Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Western blot of U2OS cells and Drp1 KI U2OS cells showing expression level of GFP-Drp1 and untagged Drp1 with two dilutions of extract loaded (1x and 2x dilution). (B) Quantification of un-tagged Drp1 and GFP-Drp1 in WT U2OS and Drp1 KI cells from western blots (normalized to tubulin level). Error bars, S.D. (C) Cell proliferation assay (Alamar blue). Three replicates taken for each time point (median shown, with error bars representing minimum and maximum). Starting density: 5000 cells/24-well plate. Representative result from two independent experiments. (D) Drp1-independent punctae in Cos7 cells. Left: merged image of a live COS7 cell transiently expressing mito-BFP (mitochondria, blue), eBFP2-PMP20 (peroxisome, gray), GFP-Drp1(green) and ER-TagRFP (ER, red). Right: insets from boxed region. Yellow arrows denote independent Drp1 punctae associating with ER. (E) Graph depicting the degree of association between independent Drp1 punctae and ER in Cos7 cells, during 3 minute movies imaged every 1.5 sec. 175 independent Drp1 puncta were analyzed from 12 ROIs from 12 cells as shown in A. Stale association 94.2%±7.6%; Partial association 2.5%±4.7%; No association 3.3%±5.4%. Scale bar, 10 μm in whole cell image; 2 μm in inset. Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Western Blot, Expressing, Proliferation Assay

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Drp1 distribution in GFP-Drp1 knock-in U2OS cells (GFP-Drp1-KI cells). Left: merged image of a live Drp1 KI cell transiently expressing mCherry-mito3 (red) and eBFP2-peroxisome (blue). Drp1 in green. Right: insets from boxed region at three time points. Yellow arrow denotes independent Drp1 puncta; blue arrow indicates peroxisome-associated Drp1; red arrow denotes mitochondrially-associated Drp1. white arrowhead denotes example of Drp1 puncta localizing at the interface of mitochondrion and peroxisome. (B) Venn diagram of Drp1 distribution in GFP-Drp1-KI cells expressing mitochondrial and peroxismal markers. Black circles denote mitochondrially associated Drp1 punctae; blue circles denote peroxisomal associated Drp1 punctae (Pex); red circles denote independent Drp1 punctae (Ind.). The percentage of Drp1 punctae in each category is average from 10 consecutive frames with 12 sec time intervals from whole-cell videos. Five cells measured (10,761 punctae). (C) Four-color imaging of a live Drp1 KI cell expressing mPlum-mito3 (Mito, gray); eBFP2-PMP20 (Peroxisome, blue); and ER-tagRFP (ER, red);Drp1 in green. Yellow arrows denote independent Drp1 puncta stably associating with ER. Video 1. (D) Time lapse montage showing de novo assembly of an independent Drp1 punctum (yellow arrow) on an ER tubule. Imaging as in panel C. (E) Graph depicting the degree of association between independent Drp1 punctae and ER during 2.5-min videos imaged every 1.6 sec. 30 ROIs from 25 GFP-Drp1-KI cells analyzed (1003 punctae). Mean values from ROIs: 76.7%±11.7% stable association between Drp1 punctae and ER (no apparent dissociation from ER in any frame); 8.9%±9.5% partial association; 14.4%±8.0% no association. Scale bar, 10 μm in whole cell image in (A); 2 μm in inset in (A), and in (C)&(D). Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Knock-In, Expressing, Imaging, Stable Transfection

