Extended Data Fig. 2: AC probes provide higher sensitivity and specificity for imaging sub-organellar F-actin accumulation.
From: Actin chromobody imaging reveals sub-organellar actin dynamics
a) Maximum projection of cells stained with phalloidin and MitoTracker do not show obvious sites of accumulation of actin on mitochondria. Scale bar: 10µm. These results were reproducible across 12 independent experiments. b) Maximum projection of cells expressing an ER marker counterstained with phalloidin do not show obvious accumulation of actin on any specific region of the ER. Scale bar: 10µm. These results were reproducible across 12 independent experiments. c) Expression of AC probes does not alter actin accumulation on mitochondria or the ER. Left graph: HeLa cells were either not transfected or transfected with AC-mito, then stained with MitoTracker and phalloidin. Pearson’s correlation coefficient between MitoTracker and phalloidin was determined for each condition. Right graph: HeLa cells were either transfected with mCherry-ER only or cotransfected with mCherry-ER and AC-ER, then stained with phalloidin. Pearson’s correlation coefficient between mCherry-ER and phalloidin was determined for each condition. N = 10 cells per condition. P-values determined by two-tailed Welch’s t-test. d) Quantification of the percentage of mitochondria or ER overlapped by AC probes, mCherry-tagged control probes, or F-actin probes. Our AC probes display more specific labeling compared to their corresponding controls or pan-actin probes. Quantification of overlap was carried out as displayed in E and described under Methods. Mitochondria overlap: AC-mito: n = 53 cells, AC-ER: n = 76 cells, mCherry-ER: n = 71 cells, F-actin: n = 26 cells. ER overlap: AC-mito: n = 20 cells, AC-ER: n = 73 cells, mCherry-mito: n = 71 cells, F-actin: n = 18 cells. P-values determined by two-tailed Welch’s t-test. e) An example of a HeLa cell expressing AC-mito and stained with MitoTracker and phalloidin. Scale bar: 10µm. The top row shows standard images of the cell. The center row shows the corresponding mitochondrial area in magenta and phalloidin or AC-mito area in green. Areas of overlap are shown in white and quantified. The lower left panel displays the area of overlap between mitochondria and phalloidin in cyan and the area of overlap between mitochondria and AC-mito in yellow. The area of overlap between the two is shown in white and quantified. The lower right panel summarizes the qunatification for this example. Green: percentage of mitochondria overlapped by phalloidin or AC-mito. Cyan: percentage of mitochondria overlapped by only phalloidin. This is the proportion of non-specific phalloidin overlap. Yellow: percentage of mitochondria overlapped by only AC-mito. This represents the proportion of actin that phalloidin lacks the sensitivity to detect. White: percentage of mitochondria overlapped by both phalloidin and AC-mito. These results were reproducible across 12 independent experiments. f) Further quantification of E across multiple cells either transfected with AC-mito and labelled with MitoTracker and phalloidin (left graph) or co-transfected with AC-ER and mCherry-ER and labelled with phalloidin (right graph). Averages and standard deviation are shown. N = 10 cells per condition. P-values determined by the two-tailed ratio paired t-test.
