Glia trigger endocytic clearance of axonal proteins to promote rodent myelination.

Axons undergo striking changes in their content and distribution of cell adhesion molecules (CAMs) and ion channels during myelination that underlies the switch from continuous to saltatory conduction. These changes include the removal of a large cohort of uniformly distributed CAMs that mediate initial axon-Schwann cell interactions and their replacement by a subset of CAMs that mediate domain-specific interactions of myelinated fibers. Here, using rodent models, we examine the mechanisms and significance of this removal of axonal CAMs. We show that Schwann cells just prior to myelination locally activate clathrin-mediated endocytosis (CME) in axons, thereby driving clearance of a broad array of axonal CAMs. CAMs engineered to resist endocytosis are persistently expressed along the axon and delay both PNS and CNS myelination. Thus, glia non-autonomously activate CME in axons to downregulate axonal CAMs and presumptively axo-glial adhesion. This promotes the transition from ensheathment to myelination while simultaneously sculpting the formation of axonal domains.

The hepatic and pancreatic tumour resection risk factors for surgical site wound infections: A meta-analysis.

A meta-analysis was conducted to measure hepatic and pancreatic tumour resection (HPTR) risk factors (RFs) for surgical site wound infections (SSWIs). A comprehensive literature inspection was conducted until February 2023, and 2349 interrelated investigations were reviewed. The nine chosen investigations included 22 774 individuals who were in the chosen investigations' starting point, 20 831 of them were with pancreatic tumours (PTs), and 1934 with hepatic tumours (HTs). Odds ratio (OR) and 95% confidence intervals (CIs) were used to compute the value of the HPTR RFs for SSWIs using dichotomous and continuous approaches, and a fixed or random model. HT patients with biliary reconstruction had significantly higher SSWI (OR, 5.81; 95% CI, 3.42-9.88, P < .001) than those without biliary reconstruction. Nevertheless, there was no significant difference between individuals with PT who underwent pancreaticoduodenectomy and those who underwent distal pancreatectomy in SSWI (OR, 1.63; 95% CI, 0.95-2.77, P = .07). HT individuals with biliary reconstruction had significantly higher SSWI compared with those without biliary reconstruction. Nevertheless, there was no significant difference between PT individuals who underwent pancreaticoduodenectomy and those who underwent distal pancreatectomy in SSWI. However, owing to the small number of selected investigations for this meta-analysis, care must be exercised when dealing with its values.

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets.

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.

Quantitative protein sensing with germanium THz-antennas manufactured using CMOS processes.

The development of a CMOS manufactured THz sensing platform could enable the integration of state-of-the-art sensing principles with the mixed signal electronics ecosystem in small footprint, low-cost devices. To this aim, in this work we demonstrate a label-free protein sensing platform using highly doped germanium plasmonic antennas realized on Si and SOI substrates and operating in the THz range of the electromagnetic spectrum. The antenna response to different concentrations of BSA shows in both cases a linear response with saturation above 20 mg/mL. Ge antennas on SOI substrates feature a two-fold sensitivity as compared to conventional Si substrates, reaching a value of 6 GHz/(mg/mL), which is four-fold what reported using metal-based metamaterials. We believe that this result could pave the way to a low-cost lab-on-a-chip biosensing platform.

Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3.

The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.

Near-Infrared-II Light Induced Mild Hyperthermia Activate Cisplatin-Artemisinin Nanoparticle for Enhanced Chemo/Chemodynamic Therapy and Immunotherapy.

Chemodynamic therapy (CDT) is an effective cancer treatment that uses Fenton reaction to induce cancer cell death. Current clinical applications of CDT are limited by the dependency of external supply of metal ions as well as low catalytic efficiency. Here, a highly efficient metal-free CDT by using endoperoxide bridge-containing artesunate as free radical-generating substance is developed. A Pt(IV) prodrug (A-Pt) containing two artesunate molecules in the axial direction is synthesized, which can be decomposed into cisplatin and artesunate under reducing intracellular environment in tumor cells. To improve the catalytic efficiency for Fenton reaction, a near-infrared-II (NIR-II) photothermal agent IR1048 is incorporated to achieve a mild hyperthermia effect. By encapsulating the A-Pt and IR1048 with human serum albumin, A-Pt-IR NP are formulated for efficient drug delivery in 4T1 tumor-bearing mice. NIR-II light irradiation of A-Pt-IR NP treated mice show accelerated Fenton reaction. In addition, A-Pt-IR NP could also induce strong immunogenic cell death, which effectively reverses the immunosuppressive tumor microenvironment, and augments antitumor immunity. This study demonstrates that A-Pt-IR NP are potent biodegradable NIR-II active chemotherapy/CDT nanomedicine for clinical translation.

Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane.

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA.

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Three-Dimensional Interfacing of Cells with Hierarchical Silicon Nano/Microstructures for Midinfrared Interrogation of In Situ Captured Proteins.