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Merged image of GFP-Drp1-KI cell expressing mCherry-mito3 (red); eBFP2-peroxisome (blue); and treated with transferrin-Alex647 (endosomes, gray). GFP-Drp1 in green. (B) Graph depicting degree of association between independent Drp1 punctae and transferrin-labeled membranes during 3 min videos imaged every 1.7 sec. 10 ROIs from 10 cells, 342 Drp1 punctae. (C) Time-lapse of inset from (A) showing independent Drp1 punctae distinct from transferrin-labeled endosomes. White arrows denote endosomes, yellow arrow denotes independent Drp1 puncta. (D) Time-lapse ROI of a GFP-Drp1-KI cell expressing mStrawberry-Rab4b (red), and ER-eBFP2 (ER, green). Drp1 in blue. Arrows defined as in C. (E) Graph depicting the degree of association between independent Drp1 punctae and Rab4b-labeled membranes during 3 min videos imaged every 1.8 sec. 10 ROIs from 10 cells, 152 Drp1 punctae. (F) Time-lapse of a Drp1 KI cell expressing mStrawberry-Rab7a (red),and ER-eBFP2 (ER, green). GFP-Drp1 is blue. Arrows defined as in C. (G) Graph depicting the degree of association between independent Drp1 punctae and Rab7a labeled membranes during 3 min videos imaged every 1.5 sec. 10 ROIs from 10 cells, 177 Drp1 punctae. Scale bar, 10 μm in (A); 5 μm in (C); 2 μm in (D) and (F). Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Expressing, Labeling

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) GFP-Drp1-KI cells transiently transfected with Tom20-mCherry (red) and eBFP2-PMP20 (peroxisomes, blue). Drp1 in green. Whole cell overlay on left, and time course of the indicated ROI on right (top, Tom20 alone. Bottom, merged image). (B) Zoom of indicated region of 0 sec time point in A, showing Drp1 and Tom20. No peroxisomes detected in this region. Yellow arrow denotes independent Drp1 puncta with no associated Tom20 signal. (C) Graph of percentage of independent Drp1 puncta overlaying with Tom20 (16 independent puncta from five ROI analyzed). One instance of overlap observed in ROI 4. Scale bar, 10 μm in whole cell in (A), 2 μm in inset in (A), 1 μm in (B). Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Transfection

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Three-color time lapse images of live GFP-Drp1-KI cell expressing mCherry-mito7 (mitochondria, red), eBFP2-PMP20 (peroxisome, blue) and Drp1 in green. An independent Drp1 puncta (yellow arrow) transfers to a mitochondrion and then translocates along the mitochondrion with no division in the observation time period. Video 2. (B) Four-color time lapse images of live GFP-Drp1-KI cell expressing mito-BFP(mitochondria, gray), mPlum-PMP20 (peroxisome, blue), ER-tagRFP (ER, red) and GFP-Drp1 in green. Yellow arrow denotes an ER-bound Drp1 puncta transferring to mitochondrion. Video 3. (C) Three-color time lapse images of live GFP-Drp1-KI cell expressing mCherry-mito7 (mitochondria, red), eBFP2-PMP20 (peroxisome, blue) and Drp1 in green. Two Drp1 punctae transfer to constriction sites, followed by division. Cells treated with ionomycin (4 μM) to stimulate mitochondrial division. Video 4. (D) Four-color time lapse images of live GFP-Drp1-KI cell expressing mito-BFP(mitochondria, red), mPlum-PMP20 (peroxisome, gray), ER-tagRFP (ER in blue) and GFP-Drp1 in green. Yellow arrow denotes an independent Drp1 puncta transferring to mitochondrion. Cells treated with ionomycin (4 μM) to stimulate mitochondrial division. Video 5. Scale bar: 2 μm in all images. Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Expressing, Transferring

