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

Abstract

AbstractThe inhibitors, CK-666 and CK-869, are widely used to probe the function of actin nucleation by the Arp2/3 complexin vitroand 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 complexes with defined subunit composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that while both inhibitors prevent linear actin filament formation when either ArpC1A- or ArpC1B-containing complexes are activated by SPIN90, inhibition of actin branching depends on iso-complex composition. Both drugs inhibited actin branch formation by VCA-activated complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express very low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and migration. We also found that CK-869 is only able to inhibit Arp3-but not Arp3B-containing iso-complexes. Our analyses 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.