained by intensity of GFP fluorescence, was high (H) or low (L). Provided FszA-GFP was expressed at low levels in ΔfszA cells, the fusion protein reduced the percentage of cells with tubular mitochondria by increasing the proportion of rods (Merge; Inset 1). FszA-GFP localized to the midpoints of mitochondria that may have been undergoing fission (Inset 1, arrowheads). When overexpressed, FszA-GFP (Inset 2, arrowheads) appeared to be unable to reduce mitochondrial tubule formation. Scale bar = 5 μm.
FIG. 6.
FIG. 6.
Localizations of FszA and FszB appeared to be independent of one another. (A) In phase-contrast images of ΔfszB cells, mitochondrial tubules appear as dark, elongated shapes. Immunofluorescence of these cells with anti-FszA revealed that FszA still localized to the mitochondria and continued to form multiple foci in the tubular organelles (arrowheads). (B) In ΔfszA cells, FszB-GFP continued to concentrate in the submitochondrial structure, the SMB (arrowheads). Scale bar = 5 μm.
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