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Endogenous Mff localization in a fixed U2OS cell by immuno-fluorescence. Cells labeled with anti-Tom20 (mitochondria, blue), anti-PMP70 (peroxisomes, gray), anti-Mff (green); and transfected with ER-TagRFP (ER, red). Left: scrambled siRNA. Right: Mff siRNA. Independent punctae, yellow arrows. (B) Graph depicting the percentage of co-localization between independent Mff punctae and ER in U2OS cells (endogenous Mff). 54 independent Mff punctae were counted from 5 ROIs from 4 cells. Mean values from ROIs: 89.3 ± 6.7% co-localized Mff with ER, 6.0 ± 6.l% not co-localized, 4.8 ± 7.3% unclear localization. (C) Live-cell time lapse of GFP-Mff-S (green) in U2OS cell also expressing mCherry-mito3 (gray); eBFP2-peroxisome (blue); and E2-Crimson-ER (red). Yellow arrows denote independent Mff punctae associating with ER; blue and gray arrows indicate peroximal and mitochondrial Mff, respectively. Video 6. (D) Graph depicting the degree of association between independent GFP-Mff-S punctae and ER from live-cell videos as in C (2.5 min videos imaged every l.5 sec). 34 ROIs from 30 U2OS cells analyzed (44l independent Mff punctae). Mean values from ROIs: 86.1 ± l7.l% stably associated Mff punctae with ER, ll.0 ± l6.8% partially associated, 4.6 ± 9.4% not associated. (E) U2OS fractionation. Left: LSP, MSP and HSP are low, medium and high-speed pellets. HSS is highspeed supernatant. Marker proteins are: ATP synthase, mitochondria; Sec63, ER; and Pmp70, peroxisomes. Right: sucrose gradient fractionation of the MSS (medium-speed supernatant). (F) Human PEX3-deficient fibroblast fractionation, similar to U2OS fractionation. Scale bar, l0 μm in whole cell image in (B); 2 μm in inset in (B) and in (D). Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Fluorescence, Labeling, Transfection, Expressing, Stable Transfection, Fractionation, Marker

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Time-lapse from region of U2OS cell expressing mCherry-mito3 (red); eBFP2-peroxisome (blue); GFP-Mff-L (green); and E2-Crimson-ER (gray). Yellow arrow denotes independent Mff puncta associating with ER tubules. (B) Graph depicting the degree of association between independent Mff-L punctae and ER during 3 min videos imaged every 2 sec. 10 ROIs from 8 U2OS cells, l67 independent Mff punctae. (C) GFP-MiD51 does not display ER-associated punctae independent of mitochondria. Left panel: merged image of a live cell expressing MiD51-GFP (green), mitoBFP (blue) and ER-tagRFP (ER, red). Right: insets. Scale bar, 2 μm in A; 10 μm in whole cell in C, and 5 μm in inset of C. Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Expressing

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Endogenous Fis1 localization in fixed U2OS cells by immuno-fluorescence. Cells labeled with anti-Tom20 (mitochondria, blue), anti-PMP70 (peroxisomes, gray), anti-Fis1 (green); and transfected with ER-TagRFP (ER, red). Left: scrambled siRNA. Right: Fis1 siRNA. Independent punctae, yellow arrows. (B) Graph depicting the percentage of co-localization between independent Fis1 punctae and ER in U2OS cells by immuno-fluorescence (endogenous Fis1). ll7 independent Fis1 punctae counted from 9 ROIs from 4 cells. Mean values from ROIs: 79.9 ± ll.3% co-localized Fis1 punctae with ER, 6.0 ± 7.4% not co-localized, l4.l ± l0.2% un-clear localization. (C) Live-cell time lapse of GFP-Fis1 in U2OS cell also expressing mCherry-mito3 (gray); eBFP2-peroxisome (blue); and E2-Crimson-ER (red). Right: individual frames from the time course of boxed region, showing independent Fis1 punctae associated with ER (yellow arrow) next to a peroxisome that is positive for Fis1 (blue arrow). (D) Graph depicting the degree of association between independent GFP-Fis1 punctae and ER from livecell videos as in C (2.5 min videos imaged every l.7 sec). l6 ROIs from l5 U2OS cells (l00 independent Fis1 punctae) analyzed. Mean values from ROIs: 78.8% ± 26.9% stably associated Fis1 punctae with ER, ll.9 ±l8.2% partially associated, 9.2 ± l4.8% not associated. Scale bar: 10 μm in whole cell image in (A) and (C); 5 μm in inset in (A); 2 μm inset in (C). Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Fluorescence, Labeling, Transfection, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Independent Mff punctae dynamics (Airyscan microscopy time lapse). Left panel showing merged image of a live U2OS cell expressing ER-tagRFP (ER, red); GFP-Mff-S (green); eBFP2-Peroxisome (blue); and mPlum-mito3 (gray). Right panel shows a time lapse series of the inset, with independent Mff punctum associating with ER then transferring to mitochondrion (yellow arrow). Blue arrow indicates peroxisomally associated Mff and white arrow denotes mitochondrial Mff. Scale bar, 2 μm in left panel of, 1 μm in inset. Time in sec. Video 7. (B) Zoom of panel A, showing heterogeneous nature of peroxisomally-associated Mff. Scale bars, 0.5 μm in all images. (C) Dot plot showing diameter of peroxisomal Mff and independent Mff punctae from Airyscan images. 14 peroxisomal Mff (0.42±0.050 μm) and 19 independent Mff punctae (0.22±0.056) analyzed.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Microscopy, Expressing, Transferring

