CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

Identifying hydrophobic protein patches to inform protein interaction interfaces

Interactions between proteins lie at the heart of numerous biological processes and are essential for the proper functioning of the cell. Although the importance of hydrophobic residues in driving protein interactions is universally accepted, a characterization of protein hydrophobicity, which informs its interactions, has remained elusive. The challenge lies in capturing the collective response of the protein hydration waters to the nanoscale chemical and topographical protein patterns, which determine protein hydrophobicity. To address this challenge, here, we employ specialized molecular simulations wherein water molecules are systematically displaced from the protein hydration shell; by identifying protein regions that relinquish their waters more readily than others, we are then able to uncover the most hydrophobic protein patches. Surprisingly, such patches contain a large fraction of polar/charged atoms and have chemical compositions that are similar to the more hydrophilic protein patches. Importantly, we also find a striking correspondence between the most hydrophobic protein patches and regions that mediate protein interactions. Our work thus establishes a computational framework for characterizing the emergent hydrophobicity of amphiphilic solutes, such as proteins, which display nanoscale heterogeneity, and for uncovering their interaction interfaces.

Protein–protein interactions play a crucial role in numerous biological processes, ranging from signal transduction and immune response to protein aggregation and phase behavior (1–3). Consequently, being able to understand, predict, and modulate protein interactions has important implications for understanding cellular processes and mitigating the progression of disease (4, 5). A necessary first step toward this ambitious goal is uncovering the interfaces through which proteins interact (6–8). In principle, identifying hydrophobic protein regions, which interact weakly with water, should be a promising strategy for uncovering protein interaction interfaces (9, 10). Indeed, the release of weakly interacting hydration waters from hydrophobic regions can drive protein interactions, as well as other aqueous assemblies (11–13). However, even when the structure of a protein is available at atomistic resolution, it is challenging to identify its hydrophobic patches because they are not uniformly nonpolar, but display variations in polarity and charge at the nanoscale. Moreover, the emergent hydrophobicity of a protein patch stems from the collective response of protein hydration waters to the nanoscale chemical and topographical patterns displayed by the patch (14–20) and cannot be captured by simply counting the number of nonpolar groups in the patch, or even through more involved additive approaches, such as hydropathy scales or surface-area models (21–28).To address this challenge, we build upon seminal theoretical advances and molecular simulation studies, which have shown that near a hydrophobic surface, it is easier to disrupt surface–water interactions and form interfacial cavities (29–34). To uncover protein regions that have the weakest interactions with water, here, we employ specialized molecular simulations, wherein protein–water interactions are disrupted by systematically displacing water molecules from the protein hydration shell (35–37). By identifying the protein patches that nucleate cavities most readily in our simulations, we are then able to uncover the most hydrophobic protein regions. Interestingly, we find that both hydrophobic and hydrophilic protein patches are highly heterogeneous and contain comparable numbers of nonpolar and polar atoms. Our results thus highlight the nontrivial relationship between the chemical composition of protein patches and their emergent hydrophobicity (24–26), and further emphasize the importance of accounting for the collective solvent response in characterizing protein hydrophobicity (16). We then interrogate whether the most hydrophobic protein patches, which nucleate cavities readily, are also likely to mediate protein interactions. Across five proteins that participate in either homodimer or heterodimer formation, we find that roughly 60 to 70% of interfacial contacts and only about 10 to 20% of noncontacts nucleate cavities. Our work thus provides a versatile computational framework for characterizing hydrophobicity and uncovering interaction interfaces of not just proteins, but also of other complex amphiphilic solutes, such as cavitands, dendrimers, and patchy nanoparticles (38–41).     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin–PFN1–polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily affects motor neurons. Symptom progression is rapid, and the disease is universally fatal. Although most cases of ALS are sporadic, ∼10% of all ALS cases are familial (FALS) due to an inheritable genetic defect (1). Identification of FALS-linked genes has informed on the biological mechanism(s) underlying ALS pathogenesis. The ∼25 known ALS proteins appear to cluster within three functional processes: cytoskeletal dynamics, protein homeostasis, and RNA processing (1, 2). These categorizations are not mutually exclusive, however, in that some ALS-linked proteins can affect more than one process. For example, mutations in the actin-binding protein profilin-1 (PFN1) were recently linked to FALS (3–6), providing further support for disrupted cytoskeletal dynamics as a pathogenic mechanism in ALS (7, 8). In addition, ALS-linked mutations destabilize the PFN1 tertiary structure and induce PFN1 aggregation, which is expected to impact cellular protein homeostasis (3, 9–13). Despite the emerging evidence that mutant PFN1 adopts a misfolded conformation, little is known about how these conformational changes affect the biological functions of PFN1. Furthermore, it has been unclear whether mutation-induced destabilization and/or altered actin dynamics contribute to PFN1-mediated ALS.Efficient actin assembly requires coordinated interactions between PFN1, actin, and cytoskeletal proteins containing polyproline (poly-Pro) motifs, such as the formin family of proteins (14, 15), Ena/Vasp (16) as well as all nucleation promoting factors (17). Monomeric actin (G-actin) is the building block of filamentous actin (F-actin) (14), and formins are a class of proteins that function in actin nucleation and elongation (18, 19). G-actin–bound PFN1 binds poly-Pro motifs within the formin homology (FH)1 domain, forming a ternary complex that facilitates incorporation of G-actin onto the FH2-bound barbed end of a growing actin filament (14). Given the pivotal role of PFN1 in actin assembly, ALS-linked mutations have been assumed to induce functional deficits related to actin binding and/or formin-mediated actin assembly (20). In support of this notion, co-immunoprecipitation (co-IP) experiments from cultured cells showed impaired binding between mutant PFN1 and actin (3). However, subsequent studies with recombinant proteins showed that PFN1 mutants retain wild-type (WT) binding affinity for actin (9). Furthermore, mutant PFN1 binds a generic poly-L-proline peptide with similar affinity as PFN1 WT, although similar binding experiments using specific poly-Pro sequences derived from biological proteins have not been reported (9). Therefore, the effects of ALS-linked mutations on PFN1 ligand-binding and function have yet to be determined.Here, we conducted an unbiased immunoprecipitation–mass spectrometry (IP-MS) study with mammalian cells to identify potential differences in the protein interactomes of PFN1 WT and several ALS-linked PFN1 variants. Most of the PFN1 protein interactions were the same across all variants tested and mainly involved cytoskeletal proteins. However, the IP-MS analyses revealed enhanced interactions between two PFN1 variants, M114T and G118V, with a subset of formin proteins. Actin polymerization assays showed that these PFN1 variants retain the ability to facilitate formin-induced actin polymerization and that this function becomes enhanced in the context of a cellular environment. In contrast, the C71G ALS variant did not exhibit enhanced binding to formins and was deficient in promoting formin-mediated actin polymerization in vitro. In fact, PFN1 C71G was the most destabilized protein in all assays performed herein, resulting in PFN1 C71G turnover by the proteasome and reduced steady-state expression.Altogether, our results demonstrate that ALS-linked mutations do not simply abrogate binding of PFN1 to actin, formins, or other cytoskeletal factors. Rather, the effect of these mutations on the actin cytoskeleton appears to be more complex than previously thought. The outcomes of our various analyses indicate that some degree of mutation-induced destabilization (i.e., caused by M114T and G118V mutations) confers enhanced flexibility within mutant PFN1 in regions that bind actin and poly-Pro sequences. PFN1 flexibility appears to positively correlate with enhanced formin-mediated actin polymerization in vitro and in cells. However, severe mutation-induced destabilization leads to unproductive interactions between PFN1 C71G and cytoskeletal proteins, resulting in loss of PFN1 function. Notably, both classes of mutation alter formin-mediated actin polymerization, which may represent a general dysregulation of actin dynamics that contributes to PFN1-mediated ALS pathogenesis.     

