Figure 4. 3D traction force mapping in fluorescent fibrin reveals that the matrix is periodically pulled in toward the constriction during parasite motility.

(A) Sequential time series images, in a single z-plane, of a tdTomato-expressing parasite moving in fibrin (boxed). Scale bar = 10 µm, timestamps in seconds. (B) Force maps from the corresponding z subvolume plane of the data shown in panel A; each map depicts the motions of the fluorescent fibrin matrix between the two consecutive time points, as indicated by the brackets. Arrow length (multiplied 24-fold for display) indicates the magnitude of matrix displacement and arrow color the directionality (see color wheel). See Video 7 for the entire time series. (C) Zoomed images showing the force maps from the boxed region of panel B, after background subtraction (see text), overlaid on the parasite images from panel A. Length of arrows indicating displacement magnitude are multiplied 15-fold for display. Note inward displacement of the matrix in the second overlay image, outward displacement (relaxation) of the matrix in the third image, and no detectable displacement vectors in the fourth image even though the parasite continues to move. (D) Zoomed overlays of the force maps (background subtracted) and images of a moving parasite from a second dataset. The inwardly displaced matrix in this example did not relax back to its original position within the time frame of the experiment. Arrow lengths indicating displacement magnitude are multiplied 15-fold for display. See Video 8 for the entire time series. Scale bars in panels C and D=5 µm. Empty red arrowheads indicate position of the constriction.

Figure 4—figure supplement 1. All displacement vectors point in towards a moving parasite.

Matrix displacement vectors from all subvolumes (49 (x) x 49 (y) x 7 (z)) in the image volume were calculated by FIDVC and projected onto the x-y, x-z and y-z planes, as indicated (axis labels are in µm). Length of arrows indicate displacement magnitude and are multiplied 10-fold for display. The data correspond to the parasite shown in the second panel of Figure 4C and frame 2 of Video 7. The moving parasite is outlined on each z slice with red circles, and the position of the constriction is indicated by either an empty arrowhead (x–y) or a black dot (y–z).

Figure 4—figure supplement 2. Pattern of pulling, holding, and release of the matrix during individual constriction events.

The number of consecutive frames in which: the matrix displaced in towards the parasite (pull); no further matrix displacement was observed (hold); and the matrix moved away from the parasite towards its original position (release) are indicated. n=9 parasites, 25 constrictions.

Figure 4—figure supplement 3. Additional examples showing that matrix displacement is directed primarily in towards the constriction in moving parasites.

Empty red arrowheads indicate position of the constriction and white arrows the direction of parasite travel. Length of black arrows indicating displacement magnitude are multiplied 15-fold for display. Scale bars = 5 µm.