In vivo single-cell CRISPR uncovers distinct TNF programmes in tumour evolution.

The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.

FGF-2-dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis.

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2-dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.

Structural evidence for the roles of divalent cations in actin polymerization and activation of ATP hydrolysis.

The structure of the actin filament is known at a resolution that has allowed the architecture of protein components to be unambiguously assigned. However, fully understanding the chemistry of the system requires higher resolution to identify the ions and water molecules involved in polymerization and ATP hydrolysis. Here, we find experimental evidence for the association of cations with the surfaces of G-actin in a 2.0-Šresolution X-ray structure of actin bound to a Cordon-Bleu WH2 motif and in previously determined high-resolution X-ray structures. Three of four reoccurring divalent cation sites were stable during molecular dynamics (MD) simulations of the filament, suggesting that these sites may play a functional role in stabilizing the filament. We modeled the water coordination at the ATP-bound Mg2+, which also proved to be stable during the MD simulations. Using this model of the filament with a hydrated ATP-bound Mg2+, we compared the cumulative probability of an activated hydrolytic water molecule approaching the γ-phosphorous of ATP, in comparison with G-actin, in the MD simulations. The cumulative probability increased in F-actin in line with the activation of actin's ATPase activity on polymerization. However, inclusion of the cations in the filament lowered cumulative probability, suggesting the rate of hydrolysis may be linked to filament flexibility. Together, these data extend the possible roles of Mg2+ in polymerization and the mechanism of polymerization-induced activation of actin's ATPase activity.

Advances in Structural Biology and the Application to Biological Filament Systems.

Structural biology has experienced several transformative technological advances in recent years. These include: development of extremely bright X-ray sources (microfocus synchrotron beamlines and free electron lasers) and the use of electrons to extend protein crystallography to ever decreasing crystal sizes; and an increase in the resolution attainable by cryo-electron microscopy. Here we discuss the use of these techniques in general terms and highlight their application for biological filament systems, an area that is severely underrepresented in atomic resolution structures. We assemble a model of a capped tropomyosin-actin minifilament to demonstrate the utility of combining structures determined by different techniques. Finally, we survey the methods that attempt to transform high resolution structural biology into more physiological environments, such as the cell. Together these techniques promise a compelling decade for structural biology and, more importantly, they will provide exciting discoveries in understanding the designs and purposes of biological machines.

Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation.

Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule.

Yersinia effector protein (YopO)-mediated phosphorylation of host gelsolin causes calcium-independent activation leading to disruption of actin dynamics.

Pathogenic Yersinia bacteria cause a range of human diseases. To modulate and evade host immune systems, these yersiniae inject effector proteins into host macrophages. One such protein, the serine/threonine kinase YopO (YpkA in Yersinia pestis), uses monomeric actin as bait to recruit and phosphorylate host actin polymerization-regulating proteins, including the actin-severing protein gelsolin, to disrupt actin filaments and thus impair phagocytosis. However, the YopO phosphorylation sites on gelsolin and the consequences of YopO-mediated phosphorylation on actin remodeling have yet to be established. Here we determined the effects of YopO-mediated phosphorylation on gelsolin and identified its phosphorylation sites by mass spectrometry. YopO phosphorylated gelsolin in the linker region between gelsolin homology domains G3 and G4, which, in the absence of calcium, are compacted but adopt an open conformation in the presence of calcium, enabling actin binding and severing. Using phosphomimetic and phosphodeletion gelsolin mutants, we found that YopO-mediated phosphorylation partially mimics calcium-dependent activation of gelsolin, potentially contributing to a reduction in filamentous actin and altered actin dynamics in phagocytic cells. In summary, this work represents the first report of the functional outcome of serine/threonine phosphorylation in gelsolin regulation and provides critical insight into how YopO disrupts normal gelsolin function to alter host actin dynamics and thus cripple phagocytosis.

The evolution of compositionally and functionally distinct actin filaments.

The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity.

Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers.

Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.

Novel actin-like filament structure from Clostridium tetani.

Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

Structural investigation of the C-terminal catalytic fragment of presenilin 1.

The gamma-secretase complex has a decisive role in the development of Alzheimer's disease, in that it cleaves a precursor to create the amyloid beta peptide whose aggregates form the senile plaques encountered in the brains of patients. Gamma-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer's disease are linked to presenilin 1, the catalytic component of gamma-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.

Exploring the pan-cancer landscape of posttranscriptional regulation.

Understanding the mechanisms underlying cancer gene expression is critical for precision oncology. Posttranscriptional regulation is a key determinant of protein abundance and cancer cell behavior. However, to what extent posttranscriptional regulatory mechanisms impact protein levels and cancer progression is an ongoing question. Here, we exploit cancer proteogenomics data to systematically compare mRNA-protein correlations across 14 different human cancer types. We identify two clusters of genes with particularly low mRNA-protein correlations across all cancer types, shed light on the role of posttranscriptional regulation of cancer driver genes and drug targets, and unveil a cohort of 55 mutations that alter systems-wide posttranscriptional regulation. Surprisingly, we find that decreased levels of posttranscriptional control in patients correlate with shorter overall survival across multiple cancer types, prompting further mechanistic studies into how posttranscriptional regulation affects patient outcomes. Our findings underscore the importance of a comprehensive understanding of the posttranscriptional regulatory landscape for predicting cancer progression.

