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.

SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin organization.

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.

The structural switch of nucleotide-free kinesin.

Kinesin-1 is an ATP-dependent motor protein that moves towards microtubules (+)-ends. Whereas structures of isolated ADP-kinesin and of complexes with tubulin of apo-kinesin and of ATP-like-kinesin are available, structural data on apo-kinesin-1 in the absence of tubulin are still missing, leaving the role of nucleotide release in the structural cycle unsettled. Here, we identified mutations in the kinesin nucleotide-binding P-loop motif that interfere with ADP binding. These mutations destabilize the P-loop (T87A mutant) or magnesium binding (T92V), highlighting a dual mechanism for nucleotide release. The structures of these mutants in their apo form are either isomorphous to ADP-kinesin-1 or to tubulin-bound apo-kinesin-1. Remarkably, both structures are also obtained from the nucleotide-depleted wild-type protein. Our results lead to a model in which, when detached from microtubules, apo-kinesin possibly occupies the two conformations we characterized, whereas, upon microtubule binding, ADP-kinesin converts to the tubulin-bound apo-kinesin conformation and releases ADP. This conformation is primed to bind ATP and, therefore, to run through the natural nucleotide cycle of kinesin-1.

Kinesin, 30 years later: Recent insights from structural studies.

Motile kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP. They share a conserved motor domain (head) which harbors both the ATP- and microtubule-binding activities. The kinesin that has been studied most moves toward the microtubule (+)-end by alternately advancing its two heads along a single protofilament. This kinesin is the subject of this review. Its movement is associated to alternate conformations of a peptide, the neck linker, at the C-terminal end of the motor domain. Recent progress in the understanding of its structural mechanism has been made possible by high-resolution studies, by cryo electron microscopy and X-ray crystallography, of complexes of the motor domain with its track protein, tubulin. These studies clarified the structural changes that occur as ATP binds to a nucleotide-free microtubule-bound kinesin, initiating each mechanical step. As ATP binds to a head, it triggers orientation changes in three rigid motor subdomains, leading the neck linker to dock onto the motor core, which directs the other head toward the microtubule (+)-end. The relationship between neck linker docking and the orientations of the motor subdomains also accounts for kinesin's processivity, which is remarkable as this motor protein only falls off from a microtubule after taking about a hundred steps. As tools are now available to determine high-resolution structures of motor domains complexed to their track protein, it should become possible to extend these studies to other kinesins and relate their sequence variations to their diverse properties.

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement.

Kinesin-1 is a dimeric ATP-dependent motor protein that moves towards microtubules (+) ends. This movement is driven by two conformations (docked and undocked) of the two motor domains carboxy-terminal peptides (named neck linkers), in correlation with the nucleotide bound to each motor domain. Despite extensive data on kinesin-1, the structural connection between its nucleotide cycle and movement has remained elusive, mostly because the structure of the critical tubulin-bound apo-kinesin state was unknown. Here we report the 2.2 Å structure of this complex. From its comparison with detached kinesin-ADP and tubulin-bound kinesin-ATP, we identify three kinesin motor subdomains that move rigidly along the nucleotide cycle. Our data reveal how these subdomains reorient on binding to tubulin and when ATP binds, leading respectively to ADP release and to neck linker docking. These results establish a framework for understanding the transformation of chemical energy into mechanical work by (+) end-directed kinesins.