(A) In the linear motor model of motility, the TgMyoA motor (TgMyoA and its associated light chains, TgMLC1 and either TgELC1 or TgELC2) is anchored to the parasite’s inner membrane complex (IMC) …
(A) Confocal imaging of a 2.25 mg/ml fluorescent fibrin gel. A maximum fluorescence intensity projection of 51 z-slices captured 0.25 µm apart is shown; scale bar = 10 µm. (B) Maximum fluorescence …
(A) Comparison of the maximum and mean speeds of parasites in 2.25 mg/ml fibrin vs. Matrigel. (B) Comparison of the proportion of parasites moving in 2.25 mg/ml fibrin vs. Matrigel. Horizontal bars …
(A) A fluorescent fibrin matrix containing wild-type parasites was imaged over 96 seconds (60 successive image volumes). FIDVC was then used to calculate the 16,807 3D fibrin displacement vectors …
The viscoelastic properties of the fibrin matrix were measured by laser trapping of 0.91 µm styrene beads. (A) The laser trap position at 1 Hz (top panel) and the resulting force as measured by the …
(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 …
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 …
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 …
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 …
(A) The time interval between constrictions during motility of individual parasites (n=99 parasites, 188 constrictions). A negative time interval (black bars) corresponds to the presence of two …
The number of time points when the constriction was present either anterior or posterior to the midway point along the parasite’s longitudinal axis were determined. Only parasites that progressed …
The top panels show the anti-TgSAG1 fluorescent signal; red empty arrowhead marks the location of the constriction. Middle panels show the Hoechst 33342-stained nucleus, which stays in focus …
(A) Brightfield images showing TgMyoA knockout and TgMIC2 knockout parasites moving within a Matrigel matrix without a detectable constriction. A wild-type parasite undergoing a typical constriction …
(A) Sequential fluorescence images in a single z-plane of a moving TgMyoA knockout parasite stained with Hoechst 33342 (to label the parasite nucleus), (B) the corresponding force maps, and (C) the …
(A) Sequential fluorescence images in a single z-plane of a moving TgMIC2 knockout parasite expressing YFP, (B) the corresponding force maps, and (C) the zoomed images showing the force maps from …
(A) Representative plots of nuclear shape (ratio of the nuclear diameters perpendicular vs. parallel to the long axis of the parasite) in one wildtype and one TgMIC2 knockout parasite (black circles …
(A) Ratio of nuclear diameter perpendicular (width) vs. parallel (length) to the long axis of the parasite in moving wild-type (WT) and TgMIC2 knockout (MIC2 KO) parasites, over time. For each …
Scale bar = 5 µm, time is shown in hr:min:sec. Single frames from this video are shown in Figure 1B.
Scale bar = 5 µm, time is shown in hr:min:sec.
Scale bar = 5 µm, time is shown in hr:min:sec. Single frames from this video are shown in Figure 1C.
51 x-y slices captured 0.25 µm apart in z were reassembled into the volumetric view shown. Dimensions of the imaged volume are shown at bottom left. See Figure 2A for maximum intensity projection of …
Scale bar = 10 µm, time is shown in hr:min:s. Single frames from this video are shown in Figure 2C.
Length of arrows indicating displacement magnitude are multiplied 10-fold for display. The parasite is outlined on each z plane with red circles. Single frames from this video displaying projections …
Arrow size corresponds to relative displacement magnitude and arrow color to displacement direction as described in Figure 4.
Arrow size corresponds to relative displacement magnitude and arrow color to displacement direction as described in Figure 4.
Scale bar = 5 µm, time is shown in hr:min:s. Single frames from time points 0:1:51.23 – 0:1:55.43 of this video are shown in Figure 5B.
Scale bar = 5 µm, time is shown in hr:min:s. Note the helical trail of shed fluorescent antibody behind the moving parasite. Single frames from this video are shown in Figure 5—figure supplement 2.
Scale bar = 5 µm, time is shown in hr:min:sec. Single frames from this video are shown in Figure 6A.
Scale bar = 5 µm, time is shown in hr:min:s. Single frames from this video are shown in Figure 6A.
Experiment | Objective | Fluorochrome(Excitation/emission wavelengths) | Image spacing in z | Exposure time per image | Number of Image stacks | Total time | Volume(x, y, z) |
---|---|---|---|---|---|---|---|
Microspheres (Matrigel) | 60× | DragonGreen (490/507–530 nm) | 41 slices, 1 µm apart | 16ms | 60 | 64 s | 225.3 µm × 84.5 µm×40 µm |
tdTomato parasites (550/579–608 nm) | |||||||
Fibrin vs Matrigel and TgMIC2 KO directionality | 20× | Hoechst 33342 (385/420–449 nm) | 41 x 1 µm | 16ms | 120 | 80 s | 665.6 µm × 249.6 µm×40 µm |
Force Mapping, WT (Fibrin) | 60× | tdTomato parasites (550/579–608 nm) | 50x0.5 µm | 16ms | 60 | 96 s | 225.3 µm × 84.5 µm×24.5 µm |
Fibrin (635/666–723 nm) | |||||||
WT, TgMyoA KO, TgMIC2 KO; Brightfield (Matrigel, fibrin) | 60× | N/A: Brightfield | 21 x 1 µm | 40ms | 360 | 302 s | 225.3 µm × 225.3 µm×20 µm |
Nuclear size vs constriction (Matrigel) | 20× | Hoechst 33342 (385/420–449 nm) | 41 x 1 µm | 16ms | 60 | 80 s | 665.6 µm × 249.6 µm×40 µm |
Anti-SAG1 Alexa 548 (550/579–608 nm) | |||||||
TgMyoA KO Force Map (Fibrin) | 60× | Hoechst 33342 (385/420–449 nm) | 50x0.5 µm | 16ms | 60 | 96 s | 225.3 µm × 84.5 µm×24.5 µm |
Fibrin (635/666–723 nm) | |||||||
TgMIC2 KO Force Map (Fibrin) | 60× | YFP cytosol (490/507–530 nm) | 50x0.5 µm | 16ms | 60 | 96 s | 225.3 µm × 84.5 µm×24.5 µm |
Fibrin (635/666–723 nm) |
The source code generates 2D and 3D quiver plots of the FIDVC data showing either all vectors or only those vectors above a background threshold.