ER tubules mark sites of mitochondrial division

Abstract

Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.

Figure 1

Mitochondrial constriction and division occurs at ER-mitochondrial contacts in yeast. (A) 3-D models (left panels) of ER (green) and mitochondria (purple) at contact domains were imaged by EM and tomography of high pressure frozen yeast cells. Middle two panels are 2-D tomographs of contact sites (second panels, ER drawn in green) and the corresponding 3-D models of each (third panels). Contact is defined as regions where the ER membrane comes within 30 nm of the mitochondrial membrane and ribosomes are excluded, and is outlined in red (third panel). Right panel is a schematic demonstrating the percentage of the mitochondrial circumference that makes contact with the ER membrane (red is contact, white is not; (19)). The diameter of each mitochondrion at positions of ER contact is shown. Note that regions where the mitochondria are constricted (models a and c) have a high percent of ER wrapping. Additional EM tomographs and analysis of constrictions are shown in Fig. S1A–B. (B) Time-lapse images of yeast cells expressing mito-dsRED and GFP-HDEL (ER). A single focal plane is shown. Arrows indicate sites of mitochondrial division. Corresponding z-series is shown in Fig. S1C. Scale bars: (A) 200 nm; (B) 2 µm.

Figure 2

Mitochondrial division occurs at ER-mitochondrial contact sites in mammalian cells. (A–D) Four examples of mitochondrial division over timecourses shown in Cos-7 cells expressing GFP-Sec61β (ER) and mito-dsRed. The site of mitochondrial division (white arrows) and the position of the newly formed mitochondrial ends (yellow arrows) are shown. Additional examples are included in Fig. S2A. Scale bars: 1 µm.

Figure 3

Dnm1/Drp1-mediated mitochondrial division occurs at ER contact sites. (A) Time-lapse images of wild type yeast cells expressing mito-CFP, GFP-HDEL (ER) and Dnm1-mCherry. A single focal plane is shown. Arrows indicate the site of mitochondrial division, which is notably marked by both ER-mitochondria contact and Dnm1. (B) Merged image of a live Cos-7 cell expressing GFP-Sec61β (ER), mito-BFP, and mCherry-Drp1. (C) Examples of cells as in (B) that show that Drp1 punctae maintain co-localization with positions of ER-mitochondrial contact over time. White arrows indicate Drp1 punctae that maintain contact with both the ER and mitochondria. Yellow arrows indicate a rare example of Drp1 that does not contact the ER. (D) Graph indicating the percentage of mitochondrial Drp1 punctae that co-localize with the ER membrane over a two-minute timecourse. (E) Examples of mitochondrial constrictions at ER contact sites marked by Drp1. Left panels show Cos-7 cells expressing mito-EGFP, BFP-KDEL (ER), and mCherry-Drp1, merged as indicated. Right panels are Linescans drawn through the mitochondria and show the relative fluorescence intensity of mitochondria (green), ER (blue) and Drp1 (red) along its length. White arrows at constrictions on images correspond to black arrows shown on the Linescan. Additional examples are shown in Fig. S4. Scale bars: (A, C, E) 1 µm; (B) 5 µm.

Figure 4

The ER localizes to mitochondrial constrictions prior to Drp1 and Mff recruitment. (A) Examples of mitochondrial constrictions at ER contacts marked by Mff in Cos-7 cells depleted of Drp1. Left and center panels show images of these cells expressing mito-dsRed, BFP-KDEL (ER), and GFP-Mff, merged as indicated. Right panels are Linescans drawn through the mitochondria and show the relative fluorescence intensity of mitochondria (red), ER (blue) and Mff (green) along their length. White arrow position at constrictions corresponds to black arrows on the Linescan. Additional examples are shown in Fig. S6. (B) Western blots with antibody against Drp1 (top panel) or Mff (bottom panel) and GAPDH demonstrate depletion of Drp1 in lysates from cells transfected with siRNA against Drp1 (as in Fig. 4A) or Mff (as in Fig. 4D) compared to control RNAi cells. (C) Table indicating the number of Mff-localized mitochondrial constrictions in Drp1-depleted cells that co-localize with ER tubules, from 23 cells. (D) As in (A), for cells depleted of Mff and expressing GFP-Sec61β (ER; green on Linescan) and mito-dsRed (red on Linescan). Scale bars: (A and C, left panels) 5 µm; (A and C, right panels) 1 µm.