Structural and functional dissection of reovirus capsid folding and assembly by the prefoldin-TRiC/CCT chaperone network

Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

Chaperones perform the essential function of folding proteins that cannot achieve a native conformation in an unassisted manner. The eukaryotic chaperonin TRiC (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) mediates the adenosine triphosphate (ATP)-dependent folding of a subset (∼10%) of newly translated cytosolic proteins (1). TRiC substrates include the cytoskeletal proteins actin and tubulin (2, 3), tumor suppressor proteins (4, 5), and aggregation-prone proteins that accumulate in neurodegenerative diseases such as Huntington disease (6, 7). TRiC has a toroidal structure formed by two rings stacked back to back, each composed of eight paralogous subunits that form a barrel with a central cavity (8, 9). The central cavity functions as a chamber within which newly translated polypeptides are sequestered in a protected protein-folding environment (10, 11). ATP binding and hydrolysis trigger cyclic changes in the conformation of TRiC between an open form in the nucleotide-free state and a closed, folding-active conformation in the ATP-hydrolysis state (12, 13). Despite its quasisymmetry, there is a high degree of specialization within the eight subunits that form TRiC, with individual subunits differing in substrate-binding capacity (14), ATP-binding and hydrolytic activity (12, 15, 16), and surface hydrophobicity (8). The diversification of these subunits is thought to contribute to the capacity of TRiC to fold a wide array of substrates with complex topologies.TRiC functions in concert with other molecular chaperones, including Hsp70 (17) and prefoldin (PFD) (18), to direct protein-folding pathways. Other chaperones, including ribosome-associated chaperones such as Hsp70, may function more promiscuously and earlier in the folding process to stabilize unfolded or partially folded proteins in conformations that can either fold without further assistance or be recognized by more specialized chaperones (19). PFD, a jellyfish-structured TRiC cochaperone (20), can also bind nascent polypeptides (18, 21). Hsp70 and PFD may direct the transfer of certain substrates to TRiC (17, 18). PFD can contribute directly to TRiC-mediated protein folding (22), but mechanisms by which cochaperones cooperate with TRiC to fold individual substrates are poorly defined.As obligate intracellular pathogens, viruses replicate within host cells and use host chaperones to fold viral polypeptides. Proteins from diverse families of viruses have been identified as TRiC substrates (23–25). TRiC performs an essential folding function in the replication of mammalian orthoreoviruses (reoviruses) (26). Reoviruses are ubiquitous, infecting most mammals early in life, and have been linked to celiac disease in humans (27). Reoviruses have a proteinaceous outer capsid composed of μ1 and σ3, which coalesce in a 1:1 stoichiometric ratio to form heterohexamers. The σ3 component of the outer capsid is an obligate TRiC substrate (26). σ3 is aggregation prone (28) and exists in multiple oligomeric forms in the cell (26). The mechanism of TRiC/σ3 binding, folding, and release is unclear. In addition, the chaperones that cooperate with TRiC to fold and assemble σ3 into a complex with its binding partner, μ1, are unknown.In this study, we identify a network of host chaperones that interact with reovirus σ3, including Hsp70, Hsp90, PFD, and TRiC. We establish a function for PFD in preventing σ3 aggregation and enhancing σ3 transfer to TRiC. Cryoelectron microscopy (cryo-EM) and cross-linking mass spectrometry (XL-MS) resolve the structure of σ3 within the TRiC folding chamber, revealing TRiC/σ3 interaction interfaces and the orientation of the σ3/μ1 oligomerization domain within TRiC. Biochemical studies establish a function for TRiC in assembling folded σ3 into a complex with μ1 through a process that is enhanced by PFD. Functional assays demonstrate that TRiC folds σ3 into its biologically active and infectious conformation. Together, this work provides mechanistic insight into the structure and function of TRiC and PFD in the folding and assembly of a heterooligomeric protein complex.     

ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14–ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA–VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid–RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

Human rhinoviruses are the cause of more than half of common colds (1). Medical visits and missed days of school and work cost tens of billions of US dollars annually (2, 3). There is currently no cure for rhinovirus infections, and the available treatments are only symptomatic. Rhinoviruses belong to the family Picornaviridae, genus Enterovirus, and are classified into species A, B, and C (4). Rhinoviruses A and B can belong to either “major” or “minor” groups, based on their utilization of intercellular adhesion molecule-1 (ICAM-1) or low-density lipoprotein receptor for cell entry (5–7). Type C rhinoviruses use CDHR3 as a receptor (8). Rhinovirus 14 belongs to the species rhinovirus B and uses ICAM-1 as a receptor. Receptors recognized by rhinoviruses and other enteroviruses can be divided into two groups based on their function in the infection process (9). Attachment receptors such as DAF, PSGL1, KREMEN1, CDHR3, and sialic acid enable the binding and endocytosis of virus particles into cells (10–13). In contrast, uncoating receptors including ICAM-1, CD155, CAR, and SCARB2 enable virus cell entry but also promote genome release from virus particles (5, 14–16).Virions of rhinoviruses are nonenveloped and have icosahedral capsids (17). Genomes of rhinoviruses are 7,000 to 9,000 nucleotide-long single-stranded positive-sense RNA molecules (1, 17). The rhinovirus genome encodes a single polyprotein that is co- and posttranslationally cleaved into functional protein subunits. Capsid proteins VP1, VP3, and VP0, originating from one polyprotein, form a protomer, 60 of which assemble into a pseudo-T = 3 icosahedral capsid. To render the virions mature and infectious, VP0 subunits are cleaved into VP2 and VP4 (18, 19). VP1 subunits form pentamers around fivefold symmetry axes, whereas subunits VP2 and VP3 form heterohexamers centered on threefold symmetry axes. The major capsid proteins VP1 through 3 have a jelly roll β-sandwich fold formed by two β-sheets, each containing four antiparallel β-strands, which are conventionally named B to I (20–22). The two β-sheets contain the strands BIDG and CHEF, respectively. The C termini of the capsid proteins are located at the virion surface, whereas the N termini mediate interactions between the capsid proteins and the RNA genome on the inner surface of the capsid. VP4 subunits are attached to the inner face of the capsid formed by the major capsid proteins. The surfaces of rhinovirus virions are characterized by circular depressions called canyons, which are centered around fivefold symmetry axes of the capsids (21).The VP1 subunits of most rhinoviruses, but not those of rhinovirus 14, contain hydrophobic pockets, which are filled by molecules called pocket factors (17, 21, 23, 24). It has been speculated that pocket factors are fatty acids or lipids (25). The pockets are positioned immediately below the canyons. The exposure of rhinoviruses to acidic pH induces expulsion of the pocket factors, which leads to the formation of activated particles and genome release (17, 26–32). The activated particles are characterized by capsid expansion, a reduction in interpentamer contacts, the release of VP4 subunits, externalization of N termini of VP1 subunits, and changes in the distribution of RNA genomes (17, 26–29, 33, 34). Artificial hydrophobic compounds that bind to VP1 pockets with high affinity inhibit infection by rhinoviruses (35, 36).ICAM-1 is an endothelial- and leukocyte-associated protein that stabilizes cell–cell interactions and facilitates the movement of leukocytes through endothelia (37). ICAM-1 can be divided into an extracellular amino-terminal part composed of five immunoglobulin domains, a single transmembrane helix, and a 29-residue–long carboxyl-terminal cytoplasmic domain. The immunoglobulin domains are characterized by a specific fold that consists of seven to eight β-strands, which form two antiparallel β-sheets in a sandwich arrangement (38–40). The immunoglobulin domains of ICAM-1 are stabilized by disulfide bonds and glycosylation (38–41). The connections between the immunoglobulin domains are formed by flexible linkers that enable bending of the extracellular part of ICAM-1. For example, the angle between domains 1 and 2 differs by 8° between molecules in distinct crystal forms (38, 42). As a virus receptor, ICAM-1 enables the virus particles to sequester at the cell surface and mediates their endocytosis (5). The structures of complexes of rhinoviruses 3, 14, and 16, and CVA21 with ICAM-1 have been determined to resolutions of 9 to 28 Å (42–46). It was shown that ICAM-1 molecules bind into the canyons at the rhinovirus surface, approximately between fivefold and twofold symmetry axes (42–46). ICAM-1 interacts with residues from all three major capsid proteins. It has been speculated that the binding of ICAM-1 triggers the transition of virions of rhinovirus 14 to activated particles and initiates genome release (45, 47). However, the limited resolution of the previous studies prevented characterization of the corresponding molecular mechanism.Here, we present the cryo-electron microscopy (cryo-EM) reconstruction of the rhinovirus 14 virion, which contains resolved density of octanucleotide segments of the RNA genome that interact with VP2 subunits. Furthermore, we show that the binding of ICAM-1 to rhinovirus 14 induces changes in its capsid and genome, which are required for subsequent virus activation and genome release at acidic pH.     

Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo–hippocampal interactions play a task-phase–dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

The neurotransmitter acetylcholine is thought to be critical for hippocampus-dependent declarative memories (1, 2). Reduction in cholinergic neurotransmission, either in Alzheimer’s disease or in experiments with cholinergic antagonists, such as scopolamine, impairs memory function (3–8). Acetylcholine may bring about its beneficial effects on memory encoding by enhancing theta rhythm oscillations, decreasing recurrent excitation, and increasing synaptic plasticity (9–11). Conversely, drugs which activate cholinergic receptors enhance learning and, therefore, are a neuropharmacological target for the treatment of memory deficits in Alzheimer’s disease (5, 12, 13).The contribution of cholinergic mechanisms in the acquisition of long-term memories and the role of the hippocampal–entorhinal–cortical interactions are well supported by experimental data (5, 12, 13). In addition, working memory or “short-term” memory is also supported by the hippocampal–entorhinal–prefrontal cortex (14–16). Working memory in humans is postulated to be a conscious process to “keep things in mind” transiently (16). In rodents, matching to sample task, spontaneous alternation between reward locations, and the radial maze task have been suggested to function as a homolog of working memory [“working memory like” (17)].Cholinergic activity is a critical requirement for working memory (18, 19) and for sustaining theta oscillations (10, 20–22). In support of this contention, theta–gamma coupling and gamma power are significantly higher in the choice arm of the maze, compared with those in the side arms where working memory is no longer needed for correct performance (23–26). It has long been hypothesized that working memory is maintained by persistent firing of neurons, which keep the presented items in a transient store in the prefrontal cortex and hippocampal–entorhinal system (27–31), although the exact mechanisms are debated (32–37). An alternative hypothesis holds that items of working memory are stored in theta-nested gamma cycles (38). Common in these models of working memory is the need for an active, cholinergic system–dependent mechanism (39–41). However, in spontaneous alternation tasks, the animals are not moving continuously during the delay, and theta oscillations are not sustained either. During the immobility epochs, theta is replaced by intermittent sharp-wave ripples (SPW-R), yet memory performance does not deteriorate. On the contrary, artificial blockade of SPW-Rs can impair memory performance (42, 43), and prolongation of SPW-Rs improves performance (44). Under the cholinergic hypothesis of working memory, such a result is unexpected.To address the relationship between cholinergic/theta versus SPW-R mechanism in spontaneous alternation, we used a G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) (45) to monitor acetylcholine (ACh) activity during memory performance in mice. In addition, we optogenetically enhanced cholinergic tone, which suppresses SPW-Rs by a different mechanism than electrically or optogenetically induced silencing of neurons in the hippocampus (43, 44). We show that cholinergic signaling in the hippocampus increases in parallel with theta power/score during walking and rapid eye movement (REM) sleep and reaches a transient minimum during SPW-Rs. Selective optogenetic stimulation of medial septal cholinergic neurons decreased the incidence of SPW-Rs during non-REM sleep (46–48), as well as during the delay epoch of a working memory task and impaired memory performance. These findings demonstrate that memory performance is supported by complementary theta and SPW-R mechanisms.     

An engineered 4-1BBL fusion protein with “activity on demand”

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Cytokines are immunomodulatory proteins that have been considered for pharmaceutical applications in the treatment of cancer patients (1–3) and other types of disease (2). There is a growing interest in the use of engineered cytokine products as anticancer drugs, capable of boosting the action of T cells and natural killer (NK) cells against tumors (3, 4), alone or in combination with immune checkpoint inhibitors (3, 5–7).Recombinant cytokine products on the market include interleukin-2 (IL-2) (Proleukin) (8, 9), IL-11 (Neumega) (10, 11), tumor necrosis factor (TNF; Beromun) (12), interferon (IFN)-α (Roferon A, Intron A) (13, 14), IFN-β (Avonex, Rebif, Betaseron) (15, 16), IFN-γ (Actimmune) (17), granulocyte colony-stimulating factor (Neupogen) (18), and granulocyte macrophage colony-stimulating factor (Leukine) (19, 20). The recommended dose is typically very low (often <1 mg/d) (21–23), as cytokines may exert biological activity in the subnanomolar concentration range (24). Various strategies have been proposed to develop cytokine products with improved therapeutic index. Protein PEGylation or Fc fusions may lead to prolonged circulation time in the bloodstream, allowing the administration of low doses of active payload (25, 26). In some implementations, cleavable polyethylene glycol polymers may be considered, yielding prodrugs that regain activity at later time points (27). Alternatively, tumor-homing antibody fusions have been developed, since the preferential concentration of cytokine payloads at the tumor site has been shown in preclinical models to potentiate therapeutic activity, helping spare normal tissues (28–34). Various antibody-cytokine fusions are currently being investigated in clinical trials for the treatment of cancer and of chronic inflammatory conditions (reviewed in refs. 2, 33, 35–37).Antibody-cytokine fusions display biological activity immediately after injection into patients, which may lead to unwanted toxicity and prevent escalation to therapeutically active dosage regimens (9, 22, 38). In the case of proinflammatory payloads (e.g., IL-2, IL-12, TNF-α), common side effects include hypotension, nausea, and vomiting, as well as flu-like symptoms (24, 39–42). These side effects typically disappear when the cytokine concentration drops below a critical threshold, thus providing a rationale for slow-infusion administration procedures (43). It would be highly desirable to generate antibody-cytokine fusion proteins with excellent tumor-targeting properties and with “activity on demand”— biological activity that is conditionally gained on antigen binding at the site of disease, helping spare normal tissues.Here we describe a fusion protein consisting of the F8 antibody specific to the alternatively spliced extra domain A (EDA) of fibronectin (44, 45) and of murine 4-1BBL, which did not exhibit cytokine activity in solution but could regain potent biological activity on antigen binding. The antigen (EDA+ fibronectin) is conserved from mouse to man (46), is virtually undetectable in normal adult tissues (with the exception of the placenta, endometrium, and some vessels in the ovaries), but is expressed in the majority of human malignancies (44, 45, 47, 48). 4-1BBL, a member of the TNF superfamily (49), is expressed on antigen-presenting cells (50, 51) and binds to its receptor, 4-1BB, which is up-regulated on activated cytotoxic T cells (52), activated dendritic cells (52), activated NK and NKT cells (53), and regulatory T cells (54). Signaling through 4-1BB on cytotoxic T cells protects them from activation-induced cell death and skews the cells toward a more memory-like phenotype (55, 56).We engineered nine formats of the F8-4-1BBL fusion protein, one of which exhibited superior performance in quantitative biodistribution studies and conditional gain of cytokine activity on antigen binding. The antigen-dependent reconstitution of the biological activity of the immunostimulatory payload represents an example of an antibody fusion protein with “activity on demand.” The fusion protein was potently active against different types of cancer without apparent toxicity at the doses used. The EDA of fibronectin is a particularly attractive antigen for cancer therapy in view of its high selectivity, stability, and abundant expression in most tumor types (44, 45, 47, 48).     

Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Hepatitis C virus (HCV) is a global disease burden, with an estimated 71 million people infected worldwide (1, 2). Roughly 75% of HCV infections become chronic (3–5) and in severe cases can result in cirrhosis or hepatocellular carcinoma (6). Viral infection can be cured at high rates by direct-acting antivirals, but multiple public health and financial barriers (7, 8), along with the possibility of reinfection or continued disease progression (7, 9, 10), have resulted in a continued rise in HCV infections. An HCV vaccine remains essential to proactively protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (11, 12). The challenges posed by HCV sequence diversity (12, 13), glycan shielding (14, 15), immunodominant nonneutralizing epitopes (16–19), and preparation of a homogeneous E1E2 antigen all contribute to the difficulty in generating protective B cell immune responses. Although multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited (20). Optimization of E1E2 to improve its immunogenicity and elicitation of bnAbs through rational design may lead to an effective B cell-based vaccine (21).HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (22–24). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (25) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (24, 26, 27) and interactions between E1 and E2 ectodomains (28–30). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and SR-B1 coreceptors as part of a multistep entry process on the surface of hepatocytes (31–34). Neutralizing antibody responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (18, 35–40). Structural knowledge of bnAb antibody–antigen interactions, which often target E2 epitopes in distinct antigenic domains B, D, or E (18, 41, 42), can inform vaccine design efforts to induce bnAb responses against flexible HCV epitopes (43–45). E1E2 bnAbs, including AR4A, AR5A (46), and others recently identified (38), are not only among the most broadly neutralizing (35) but also represent E1E2 quaternary epitopes unique to antibody recognition of HCV.Although much is known about bnAb responses to E1E2 glycoproteins, induction of B cell-based immunity with an E1E2-based vaccine immunogen (47–49) has remained difficult. The inherent hydrophobicity of E1 and E2 TMDs (24, 50) may impede uniform production of an immunogenic E1E2 heterodimer that could be utilized for both vaccine development and E1E2 structural studies. Although partial E1 and E2 structures have been determined (39, 51–54), many other enveloped viruses have structures of a complete and near-native glycoprotein assembly (55–59), providing a basis for rational vaccine design (60–62). Viral glycoproteins of influenza hemagglutinin (63), respiratory syncytial virus (RSV) (55), severe acute respiratory syndrome coronavirus 2 (64), and others (65, 66) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. HIV gp120–gp41 proteins have been designed as soluble SOSIP trimers in part by introducing a furin cleavage site to facilitate native-like assembly when cleaved by the enzyme (56, 67). Previously described E1E2 glycoprotein designs include covalently linked E1 and E2 ectodomains (68, 69), E1E2 with TMDs intact and an immunoglobulin G (IgG) Fc tag for purification (70), as well as E1 and E2 ectodomains with a cleavage site (68), which presented challenges for purification either due to intracellular expression or to high heterogeneity. Two recently described scaffolded E1E2 designs, while promising, have not been shown to engage monoclonal antibodies (mAbs) that recognize the native E1E2 assembly, though they were engaged by E1-specific and E2-specific mAbs, as well as coreceptors that recognize E2 (71). Therefore, these presentations of E1E2 glycoproteins may not represent a native and immunogenic heterodimeric assembly, and thus their potential as vaccine candidates remains unclear.Here, we describe the design of a secreted E1E2 glycoprotein (sE1E2) that mimics both the antigenicity in vitro and the immunogenicity in vivo of the native heterodimer through the scaffolding of E1E2 ectodomains. In testing our designs, we found that both replacing E1E2 TMDs with a leucine zipper scaffold and inserting a furin cleavage site between E1 and E2 enabled secretion and native-like sE1E2 assembly. We assessed the size, heterogeneity, antigenicity, and immunogenicity of this construct (identified as sE1E2.LZ) in comparison with full-length membrane-bound E1E2 (mbE1E2). sE1E2.LZ binds a broad panel of bnAbs to E2 and E1E2, as well as coreceptor CD81, providing evidence of assembly into a native-like heterodimer. An immunogenicity study indicated that sera of mice injected with sE1E2.LZ neutralize HCV pseudoparticles at levels comparable to sera from mice immunized with mbE1E2. This sE1E2 design is a form of the native E1E2 heterodimer that both improves upon current designs and represents a platform for structural characterization and engineering of additional HCV vaccine candidates.     

Tropospheric carbonyl sulfide mass balance based on direct measurements of sulfur isotopes

Robust estimates for the rates and trends in terrestrial gross primary production (GPP; plant CO₂ uptake) are needed. Carbonyl sulfide (COS) is the major long-lived sulfur-bearing gas in the atmosphere and a promising proxy for GPP. Large uncertainties in estimating the relative magnitude of the COS sources and sinks limit this approach. Sulfur isotope measurements (³⁴S/³²S; δ³⁴S) have been suggested as a useful tool to constrain COS sources. Yet such measurements are currently scarce for the atmosphere and absent for the marine source and the plant sink, which are two main fluxes. Here we present sulfur isotopes measurements of marine and atmospheric COS, and of plant-uptake fractionation experiments. These measurements resulted in a complete data-based tropospheric COS isotopic mass balance, which allows improved partition of the sources. We found an isotopic (δ³⁴S ± SE) value of 13.9 ± 0.1‰ for the troposphere, with an isotopic seasonal cycle driven by plant uptake. This seasonality agrees with a fractionation of −1.9 ± 0.3‰ which we measured in plant-chamber experiments. Air samples with strong anthropogenic influence indicated an anthropogenic COS isotopic value of 8 ± 1‰. Samples of seawater-equilibrated-air indicate that the marine COS source has an isotopic value of 14.7 ± 1‰. Using our data-based mass balance, we constrained the relative contribution of the two main tropospheric COS sources resulting in 40 ± 17% for the anthropogenic source and 60 ± 20% for the oceanic source. This constraint is important for a better understanding of the global COS budget and its improved use for GPP determination.