Label-free optical detection of biomolecules is currently limited by a lack of specificity rather than sensitivity. To exploit the much more characteristic refractive index dispersion in the mid-infrared (IR) regime, we have engineered three-dimensional IR-resonant silicon micropillar arrays (Si-MPAs) for protein sensing. By exploiting the unique hierarchical nano- and microstructured design of these Si-MPAs attained by CMOS-compatible silicon-based microfabrication processes, we achieved an optimized interrogation of surface protein binding. Based on spatially resolved surface functionalization, we demonstrate controlled three-dimensional interfacing of mammalian cells with Si-MPAs. Spatially controlled surface functionalization for site-specific protein immobilization enabled efficient targeting of soluble and membrane proteins into sensing hotspots directly from cells cultured on Si-MPAs. Protein binding to Si-MPA hotspots at submonolayer level was unambiguously detected by conventional Fourier transform IR spectroscopy. The compatibility with cost-effective CMOS-based microfabrication techniques readily allows integration of this novel IR transducer into fully fledged bioanalytical microdevices for selective and sensitive protein sensing.

Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene-induced energy transfer.

Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a 'closed' and an 'open' conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution.

Proteins are part of the building blocks of life and are essential for structure, function and regulation of every cell, tissue and organ of the body. Proteins adopt different conformations to work efficiently within the various environments of a cell. They can also switch between shapes. One way to monitor how proteins change their shapes involves energy transfer. This approach can measure how close two proteins, or two parts of the same protein, are, by using dye labels that respond to each other when they are close together. For example, in a method called FRET, one dye label absorbs light and transfers the energy to the other label, which emits it as a different color of light. However, FRET only works over short distances (less than 10nm apart or 1/100,000th of a millimeter), so it is not useful for larger proteins. Here, Füllbrunn, Li et al. developed a method called GIET that uses graphene to analyze the dynamic structures of proteins on membrane surfaces. Graphene is a type of carbon nanomaterial that can absorb energy from dye labels and could provide a way to study protein interactions over longer distances. Graphene was deposited on a glass surface where it was coated with single layer of membrane, which could then be used to capture specific proteins. The results showed that GIET worked over longer distances (up to 30 nm) than FRET and could be used to study proteins attached to the membrane around graphene. Füllbrunn, Li et al. used it to examine a specific complex of proteins called HOPS, which is linked to multiple diseases, including Ebola, measuring distances between the head or tail of HOPS and the membrane to understand protein shapes. This revealed that HOPS adopts an upright position on membranes and alternates between open and closed shapes. The study of Füllbrunn, Li et al. highlights the ability of GIET to address unanswered questions about the function of protein complexes on membrane surfaces and sheds new light on the structural dynamics of HOPS in living cells. As it allows protein interactions to be studied over much greater distances, GIET could be a powerful new tool for cell biology research. Moreover, graphene is also useful in electron microscopy and both approaches combined could achieve a detailed structural picture of proteins in action.

Surface Modification of Polycaprolactone Scaffold With Improved Biocompatibility and Controlled Growth Factor Release for Enhanced Stem Cell Differentiation.

Polycaprolactone (PCL) has been widely used as a scaffold material for tissue engineering. Reliable applications of the PCL scaffolds require overcoming their native hydrophobicity and obtaining the sustained release of signaling factors to modulate cell growth and differentiation. Here, we report a surface modification strategy for electrospun PCL nanofibers using an azide-terminated amphiphilic graft polymer. With multiple alkylation and pegylation on the side chains of poly-L-lysine, stable coating of the graft polymer on the PCL nanofibers was achieved in one step. Using the azide-alkyne "click chemistry", we functionalized the azide-pegylated PCL nanofibers with dibenzocyclooctyne-modified nanocapsules containing growth factor, which rendered the nanofiber scaffold with satisfied cell adhesion and growth property. Moreover, by specific immobilization of pH-responsive nanocapsules containing bone morphogenetic protein 2 (BMP-2), controlled release of active BMP-2 from the PCL nanofibers was achieved within 21 days. When bone mesenchyme stem cells were cultured on this nanofiber scaffold, enhanced ossification was observed in correlation with the time-dependent release of BMP-2. The established surface modification can be extended as a generic approach to hydrophobic nanomaterials for longtime sustainable release of multiplex signaling proteins for tissue engineering.

Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo.

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.

Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.

Expression of MACC1 protein in gastric cancer and its effect on proliferation and invasion of gastric cancer cells.

