Optogenetic actin network assembly on lipid bilayer uncovers the network density-dependent functions of actin-binding proteins
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
The actin cytoskeleton forms a mesh-like network that drives cellular deformations. The network property is defined by the network density and the species of actin-binding proteins. However, the relationship between the network density, the penetration ability of actin-binding proteins into the network, and resulting network dynamics remains elusive. Here, we report an in vitro optogenetic system, named OptoVCA, which induces Arp2/3 complex-mediated actin network assembly on a lipid membrane. By changing the illumination power, duration, and pattern, the OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine the effects of the network density on two representative actin-binding proteins, myosin and ADF/cofilin. We find that the penetration of myosin filaments into the network is strictly inhibited by only a several-fold increase in network density due to the steric hindrance. Furthermore, penetrated myosin filaments induce directional actin flow when the network has a density gradient. On the other hand, ADF/cofilin penetrates into the network regardless of network density. However, network disassembly is dramatically inhibited by only a several-fold increase in network density. Thus, the OptoVCA contributes to understanding cell mechanics by examining the network density-dependent effects on actin-binding proteins.
Article activity feed
-
Finally, we confirmed that this actin polymerization is mediated by the Arp2/3 complex; the cells treated with CK-666, an Arp2/3 complex inhibitor, showed a relatively weaker increase in the cortical Lifeact signal compared to the DMSO-treated control cells during illumination (Fig. 1f, 1g), although the translocation dynamics of SspB-mScarlet-I-VCA were nearly identical between the two conditions
I found this result a bit puzzling with respect to the maintenance of a strong cortical network in the presence of CK-666 (which could be due to timing of the experiment if there was insufficient time for branched network turnover and inhibition of newly nucleated actin branches) but then began to wonder if part of the issue is due to this being done in trypsinized cells shortly after replating -- I'm sure I'm not thinking of something …
Finally, we confirmed that this actin polymerization is mediated by the Arp2/3 complex; the cells treated with CK-666, an Arp2/3 complex inhibitor, showed a relatively weaker increase in the cortical Lifeact signal compared to the DMSO-treated control cells during illumination (Fig. 1f, 1g), although the translocation dynamics of SspB-mScarlet-I-VCA were nearly identical between the two conditions
I found this result a bit puzzling with respect to the maintenance of a strong cortical network in the presence of CK-666 (which could be due to timing of the experiment if there was insufficient time for branched network turnover and inhibition of newly nucleated actin branches) but then began to wonder if part of the issue is due to this being done in trypsinized cells shortly after replating -- I'm sure I'm not thinking of something obvious but why not just grow and image directly on chamber slides to preserve the cortical actin network in a more native state?
Regardless, I also wanted to mention that I found this to be an very elegant study using an approach that indeed can illuminate some important properties of actin binding protein behavior.
-
-
-