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Left: Merged confocal image of a live GFP-Drp1-KI cell expressing mito-BFP(gray), eBFP2-peroxisome (gray), mStrawberry-Mff-S(red) and pLVX-E2-Crimson-ER (blue). Drp1 in green. Right: Time lapse confocal images of boxed region show example of a Drp1 puncta maturing from an independent Mff puncta (yellow arrows). Video 8. (B) Independent Mff punctae in scramble siRNA treated (left) and Drp1 siRNA treated cells (right). Left: merged image of live U2OS cells transiently expressing GFP-Mff-S(Mff, green), eBFP2-PMP20 (Pex, blue) and mCherry-mito7 (Mito, red). Right: insets from boxed regions in whole cell image. Yellow arrows denote independent Mff punctae. (C) Density of independent Mff punctae in control and Drp1 siRNA-treated U2OS cells, quantified from live cell images of GFP-Mff as (B). Units, number of independent Mff punctae per μm 2 in the ROI. 368 independent punctae from nine control cell ROIs and 106 punctae from nine Drp1 KD cell ROIs. *** denotes p value < 0.0001 by student t-test. (D) Density of independent Mff punctae in control and Drp1 siRNA-treated U2OS cells, quantified from fixed cell immuno-fluorescence of endogenous Mff. Units, number of Mff punctae per μm 2 in ROI. 643 independent punctae from five control cell ROIs and 153 puncta from seven Drp1 KD cell ROIs. *** denotes p value < 0.0005 by student t-test. Scale bar: 10 μm in whole cell images; 2 μm in insets. Time in sec

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Expressing, Control, Fluorescence

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Schematic cartoon of rapamycin-induced Mff recruitment either to OMM (left) or ER (right). “Mff” refers to the cytoplasmic portion of Mff-S. (B) Dynamics of GFP-Mff-FRB translocation to mitochondria upon rapamycin treatment in Mff KO cells. Live cell images of cell transfected with AKAP-FKBP12 (red), GFP-Mff-Cyto-FRB (green), eBFP2-PMP20 (peroxisomes, blue) and mitoBFP (Mitochondria, blue). Rapamycin (final concentration: 10μM) added at time 0. (C) Dynamics of GFP-Mff-Cyto translocation to ER upon rapamycin in rapamycin treatment in Mff KO cells. Live cell images of cells transfected with Sac1-FKBP12 (ER, blue), GFP-Mff-Cyto-FRB (green), and mCherry-mito7 (mitochondria, red). The lower green panel represents GFP-MFF-CytoFRB signal that has been thresholded to remove the cytoplasmic signal. Rapamycin (final concentration: 10μM) added at time 0. (D) Rapamycin-induced mitochondrial division rates in control U2OS cells (l6 ROIs from l5 cells); Mff KO cells (2l ROIs from 2l cells) (P=0.0000l, ***); Mff KO cells transfected with mitochondria-targeted Mff (34 ROIs from 30 cells) (P=0.0l79,*); Mff KO cells transfected with ER-targeted Mff (20 ROIs from l7 cells) (P=0.0049,***); or Mff KO cells transfected with both mitochondria‐ and ER-targeted Mff (34 ROIs from 30 cells)(P=0.4l8l). Statistical analysis based on comparison to control cells by student t-test. (E) Western blot showing Mff and Drp1 expression levels in WT cells, Mff-KO cells, and Mff-KO cells transfected with either the Mff-FRB construct + the mitochondrially-targeted FKBPl2 construct (Mff KO + Mff-mito) or the Mff-FRB construct + the mitochondrially-targeted FKBPl2 construct + the ER-targeted FKBPl2 construct (Mff KO + Mff-ER & Mff-mito). Tubulin and myosin IIA are loading controls. Endogenous Mff runs as a doublet below 37 kDa, whereas the Mff-FRB construct runs at the 37 kDa marker. Scale bar: l0 μm in whole cell images in (B, C);2 μm in insets in (B, C). Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Translocation Assay, Transfection, Concentration Assay, Control, Comparison, Western Blot, Expressing, Construct, Marker