Inhibition of AXL receptor tyrosine kinase enhances brown adipose tissue functionality in mice.

The current obesity epidemic and high prevalence of metabolic diseases necessitate efficacious and safe treatments. Brown adipose tissue in this context is a promising target with the potential to increase energy expenditure, however no pharmacological treatments activating brown adipose tissue are currently available. Here, we identify AXL receptor tyrosine kinase as a regulator of adipose function. Pharmacological and genetic inhibition of AXL enhance thermogenic capacity of brown and white adipocytes, in vitro and in vivo. Mechanistically, these effects are mediated through inhibition of PI3K/AKT/PDE signaling pathway, resulting in induction of nuclear FOXO1 localization and increased intracellular cAMP levels via PDE3/4 inhibition and subsequent stimulation of the PKA-ATF2 pathway. In line with this, both constitutive Axl deletion as well as inducible adipocyte-specific Axl deletion protect animals from diet-induced obesity concomitant with increases in energy expenditure. Based on these data, we propose AXL receptor as a target for the treatment of obesity.

Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer.

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence.

Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes.

MraY translocase catalyzes the first committed membrane-bound step of bacterial peptidoglycan synthesis leading to the formation of lipid I. The essential membrane protein therefore has a high potential as target for drug screening approaches to develop antibiotics against gram-positive as well as gram-negative bacteria. However, the production of large integral membrane proteins in conventional cellular expression systems is still very challenging. Cell-free expression technologies have been optimized in recent times for the production of membrane proteins in the presence of detergents (D-CF), lipids (L-CF), or as precipitates (P-CF). We report the development of preparative scale production protocols for the MraY homologues of Escherichia coli and Bacillus subtilis in all three cell-free expression modes followed by their subsequent quality evaluation. Although both proteins can be cell-free produced at comparable high levels, their requirements for optimal expression conditions differ markedly. B. subtilus MraY was stably folded in all three expression modes and showed highest translocase activities after P-CF production followed by defined treatment with detergents. In contrast, the E. coli MraY appears to be unstable after post- or cotranslational solubilization in detergent micelles. Expression kinetics and reducing conditions were identified as optimization parameters for the quality improvement of E. coli MraY. Most remarkably, in contrast to B. subtilis MraY the E. coli MraY has to be stabilized by lipids and only the production in the L-CF mode in the presence of preformed liposomes resulted in stable and translocase active protein samples.

Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening.

Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

A pan-cancer metabolic atlas of the tumor microenvironment.

Tumors are heterogeneous cellular environments with entwined metabolic dependencies. Here, we use a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. We identify metabolic genes and processes recurrently altered in cancer cells across tumor types, highlighting pan-cancer upregulation of deoxythymidine triphosphate (dTTP) production. In contrast, the tryptophan catabolism rate-limiting enzymes IDO1 and TDO2 are highly overexpressed in stroma, raising the hypothesis that kynurenine-mediated suppression of antitumor immunity may be predominantly constrained by the stroma. Oxidative phosphorylation is the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the Warburg effect may be less pronounced in cancer cells in vivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism.

Filament structure, organization, and dynamics in MreB sheets.

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several mum long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

MicroRNA-7a2 Regulates Prolactin in Developing Lactotrophs and Prolactinoma Cells.

Prolactin production is controlled by a complex and temporally dynamic network of factors. Despite this tightly coordinated system, pathological hyperprolactinemia is a common endocrine disorder that is often not understood, thereby highlighting the need to expand our molecular understanding of lactotroph cell regulation. MicroRNA-7 (miR-7) is the most highly expressed miRNA family in the pituitary gland and the loss of the miR-7 family member, miR-7a2, is sufficient to reduce prolactin gene expression in mice. Here, we used conditional loss-of-function and gain-of-function mouse models to characterize the function of miR-7a2 in lactotroph cells. We found that pituitary miR-7a2 expression undergoes developmental and sex hormone-dependent regulation. Unexpectedly, the loss of mir-7a2 induces a premature increase in prolactin expression and lactotroph abundance during embryonic development, followed by a gradual loss of prolactin into adulthood. On the other hand, lactotroph development is delayed in mice overexpressing miR-7a2. This regulation of lactotroph function by miR-7a2 involves complementary mechanisms in multiple cell populations. In mouse pituitary and rat prolactinoma cells, miR-7a2 represses its target Raf1, which promotes prolactin gene expression. These findings shed light on the complex regulation of prolactin production and may have implications for the physiological and pathological mechanisms underlying hyperprolactinemia.

Pan-Cancer Analysis of Ligand-Receptor Cross-talk in the Tumor Microenvironment.

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is a key to tumor progression. Here, we deconvoluted bulk tumor transcriptomes to infer cross-talk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. This approach recovered known transcriptional hallmarks of cancer and stromal cells and was concordant with single-cell, in situ hybridization and IHC data. Inferred autocrine cancer cell interactions varied between tissues but often converged on Ephrin, BMP, and FGFR-signaling pathways. Analysis of immune checkpoints nominated interactions with high levels of cancer-to-immune cross-talk across distinct tumor types. Strikingly, PD-L1 was found to be highly expressed in stromal rather than cancer cells. Overall, our study presents a new resource for hypothesis generation and exploration of cross-talk in the TME. SIGNIFICANCE: This study provides deconvoluted bulk tumor transcriptomes across multiple cancer types to infer cross-talk in the tumor microenvironment.