A circular zone of attachment to the extracellular matrix provides directionality to the motility of Toxoplasma gondii in 3D
- Department of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine, United States
- Department of Molecular Physiology and Biophysics, University of Vermont Larner College of Medicine, United States
Figures
Figure 1
Parasites moving in 3D deform the surrounding Matrigel and undergo periodic constrictions.
(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) …
Figure 2 with 1 supplement
Parasite motility within a 3D fibrin matrix.
(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 …
Figure 2—figure supplement 1
Quantitative comparison of parasite motility in fluorescent fibrin vs. Matrigel.
(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 …
Figure 3 with 1 supplement
Determining threshold of detection in 3D traction force mapping.
(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 …
Figure 3—figure supplement 1
Rheological properties of the fibrin matrix.
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 …
Figure 4 with 3 supplements
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 …
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 …
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 …
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 …
Figure 5 with 2 supplements
The constrictions are tightly linked with motility and are sufficiently narrow to deform the parasite nucleus.
(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 …
Figure 5—figure supplement 1
Parasites move faster after the constriction has past the midway point on the parasite’s longitudinal axis.
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 …
Figure 5—figure supplement 2
Fluorescent antibody against TgSAG1 is depleted from the parasite surface anterior to the constriction and shed into the Matrigel matrix.
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 …
Figure 6 with 2 supplements
3D motility and force mapping of parasites lacking TgMyoA or TgMIC2.
(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 …
Figure 6—figure supplement 1
The small number of TgMyoA knockout parasites that move more than one body length produce no detectable force on the fibrin matrix.
(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 …
Figure 6—figure supplement 2
Moving TgMIC2 knockout parasites produce no detectable force on the fibrin matrix.
(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 …
Figure 7 with 1 supplement
Knockout of TgMIC2 results in the loss of the constriction and less directional motility.
(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 …
Figure 7—figure supplement 1
In parasites lacking TgMIC2, the nuclei do not change shape during 3D motility and the trajectories are less straight than those of wild-type parasites.
(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 …
Videos
Video 1
Maximum intensity projection in z of microspheres (green) being displaced due to parasite (red) movement within the Matrigel.
Scale bar = 5 µm, time is shown in hr:min:sec. Single frames from this video are shown in Figure 1B.
Video 2
Microsphere (green) being displaced towards a parasite (red) moving in Matrigel within in a single z plane.
Scale bar = 5 µm, time is shown in hr:min:sec.
Video 3
Brightfield imaging of a parasite undergoing a single constriction.
Scale bar = 5 µm, time is shown in hr:min:sec. Single frames from this video are shown in Figure 1C.
Video 4
Confocal fluorescence imaging of a 2.25 mg/ml fluorescent fibrin gel.
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 …
Video 5
Deformation of fluorescent fibrin (green) by a parasite (red; bottom) as it moves up through the image stack.
Scale bar = 10 µm, time is shown in hr:min:s. Single frames from this video are shown in Figure 2C.
Video 6
3D displacement map surrounding the moving parasite shown in the second panel of Figure 4C and frame 2 of Video 7.
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 …
Video 7
x-y displacement map overlaid on the moving parasite shown in Figure 4A–C.
Arrow size corresponds to relative displacement magnitude and arrow color to displacement direction as described in Figure 4.
Video 8
x-y displacement map overlaid on the moving parasite shown in Figure 4D.
Arrow size corresponds to relative displacement magnitude and arrow color to displacement direction as described in Figure 4.
Video 9
Brightfield imaging of a parasite undergoing two constrictions at the same time.
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.
Video 10
Parasite labeled with Alexa546-conjugated anti-TgSAG1 (red) and Hoechst 33342 (blue) undergoing a constriction.
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.
Video 11
Brightfield imaging of a moving TgMyoA knockout parasite.
Scale bar = 5 µm, time is shown in hr:min:sec. Single frames from this video are shown in Figure 6A.
Video 12
Brightfield imaging of a moving TgMIC2 knockout parasite.
Scale bar = 5 µm, time is shown in hr:min:s. Single frames from this video are shown in Figure 6A.
Tables
Table 1
Imaging parameters for the different experiments described.
| 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) |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/85171/elife-85171-mdarchecklist1-v2.docx
-
Source code 1
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.
- https://cdn.elifesciences.org/articles/85171/elife-85171-code1-v2.zip