To detect the expression of metastasis-associated colon cancer gene 1 (MACC1) protein in gastric cancer tissues, and analyze its relationship with clinicopathological parameters of gastric cancer and its effect on proliferation and invasion of gastric cancer cells. METHODS: 71 patients with gastric cancer in Fifth Hospital in Wuhan from June 2014 to March 2018 were selected as research subjects. Western blot was used to detect the expression of MACC1 in gastric cancer tissue and normal gastric mucosa tissue, and gastric cancer cell SGC7901 was transfected. Transfection group (transfected with MACC1-siRNA), negative control group (transfected with siRNA-NC) and blank control group (untreated cells) were set up. After transfection, the expressions of MACC1 protein and mRNA in the 3 groups were detected by Western blot and qRT-PCR methods, the cell proliferation was detected by MTT method, and the invasion ability of cells in vitro was detected by Transwell chamber. RESULTS: The expression of MACC1 protein in gastric cancer tissue was higher than the control group (P< 0.05). The expression of MACC1 protein in gastric cancer was related to the differentiation degree, infiltration depth, lymph node metastasis and different stages of gastric cancer (P< 0.05). After transfection, the expressions of MACC1 protein and mRNA in the transfection group was significantly lower than the negative control group and blank group (P< 0.05). There was no significant difference in cell viability between the blank group and negative control group at each time point (P> 0.05). CONCLUSION: MACC1 was highly expressed in gastric cancer tissues. The expression of MACC1 was related to the differentiation degree, infiltration depth, lymph node metastasis and staging of gastric cancer. Down-regulation of MACC1 could inhibit the proliferation and invasion of gastric cancer cells. This study provided a certain biological basis for early clinical prediction, diagnosis and treatment of gastric cancer.

Self-assembly of robust gold nanoparticle monolayer architectures for quantitative protein interaction analysis by LSPR spectroscopy.

Localized surface plasmon resonance (LSPR) detection offers highly sensitive label-free detection of biomolecular interactions. Simple and robust surface architectures compatible with real-time detection in a flow-through system are required for broad application in quantitative interaction analysis. Here, we established self-assembly of a functionalized gold nanoparticle (AuNP) monolayer on a glass substrate for stable, yet reversible immobilization of Histidine-tagged proteins. To this end, one-step coating of glass substrates with poly-L-lysine graft poly(ethylene glycol) functionalized with ortho-pyridyl disulfide (PLL-PEG-OPSS) was employed as a reactive, yet biocompatible monolayer to self-assemble AuNP into a LSPR active monolayer. Site-specific, reversible immobilization of His-tagged proteins was accomplished by coating the AuNP monolayer with tris-nitrilotriacetic acid (trisNTA) PEG disulfide. LSPR spectroscopy detection of protein binding on these biocompatible functionalized AuNP monolayers confirms high stability under various harsh analytical conditions. These features were successfully employed to demonstrate unbiased kinetic analysis of cytokine-receptor interactions. Graphical abstract.

Close-packed silane nanodot arrays by capillary nanostamping coupled with heterocyclic silane ring opening.

We report the parallel generation of close-packed ordered silane nanodot arrays with nanodot diameters of few 100 nm and nearest-neighbor distances in the one-micron range. Capillary nanostamping of heterocyclic silanes coupled with ring-opening triggered by hydroxyl groups at the substrate surfaces yields nanodots consisting of silane monolayers with exposed terminal functional groups. Using spongy mesoporous silica stamps with methyl-terminated mesopore walls inert towards the heterocyclic silanes, we could manually perform multiple successive stamping cycles under ambient conditions without interruptions for ink refilling. Further functionalizations include the synthesis of polymer nanobrushes on the silane nanodots by surface-initiated atom-transfer radical polymerization. Proteins-of-interest fused to the HaloTag were site-specifically captured to silane nanodots functionalized by copper-free reactions with azide derivatives. Thus, bioorthogonal functionalization for bioanalytics with a spatial resolution in the one-micron range may be realized on solid supports compatible with fluorescence-based optical microscopy. The feature sizes of the silane nanodot arrays match well the length scales characteristic of a variety of biomolecular submicroscopic organizations in living cells, thus representing a compromise between miniaturization and the resolution limit of optical microscopy for sensitive high-throughput bioanalytics.

Near-native, site-specific and purification-free protein labeling for quantitative protein interaction analysis by MicroScale Thermophoresis.

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques. To this end, we exploited the high-affinity interaction of tris-NTA with oligohistidine-tags, which are popular for purification, immobilization or detection of recombinant proteins. We used various DYE-tris-NTA conjugates to successfully label His-tagged proteins that were either purified or a component of cell lysate. The RED-tris-NTA was identified as the optimal dye conjugate with a high affinity towards oligohistidine-tags, a high fluorescence signal and an optimal signal-to-noise ratio in MST binding experiments. Owing to its emission in the red region of the spectrum, it also enables reliable measurements in complex biological matrices such as cell lysates allowing a more physiologically realistic assessment and eliminating the need for protein purification.

Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.

Surrogate Wnt agonists that phenocopy canonical Wnt and ß-catenin signalling.

Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing ß-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic ß-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.