The Earth system is going through rapid changes as the climate warms and CO₂ level rises. This rise in CO₂ is mitigated by plant uptake; hence, it is important to estimate global and regional photosynthesis rates and trends (1). Yet, robust tools for investigating these processes at a large scale are scarce (2). Recent studies suggest that carbonyl sulfide (COS) could provide an improved constraint on terrestrial photosynthesis (gross primary production, GPP) (2–12). COS is the major long-lived sulfur-bearing gas in the atmosphere and the main supplier of sulfur to the stratospheric sulfate aerosol layer (13), which exerts a cooling effect on the Earth’s surface and regulates stratospheric ozone chemistry (14).During terrestrial photosynthesis, COS diffuses into leaf stomata and is consumed by photosynthetic enzymes in a similar manner to CO₂ (3–5). Contrary to CO₂, COS undergoes rapid and irreversible hydrolysis mainly by the enzyme carbonic-anhydrase (6, 7). Thus, COS can be used as a proxy for the one-way flux of CO₂ removal from the atmosphere by terrestrial photosynthesis (2, 8–11). However, the large uncertainties in estimating the COS sources weaken this approach (10–12, 15). Tropospheric COS has two main sources: the oceans and anthropogenic emissions, and one main sink–terrestrial plant uptake (8, 10–13). Smaller sources include biomass burning, soil emissions, wetlands, volcanoes, and smaller sinks include OH destruction, stratospheric destruction, and soil uptake (12). The largest source of COS to the atmosphere is the ocean, both as direct COS emission, and as indirect carbon disulfide (CS₂) and dimethylsulfide (DMS) emissions that are rapidly oxidized to COS (10, 16–20). Recent studies suggest oceanic COS emissions are in the range of 200–4,000 GgS/y (19–22). The second major COS source is the anthropogenic source, which is dominated by indirect emissions derived from CS₂ oxidation, mainly from the use of CS₂ as an industrial solvent. Direct emissions of COS are mainly derived from coal and fuel combustion (17, 23, 24). Recent studies suggest that anthropogenic emissions are in the range of 150–585 GgS/y (23, 24). The terrestrial plant uptake is estimated to be in the range of 400–1,360 GgS/y (11). Measurements of sulfur isotope ratios (δ³⁴S) in COS may be used to track COS sources and thus reduce the uncertainties in their flux estimations (15, 25–27). However, the isotopic mass balance approach works best if the COS end members are directly measured and have a significantly different isotopic signature. Previous δ³⁴S measurements of atmospheric COS are scarce and there have been no direct measurements of two important components: the δ³⁴S of oceanic COS emissions, and the isotopic fractionation of COS during plant uptake (15, 25–27). In contrast to previous studies that used assessments for these isotopic values, our aim was to directly measure the isotopic values of these missing components, and to determine the tropospheric COS δ³⁴S variability over space and time.     

Chemokine-biased robust self-organizing polarization of migrating cells in vivo

To study the mechanisms controlling front-rear polarity in migrating cells, we used zebrafish primordial germ cells (PGCs) as an in vivo model. We find that polarity of bleb-driven migrating cells can be initiated at the cell front, as manifested by actin accumulation at the future leading edge and myosin-dependent retrograde actin flow toward the other side of the cell. In such cases, the definition of the cell front, from which bleb-inhibiting proteins such as Ezrin are depleted, precedes the establishment of the cell rear, where those proteins accumulate. Conversely, following cell division, the accumulation of Ezrin at the cleavage plane is the first sign for cell polarity and this aspect of the cell becomes the cell back. Together, the antagonistic interactions between the cell front and back lead to a robust polarization of the cell. Furthermore, we show that chemokine signaling can bias the establishment of the front-rear axis of the cell, thereby guiding the migrating cells toward sites of higher levels of the attractant. We compare these results to a theoretical model according to which a critical value of actin treadmilling flow can initiate a positive feedback loop that leads to the generation of the front-rear axis and to stable cell polarization. Together, our in vivo findings and the mathematical model, provide an explanation for the observed nonoriented migration of primordial germ cells in the absence of the guidance cue, as well as for the directed migration toward the region where the gonad develops.

Establishing and maintaining polarity is essential for a range of different cellular functions (1). This requirement is particularly critical for single-cell migration, a process that depends on the definition of a front-rear axis, which, in turn, dictates the direction of movement. This important issue has previously been investigated primarily in cells such as Dictyostelium discoideum, leukocytes, fibroblasts, and neural crest cells (2–7), cell types that employ actin polymerization to generate physical force to advance their leading edge in a form of lamellipodia. These studies suggest that polarity is generated and stabilized by processes of local positive feedback control loops, long-range negative feedback loops, and by control of protrusion formation through membrane tension, thereby facilitating forward movement of the cells (8–11).In contrast to actin-powered lamellipodia-based migration, a different cell migration strategy exhibited by diverse cell types in physiological contexts, as well as in pathological conditions is characterized by the generation of bleb-type protrusions at the cell front (12–14). Blebs are spherical in shape and are powered by hydrostatic pressure and cytoplasmic flow (15–17). Here, a net forward movement is achieved via polarized bleb formation driven by inflow of cytoplasm and concomitant retraction of the opposite side of the cell. Several molecular processes have been found to contribute to the generation of blebs. Most notably, actomyosin contractility is required to generate the intracellular hydrostatic pressure and to induce breaks in the cell cortex that promote the separation of the plasma membrane from the underlying actin filaments (18). This process is therefore controlled by the balance between contractility, membrane tension, and membrane-cortex attachment (15–17, 19). Nevertheless, little is known about the mechanisms responsible for defining the front of blebbing cells and establishing the rear. Similarly, the nature of the interactions between the front and the rear is not well understood in this context. A theoretical model that describes these interactions is the universal coupling of speed and cell persistence (UCSP) model (20). An important component in this model for establishing cell polarization is the net actin retrograde flow away from the side of the cell that becomes the cell front. According to this model, the front can be maintained at this location by a positive feedback between the actin flow and the flow of inhibitors of actin polymerization away from it (21). Experimentally, in the context of cell migration, actin retrograde flow was first shown to be important in processes of focal-adhesion-dependent motility. The flow results from a combination of actin polymerization against the cell membrane at the leading edge and nonmuscle myosin II activity (22–24).An important model for studying bleb-driven motility in an in vivo context is that of zebrafish primordial germ cells (PGCs) (25). Zebrafish PGCs migrate within the embryo employing blebs and are directed by the chemokine Cxcl12a and its receptor Cxcr4b (26). When guided by the chemokine as well as when migrating in random directions in its absence, PGCs alternate between two distinct modes of behavior namely “run” and “tumbling” (27). During run phases, PGCs extend blebs in the direction of movement and actively migrate, while during the tumbling phases the cells are morphologically apolar, form blebs in all directions, and are immotile (27). This periodic sequence of events where PGCs lose and regain polarity makes these cells an excellent model for studying the establishment and maintenance of cell polarity of blebbing cells and for determining the role of chemokine signaling in this process.Determining the precise course of events leading to cell motility and the interdependency of different events that contribute to the polarization of migrating cells in vivo is challenging. Nevertheless, by studying PGC migration within early zebrafish embryos we could describe the molecular cascade leading to cell polarization. We found that polarization is initiated by a biased actin polymerization at one aspect of the cell. This initial symmetry-breaking event determines and orients the front-rear axis of the cell by directing streaming of specific proteins to the future back. Proteins such as Ezrin and extended synaptotagmin-like 2a that are transported to the cell back can inhibit the formation of blebs, thereby specifying the rear of the cell. At the same time, the transport of these proteins away from the putative cell front makes blebbing more likely to occur there. In addition, we provide evidence suggesting that blebbing enhances actin polymerization at the front, thereby establishing a positive feedback with the retrograde actin flow. The combination of the positive and negative feedback reinforces the initial cell polarization event to establish a persistent polarity. Furthermore, while this cascade can be initiated at random positions around the cell perimeter, the primary polarization event can be biased by chemokine signals, thereby directing cell migration toward specific regions in the embryo where the attractant is expressed. Last, our experimental observations could be described by the set of equations based on the UCSP theoretical model. This model predicts that persistent cell polarization can be established above a critical level of actin polymerization-induced treadmilling, which breaks the cell symmetry by advection of bleb-inhibiting proteins to the other aspect of the cell, thereby maintaining the polarized actin flow.     

Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.     

