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ER-mitochondria Contacts Couple mtDNA Synthesis With Mitochondrial Division in Human Cells

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ER-mitochondria Contacts Couple mtDNA Synthesis With Mitochondrial Division in Human Cells

Samantha C Lewis et al. Science.

Abstract

Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

Figures

Figure 1
Figure 1. Mitochondrial DNA nucleoids are spatially linked to mitochondria-ER contacts in human cells
(A) Left panels show a merged image of a live U2OS cell expressing mito-BFP, TFAM-GFP and Sec61b-mCherry (ER). Right panel shows the pixel intensity of mito-BFP, TFAM-GFP and Sec61b-mCherry from a line scan drawn along the mitochondrial tubule (dotted line), arrows indicate nucleoid positions. (B) Time-lapse images of a U2OS cell expressing mito-BFP, TFAM-GFP and mRuby-KDEL (ER), a single plane is shown. Arrow indicates a site of persistent co-localization between a TFAM-GFP labeled nucleoid and an ER-mitochondria contact. (C) Number of mitochondrial divisions in U2OS cells spatially linked to TFAM-GFP labeled nucleoids, from 43 cells. (D) Time-lapse images of mitochondrial division (marked by arrow) spatially linked to a TFAM-labeled nucleoid focus in a U2OS cell. (E) Graph indicating the displacement of picogreen-labeled nucleoids in live Cos7 cells over 12.5 minutes as a function of their intra-mitochondrial position. Data are mean +/− SD. (F) Merged image of a live U2OS cell expressing mito-BFP and Sec61b-mCherry (ER). Right panels show examples of mitochondrial constrictions co-localized with ER tubules. (G) Graph indicating the percentage of persistent mitochondrial-ER co-localizations that become sites of mitochondrial constriction or division over 5 minutes in live U2OS cells. Scale bars: (A, B, D, F inset) 2 um; (F) 10 um.
Figure 2
Figure 2. POLG2-GFP is specifically recruited to replicating nucleoids in live cells
(A) A live U2OS cell expressing mito-BFP and POLG2-GFP was imaged (left panel), then stained with Picogreen DNA dye and re-imaged ten minutes later (right panel). Inset is 488 channel signal intensity in an example mitochondrion, left panel, and the same organelle after Picogreen staining, right panel. Magnified 2X. (B) The number of mitochondrial POLG2-GFP (n= total 441 foci from 10 cells) or TFAM-GFP foci (n=5,182 foci from 10 cells) per U2OS cell (***p<0.001, two-tailed t test). Data are mean +/− SD. (C) Representative image of a U2OS cell expressing POLG2-GFP and pulse-labeled with 50mM EdU, fixed and stained with DAPI (DNA, blue), Mitotracker (mitochondria, red), anti-GFP AlexaFluor488 conjugate antibody (POLG2-GFP, green), and ClickIt Edu-AlexaFluor647 (nascent DNA, magenta). (D) Observed co-localization between mitochondrial POLG2-GFP and EdU foci in fixed U2OS cells labeled as in C, from 15 cells. Scale bars: (A, C) 10 um, (C inset) 5 um.
Figure 3
Figure 3. Replicating nucleoids mark sites of ER-mediated division
(A) Representative time-lapse images of a U2OS cell expressing mito-BFP, mRuby-KDEL (ER) and POLG2-GFP demonstrating mitochondrial division at a mitochondrial-ER contact site spatially linked to POLG2-labeled nucleoid. (B) Percentage of ERMD events (marked by mRuby-KDEL and mito-BFP as in E) in live U2OS cells that occurred within one micron of a POLG2-GFP labeled nucleoid (n = 15 events from 7 cells, **p<.01, Fisher’s exact test). Significantly more division events occurred at pre-existing POLG2-GFP foci (73.3%) than expected by random chance at stable ER-mitochondria contacts (22.1%). Scale bar: 2um.
