PMC

a,b, Mitochondrial division was imaged live in Sk-Mel2 (14 events from 144 cells). c,d, and PtK cells (26 events from 254 cells). Highlighted insets show the spatiotemporal dynamics of Drp1 (arrows) and Dyn2 (arrowheads) prior to and during mitochondrial division (a,c), Line-scan analysis of mean fluorescence intensity (MFI) verifying Drp1 and Dyn2 dynamics (b,d). e, A cartoon schematic depicts the frame prior to division that determines the presence of division factors (Dyn2 and Drp1) at the site of division, and the frame after division that determines the interactions of division factors with the ends of daughter mitochondria, which are summarized in f. The dashed line in (e) represents area analyzed by line-scan. g, Lifetimes of Drp1 and Dyn2 puncta on mitochondria before and after division were analyzed from 21 division events. Error bars represent s.e.m.. b,d, Scale bars for whole cell panels and the inset panel are 10 μm and 1 μm, respectively.

a, Representative example of mitochondrial division (6 events from 108 cells) in COS-7 cells expressing mito-BFP (gray), mCh-Drp1 (red), and Dyn2-mNeon (green) (). The highlighted insets show the temporal and spatial dynamics of Drp1 (arrows) and Dyn2 (arrowheads) prior to and during mitochondrial division. Scale bars for whole cell panels and the inset panel are 10 μm and 1 μm, respectively. b, The cartoon schematic identifies two temporal moments of interest with respect to Drp1, Dyn2, and mitochondrial dynamics, the frame prior to and the frame after division, with a dashed line that identifies the region that was analyzed by line-scan. c, Line-scan analysis of Drp1 and Dyn2 leading up to and following mitochondrial division. d, Representative example mitochondrial division (27 events from 49 cells) in PtK1 cells expressing mito-BFP (gray), mCh-Drp1 (red), and GFP-Mff (green). The highlighted insets show the temporal and spatial dynamics of Drp1 (arrows) and Mff (arrowheads) prior to and during mitochondrial division. Scale bars for whole cell panels and the inset panel are 10 μm and 1 μm, respectively. e, A cartoon schematic identifies the two temporal moments of interest with respect to Mff, Drp1, and mitochondrial dynamics. F, Line-scan analysis of Mff and Drp1 leading up to and following mitochondrial division. Note, that the interaction between Drp1 and its adaptor, Mff, is maintained throughout the process of mitochondrial division, in contrast, Dyn2 associates with only one daughter mitochondrion.

Cortical enrichment of GFP-INCENP at the division plane requires MKlp2 and INCENP-actin binding. (A) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a HeLa cell expressing GFP-INCENP treated with control siRNA. Pink arrowhead denotes cortical GFP-INCENP fluorescence; yellow arrowhead marks GFP-INCENP fluorescence on a midzone bundle. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with control siRNA. Asterisks mark peaks of GFP-INCENP localized to midzone bundles. n = 10 cells. (B) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a cell expressing GFP-INCENP treated with MKlp2 siRNA. Pink arrowhead denotes cortical GFP-INCENP fluorescence. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with MKlp2 siRNA. (C) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a HeLa cell expressing GFP-INCENP CR treated with control siRNA. Pink arrowhead denotes cortical GFP-INCENP CR fluorescence; yellow arrowhead marks GFP-INCENP CR fluorescence on a midzone bundle. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP CR fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with control siRNA. Asterisks mark peaks of GFP-INCENP CR localized to midzone bundles. n = 10 cells. (D) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a HeLa cell expressing GFP-INCENP CR treated with MKlp2 siRNA. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP CR fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with MKlp2 siRNA. n = 9 cells. (E) Quantification of cortical peak intensities of GFP-INCENP or GFP-INCENP CR from line scans across the YZ projection as described in A–D. Three adjacent peak intensity values were averaged for each individual cell. n ≥ 9 cells,*p < 0.05.

Altered expression of the Flii gene results in proliferating basal cells adopting different cell division patterns during late embryonic development. (A) Representative image of proliferating basal cell going through asymmetric division with division plane perpendicular to the basement membrane, (B) symmetric division with division plane parallel to the basement membrane and (C) uncategorized division with division plane diagonal to the basement membrane. Triple immunofluorescent staining of PCNA (pseudo-stained yellow) identifying proliferating cell, pH3 (pseudo-stained red) identifying mitotic chromosomes during metaphase of proliferation and γ-tub (pseudo-stained green) identifying centrosomal axis. Nuclear DAPI staining is shown as pseudo-stained blue. The epidermal-dermal junction is shown by white dash lines. Scale bars, 5 μm. (D) Cartoon summarizing the three division categories. (E) Quantification of proliferating basal cells going through the asymmetric division with perpendicular division plane and (F) symmetric division with parallel division plane and as % of total proliferating events with an average of 100 proliferating events per group at E16, E17, E19 and P21 in Flii^+/−, Flii knockdown; WT, wild-type; FIii^Tg/Tg, Flii overexpressing mouse skin. N = 6. Mean ± SEM.