Effects of Fcj1-Mos1 and mitochondrial division on aggregation of mitochondrial DNA nucleoids and organelle morphology

Abstract

Mitochondrial DNA (mtDNA) is packaged into DNA-protein complexes called nucleoids, which are distributed as many small foci in mitochondria. Nucleoids are crucial for the biogenesis and function of mtDNA. Here, using a yeast genetic screen for components that control nucleoid distribution and size, we identify Fcj1 and Mos1, two evolutionarily conserved mitochondrial proteins that maintain the connection between the cristae and boundary membranes. These two proteins are also important for establishing tubular morphology of mitochondria, as mitochondria lacking Fcj1 and Mos1 form lamellar sheets. We find that nucleoids aggregate, increase in size, and decrease in number in fcj1 and mos1 cells. In addition, Fcj1 form punctate structures and localized adjacent to nucleoids. Moreover, connecting mitochondria by deleting the DNM1 gene required for organelle division enhances aggregation of mtDNA nucleoids in fcj1 and mos1 cells, whereas single deletion of DNM1 does not affect nucleoids. Conversely, deleting F1Fo-ATP synthase dimerization factors generates concentric ring-like cristae, restores tubular mitochondrial morphology, and suppresses nucleoid aggregation in these mutants. Our findings suggest an unexpected role of Fcj1-Mos1 and organelle division in maintaining the distribution and size of mtDNA nucleoids.

FIGURE 1:

Fcj1 and Mos1 are required for mtDNA nucleoid size. (A) WT, fcj1Δ, mos1Δ, fcj1Δmos1Δ, aim5∆, aim13∆, aim37∆, and mos2∆ cells expressing Su9-RFP (Mt) were grown in SGalSuc medium to early log phase and stained with DAPI. Cells were examined by DIC and fluorescence microscopy. Dotted lines outline cells based on DIC images. (B) Quantification of cells with larger mtDNA nucleoids. Cells that have nucleoids with diameter of 0.5–1.0 μm (white) and >1.0 μm (gray) were scored. At least 200 cells were examined in each experiment (n = 3). (C) WT and fcj1Δ cells expressing Mmm1-GFP were grown to log phase in SGalSuc medium and stained with DAPI for 15 min. Cells were viewed by fluorescence microscopy. (D) WT, fcj1Δ, mos1Δ, and fcj1Δmos1Δ cells expressing Abf2-GFP were stained with DAPI.

FIGURE 2:

Fcj1p-GFP forms puncate structures next mtDNA nucleoids. (A) GFP fusion proteins of Fcj1, Mos1, and Ups1 were expressed by integrating the GFP gene at their 3′ end in chromosomes. The GFP strains were transformed with Su9-RFP (Mt), grown to log phase, and stained with DAPI. Boxed regions show magnified images. (B) Fcj1-GFP was expressed in WT, mos1∆, aim5∆, aim13∆, aim37∆, and mos2∆ cells. The GFP strains were transformed with matrix-targeted Su9-RFP (Mt). Cells were grown to log phase, stained with DAPI, and observed by fluorescence microscopy. Boxed regions show magnified images.

FIGURE 3:

Round, hollow mitochondria are formed in fcj1Δ, mos1Δ, and fcj1Δmos1Δ cells that contain large mtDNA nucleoids. (A) WT, fcj1Δ, mos1Δ, and fcj1Δmos1Δ cells expressing Su9-RFP were grown in SGalSuc medium to early log phase and stained with DAPI. Cells were examined by DIC and fluorescence microscopy. Large spherical mitochondria are indicated by asterisks. (B) Quantification of cells that contain large, hollow mitochondria. At least 600 cells were examined in each experiment (n = 3). (C) Large, hollow mitochondria were analyzed by several mitochondrial markers, including OM45-GFP for the outer membrane (OM), Ups1-GFP for the IMS, and MitoTracker for the IM, in fcj1∆mos1∆ cells. mtDNA was labeled by either DAPI (with OM45p-GFP and Ups1p-GFP) or Abf2-GFP (with MitoTracker). (D) The ER, vacuoles, and the cytosol were marked by Sec63-GFP, FM4-64, and GFP, respectively. (E) WT and fcj1Δmos1Δ cells with (rho⁺) or without (rho⁰) mtDNA expressing Su9-RFP were examined by fluorescence microscopy. Dotted lines outline cells based on DIC images. Large, spherical mitochondria are indicated by an arrowhead. (F) Quantification of cells containing large, hollow mitochondria. At least 600 cells were examined in each experiment (n = 3).

FIGURE 4:

Loss of Atp20 and Atp21 rescues nucleoid defects in fcj1∆mos1∆ cells. (A) WT, atp20∆, atp21∆, fcj1∆mos1∆, fcj1∆mos1∆atp20∆, and fcj1∆mos1∆atp21∆ cells expressing Su9-RFP were stained with DAPI. (B, C) Quantification of cells that contain increased size of mtDNA nucleoids (B) and mitochondrial morphology (C). At least 200 cells were examined in each experiment (n = 3).

FIGURE 5:

Connecting mitochondria enhances aggregation of nucleoids in fcj1∆, mos1∆, and fcj1∆mos1∆ cells. (A) dnm1∆, dnm1∆fcj1∆, dnm1∆mos1∆, and dnm1∆fcj1∆mos1∆ cells expressing Su9-RFP were stained with DAPI. (B) Quantification of cells that contain increased size of mtDNA nucleoids. At least 150 cells were examined in each experiment (n = 3). (C) Quantification of average nucleoid diameter in the indicated cells. At least 150 nucleoids were examined in each experiment (n = 3). (D) Serial dilutions of cells were spotted onto YPD and YPGE medium and incubated at 30°C for 2 and 5 d, respectively. (E) Quantification of cells that contain mtDNA. At least 600 cells were examined in each experiment (n = 3). (F) Quantification of cells that contain increased size of nucleoids. At least 150 cells were examined in each experiment (n = 3).

FIGURE 6:

Ultrastructural analysis of mitochondria. (A, B) Electron micrographs of mitochondria in WT and the indicated mutants. Boxed regions show magnified images in B. An arrow indicates a vacuole in B. (C) Quantification of the density of crista junctions and concentric ring-like cristae relative to the length of mitochondrial perimeter. At least 22 mitochondria were analyzed for each genotype.