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. 2019 Dec 16;29(24):4169–4182.e4. doi: 10.1016/j.cub.2019.10.036

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© 2019 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

WASP Does Not Require Active Rac to Recruit the Arp2/3 Complex and Trigger Actin Polymerization on Puncta

(A) Live imaging of migrating wasA⁻ cells co-expressing GFP-WASP (rescue) and mRFPmars2-ArpC4. WASP puncta are evenly distributed at the rear and are all Arp2/3 complex positive. The area indicated by the square is highlighted within insets. Scale bar represents 10 μm.

(B) GFP-WASP appears on a punctum (t = 0 s, cyan arrow) alongside mRFPmars2-ArpC4 and then synchronously disappears (t = 9 s).

(C) Live imaging of migrating wasA⁻ cells co-expressing GFP-WASP^ΔCRIB and mRFPmars2-ArpC4. All WASP^ΔCRIBpuncta cluster within the enlarged rear (square). As highlighted (insets), all GFP-WASP^ΔCRIB spots are Arp2/3 complex positive. Scale bar represents 10 μm.

(D) GFP-WASP^ΔCRIB appears as a punctum (t = 0 s, cyan arrow) along with mRFPmars2-ArpC4. WASP^ΔCRIB remains within spots (alongside the Arp2/3 complex) and then synchronously disappears (t = 117 s).

(E) Live imaging of migrating wasA⁻ cells co-expressing GFP-WASP^∗∗CRIB and mRFPmars2-ArpC4. All WASP^∗∗CRIBpuncta cluster within the enlarged rear (square). As shown in insets, all WASP^ΔCRIB spots are Arp2/3 complex positive. Scale bar represents 10 μm.

(F) GFP-WASP^∗∗CRIB appears as a punctum (t = 0 s, cyan arrow) alongside mRFPmars2-ArpC4. They disappear synchronously (t = 27 s).

(G) Lifetime of wild-type and WASP CRIB mutants’ puncta. wasA⁻/GFP-WASP (n = 52 puncta): 7.2 ± 0.5 s; wasA⁻/GFP-WASP^ΔCRIB (n = 28 puncta): 53.4 ± 16.6 s; wasA⁻/GFP-WASP^∗∗CRIB (n = 24 puncta): 35.8 ± 5.1 s; means ± SEM. Kruskal-Wallis test, wasA⁻/GFP-WASP versus wasA⁻/GFP-WASP^ΔCRIB = p < 0.0001; wasA⁻/GFP-WASP versus wasA⁻/GFP-WASP^∗∗CRIB = p < 0.0001).

(H) Live imaging of migrating wasA⁻ cells expressing LifeAct-mRFPmars2 and GFP-WASP (rescue, left), GFP-WASP^ΔCRIB (center), and GFP-WASP^∗∗CRIB (right). All puncta generated by wild-type WASP or WASP CRIB are F-actin positive. Examples indicated by squares are highlighted within insets. Scale bars represent 10 μm. Related to Video S2.

(I) Live imaging of wasA⁻ cells expressing GFP-WASP and mRFPmars2-ArpC4 treated with Rac inhibitor (EHT1864). Before EHT1864 addition (t = −10 s), cells are polarized and WASP generates Arp2/3-complex-positive puncta (square). Shortly after treatment (t = 0 s), cells become rounder. As highlighted by squares, WASP generates Arp2/3-complex-positive puncta even after 10 min from treatment. Scale bar represents 10 μm.

See also Figure S3.