Cortactin stabilizes actin branches by bridging activated Arp2/3 to its nucleated actin filament.

Regulation of the assembly and turnover of branched actin filament networks nucleated by the Arp2/3 complex is essential during many cellular processes, including cell migration and membrane trafficking. Cortactin is important for actin branch stabilization, but the mechanism by which this occurs is unclear. Given this, we determined the structure of vertebrate cortactin-stabilized Arp2/3 actin branches using cryogenic electron microscopy. We find that cortactin interacts with the new daughter filament nucleated by the Arp2/3 complex at the branch site, rather than the initial mother actin filament. Cortactin preferentially binds activated Arp3. It also stabilizes the F-actin-like interface of activated Arp3 with the first actin subunit of the new filament, and its central repeats extend along successive daughter-filament subunits. The preference of cortactin for activated Arp3 explains its retention at the actin branch and accounts for its synergy with other nucleation-promoting factors in regulating branched actin network dynamics.

The stabilization of Arp2/3 complex generated actin filaments.

The Arp2/3 complex, which generates both branched but also linear actin filaments via activation of SPIN90, is evolutionarily conserved in eukaryotes. Several factors regulate the stability of filaments generated by the Arp2/3 complex to maintain the dynamics and architecture of actin networks. In this review, we summarise recent studies on the molecular mechanisms governing the tuning of Arp2/3 complex nucleated actin filaments, which includes investigations using microfluidics and single-molecule imaging to reveal the mechanosensitivity, dissociation and regeneration of actin branches. We also discuss the high-resolution cryo-EM structure of cortactin bound to actin branches, as well as the differences and similarities between the stability of Arp2/3 complex nucleated branches and linear filaments. These new studies provide a clearer picture of the stabilisation of Arp2/3 nucleated filaments at the molecular level. We also identified gaps in our understanding of how different factors collectively contribute to the stabilisation of Arp2/3 complex-generated actin networks.

Regeneration of actin filament branches from the same Arp2/3 complex.

Branched actin filaments are found in many key cellular structures. Branches are nucleated by the Arp2/3 complex activated by nucleation-promoting factor (NPF) proteins and bound to the side of preexisting "mother" filaments. Over time, branches dissociate from their mother filament, leading to network reorganization and turnover, but this mechanism is less understood. Here, using microfluidics and purified proteins, we examined the dissociation of individual branches under controlled biochemical and mechanical conditions. We observe that the Arp2/3 complex remains bound to the mother filament after most debranching events, even when accelerated by force. Strikingly, this surviving Arp2/3 complex readily nucleates a new actin filament branch, without being activated anew by an NPF: It simply needs to exchange its nucleotide and bind an actin monomer. The protein glia maturation factor (GMF), which accelerates debranching, prevents branch renucleation. Our results suggest that actin filament renucleation can provide a self-repair mechanism, helping branched networks to sustain mechanical stress in cells over extended periods of time.

Palisade structure in intact vaccinia virions.

Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24-66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.

The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus.

IMPORTANCE: Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host's transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.

ARPC5 deficiency leads to severe early-onset systemic inflammation and mortality.

The Arp2/3 complex drives the formation of branched actin networks that are essential for many cellular processes. In humans, the ARPC5 subunit of the Arp2/3 complex is encoded by two paralogous genes (ARPC5 and ARPC5L) with 67% identity. Through whole-exome sequencing, we identified a biallelic ARPC5 frameshift variant in a female child who presented with recurrent infections, multiple congenital anomalies, diarrhea and thrombocytopenia, and suffered early demise from sepsis. Her consanguineous parents also had a previous child who died with similar clinical features. Using CRISPR/Cas9-mediated approaches, we demonstrate that loss of ARPC5 affects actin cytoskeleton organization and function in vitro. Homozygous Arpc5-/- mice do not survive past embryonic day 9 owing to developmental defects, including loss of the second pharyngeal arch, which contributes to craniofacial and heart development. Our results indicate that ARPC5 is important for both prenatal development and postnatal immune signaling, in a non-redundant manner with ARPC5L. Moreover, our observations add ARPC5 to the list of genes that should be considered when patients present with syndromic early-onset immunodeficiency, particularly if recessive inheritance is suspected.

ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells.

CD4 T cell activation induces nuclear and cytoplasmic actin polymerization via the Arp2/3 complex to activate cytokine expression and strengthen T cell receptor (TCR) signaling. Actin polymerization dynamics and filament morphology differ between nucleus and cytoplasm. However, it is unclear how the Arp2/3 complex mediates distinct nuclear and cytoplasmic actin polymerization in response to a common stimulus. In humans, the ARP3, ARPC1, and ARPC5 subunits of the Arp2/3 complex exist as two different isoforms, resulting in complexes with different properties. Here, we show that the Arp2/3 subunit isoforms ARPC5 and ARPC5L play a central role in coordinating distinct actin polymerization events in CD4 T cells. While ARPC5L is heterogeneously expressed in individual CD4 T cells, it specifically drives nuclear actin polymerization upon T cell activation. In contrast, ARPC5 is evenly expressed in CD4 T cell populations and is required for cytoplasmic actin dynamics. Interestingly, nuclear actin polymerization triggered by a different stimulus, DNA replication stress, specifically requires ARPC5 but not ARPC5L. TCR signaling but not DNA replication stress induces nuclear actin polymerization via nuclear calcium-calmodulin signaling and N-WASP. Diversity in the molecular properties and individual expression patterns of ARPC5 subunit isoforms thus tailors Arp2/3-mediated actin polymerization to different physiological stimuli.

A succession of two viral lattices drives vaccinia virus assembly.

During its cytoplasmic replication, vaccinia virus assembles non-infectious spherical immature virions (IV) coated by a viral D13 lattice. Subsequently, IV mature into infectious brick-shaped intracellular mature virions (IMV) that lack D13. Here, we performed cryo-electron tomography (cryo-ET) of frozen-hydrated vaccinia-infected cells to structurally characterise the maturation process in situ. During IMV formation, a new viral core forms inside IV with a wall consisting of trimeric pillars arranged in a new pseudohexagonal lattice. This lattice appears as a palisade in cross-section. As maturation occurs, which involves a 50% reduction in particle volume, the viral membrane becomes corrugated as it adapts to the newly formed viral core in a process that does not appear to require membrane removal. Our study suggests that the length of this core is determined by the D13 lattice and that the consecutive D13 and palisade lattices control virion shape and dimensions during vaccinia assembly and maturation.

Regulation of branched versus linear Arp2/3-generated actin filaments.

Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides of pre-existing "mother" filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates "linear" actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N-WASP, and WASH destabilize existing branches, as well as SPIN90-Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.

Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection.

Intracellular mature viruses (IMVs) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMVs can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMVs, like intracellular enveloped viruses (IEVs), the second form of vaccinia that are wrapped in Golgi-derived membranes, recruit kinesin-1 and undergo anterograde transport. In vitro reconstitution of virion transport in infected cell extracts revealed that IMVs and IEVs move toward microtubule plus ends with respective velocities of 0.66 and 0.56 µm/s. Quantitative imaging established that IMVs and IEVs recruit an average of 139 and 320 kinesin-1 motor complexes, respectively. In the absence of kinesin-1, there was a near-complete loss of in vitro motility and reduction in the intracellular spread of both types of virions. Our observations demonstrate that kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to elucidate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.

Characterization of Resistance to the Mexican Rice Borer (Lepidoptera: Crambidae) among Sugarcane Cultivars.

