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Mammalian Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs). Although TLRs are clearly involved in the detection of bacteria and viruses, relatively little is known about their function in the innate response to eukaryotic microorganisms. Here we identify a profilin-like molecule from the protozoan parasite Toxoplasma gondii that generates a potent interleukin-12 (IL-12) response in murine DCs that is dependent on myeloid differentiation factor 88. T. gondii profilin activates DCs through TLR11 and is the first chemically defined ligand for this TLR. Moreover, TLR11 is required in vivo for parasite-induced IL-12 production and optimal resistance to infection, thereby establishing a role for the receptor in host recognition of protozoan pathogens.

Constriction of the cytokinetic ring, a circular structure of actin filaments, is an essential step during cell division. Mechanical forces driving the constriction are attributed to myosin motor proteins, which slide actin filaments along each other. However, in multiple organisms, ring constriction has been reported to be myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that anillin, a non­motor actin crosslinker, indispensable during cytokinesis, autonomously propels the contractility of actin bundles. Anillin generates contractile forces of tens of pico-Newtons to maximise the lengths of overlaps between bundled actin filaments. The contractility is enhanced by actin disassembly. When multiple actin filaments are arranged into a ring, this contractility leads to ring constriction. Our results indicate that passive actin crosslinkers can substitute for the activity of molecular motors to generate contractile forces in a variety of actin networks, including the cytokinetic ring. Cytokinetic ring constriction during cell division requires actin but curiously is independent of myosin in many organisms. Here, the authors show that anillin, a protein enriched in the contractile ring, is a non-motor actin crosslinker that generates contractile force in lieu of a molecular motor.

The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins-TssA, TssE, TssF, TssG, TssK and VgrG-are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.

Cholangiocarcinoma (CCA) is associated with high mortality rates because of its resistance to conventional gemcitabine-based chemotherapy. Hydroxy-methyl-glutaryl-coenzyme A reductase inhibitors (statins) reportedly exert anti-cancer effects in CCA and lower the risk of CCA; however, the underlying mechanism of these effects remains unclear. The proliferative and oncogenic activities of the transcriptional co-activator Yes-associated protein (YAP) are driven by its association with the TEA domain (TEAD) of transcription factors; thereby, upregulating genes that promote cell growth, inhibit apoptosis, and confer chemoresistance. This study investigated the effects of atorvastatin in combination with gemcitabine on the progression of human CCA associated with YAP oncogenic regulation. Both atorvastatin and gemcitabine concentration-dependently suppressed the proliferation of HuCCT-1 and KKU-M213 human CCA cells. Moreover, both agents induced cellular apoptosis by upregulating the pro-apoptotic marker BAX and downregulating the anti-apoptotic markers MCL1 and BCL2. Atorvastatin also significantly decreased the mRNA expression of the TEAD target genes CTGF, CYR61, ANKRD1, and MFAP5 in both CCA cell lines. A xenograft tumor growth assay indicated that atorvastatin and gemcitabine potently repressed human CCA cell-derived subcutaneous tumor growth by inhibiting YAP nuclear translocation and TEAD transcriptional activation. Notably, the anti-cancer effects of the individual agents were significantly enhanced in combination. These results indicate that gemcitabine plus atorvastatin could serve as a potential novel treatment option for CCA.