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Like most pathways in the mature central nervous system (CNS), the optic nerve cannot regenerate once damaged due in part to cell-extrinsic suppressors of axon growth (1, 2) and the low intrinsic growth capacity of adult retinal ganglion cells (RGCs), the projection neurons of the eye (3–5). Consequently, traumatic or ischemic optic nerve injury or degenerative diseases such as glaucoma lead to irreversible visual losses. Experimentally, some degree of regeneration can be induced by intraocular inflammation or growth factors expressed by inflammatory cells (6–10), altering the cell-intrinsic growth potential of RGCs (3–5), enhancing physiological activity (11, 12), chelating free zinc (13, 14), and other manipulations (15–19). However, the extent of regeneration achieved to date remains modest, underlining the need for more effective therapies.Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for glaucoma and other ocular diseases (20–23). Activation of the downstream signal transduction cascade requires CNTF to bind to CNTF receptor-α (CNTFRα) (24), which leads to recruitment of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor-β (LIFRβ) to form a tripartite receptor complex (25). CNTFRα anchors to the plasma membrane through a glycosylphosphatidylinositol linkage (26) and can be released and become soluble through phospholipase C-mediated cleavage (27). CNTF has been reported to activate STAT3 phosphorylation in retinal neurons, including RGCs, and to promote survival, but it is unknown whether these effects are mediated by direct action of CNTF on RGCs via CNTFRα (28). Our previous studies showed that CNTF promotes axon outgrowth from neonate RGCs in culture (29) but fails to do so in cultured mature RGCs (8) or in vivo (6). Although some studies report that recombinant CNTF (rCNTF) can promote optic nerve regeneration (20, 30, 31), others find little or no effect unless SOCS3 (suppressor of cytokine signaling-3), an inhibitor of the Jak-STAT pathway, is deleted in RGCs (5, 6, 32). In contrast, multiple studies show that adeno-associated virus (AAV)-mediated expression of CNTF in RGCs induces strong regeneration (33–40). The basis for the discrepant effects of rCNTF and CNTF gene therapy is unknown but is of considerable interest in view of the many promising clinical and preclinical outcomes obtained with CNTF to date.Because intravitreal virus injections induce inflammation (41), we investigated the possibility that CNTF, a known immune modulator (42–44), might act by elevating expression of other immune-derived factors. We report here that the beneficial effects of CNTF gene therapy in fact require immune system activation and elevation of C-C motif chemokine ligand 5 (CCL5). Depletion of neutrophils, global knockout (KO) or RGC-selective deletion of the CCL5 receptor CCR5, or a CCR5 antagonist all suppress the effects of CNTF gene therapy, whereas recombinant CCL5 (rCCL5) promotes axon regeneration and increases RGC survival. These studies point to CCL5 as a potent monotherapy for optic nerve regeneration and to the possibility that other applications of CNTF and other forms of gene therapy might similarly act indirectly through other factors.     

The β-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large β-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs⁻ mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro−Ala−Ala−Arg (PAAR) repeat domains, which bind the β-spike of trimeric valine−glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact β-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs⁺ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the β-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact β-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Bacteria use many strategies to compete against other microorganisms in the environment. Research over the past 15 y has uncovered several distinct mechanisms by which bacteria deliver inhibitory toxins directly into neighboring competitors (1–8). Cell contact-dependent competition systems have been characterized most extensively in Gram-negative bacteria, and the most widespread mechanism is mediated by the type VI secretion system (T6SS) (9). T6SSs are multiprotein complexes related in structure and function to the contractile tails of Myoviridae bacteriophages. T6SS loci vary considerably between bacterial species, but all encode 13 core type VI secretion (Tss) proteins that are required to build a functional apparatus. TssJ, TssL, and TssM form a multimeric complex that spans the cell envelope and serves as the secretion conduit. The phage-like baseplate is composed of TssE, TssF, TssG, and TssK proteins, which form a sixfold symmetrical array surrounding a central “hub” of trimeric valine−glycine repeat protein G (VgrG/TssI). VgrG is structurally homologous to the gp27−gp5 tail spike of phage T4 (10, 11). The T4 tail spike is further acuminated with gp5.4, a small protein that forms a sharpened apex at the tip of the gp5 spike (12). Proline−alanine−alanine−arginine (PAAR) repeat proteins form an orthologous structure on VgrG; and PAAR is thought to facilitate penetration of the target cell outer membrane (13). The T6SS duty cycle begins when the baseplate docks onto TssJLM at the cytoplasmic face of the inner membrane (14). The baseplate then serves as the assembly origin for the contractile sheath and inner tube. The sheath is built from TssB−TssC subunits, and the tube is formed by stacked hexameric rings of hemolysin-coregulated protein (Hcp/TssD). TssA coordinates this assembly process to ensure that the sheath and tube are polymerized at equivalent rates (15). After elongating across the width of the cell, the sheath undergoes rapid contraction to expel the PAAR•VgrG-capped Hcp tube through the transenvelope complex. The ejected tube impales neighboring cells and delivers a variety of toxic effector proteins into the target. After firing, the contracted sheath is disassembled by the ClpV (TssH) ATPase (16), and the recycled TssBC subunits are used to support additional rounds of sheath assembly and contraction.T6SSs were originally identified through their ability to intoxicate eukaryotic host cells (17), and VgrG proteins were the first effectors to be recognized. VgrG-1 from Vibrio cholerae V52 carries a C-terminal domain that cross-links actin and blocks macrophage phagocytosis (10). Similarly, the VgrG1 protein from Aeromonas hydrophila American Type Culture Collection (ATCC) 7966 carries a C-terminal actin adenosine 5′-diphosphate (ADP) ribosyltransferase domain that disrupts the host cytoskeleton (18). Although the T6SS clearly plays a role in pathogenesis, most of the systems characterized to date deliver toxic effectors into competing bacteria. Because antibacterial effectors are potentially autoinhibitory, these latter toxins are invariably encoded with specific immunity proteins. Antibacterial effectors commonly disrupt the integrity of the bacterial cell envelope. VgrG-3 from V. cholerae carries a lysozyme-like domain that degrades the peptidoglycan cell wall (19, 20). Other peptidoglycan-cleaving amidase toxins are packaged within the lumen of Hcp hexamers for T6SS-mediated delivery (21–24). Phospholipase toxins collaborate with peptidoglycan degrading enzymes to lyse target bacteria (25–27). Other T6SS effectors act in the cytosol to degrade nucleic acids and nicotinamide adenine dinucleotide cofactors (3, 28, 29). Most recently, Whitney and coworkers described a novel T6SS effector that produces the inhibitory nucleotide ppApp (30). These latter toxins are commonly delivered through noncovalent interactions with VgrG. Many effectors contain PAAR domains, which enable direct binding to the C-terminal β-spike of VgrG (13), whereas others are indirectly tethered to VgrG through adaptor proteins (31–33). This combinatorial strategy allows multiple different toxins to be delivered with each firing event.Rearrangement hotspot (Rhs) proteins are potent effectors deployed by many T6SS⁺ bacteria (3, 34–37). T6SS-associated Rhs effectors range from ∼150 kDa to 180 kDa in mass and carry highly variable C-terminal toxin domains. The N-terminal region of Rhs proteins often contains two predicted transmembrane (TM) helix regions followed by a PAAR domain. The central region is composed of many Rhs/YD-peptide repeats, which form a β-cage structure that fully encapsulates the toxin domain (38). Genes coding for Rhs were first identified in Escherichia coli K-12 as elements that promote chromosomal duplication (39, 40). This genomic rearrangement was the result of unequal recombination between the rhsA and rhsB loci, which share 99.4% sequence identity over some 3,700 nucleotides. Subsequently, Hill and coworkers recognized that rhs genes are genetic composites (41), and that the variable C-terminal extension domains inhibit cell growth (42). Although E. coli K-12 encodes four full-length Rhs proteins, it lacks a T6SS, and there is no evidence that it deploys Rhs in competition. However, other Rhs/YD-peptide repeat proteins are known to deliver toxins in a T6SS-independent manner. Gram-positive bacteria export antibacterial YD-repeat proteins through the Sec pathway (3), and the tripartite insecticidal toxin complexes released by Photorhabdus and Yersinia species contain subunits with Rhs/YD repeats (38, 43). Thus, the Rhs encapsulation structure has been incorporated into at least three different toxin delivery platforms.Here, we report that Rhs effectors are critical for the activity of the T6SS-1 locus of Enterobacter cloacae ATCC 13047 (ECL). ECL encodes two Rhs effectors—RhsA and RhsB—which are each exported in a constitutive manner by T6SS-1 (35). Deletion of either rhs gene has little effect on T6SS-1 activity, but mutants lacking both rhsA and rhsB are defective for Hcp1 secretion and no longer inhibit target bacteria. Although ∆rhsA ∆rhsB mutants lose T6SS-1−mediated inhibition activity, they still assemble and fire contractile sheaths at a significantly reduced frequency. We further show that truncated RhsA that retains the PAAR domain still interacts with cognate VgrG2, but the resulting complex does not support Hcp1 secretion or target-cell killing. Full T6SS-1 function requires wild-type Rhs effectors that retain the N-terminal TM helices and PAAR domain together with an intact β-cage. These findings suggest that the Rhs β-cage mediates a quality control checkpoint on T6SS-1 assembly to ensure that VgrG is loaded with a toxic effector prior to export.     