Cultivar resistance is an essential management strategy for the Mexican rice borer, Eoreuma loftini (Dyar), in sugarcane in the USA, but resistance mechanisms are poorly understood. Resistance was evaluated among Louisiana's (USA) commercial sugarcane cultivars and experimental clones through field screenings, greenhouse trials, and a diet incorporation assay. Cultivars L 01-299 and HoCP 85-845 had the lowest borer injury levels, while HoCP 00-950 and L 12-201 were among the most heavily injured in field and greenhouse trials. The variability of results between the two field trials suggests that a genotype × environment interaction might affect the expression of resistance. Oviposition did not differ among evaluated cultivars in the greenhouse choice study. Results from the no-choice experiment showed that neonatal establishment differed among cultivars by up to 3-fold. In a diet incorporation assay, all cultivars reduced larval weight up to 86.5% and increased days to pupation by 1.8-fold relative to the diet-only control. Collectively, these results suggest that Louisiana's sugarcane breeding germplasm contains various resistance levels to E. loftini, emphasizing the importance of screening cultivars before they are released to growers. Future studies should try to determine the influence of environmental factors on resistance expression.

The relative binding position of Nck and Grb2 adaptors impacts actin-based motility of Vaccinia virus.

Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to assemble complex signalling networks. The concept of phase separation has recently changed our appreciation of multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now investigated this parameter in the operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3 complex-mediated actin polymerisation. Manipulating the position of pTyr motifs in A36 and the unrelated p14 from Orthoreovirus, we find that only specific spatial arrangements of Nck and Grb2 binding sites result in robust N-WASP recruitment, Arp2/3 complex driven actin polymerisation and viral spread. This suggests that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are frequently conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those undergoing phase transitions.

Field Assessment of Oryzophagus oryzae (Coleoptera: Curculionidae) Preference and Performance on Selected Rice Cultivars.

Plant resistance is a key strategy for the management of Oryzophagus oryzae (Costa Lima) (Coleoptera: Curculionidae), an important pest in South American rice paddies. The present study investigated the resistance of rice cultivars in terms of feeding and oviposition preference, growth, development, and biological performance of O. oryzae under natural conditions of field infestation during two consecutive rice seasons. There were no effects of the six cultivars on the feeding and oviposition preferences of O. oryzae as evaluated 5, 8, and 11 d After Flooding (DAF) of the plots, indicating the absence of antixenosis. Cultivars did not differ in terms of egg viability and larval density of first instars on the roots at 15 DAF. Significant differences were found 25 and 35 DAF when larval density per sample was high on 'BRS Pampa CL' (up to 24.5), intermediate on 'BRS Querência' and 'BRS Ligeirinho' (up to 16.1), and low on 'BRS Atalanta', 'BRS Firmeza', and 'Dawn' (up to 8.8). The cultivars 'BRS Atalanta', 'BRS Firmeza', and 'Dawn' caused malnutrition and inhibition of larval growth. These effects, typical of antibiosis, resulted in delayed pupation and emergence of adults; in addition, emerged females had body weight decreased strongly. The cultivars BRS Pampa CL, BRS Querência, and BRS Ligeirinho are susceptible, resulting in high larval populations and more suitable development of O. oryzae; antibiosis, as indicated for 'BRS Atalanta', 'BRS Firmeza', and 'Dawn', probably is the key mechanism of rice resistance to O. oryzae.

Circumpolar ocean stability on Mars 3 Gy ago.

What was the nature of the Late Hesperian climate, warm and wet or cold and dry? Formulated this way the question leads to an apparent paradox since both options seem implausible. A warm and wet climate would have produced extensive fluvial erosion but few valley networks have been observed at the age of the Late Hesperian. A too cold climate would have kept any northern ocean frozen most of the time. A moderate cold climate would have transferred the water from the ocean to the land in the form of snow and ice. But this would prevent tsunami formation, for which there is some evidence. Here, we provide insights from numerical climate simulations in agreement with surface geological features to demonstrate that the Martian climate could have been both cold and wet. Using an advanced general circulation model (GCM), we demonstrate that an ocean can be stable, even if the Martian mean surface temperature is lower than 0 °C. Rainfall is moderate near the shorelines and in the ocean. The southern plateau is mostly covered by ice with a mean temperature below 0 °C and a glacier return flow back to the ocean. This climate is achieved with a 1-bar CO2-dominated atmosphere with 10% H2 Under this scenario of 3 Ga, the geologic evidence of a shoreline and tsunami deposits along the ocean/land dichotomy are compatible with ice sheets and glacial valleys in the southern highlands.