Symmetry breaking in reconstituted actin cortices
- Technion–Israel Institute of Technology, Israel
Figures
Figure 1 with 3 supplements
Reconstitution of actin cortices within water-in-oil emulsions.
(A) Spinning disk confocal images of bodipy-conjugated ActA (left) and rhodamine-labeled actin (right) in a water-in-oil emulsion. The bodipy-conjugated ActA localizes to the water–oil interface, …
Figure 1—figure supplement 1
Actin and ActA distributions in water-in-oil emulsions.
The fluorescence intensity cross-sections of rhodamine-labeled actin (top) and bodipy-conjugated ActA (bottom) extracted from spinning disk confocal images (inset) are plotted as a function of …
Figure 1—figure supplement 2
Analysis of photobleaching experiments.
FRAP experiments were performed on reconstituted cortices at 30°C using a scanning confocal microscope (‘Materials and methods’). A region of the cortex at the bottom of an emulsion was bleached and …
Figure 1—figure supplement 3
Actin cortices formed with different types of labeled actin.
Spinning disk confocal images of actin cortices prepared with different types of labeled actin, as indicated. Similar behavior was observed in all cases. Scale bars: 10 µm.
Figure 2 with 6 supplements
Temperature-dependent symmetry breaking in reconstituted actin cortices.
(A and B) Spinning disk confocal images of bodipy-conjugated ActA (top left) and rhodamine-labeled actin (top right) in water-in-oil emulsions incubated at 20°C (A) or at 30°C (B). A polar actin …
Figure 2—figure supplement 1
Actin and ActA distributions in water-in-oil emulsions at 20°C.
The fluorescence intensity cross-sections of rhodamine-labeled actin (top) and bodipy-conjugated ActA (bottom) extracted from spinning disk confocal images (inset) are plotted as a function of …
Figure 2—figure supplement 2
Analysis of photobleaching experiments at 20°C.
FRAP experiments were performed on reconstituted cortices at 20°C using a scanning confocal microscope (‘Materials and methods’). A region of the cortex at the bottom of an emulsion was bleached and …
Figure 2—figure supplement 3
The actin cap remains stable following symmetry breaking.
(A) Spinning disk confocal images from a time lapse video following the dynamics of cap evolution over 2 hr. Once a stable cap is formed, its position remains relatively stable over time. (B) A …
Figure 2—figure supplement 4
A temperature shift from 30°C to 20°C leads to symmetry breaking.
Samples were incubated for 30 min at 30°C to generate homogenous cortices, and then moved to 20°C. (A) Spinning disk confocal images of the actin distribution in representative emulsions at …
Figure 2—figure supplement 5
The temperature–dependent behavior of artificial cortices as a function of ActA concentration.
Artificial actin cortices were prepared with different ActA concentrations which were higher (3 µM) or lower (0.5 µM) than the typical ActA concentration used (1.5 µM). (A and B) Bar plots showing …
Figure 2—figure supplement 6
ActA dynamics at the interface.
Scanning confocal images showing the ActA distribution in an emulsion incubated at 20°C. A small patch of the ActA at the interface was photobleached and the recovery of the fluorescence signal was …
Figure 3
The effect of myosin and crosslinkers on symmetry breaking.
(A) Spinning disk confocal images of actin cortices in water-in-oil emulsions made with myosin–depleted (upper panel) or mock-depleted (lower panel) extracts and incubated at 20°C. Myosin depletion …
Figure 4 with 1 supplement
Cell-like behavior of reconstituted actin cortices.
(A) Scanning confocal images from a time-lapse video (Video 4) showing local detachment of an actin cortex from the interface followed by regrowth of a new cortex within minutes. The time after …
Figure 4—figure supplement 1
Actin and ActA distributions in a deformed droplet.
The fluorescence intensity of rhodamine-labeled actin (top) and bodipy-conjugated ActA (bottom) extracted from spinning disk confocal images of the deformed emulsion shown in Figure 4B (insets) are …
Videos
Video 1
Cortical recovery after photobleaching.
This video shows scanning confocal images (at a single z-plane) of rhodamine-labeled actin within a water-in-oil emulsion incubated at 30°C. A small region of the cortex was photobleached using a …
Video 2
Actin cortical flow during symmetry breaking.
This video shows spinning disc confocal images (at a single z-plane) of rhodamine-labeled actin within a water-in-oil emulsion incubated at 20°C. The actin signal reflects the distribution of actin …
Video 3
Cortical recovery after photobleaching in an asymmetric actin cap.
This video shows scanning confocal images (at a single z-plane) of rhodamine-labeled actin within a water-in-oil emulsion incubated at 20°C. A small region at the tip of the actin cap was …
Video 4
Cytoskeletal forces in the actin cortex lead to bleb-like cortex detachment and shape deformation.
This video shows scanning confocal images (at a single z-plane) of bodipy-conjugated ActA (left), rhodamine-labeled actin (center) and an overlay (right), within a large water-in-oil emulsion …
