The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast

Abstract

The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast. Here we show that dnm1 mutations convert the mitochondrial compartment into a planar 'net' of interconnected tubules. We propose that this net morphology results from a defect in mitochondrial fission. Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division. The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion. dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains. These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.

Figure 1. Mitochondrial membranes form nets in dnm1 mutant cells

Morphology of GFP-labelled mitochondrial membranes (pDO12) in b, wild-type (strain JSY3096), d, f, dnm1Δ (JSY3097), h, mdm20Δ (JSY3094), and j, dnm1Δ mdm20Δ (JSY3095) cells at 25 °C. The corresponding differential interference contrast images are shown in a, c, e, g, i. The four strains are sister spores from a single tetrad. Scale bar represents 5 μm.

Figure 2

Mitochondrial-membrane morphology in dnm1Δ and dnm1Δ mdm20Δ mutant cells.

Figure 3. Transmission electron microscopy and serial reconstruction of a mitochondrial net

a, Section through a mitochondrial net (m) in a dnm1Δ mdm20Δ cell (strain JSY3095). Black arrowheads mark ‘holes’ in the mitochondrial compartment. b, Computer reconstruction of a mitochondrial net (grey and white) generated from 12 serial sections, including the image shown in a. Scale bars represent 1 μm.

Figure 4. Dnm1 localizes to mitochondrial constriction sites and mitochondrial tips

The distribution of the Dnm1–HA_cp protein (black arrowheads; strain JSY1781) was determined by immunogold labelling of ultrathin cryosections. Gold particles were found concentrated at the tips of mitochondrial tubules proximal to the plasma membrane (b, c, f), at the tips of mitochondrial tubules that may be newly divided (d, e, h), and at putative mitochondrial constriction/division sites (i–l). g, Gold particles are also detected on mitochondria at sites that are not constricted. a, No mitochondrial labelling was detected in cells lacking the Dnm1–HA_cp protein (strain JSY3097). m, mitochondria; pm, plasma membrane; v, vacuole. Scale bars represent 0.1 μm.

Figure 5. dnm1 mutations block mitochondrial fragmentation in fzo1-1 cells

Morphology of GFP-labelled mitochondrial membranes (carrying plasmid pVT100UGFP) in b, wild-type (strain JSY3162), d, fzo1-1 (JSY3161), f, dnm1Δ (JSY3164), and h, j, dnm1Δ fzo1-1 (JSY3163) cells at 37 °C. The corresponding differential interference contrast images are shown in a, c, e, g, i. The four strains are sister spores from a single tetrad. Scale bar represents 5 μm.

Figure 6. Mitochondrial-membrane morphology in dnm1 and dnm1 fzo1-1 mutant cells

The asterisk indicates that 5% of dnm1Δ fzo1-1 cells contain visible nets at 37 °C.

Figure 7. Mitochondrial fusion is not restored in dnm1 fzo1-1 cells

Wild-type (a–d), fzo1-1 (e–h), dnm1Δ (i–l) and dnm1Δ fzo1-1 (m–p) cells of opposite mating type were labelled with either mito-GFP (b, f, j, n) or MitoTracker CMXRos (c, g, k, o) and mated at 37 °C. Images of zygotes were obtained by confocal microscopy and mitochondrial fusion was assessed by examining the overlayed mito-GFP and MitoTracker images (d, h, l, p). The ‘z’ in i designates the zygote visualized in j–l. Scale bars represent 2 μm.