Figure 4
Figure 4. Nascent mtDNA is segregated to daughter mitochondria by division
(A) Representative image of live U2OS cells expressing mito-BFP and TFAM-GFP, (top panels) or POLG2-GFP (bottom panels). (B) Significant spatial enrichment of the total population of POLG2-GFP foci within one micron of mitochondrial tips, as compared to TFAM-GFP foci (in dark grey) in live U2OS cells as labeled and imaged in (A); ***p<.001, two-tailed t test. (C) Left panel: In live U2OS cells, the number of TFAM-GFP foci per mitochondrion, but not POLG2-GFP foci, is correlated with mitochondrion length. Linear regression with best-fit line is shown. For TFAM-GFP, adjusted R2 = 0.86, Pearson’s R = 0.92 (****p<.0001). For POLG2-GFP, adjusted R2= 0.15, Pearson’s R= 0.41 (n.s.). Right panel: In fixed Arpe19 cells, the number of Picogreen foci per mitochondrion, but not Edu foci, is correlated with mitochondrion length. Linear regression as in A. For Picogreen, adjusted R2= 0.68, Pearson’s R=0.82 (****p<.0001). For Edu, adjusted R2 =0.07, Pearson’s R= 0.31 (n.s.). (D) Top panel, diagram of Edu pulse-chase experiments in Arpe19 cells. Bottom, empirical cumulative distribution analysis of Edu focus position along mitochondria, demonstrating depletion of pulse-labeled nascent mtDNA from mitochondrial tips over time, towards a simulated random distribution (***p<.001, **p<.01, Kolmogorov-Smirnov test). Scale bar: 5 um.
Figure 5
Figure 5. Replicating nucleoids mark division sites prior to mitochondrial constriction or DRP1 recruitment
(A) Top panel, time-lapse images of a U2OS cell expressing mito-BFP, mCherry-DRP1 and POLG2-GFP. Arrow indicates site of division. Bottom panel, linescan drawn along the mitochondrial tubule to show relative fluorescence intensity of mitochondria, DRP1 division machinery and POLG2 signal for timepoints t = 0s (pre-constriction), t = 45s (post-constriction), and t = 90s (post-division). Scale bar: 1 um. (B) Table indicating the percentage of mitochondrial divisions (marked by mCherry-DRP1 as in A) that occur within one micron of a POLG2-GFP focus, in live U2OS cells (n = 26 events from 22 cells, **p<.01, Fisher’s exact test).
Figure 6
Figure 6. ER tubules license mtDNA synthesis and are required for nucleoid distribution
(A) ER network morphologies in representative Cos7 cells expressing fluorescently tagged ER membrane proteins: Sec61b-GFP, upper left; RTN4A-GFP, upper right; CLIMP63-GFP over-expression, lower left; or Rtn4A-GFP and Climp63-mCherry double over-expression, lower right. Inset regions magnified 5X. (B) Quantification of the number of mitochondrial EdU foci per fixed Cos7 cell following a 4 hour pulse of 50mM EdU, in cells labeled with Mitotracker Red and indicated ER markers, n = 15+ cells per condition (***p<.001, **p<.01, two-tailed t test). (C) Quantification of fluorescence intensity of mitochondrial Edu foci, n = 600+ foci from 15+ cells per condition. (D) Image of fixed Cos7 cell over-expressing CLIMP63-mCherry (ER) following a 4 hour pulse of 50mM EdU in cells labeled with Mitotracker Red. (E) Image of a live Cos7 cell expressing mito-BFP (mitochondria), TFAM-GFP (nucleoids), and over-expressing CLIMP63-mCherry (ER). Left, full field view of entire cell; right, examples of aggregated nucleoids associated with sheet-like ER (top panels) and distributed nucleoids associated with reticular ER (bottom panels). Scale bars: (A) 10 um, (D, E) 10 um, (D inset, E inset) 2 um.

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