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

There is considerable interest in the design of novel protein assemblies, for example, to develop cryogenic electron microscopy (cryo-EM) scaffolds to aid in structure determination (1, 2) and protein nanoparticle vaccines (3, 4). Many studies have utilized 1) genetic fusion of α-helices at N and C termini or 2) protein interface design to drive assembly of symmetric protein homo-oligomers into a larger symmetric nanoparticle or lattice. The genetic fusion route was originally demonstrated with the creation of a tetrahedral protein nanocage and fiber (5) and has since been used to generate two-dimensional (2D) layers (6), a porous cube (7), additional tetrahedra (8, 9), octahedra (10), and icosahedra (11, 12). Strengths of the fusion approach are that it is relatively straightforward, not inherently destabilizing, and guarantees the intended stoichiometry and specificity between fusion partners. Despite its simplicity and success, several aspects of genetic fusion complicate its use. Genetic fusion is best applied with α-helical termini, as this imparts some rigidity between the fused domains, but termini are not always α-helical or physically accessible. Furthermore, the number of possible sequence alignments between any given pair of building blocks is finite and this, in turn, limits the number of geometric solutions for a desired symmetric architecture. This can complicate or preclude the use of fusion methods in assembly design, especially when a particular protein building block is required for a downstream application. Finally, flexibility is often introduced at the point of fusion, even with α-helical linkers and certainly with disordered linkers. In the best cases, model deviations are subtle (8, 13); however, varying levels of unintended assembly products are also commonly observed. In contrast, the noncovalent protein interface design approach bypasses considerations of termini and their alignment. Interface design is compatible with the free rotation and translation of building blocks along their axes (14), so the set of valid geometric solutions is technically unlimited. The drawback to interface design is that it requires the creation of a new soluble and specific interface without destabilizing either protein. Like fusion, the interface design route to the protein nanomaterial production has achieved considerable success (4, 15–20).Among applications for genetic fusion is the creation of cryo-EM scaffolds (1, 2, 21–23): If a small target protein can be immobilized and rigidly bound onto a larger symmetric assembly, EM particle images can be more readily aligned and classified, and structures too small to analyze alone could then become amenable to structure determination. Yeates et al. demonstrated the potential of this approach by fusing a designed ankyrin repeat protein (DARPin) (24, 25) to the outside of a previously designed protein nanocage (1); the bound green fluorescent protein (GFP) target was resolved at 3.8 Å resolution with only a single α-helical fusion anchoring the DARPin.     

Circular swimming motility and disordered hyperuniform state in an algae system

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae (Effrenium voratum), which swim in circles at the air–liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

Active matter exists over a wide range of spatial and temporal scales (1–6) from animal groups (7, 8) to robot swarms (9–11), to cell colonies and tissues (12–16), to cytoskeletal extracts (17–20), to man-made microswimmers (21–25). Constituent particles in active matter systems are driven out of thermal equilibrium at the individual level; they interact to develop a wealth of intriguing collective phenomena, including clustering (13, 22, 24), flocking (11, 26), swarming (12, 13), spontaneous flow (14, 20), and giant density fluctuations (10, 11). Many of these observed phenomena have been successfully described by particle-based or continuum models (1–6), which highlight the important roles of both individual motility and interparticle interactions in determining system dynamics.Current active matter research focuses primarily on linearly swimming particles which have a symmetric body and self-propel along one of the symmetry axes. However, a perfect alignment between the propulsion direction and body axis is rarely found in reality. Deviation from such a perfect alignment leads to a persistent curvature in the microswimmer trajectories; examples of such circle microswimmers include anisotropic artificial micromotors (27, 28), self-propelled nematic droplets (29, 30), magnetotactic bacteria and Janus particles in rotating external fields (31, 32), Janus particle in viscoelastic medium (33), and sperm and bacteria near interfaces (34, 35). Chiral motility of circle microswimmers, as predicted by theoretical and numerical investigations, can lead to a range of interesting collective phenomena in circular microswimmers, including vortex structures (36, 37), localization in traps (38), enhanced flocking (39), and hyperuniform states (40). However, experimental verifications of these predictions are limited (32, 35), a situation mainly due to the scarcity of suitable experimental systems.Here we address this challenge by investigating marine algae Effrenium voratum (41, 42). At air–liquid interfaces, E. voratum cells swim in circles via two eukaryotic flagella: a transverse flagellum encircling the cellular anteroposterior axis and a longitudinal one running posteriorly. Over a wide range of densities, circling E. voratum cells self-organize into disordered hyperuniform states with suppressed density fluctuations at large length scales. Hyperuniformity (43, 44) has been considered as a new form of material order which leads to novel functionalities (45–49); it has been observed in many systems, including avian photoreceptor patterns (50), amorphous ices (51), amorphous silica (52), ultracold atoms (53), soft matter systems (54–61), and stochastic models (62–64). Our work demonstrates the existence of hyperuniformity in active matter and shows that hydrodynamic interactions can be used to construct hyperuniform states.     

Photosynthesis tunes quantum-mechanical mixing of electronic and vibrational states to steer exciton energy transfer

Photosynthetic species evolved to protect their light-harvesting apparatus from photoxidative damage driven by intracellular redox conditions or environmental conditions. The Fenna–Matthews–Olson (FMO) pigment–protein complex from green sulfur bacteria exhibits redox-dependent quenching behavior partially due to two internal cysteine residues. Here, we show evidence that a photosynthetic complex exploits the quantum mechanics of vibronic mixing to activate an oxidative photoprotective mechanism. We use two-dimensional electronic spectroscopy (2DES) to capture energy transfer dynamics in wild-type and cysteine-deficient FMO mutant proteins under both reducing and oxidizing conditions. Under reducing conditions, we find equal energy transfer through the exciton 4–1 and 4–2-1 pathways because the exciton 4–1 energy gap is vibronically coupled with a bacteriochlorophyll-a vibrational mode. Under oxidizing conditions, however, the resonance of the exciton 4–1 energy gap is detuned from the vibrational mode, causing excitons to preferentially steer through the indirect 4–2-1 pathway to increase the likelihood of exciton quenching. We use a Redfield model to show that the complex achieves this effect by tuning the site III energy via the redox state of its internal cysteine residues. This result shows how pigment–protein complexes exploit the quantum mechanics of vibronic coupling to steer energy transfer.

