Analysis of functional surfaces on the actin nucleation promoting factor Dip1 required for Arp2/3 complex activation and endocytic actin network assembly.

Arp2/3 complex nucleates branched actin filaments that drive processes like endocytosis and lamellipodial protrusion. WISH/DIP/SPIN90 (WDS) proteins form a class of Arp2/3 complex activators or nucleation promoting factors (NPFs) that, unlike WASP family NPFs, activate Arp2/3 complex without requiring preformed actin filaments. Therefore, activation of Arp2/3 complex by WDS proteins is thought to produce the initial actin filaments that seed branching nucleation by WASP-bound Arp2/3 complexes. However, whether activation of Arp2/3 complex by WDS proteins is important for the initiation of branched actin assembly in cells has not been directly tested. Here, we used structure-based point mutations of the Schizosaccharomyces pombe WDS protein Dip1 to test the importance of its Arp2/3-activating activity in cells. Six of thirteen Dip1 mutants caused severe defects in Arp2/3 complex activation in vitro, and we found a strong correlation between the ability of mutants to activate Arp2/3 complex and to rescue endocytic actin assembly defects caused by deleting Dip1. These data support a model in which Dip1 activates Arp2/3 complex to produce actin filaments that initiate branched actin assembly at endocytic sites. Dip1 mutants that synergized with WASP in activating Arp2/3 complex in vitro showed milder defects in cells compared to those that did not, suggesting that in cells the two NPFs may coactivate Arp2/3 complex to initiate actin assembly. Finally, the mutational data reveal important complementary electrostatic contacts at the Dip1-Arp2/3 complex interface and corroborate the previously proposed wedge model, which describes how Dip1 binding triggers structural changes that activate Arp2/3 complex.

Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks.

The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate 'seed' filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

Reconstitution reveals two paths of force transmission through the kinetochore.

Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a 'parts list' of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.

Single-Turnover Activation of Arp2/3 Complex by Dip1 May Balance Nucleation of Linear versus Branched Actin Filaments.

Arp2/3 complex nucleates branched actin filaments important for cellular motility, endocytosis, meiosis, and cellular differentiation [1-4]. Wiskott-Aldrich syndrome proteins (WASPs), the prototypical Arp2/3 complex activators, activate Arp2/3 complex only once it is bound to the side of an actin filament [5, 6]. This ensures WASP-activated Arp2/3 complex only nucleates branched actin filaments but means branched actin networks must be seeded with an initial preformed filament. Dip1 and other WISH/DIP/SPIN90 family proteins activate Arp2/3 complex without preformed filaments [7], creating seed filaments that activate WASP-bound Arp2/3 complex [8]. Importantly, Dip1-mediated activation of Arp2/3 complex creates linear filaments instead of branches [7]. Cells may therefore need to limit Dip1 activity relative to WASP to preserve the dendritic nature of actin networks, although it is unclear whether such regulatory mechanisms exist. Here, we use total internal reflection fluorescence (TIRF) microscopy to show that Dip1 causes actin assembled with WASP and Arp2/3 complex to form disconnected networks with many linear filaments rather than highly branched arrays. We discover a key biochemical difference between Dip1 and WASP that may limit linear filament nucleation in cells; although WASP must be released for nucleation, Dip1 stays associated with Arp2/3 complex on the pointed ends of nucleated actin filaments, so Dip1 is consumed in the reaction. Using live-cell imaging of fission yeast, we provide evidence that Dip1 is a single-turnover activator of Arp2/3 complex in vivo, revealing a mechanism by which Dip1 can initiate branched actin networks at endocytic sites without disrupting their branched architectures.

Dip1 Co-opts Features of Branching Nucleation to Create Linear Actin Filaments that Activate WASP-Bound Arp2/3 Complex.