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

Large‐scale cancer genome sequencing has uncovered thousands of gene mutations, but distinguishing tumor driver genes from functionally neutral passenger mutations is a major challenge. We analyzed 800 cancer genomes of eight types to find single‐nucleotide variants (SNVs) that precisely target phosphorylation machinery, important in cancer development and drug targeting. Assuming that cancer‐related biological systems involve unexpectedly frequent mutations, we used novel algorithms to identify genes with significant phosphorylation‐associated SNVs (pSNVs), phospho‐mutated pathways, kinase networks, drug targets, and clinically correlated signaling modules. We highlight increased survival of patients with TP53 pSNVs, hierarchically organized cancer kinase modules, a novel pSNV in EGFR , and an immune‐related network of pSNVs that correlates with prolonged survival in ovarian cancer. Our findings include multiple actionable cancer gene candidates ( FLNB , GRM1 , POU2F1 ), protein complexes (HCF1, ASF1), and kinases (PRKCZ). This study demonstrates new ways of interpreting cancer genomes and presents new leads for cancer research. Phosphorylation sites of human proteins are frequently mutated in cancer. Statistical analysis of phosphorylation‐associated single nucleotide variants (pSNVs) predicts novel cancer drivers and phospho‐mutation mechanisms in known cancer genes. Synopsis Phosphorylation sites of human proteins are frequently mutated in cancer. Statistical analysis of phosphorylation‐associated single nucleotide variants (pSNVs) predicts novel cancer drivers and phospho‐mutation mechanisms in known cancer genes. We designed the ActiveDriver method to identify significantly mutated signaling regions in proteins. ActiveDriver is complementary to standard frequency‐based methods of mutation significance and helps interpret rare, but site‐specific mutations. Analysis of somatic mutations in 800 cancer genomes reveals dozens of known and novel cancer genes, including potential drivers that are apparent only when integrating multiple cancer types. Pathway and network analysis identifies systems with significantly enriched pSNVs, including kinase modules and protein complexes. Clinical data analysis identifies phospho‐mutations of TP53 that correlate with prolonged patient survival in ovarian and brain cancer. Kinase network analysis highlights multiple survival‐associated signaling modules with pSNVs.

Filamins were discovered as the first family of non-muscle actin-binding protein. They are lage cytoplasmic proteins that cross-link cortical actin into a dynamic three-dimensional structure. Filamins have also been reported to interact with a large number of cellular proteins of great functional diversity, suggesting that they are unusually versatile signalling scaffolds. More recently, genetic mutations in filamin A and B have been reported to cause a wide range of human diseases, suggesting that different diseases highlight distinct filamin interactions.

Mechanosensing by the actin cytoskeleton Living cells need to respond to mechanical forces for many essential biological functions. This mechanosensing activity is thought to be a property of the actin cytoskeleton, but no specific mechanisms have yet been identified. In this study, Ehrlicher et al . identify the actin-binding protein filamin A (FLNa) as a central mechanotransduction element. In a minimal reconstituted system, ligand binding to filamin is affected by mechanical forces, causing certain binding partners to dissociate and others to adhere more strongly. This selectivity may provide a direct molecular link between physical forces and biological activity. Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders 1 , 2 , 3 , 4 such as cardiac failure 5 and pulmonary injury 6 . The actin cytoskeleton’s connectivity enables it to transmit forces rapidly over large distances 7 , implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) 8 , 9 as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with β-integrin tails connecting to the actin cytoskeleton by binding directly to filamin 4 . FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation 10 . Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching 11 , we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases β-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.

Cryo-EM structures of the pre- and postcontraction states of Pseudomonas aeruginosa R-type pyocins provide details of the conformational changes between the tube and sheath that take place during contraction. R-type pyocins are representatives of contractile ejection systems, a class of biological nanomachines that includes, among others, the bacterial type VI secretion system (T6SS) and contractile bacteriophage tails. We report atomic models of the Pseudomonas aeruginosa precontraction pyocin sheath and tube, and the postcontraction sheath, obtained by cryo-EM at 3.5-Å and 3.9-Å resolutions, respectively. The central channel of the tube is negatively charged, in contrast to the neutral and positive counterparts in T6SSs and phage tails. The sheath is interwoven by long N- and C-terminal extension arms emanating from each subunit, which create an extensive two-dimensional mesh that has the same connectivity in the extended and contracted state of the sheath. We propose that the contraction process draws energy from electrostatic and shape complementarities to insert the inner tube through bacterial cell membranes to eventually kill the bacteria.

Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20–21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.