Science1 publisher2 min readPublished
Deleting two proteins at once places profilin above Arp2/3 at the cell's leading edge
Klemens Rottner's group at the Helmholtz Center for Infection Research disrupted lamellipodial proteins singly and in combination, and profilin still directed the Arp2/3 branching complex in cells lacking Ena/VASP. The result picks a side in a long-running dispute.
The Scientist · Science desk
What happened
- Klemens Rottner's group at the Helmholtz Center for Infection Research used CRISPR/Cas9 to disrupt lamellipodial proteins alone and in combination, then tracked lamellipodium formation and where the remaining proteins went.
- The knockouts put profilin upstream of the Arp2/3 branching complex, promoting Arp2/3 activity while counteracting the Ena/VASP polymerases, with Ena/VASP and capping protein holding each other back.
- Earlier studies had argued both that profilin funnels actin monomers away from Arp2/3 toward formin and Ena/VASP structures, and the reverse.
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Why it matters
- constraint Anyone simulating branched actin growth now has to place profilin above Arp2/3 and keep its capping-protein effect on a separate line, since the monomer-funnelling account no longer fits the knockout data.
- decision For anyone hoping to slow metastasis or immune cell trafficking through this network, the control point is now identified, and the hard part is restricting it to one cell type, because wound closure and axon growth run on the same machinery.
- precedent Combinatorial knockouts resolved a dispute that single knockouts had kept alive for years, and cytoskeleton papers making ordering claims will be expected to meet that bar.
The double knockouts are where the argument gets settled, because a single knockout can be read two ways. Take away profilin and the Arp2/3 complex is no longer properly positioned in the lamellipodium. Two stories predict that. In one, profilin prepares actin monomers and feeds branching by Arp2/3 [4]. In the other, profilin ties up the competing polymerases, Ena/VASP and the formins, and monomers reach Arp2/3 by default [7]. Published work has argued both directions [13].
So the team removed Ena/VASP first, then asked what profilin was still doing. Profilin was still required for Arp2/3 localization in those cells [10]. Its effect on capping protein was not: in the absence of Ena/VASP, that effect vanished [11]. Profilin's Arp2/3 arm works independently of Ena/VASP, and its capping-protein arm runs through it [20]. That separation is what the authors lean on in calling profilin a master regulator of Arp2/3-dependent actin networks [12].
Rottner said the role of profilin in these processes "has been controversial, but our results could largely clarify previous inconsistencies in the literature" [15]. Of the outcome, he said: "We are now clarifying this controversy by clearly showing profilin to operate upstream of Arp2/3 complex in lamellipodia and also how" [14].
The mathematical model, built with Martin Falcke at the Max Delbrück Center Berlin, reproduced all of the experimental results, and it also implied that Ena/VASP and capping protein have to compete for a common recruitment mechanism in lamellipodia [17].
HZI says the insights pave the way for defining how such processes change in diseases and how they might be modulated in future [18]. The experiments behind that are gene disruptions in cells plus modeling, and the account covers no animal work, disease model or compound [19]. HZI did not say which cell type was used or how large any effect was [22]. The same branched-network machinery moves immune cells to sites of action, closes epithelial wounds, extends axons and carries cancer cells through the body during metastasis [1].
What to watch
- A direct binding or structural test of whether Ena/VASP and capping protein really share one recruitment site at the lamellipodial edge.
- Whether the profilin-above-Arp2/3 hierarchy holds in primary immune cells and in animals, beyond the edited cells used here.
- Whether other groups reproduce the double-knockout result, since the earlier literature supported both directions.