Arf GTPase activates the WAVE regulatory complex through a distinct binding site.

Cross-talk between Rho- and Arf-family guanosine triphosphatases (GTPases) plays an important role in linking the actin cytoskeleton to membrane protrusions, organelle morphology, and vesicle trafficking. The central actin regulator, WAVE regulatory complex (WRC), integrates Rac1 (a Rho-family GTPase) and Arf signaling to promote Arp2/3-mediated actin polymerization in many processes, but how WRC senses Arf signaling is unknown. Here, we have reconstituted a direct interaction between Arf and WRC. This interaction is greatly enhanced by Rac1 binding to the D site of WRC. Arf1 binds to a previously unidentified, conserved surface on the Sra1 subunit of WRC, which, in turn, drives WRC activation using a mechanism distinct from that of Rac1. Mutating the Arf binding site abolishes Arf1-WRC interaction, disrupts Arf1-mediated WRC activation, and impairs lamellipodia formation and cell migration. This work uncovers a new mechanism underlying WRC activation and provides a mechanistic foundation for studying how WRC-mediated actin polymerization links Arf and Rac signaling in cells.

Structures reveal a key mechanism of WAVE regulatory complex activation by Rac1 GTPase.

The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ~3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2-central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.

Structure of Arp2/3 complex at a branched actin filament junction resolved by single-particle cryo-electron microscopy.

Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, and multiple classes of nucleation promoting factors (NPFs) stimulate its nucleation activity. When activated by WASP family NPFs, the complex must bind to the side of a preexisting (mother) filament of actin to complete the nucleation process, ensuring that WASP-mediated activation creates branched rather than linear actin filaments. How actin filaments contribute to activation is currently not understood, largely due to the lack of high-resolution structures of activated Arp2/3 complex bound to the side of a filament. Here, we present the 3.9-Å cryo-electron microscopy structure of the Arp2/3 complex at a branch junction. The structure reveals contacts between Arp2/3 complex and the side of the mother actin filament that likely stimulate subunit flattening, a conformational change that allows the actin-related protein subunits in the complex (Arp2 and Arp3) to mimic filamentous actin subunits. In contrast, limited contact between the bottom half of the complex and the mother filament suggests that clamp twisting, a second major conformational change observed in the active state, is not stimulated by actin filaments, potentially explaining why actin filaments are required but insufficient to trigger nucleation during WASP-mediated activation. Along with biochemical and live-cell imaging data and molecular dynamics simulations, the structure reveals features critical for the interaction of Arp2/3 complex with actin filaments and regulated assembly of branched actin filament networks in cells.

Unique Structural Features of the Mitochondrial AAA+ Protease AFG3L2 Reveal the Molecular Basis for Activity in Health and Disease.

Mitochondrial AAA+ quality-control proteases regulate diverse aspects of mitochondrial biology through specialized protein degradation, but the underlying mechanisms of these enzymes remain poorly defined. The mitochondrial AAA+ protease AFG3L2 is of particular interest, as genetic mutations localized throughout AFG3L2 are linked to diverse neurodegenerative disorders. However, a lack of structural data has limited our understanding of how mutations impact enzymatic function. Here, we used cryoelectron microscopy (cryo-EM) to determine a substrate-bound structure of the catalytic core of human AFG3L2. This structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins. Many disease-relevant mutations localize to these unique structural features of AFG3L2 and distinctly influence its activity and stability. Our results provide a molecular basis for neurological phenotypes associated with different AFG3L2 mutations and establish a structural framework to understand how different members of the AAA+ superfamily achieve specialized biological functions.

Statistic Experience Based Adaptive One-Shot Detector (EAO) for Camera Sensing System.

Object detection in a camera sensing system has been addressed by researchers in the field of image processing. Highly-developed techniques provide researchers with great opportunities to recognize objects by applying different algorithms. This paper proposes an object recognition model, named Statistic Experience-based Adaptive One-shot Detector (EAO), based on convolutional neural network. The proposed model makes use of spectral clustering to make detection dataset, generates prior boxes for object bounding and assigns prior boxes based on multi-resolution. The model is constructed and trained for improving the detection precision and the processing speed. Experiments are conducted on classical images datasets while the results demonstrate the superiority of EAO in terms of effectiveness and efficiency. Working performance of the EAO is verified by comparing it to several state-of-the-art approaches, which makes it a promising method for the development of the camera sensing technique.

Dissecting Substrate Specificities of the Mitochondrial AFG3L2 Protease.

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degradation products from multiple substrates using mass spectrometry, we construct a peptidase specificity profile that displays constrained product lengths and is dominated by the identity of the residue at the P1' position, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, discriminating between potential substrates by recognizing accessible degron sequences and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

Structural basis for inhibition of the deadenylase activity of human CNOT6L.

Human CNOT6L/CCR4, a member of the endonuclease-exonuclease-phosphatase (EEP) family enzymes, is one of the two deadenylase enzymes in the conserved CCR4-NOT complex. Here, we report inhibitor-bound crystal structures of the human CNOT6L nuclease domain in complex with the nucleotide CMP and the aminoglycoside neomycin. Deadenylase activity assays show that nucleotides are effective inhibitors of both CNOT6L and CNOT7, with AMP more effective than other nucleotides, and that neomycin is a weak deadenylase inhibitor. Structural analysis shows that all inhibitors occupy the substrate and magnesium-binding sites of CNOT6L, suggesting that inhibitors compete with both substrate and divalent magnesium ions for overlapping binding sites.