When activated by Wiskott-Aldrich syndrome proteins (WASP), Arp2/3 complex nucleates branched actin filaments important for processes like cellular motility and endocytosis [1]. WASP-mediated activation of Arp2/3 complex requires a preformed actin filament, ensuring that activation by WASP creates branched instead of linear filaments. However, this biochemical requirement also means that assembly of branched actin networks must be primed with an initial seed filament [2-4]. We recently described a class of activators called WISH/DIP/SPIN90 (WDS) proteins, which, unlike WASP, activate Arp2/3 complex without a preformed filament [4]. Although this property may allow WDS proteins to serve as seed filament generators, it is unknown whether actin filaments nucleated by WDS-activated Arp2/3 complex can activate WASP-bound Arp2/3 complex. Further, despite their potential importance as branched actin network initiators, little is known about how WDS proteins turn on Arp2/3 complex. Here, we use two-color single-molecule total internal reflection fluorescence (TIRF) microscopy to show that Dip1, the S. pombe WDS protein [5], co-opts features of branching nucleation to activate Arp2/3 complex. Specifically, it activates Arp2/3 complex to nucleate linear filaments analogous to the branch created by WASP-mediated activation. The barbed ends of Dip1-Arp2/3 nucleated filaments are free to elongate, and their pointed ends remain anchored to Dip1-bound Arp2/3 complex. The linear filaments nucleated by Dip1-bound Arp2/3 complex activate WASP-bound Arp2/3 complex as potently as spontaneously nucleated or branched actin filaments. These observations provide important insights into the regulation of Arp2/3 complex by its activators and the molecular basis for initiation of branched actin networks.

Structure of the nucleation-promoting factor SPIN90 bound to the actin filament nucleator Arp2/3 complex.

Unlike the WASP family of Arp2/3 complex activators, WISH/DIP/SPIN90 (WDS) family proteins activate actin filament nucleation by the Arp2/3 complex without the need for a preformed actin filament. This allows WDS proteins to initiate branched actin network assembly by providing seed filaments that activate WASP-bound Arp2/3 complex. Despite their important role in actin network initiation, it is unclear how WDS proteins drive the activating steps that require both WASP and pre-existing actin filaments during WASP-mediated nucleation. Here, we show that SPIN90 folds into an armadillo repeat domain that binds a surface of Arp2/3 complex distinct from the two WASP sites, straddling a hinge point that may stimulate movement of the Arp2 subunit into the activated short-pitch conformation. SPIN90 binds a surface on Arp2/3 complex that overlaps with actin filament binding, explaining how it could stimulate the same structural rearrangements in the complex as pre-existing actin filaments. By revealing how WDS proteins activate the Arp2/3 complex, these data provide a molecular foundation to understand initiation of dendritic actin networks and regulation of Arp2/3 complex by its activators.

Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments.

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the kinetochore-microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.

Interactions with actin monomers, actin filaments, and Arp2/3 complex define the roles of WASP family proteins and cortactin in coordinately regulating branched actin networks.

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP.

Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ∼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation. DOI:http://dx.doi.org/10.7554/eLife.00884.001.

Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo.

Insertions within the actin core of actin-related protein 3 (Arp3) modulate branching nucleation by Arp2/3 complex.

The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp2 and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a "cryptic dimer" that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Here we characterize three key insertions within the actin core of Arp3 and show that each one plays a distinct role in modulating Arp2/3 function. The ß4/ß5 insert mediates interactions of Arp2/3 complex with actin filaments and "dampers" the nucleation activity of the complex. The Arp3 hydrophobic plug plays an important role in maintaining the integrity of the complex but is not absolutely required for formation of the daughter filament nucleus. Deletion of the αK/ß15 insert did not constitutively activate the complex, as previously hypothesized. Instead, it abolished in vitro nucleation activity and caused defects in endocytic actin patch assembly in fission yeast, indicating a role for the αK/ß15 insert in the activated state of the complex. Biochemical characterization of each mutant revealed steps in the nucleation pathway influenced by each Arp3-specific insert to provide new insights into the structural basis of activation of the complex.

Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins.

VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6(1)22, P2(1)2(1)2(1) and P2(1), respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source.

Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35.

Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.