Fig 1.
Bleb formation can be identified by cortex-to-membrane positioning.
At T1, the membrane detaches from the cortex, initiating a bleb. At T2, an actin scar in the original location of the cortex disassembles as the cortex begins to reform at the new location of the membrane. At T3, the cortex is bound to the membrane in the bleb.
Fig 2.
The under agarose assay simultaneously visualized cortex-to-membrane positions of a putative bleb.
A single time point of a wild-type cell crawling under 0.5 percent agarose gel is shown. Left, LifeAct-GFP labeled the cortex’s position as well as other actin structures. Center, the RITC-Dextran in the gel was blocked by the cell, providing the cell’s edge and, therefore, membrane position. Right, a merge of the two channels provided cortex-to-membrane position where LifeAct-GFP and RITC-Dextran were false colored green and red, respectively. A putative bleb lacking actin structures was observed observed (white arrow).
Fig 3.
ImageJ intensity plots quantitatively confirm membrane and cortex positions of a putative bleb.
A single time point of a wild-type cell crawling under 0.5 percent agarose gel from Fig 2 with respective intensity plots of the signals along the drawn line (white line) for each channel is shown. Left, the LifeAct-GFP signal (dashed line) indicated the cortex’s position (white arrowhead) at 1.1 microns. Center, the RITC-Dextran signal (solid line) indicated the membrane’s position (black arrow) at 2.9 microns where the cell’s membrane ends and the agarose begins. Right, the merge of the two channels shows membrane detachment from the cortex by a distance of 1.8 microns.
Fig 4.
ImageJ intensity plots confirmed cortex-to-membrane positions throughout bleb formation of a putative bleb.
Merges of GFP and RITC time points representative of steps in bleb formation (T0-T3) with their respective intensity plots (dashed and solid lines) from the same cell in Fig 2 confirmed bleb formation quantitatively in real-time along the line drawn (white). Cortex positions (white arrowhead), the actin scar characteristic (black arrowhead), and membrane (black arrow) positions are indicated.
Fig 5.
Image processing: Microscopy → Digitization Flowchart. (A) The original microscopy output. (B) The cropped image. Only unnecessary background was removed. (C) The result of FillingTransform. Very small dark features were blended with adjacent areas to form a more uniformly gray cell interior. (D) ImageAdjust rescaled the brightness component within the unit interval, [0, 1]. (E) The blurring operation modified areas where high intensity and low intensity were adjacent. (F) Binerize reset the image pixels into 0 or 1. The Binerize parameter set the threshold determining which pixels were set to white (1) and black (0). (G) The filling transform was reapplied. (H) DeleteSmallComponents removed any unwanted artifacts, white areas inside the black and black inside the white. This ensured there is only one edge to detect. (I) EdgeDetect identified the edge between the black and white regions. Scale bars identify 10 micron lengths.
Fig 6.
Creation of B-splines and equal arc length points.
(A) Three views of a cell: the microscopy image, the edge detect image, the B-spline plot overlaying the microscopy image. (B) The entire cell followed by two views of the boundary segment. The ROI view shows points on the B-spline at equal parameter increments, t = 0.5, and the same segment with points at equal arc length intervals, δ = 0.5 pixel units and ϵ = 0.001 pixel units. Large scale bars identify 10 micron lengths; small scale bars identify 1 micron.
Fig 7.
Applying Delauney triangulation to determine cell and sub cellular areas.
(A) The area of a cell. A cell triangulated by the Delaunay procedure. The convex hull is included within the triangulation. The image on the right shows the result of removing the external triangles. The sum of the triangle areas in this image estimates the cell area. (B) The area of a belb. The two images on the left show the cell before and after the bleb. The next pair (above and below) show the cell by plotting the equi-spaced list of points on the boundary. On the right we see the region of the bleb before and after the event. These two images are overlaid in the lower right. This is a geometric view of the bleb. Finally, we have triangulated the bleb in preparation to estimating its area. Large scale bars identify 10 micron lengths; small scale bars identify 1 micron.
Fig 8.
Curvature and relative curvature.
(A) Cell boundary with positive (blue) and negative (red) curvature values identified. This plot demonstrates the chaotic nature of raw curvature measurements. (B) The relative curvature between the points a and b. The fitting circle passes through c. The relative curvature value is −0.05097. Scale bars are 10 microns and 1 micron for the ROI.
Table 1.
Bleb distribution: Wild type under 0.7% agarose.
Fig 9.
Relative curvature and blebbing.
(A) Histogram showing the distribution of blebbing sites by local relative curvature. The mean relative curvature for concave blebbing sites is 0.9 with standard deviation 0.11. (B) Examples of relative curvature. The first is 0.09 the mean relative curvature identified in the histogram. The second is 0.2 = 0.09 + 0.11, the mean plus one standard deviation.