Computational methods for identifying cells with a stellate morphology: adaptive corner detection and a Fourier analysis. (A and B) Selected images of S2 cells displaying normal and stellate (Wave/Scar depleted) phenotypes. Bars, 12 µm. (C and D) An adaptive corner detection algorithm (He, X.C., and N.H.C. Yung. 2004. Proceedings of The Pattern Recognition 17th International Conference) applied to the same images as in A and B, where cell area was identified by thresholding. Detected corners are depicted by red boxes. (E) Quantification of the number of corners detected on the specific cells in C and D (blue bars) and means for Wave/Scar-depleted stellate cells and wild-type cells (red bars, n = 144 Wave/Scar depleted; n = 150 wild type). (F and G) A new method for analyzing the topology of the cell perimeter using power spectral analysis. The technique first traces the cell perimeter to create a matrix of all perimeter pixels and measures the distance from the cell centroid to each of these pixels. The insets show the distance from centroid values for the 140 perimeter pixels between the lines labeled D1 and D140. The Wave/Scar cell has been resized (not depicted) so that the perimeters of the two cells are both equal to ∼500. These values are Fourier transformed, and the complex modulus was calculated to yield the power spectrum for the perimeter of the cell. This power spectrum can then be binned into discrete frequency ranges and used to compare the membrane topology of cells by analyzing which frequencies are enriched in each phenotype. (H) Quantification of the integrated value of the power spectrum from frequencies 12–16 (Fig. S1) on the specific cells in F and G (blue bars) and means per plate well for Wave/Scar-depleted stellate cells and wild-type cells (red bars). Error bars indicate mean ± SD.

Gene knockdowns that impair cell spreading. (A) Domain schematics of CG9175 (Dm Sec12), CG8465, and CG15231. (B) Representative fields of cells with the indicated gene knockdowns. This phenotype can be detected quantitatively by calculating the total actin staining intensity divided by the total surface area of the cell (Table II). The inset shows a wild-type cell. Bar, 20 µm. (C) CG8465-GFP (left) and Sar1-mCherry (an ER-resident protein; middle) expressed in the same cell. (D) GFP-CG9175 (left) and Sar1-mCherry (middle) expressed in the same cell. (E) Selected frames from time-lapse video of CG15231-GFP. The arrows follow the position of a punctum that can be tracked over time. At 3 s, the punctum disappears, and a radial burst of fluorescence (within dashed line) is seen, likely reflecting plasma membrane fusion and secretion into the medium (Video 1). (C–E) Bars, 5 µm. (F) Secretion of 15231-GFP into the media under the listed RNAi conditions (see Materials and methods). Secretion was measured by immunoblot analysis 12 h after induction and compared ratiometrically with tubulin levels (as a control for total protein; Fig. S1). Error bars indicate mean ± SD. (G) Quantitative rates of FRAP. Curves are means for at least 10 cells.

Work flow for performing and analyzing a whole genome RNAi screen for the spreading of Drosophila S2 cells on Con A–coated surfaces. RNAi treatment was preformed in 96-well format (top left), with one unique dsRNA corresponding to one Drosophila gene per well. After 5 d of treatment, cells were plated on Con A and stained for actin, tubulin, and DNA and subjected to high throughput imaging (top right). After acquisition of the image dataset, images were segmented by a watershed transform to identify individual cells (bottom right). A multitude of parameters were extracted from the individual cells (bottom left). For details see Materials and methods. Bars, 10 µm.

CK1α knockdown produces a stellate phenotype. (A–C) Images of representative fields of cells with no treatment (A), Scar RNAi (B), and CK1α RNAi (C). (D) Quantification of actin retrograde flow rates in GFP-actin cells with the indicated gene depleted. Retrograde flow rates were calculated from kymograph analysis of time-lapse total internal reflection fluorescence images (mean and SEM; n = 45 measurements for each condition). Bar, 20 µm.