CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes.

The inhibitors, CK-666 and CK-869, are widely used to probe the function of Arp2/3 complex mediated actin nucleation in vitro and in cells. However, in mammals, the Arp2/3 complex consists of 8 iso-complexes, as three of its subunits (Arp3, ArpC1, ArpC5) are encoded by two different genes. Here, we used recombinant Arp2/3 with defined composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that both inhibitors prevent linear actin filament formation when ArpC1A- or ArpC1B-containing complexes are activated by SPIN90. In contrast, inhibition of actin branching depends on iso-complex composition. Both drugs prevent actin branch formation by complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and cell migration. CK-869 also only inhibits Arp3- but not Arp3B-containing iso-complexes. Our findings have important implications for the interpretation of results using CK-666 and CK-869, given that the relative expression levels of ArpC1 and Arp3 isoforms in cells and tissues remains largely unknown.

Use of CK-548 and CK-869 as Arp2/3 complex inhibitors directly suppresses microtubule assembly both in vitro and in vivo.

Two types of Arp2/3 complex inhibitors, CK-666/636 and CK-548/869, are commonly used to study Arp2/3 complex-dependent actin assembly both in vitro and in vivo. However, we found that CK-548 and CK-869 directly suppress microtubule (MT) assembly independent of the actin cytoskeleton. Treatment of cultured mammalian cells with 50 µM CK-869 dramatically decreased MT networks and, instead, accumulated tubulin at the cell periphery, as did nocodazole that inhibits MT assembly. An in vitro MT-sedimentation assay revealed that CK-548 and CK-869 significantly suppressed MT polymerization. In budding yeast, although CK-548 and CK-869 are reported to lack binding abilities in the yeast Arp3, CK-548 treatment decreased cytoplasmic MT at several tens of micromolar concentrations. In addition, we found that the effects of CK-548 and CK-869 on MT assembly varied according to species. We propose that CK-548 and CK-869 are not suitable for studying the cytoskeleton in living cells.