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. 2019 Jul 3;8:e42695. doi: 10.7554/eLife.42695

Figure 2. pLAT → N-WASP condensates bind to and move with moving actin filaments.

(A) TIRF microscopy images of pLAT → Sos1 condensates (left two columns), and pLAT → N-WASP condensates (middle two columns), and pLAT→ WASP (right two columns), formed in an actin network before (t = 0 min) and after (t = 2 min) addition of myosin II. pLAT condensates are green and actin is magenta in merge. Scale bar = 5 μm. (B–D) STICS analysis of actin and condensate movement. (B) Representative map of actin (magenta) and pLAT condensate (green) vector fields. Lower panels show magnification of box regions in upper panels. (C) Condensate speed vs. actin speed at same position. Condensate composition indicated above each heat map. Heat map indicates frequency in each bin, that is counts in each bin normalized by total number of counts. (D) Distribution of the angle between actin and condensate movement vectors for pLAT → Sos1 (blue), pLAT → N-WASP (gold), randomized pLAT → Sos1 (red) and randomized pLAT → N-WASP (purple) (see Materials and methods for randomization). P-values are for indicated distribution comparisons via Kolmogorov-Smirnov test. Data in (C) and (D) are pooled from 15 fields of view from three independent experiments (5 FOV per experiment).

Figure 2.

Figure 2—figure supplement 1. In actomyosin contractile assays, imaging conditions were chosen such that photo-damage was minimized to avoid artifactual results.

Figure 2—figure supplement 1.

(A) TIRF microscopy images of LAT condensate movement following myosin II-induced actin contraction inside or outside the field of view (FOV) when care was not taken to minimize photo-damage. (left) Representative image of pLAT → N-WASPNeutral condensate (green) wetting of rhodamine-actin filaments (magenta) prior to induced actin contraction. (center) Image of same FOV after imaging during myosin II-induced actin contraction. (right) Image of region outside of FOV (i.e. not imaged during contraction) obtained immediately following actin contraction. Comparing center and right images indicates that photo-damage can increase interactions between condensates and actin, leading to artifactual co-localization following contraction. (B) When imaging is performed with low laser power (as done in our experiments), regions inside and outside of the FOV during actin contraction show similar co-localization of condensates with actin asters. Figure shows representative images for a variety of pLAT condensates types captured in the FOV prior to myosin II-induced actin contraction (left), in the FOV after myosin II-induced contraction (center), and outside of the FOV after contraction (right).