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Left: merged image of a live GFP-Drp1-KI cell before ionomycin treatment, transiently expressing mPlum-mito3 (gray), eBFP2-peroxisome (blue), and ER-tagRFP (ER, red). Drp1 in green. Right: inset from boxed region before (top) and after (bottom) ionomycin treatment (4 μM, l0 min). Yellow arrows denote independent Drp1 maturing upon ionomycin treatment. Video 9. (B) Similar experiment as in A, except cells were pre-treated for l0min with l μM LatA. Video l0. (C) Quantification of independent Drp1 punctae number in response to vehicle treatment (DMSO), ionomycin treatment and LatA pre-treatment followed by ionomycin treatment. 6 ROIs from 6 DMSO treated cells, l6 ROIs from l4 ionomycin treated cells, and 8 ROIs from 6 LatA pre-treated/ionomycin treated cells. Punctae per ROI normalized to l at time of ionomycin addition. Error bar, S.E.M. Arrow indicates time point where ionomycin was added during imaging (time 0). Scale bar: l0 μm in left panels; 2 μm in right panels. Time in sec.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Expressing, Imaging

Journal: bioRxiv

Article Title: Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

doi: 10.1101/190538

Figure Lengend Snippet: (A) Two examples of independent Drp1 punctae maturation in response to ionomycin. Time-lapse images of live GFP-Drp1-KI cell as in . Time indicates sec after ionomycin treatment. Fluorescence intensity levels modulated uniformly across timecourse so that final fluorescence is in linear range (resulting in time 0 fluorescence being undetectable as displayed). Scale bars, l μm. Time in sec. (B) Quantification of mean independent Drp1 punctum intensity in un-stimulated or ionomycin treated conditions. Seven independent Drp1 punctae from un-stimulated cells and eight independent Drp1 punctae from ionomycin treated analyzed. Error bars, S.D. (C) Effect of INF2 KD on independent Drp1 punctae in GFP-Drp1-KI cells transfected with mCherry-mito7 (mitochondria, red) and eBFP2-peroxisome (blue). Drp1 in green. Top is control siRNA, bottom is INF2 siRNA. Right: zoomed images of boxed regions indicated by numbers. Scale bar: l0 μm in left panels; 2 μm in right panels (insets). (D) Quantification of independent Drp1 punctae density in control (scrambled siRNA) and INF2 siRNA cells. l74 independent punctae from seven control cells, 45 independent punctae from nine INF2 siRNA cells. Density expressed as number of independent Drp1 punctae per area of ROI (in μm 2 ). *** denotes p < 0.00l by student’s t-test.

Article Snippet: Tom20-mCherry was previously described in( ). eBFP-Peroxisome was constructed by replacing the CFP sequence of CFP-Peroxisome containing peroxisomal targeting signal 1 (PTS1) (Addgene #54548) with eBFP2, cut from eBFP2 ‐Mito7 (Addgene #55248) with BsrGI/BamHI. mPlum-mito3 was purchased from Addgene (#55988).

Techniques: Fluorescence, Transfection, Control