Photosynthetic organisms convert solar photons into chemical energy by taking advantage of the quantum mechanical nature of their molecular systems and the chemistry of their environment (1–4). Antenna complexes, composed of one or more pigment–protein complexes, facilitate the first steps in the photosynthesis process: They absorb photons and determine which proportion of excitations to move to reaction centers, where charge separation occurs (4). In oxic environments, excitations can generate highly reactive singlet oxygen species. These pigment–protein complexes can quench excess excitations in these environments with molecular moieties such as quinones and cysteine residues (1, 5–7).The Fenna–Matthews–Olson (FMO) complex, a trimer of pigment–protein complexes found in the green sulfur bacterium Chlorobaculum tepidum (8), has emerged as a model system to study the photophysical properties of photosynthetic antenna complexes (9–19). Each subunit in the FMO complex contains eight bacteriochlorophyll-a site molecules (Protein Data Bank, ID code: 3ENI) that are coupled to form a basis of eight partially delocalized excited states called excitons (Fig. 1) (20–23). Previous experiments on FMO have observed the presence of long-lived coherences in nonlinear spectroscopic signals at both cryogenic and physiological temperatures (11, 13). The coherent signals are thought to arise from some combination of electronic (24–26), vibrational (16–18), and vibronic (27) coherences in the system (28–30). One previous study reported that the coherent signals in FMO remain unchanged upon mutagenesis of the protein, suggesting that the signals are ground state vibrational coherences (17). Others discuss the role of vibronic coupling, where electronic and nuclear degrees of freedom become coupled (29). Other dimeric model systems have demonstrated the regimes in which these vibronically coupled states produce coherent or incoherent transport and vibronic coherences (31–33). Recent spectroscopic data has suggested that vibronic coupling plays a role in driving efficient energy transfer through photosynthetic complexes (27, 31, 33, 34), but to date there is no direct experimental evidence suggesting that biological systems use vibronic coupling as part of their biological function.Open in a separate windowFig. 1.(Left) Numbered sites and sidechains of cysteines C353 and C49 in the FMO pigment–protein complex (PDB ID code: 3ENI) (20). (Right) Site densities for excitons 4, 2, and 1 in reducing conditions with the energy transfer branching ratios for the WT oxidized and reduced protein. The saturation of pigments in each exciton denotes the relative contribution number to the exciton. The C353 residue is located near excitons 4 and 2, which have most electron density along one side of the complex, and other redox-active residues such as the Trp/Tyr chain. C353 and C49 surround site III, which contains the majority of exciton 1 density. Excitons 2 and 4 are generally delocalized over sites IV, V, and VII.It has been shown that redox conditions affect excited state properties in pigment-protein complexes, yet little is known about the underlying microscopic mechanisms for these effects (1, 9). Many commonly studied light-harvesting complexes—including the FMO complex (20), light-harvesting complex 2 (LH2) (35), the PC645 phycobiliprotein (36), and the cyanobacterial antenna complex isiA (37)—contain redox-active cysteine residues in close proximity to their chromophores. As the natural low light environment of C. tepidum does not necessitate photoprotective responses to light quantity and quality, its primary photoprotective mechanism concerns its response to oxidative stress. C. tepidum is an obligate anaerobe, but the presence of many active anoxygenic genes such as sodB for superoxide dismutase and roo for rubredoxin oxygen oxidoreductase (38) suggests that it is frequently exposed to molecular oxygen (7, 39). Using time-resolved fluorescence measurements, Orf et al. demonstrated that two cysteine residues in the FMO complex, C49 and C353, quench excitons under oxidizing conditions (1), which could protect the excitation from generating reactive oxygen species (7, 40–42). In two-dimensional electronic spectroscopy (2DES) experiments, Allodi et al. showed that redox conditions in both the wild-type and C49A/C353A double-mutant proteins affect the ultrafast dynamics through the FMO complex (9, 43). The recent discovery that many proteins across the evolutionary landscape possess chains of tryptophan and tyrosine residues provides evidence that these redox-active residues may link the internal protein behavior with the chemistry of the surrounding environment (41, 43).In this paper, we present data showing that pigment–protein complexes tune the vibronic coupling of their chromophores and that the absence of this vibronic coupling activates an oxidative photoprotective mechanism. We use 2DES to show that a pair of cysteine residues in FMO, C49 and C353, can steer excitations toward quenching sites in oxic environments. The measured reaction rate constants demonstrate unusual nonmonotonic behavior. We then use a Redfield model to determine how the exciton energy transfer (EET) time constants arise from changing chlorophyll site energies and their system-bath couplings (44, 45). The analysis reveals that the cysteine residues tune the resonance between exciton 4–1 energy gap and an intramolecular chlorophyll vibration in reducing conditions to induce vibronic coupling and detune the resonance in oxidizing conditions. This redox-dependent modulation of the vibronic coupling steers excitations through different pathways in the complex to change the likelihood that they interact with exciton quenchers.     

Addiction to Golgi-resident PI4P synthesis in chromosome 1q21.3–amplified lung adenocarcinoma cells

A chromosome 1q21.3 region that is frequently amplified in diverse cancer types encodes phosphatidylinositol (PI)-4 kinase IIIβ (PI4KIIIβ), a key regulator of secretory vesicle biogenesis and trafficking. Chromosome 1q21.3–amplified lung adenocarcinoma (1q-LUAD) cells rely on PI4KIIIβ for Golgi-resident PI-4-phosphate (PI4P) synthesis, prosurvival effector protein secretion, and cell viability. Here, we show that 1q-LUAD cells subjected to prolonged PI4KIIIβ antagonist treatment acquire tolerance by activating an miR-218-5p–dependent competing endogenous RNA network that up-regulates PI4KIIα, which provides an alternative source of Golgi-resident PI4P that maintains prosurvival effector protein secretion and cell viability. These findings demonstrate an addiction to Golgi-resident PI4P synthesis in a genetically defined subset of cancers.

The term “oncogene addiction” was coined to describe cancer cells’ exquisite dependence on individual oncogenes to sustain the malignant phenotype (1, 2). Examples include the BCR-ABL oncogene produced by a chromosome 9:22 translocation in chronic myelogenous leukemia and the somatically mutated EGFR oncogene in lung adenocarcinoma (LUAD) (3–5). In both cancer types, the mutant kinases are bona fide oncogenes in vitro and in vivo (6, 7). Although patients treated with selective kinase inhibitors attain profound clinical responses (8), chronic exposure of patients to targeted therapeutics is followed by disease relapse owing to an almost universal reactivation of mutant kinase activity, demonstrating that most cancers retain an underlying addiction to oncogene-induced signaling pathways (2, 9). Elucidating the molecular underpinnings of oncogene reactivation may lead to improved therapeutic strategies.Heightened secretion of protumorigenic effector proteins promotes metastasis and acquired resistance to targeted therapeutics (10, 11). The conventional secretory pathway directs the transport of secretory vesicles from the endoplasmic reticulum to the plasma membrane via the Golgi apparatus (12). Tensile forces exerted on Golgi membranes activate secretory vesicle biogenesis and are mediated by a Golgi phosphoprotein-3 (GOLPH3)/F-actin protein complex (13). GOLPH3 binds to phosphatidylinositol (PI)-4-phosphate (PI4P), which tethers GOLPH3 to Golgi membranes and is generated by the Golgi-resident PI-4 kinases PI4KIIα and PI4KIIIβ (10, 13, 14).A chromosome 1q21.3 region that is frequently amplified in diverse cancer types encodes PI4KIIIβ (11, 15). Chromosome 1q21.3–amplified LUAD (1q-LUAD) cells undergo apoptosis following treatment with small-molecule PI4KIIIβ antagonists or depletion of PI4KIIIβ–dependent secreted proteins (11), establishing that PI4KIIIβ–dependent secreted proteins are prosurvival effectors in 1q-LUAD cells. On the basis of this conceptual framework, here, we postulated that chromosome 1q21.3 amplifications confer an addiction to Golgi-resident PI-4 kinase activity.