PMC

HeLa cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 and stained for ELMOD2 (green, left) and cytochrome c (red, middle). ELMOD2 staining overlaps extensively with that of cytochrome c (Merge, right).

HeLa cells were transfected with plasmids directing expression of ARL2, ARL2[T30N], ARL2[K71R], ARL2[Q70L], or with ARL2 siRNA #1. Cells were fixed 48 hours later and stained for α-tubulin, as described under Materials and Methods. Representative confocal images are shown, though the extent of microtubule density loss with ARL2[T30N] and ARL2[Q70L] vary and is more severe with the latter.

(A) HeLa cells were transfected with either no siRNA (control), each of two individual siRNAs (siRNA-1 and siRNA-2), or a pool of four siRNAs (siRNA pool) and collected 48 hours after transfection for ATP determinations, as described under Materials and Methods. A single or double asterisk indicate statistically significant differences at p<0.05 and p<0.01, respectively. (B) HeLa cells were transfected with ARL2, ARL2[T30N], or ARL2[K71R] and assayed for ATP levels as in (A).

HeLa cells were fixed in 4% paraformaldehyde prior to permeabilization in either 0.02% (two upper rows) or 0.1% w/v digitonin (bottom row) for 10 minutes at room temperature. Cells were then processed for imaging using dual labeling for ARL2 (green, left) and either cytochrome c (top row, middle panel) or HSP60 (lower two rows, middle panels), as markers of the IMS and matrix, respectively. Merged images are shown on the far right in each case.

HeLa cells were co-transfected with either GFP or GFP-DRP1[K38A] and empty vector, ARL2, or ARL2[T30N]. (A) Cells were fixed 24 hours post-transfection and immunostained for HSP60. Representative GFP positive cells are shown. Insets show magnified regions from the cell periphery where the lower densities better highlight mitochondrial morphology. Cells expressing wild type ARL2 have similar mitochondrial morphology to cells transfected with empty vector (not shown). (B) Cells expressing GFP-DRP1[K38A] were scored for fragmented mitochondria, using the criteria illustrated in . Cells were quantified from three independent experiments.

(A) HeLa cells were either mock transfected (Control, left panels in A and B), or transfected with the SmartPool of ARL2 siRNAs (right panels in A and B), and the next day were fixed and stained for cytochrome c, as described under Materials and Methods. Cytochrome c staining is shown in red and overlaid onto phase contrast images, along with nuclear (Hoechst) stain, shown in blue. Representative cells are shown. (B) Cytochrome c staining is shown in white, with the cell borders outlined in red to highlight the perinuclear clustering in cells lacking ARL2 activity.

(A) HeLa cells were transfected with a SmartPool or individual siRNAs directed against ELMOD2 and two days later cells were harvested, total protein determined, and equal amounts (40 µg) of total cellular homogenates were resolved in denaturing polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and probed with our rabbit polyclonal antibody to ELMOD2 (upper panel) or α-tubulin (lower panel). Only the relevant regions of the gels are shown. Asterisk indicates a non-specific band, migrating slightly faster than ELMOD2, which is not competable with antigen. (B) HeLa cells were transfected with either no siRNA (Mock) or siRNA directed against ELMOD2 and two days later cells were fixed and stained with Mitotracker and Hoechst DNA stain. Representative cells are shown. Note the proximity of mitochondrial staining to the nuclear stain and the lack of peripheral mitochondrial staining in ELMOD2 knockdown cells.

HeLa cells were transfected with plasmids directing expression of mito-GFP or mito-GFP plus ARL2 siRNAs. After 24 hours cells displaying normal appearing mitochondria were imaged over time, as described in Materials and Methods. Examples of control (A) and knockdown (B) cells are shown, with mito-GFP fluorescence in the top panels, blow ups of the boxed areas bottom left, and kymographs from that region shown as a function of time below right. Colored lines showing tracking of individual mitochondria are shown in the bottom left of each panel with different colors indicating different particles tracked. (C) Velocities of organelle movements were determined as described under Materials and Methods and averaged over the entire eight hour recording period. HeLa cells were depleted of ARL2 by siRNA (solid bars) or transfected to over-express wild type (ARL2) or the dominant negative mutant (ARL2[T30N] (open bars)). Lysosome movements were recorded with time lapse imaging in control or ARL2 siRNA cells by incubating cells with LysoTracker Red 30 minutes prior to imaging. Double asterisks indicate statistically significant differences (p<0.01).

(A) HeLa cells were transfected with either no siRNA (control), each of two individual, sequence-independent siRNAs (siRNA-1 and siRNA-2), or a pool of four siRNAs (siRNA pool) and harvested 48 hours after transfection. Equal amounts (25 µg) of total cellular homogenates were resolved in denaturing polyacrylamide gels. The immunoblot was probed with rabbit polyclonal antibody to ARL2 (upper panel) or actin (lower panel). Only the relevant regions of the gels are shown. (B) HeLa cells were transfected with plasmids directing expression of either ARL2 (left), or ARL2[T30N] (right), and stained for cytochrome c, as described under Materials and Methods. Representative widefield images with deconvolution of z-stacks are shown. (C) HeLa cells were fixed and stained with an HSP60 antibody and data captured using confocal microscopy. Examples of cells from a mock transfected cell population showing tubular (mostly tubular mitochondria), intermediate (mix of tubular and spherical mitochondria), and fragmented (almost all spherical) mitochondria are shown. (D) HeLa cells were co-transfected with mito-GFP and either ARL2 or ARL2[T30N] (for overexpression, scored at 24 hours), or mito-GFP plus ARL2 SmartPool or no siRNA (for knockdown, scored at 48 hours). Mito-GFP expressing cells were scored using the criteria in C, and are expressed as percentages of the transfected cell population. At least 200 cells per condition in three independent experiments were scored. Differences between control and experimental were significant to p<0.01 in the tubular and fragmented categories for both ARL2[T30N] and ARL2 siRNA.