ИСТИНА

Dynamic reorganization of actin cytoskeleton networks is vital to the proper formation of organ systems during embryogenesis, and to such processes as wound healing, neuronal activity, and inflammatory responses. These dynamic rearrangements of actin filamentous networks are controlled by the activities of numerous actin-binding proteins (ABPs). One of the important ABP, Arp2/3 complex directly binds to the side of existing actin filament to nucleate formation of a daughter filament. This ‘branched nucleation’ activity of the Arp2/3 complex is essential for cell motility, endocyosis, and intracellular transport of specific organelles and vesicles in virtually all eukaryotic cell types. On the other hand, it is unclear how the densely branched networks of filaments are subsequently ‘pruned’ or debranched during phases of rapid actin network turnover, which is equally essential in vivo. Further, still not well understood is what conformational changes in Arp2/3 complex are associated with inactivation state. Here we use single particle EM to determine the 3D structures of Arp2/3 complex bound to two different inhibitors: Gmf1 and Arpin. Arp2/3 complex bound to each of these ligands was isolated by affinity chromatography, applied to EM grids negative stained, and analyzed on a JEOL 2100 electron microscope at 200 kV. Projections of Arp2/3 complexes were captured by CCD camera (Gatan) at 8 Mpix resolution and 3D reconstructions were generated using random Conical Tilt method implemented in EMAN2.1, with subsequent refinement in IMAGIC-4D. Our results show that GMF-bound Arp2/3 complex exists in two inactive conformations, suggestive of two separate Gmf binding sites on the complex (Fig. 1), as was proposed earlier (1). 100% of Arp2/3 complexes bound to Arpin adopted an inactive conformation (Fig. 2). The binding site for Arpin is overlapping with the binding site for VCA domains (2), suggesting a mechanism for Arpin inhibition of Arp2/3 complex (Fig. 3). Additionally, we have demonstrated that different cellular factors, working in concert, like Crn1 and Gmf1, have an increasing inhibitory effect, implicated in more Arp2/3 complexes in inactive conformation (Fig. 2B). Thus our results provided an improved level of mechanistic understanding of Arp2/3 complex regulation by determining the conformations (Fig. 4) of an inactive complex.