High Refractive Index Silicone Gels for Simultaneous Total Internal Reflection Fluorescence and Traction Force Microscopy of Adherent Cells

Substrate rigidity profoundly impacts cellular behaviors such as migration, gene expression, and cell fate. Total Internal Reflection Fluorescence (TIRF) microscopy enables selective visualization of the dynamics of substrate adhesions, vesicle trafficking, and biochemical signaling at the cell-substrate interface. Here we apply high-refractive-index silicone gels to perform TIRF microscopy on substrates with a wide range of physiological elastic moduli and simultaneously measure traction forces exerted by cells on the substrate

A mutant O-GlcNAcase enriches Drosophila developmental regulators

YesProtein O-GlcNAcylation is a reversible post-translational modification of serines/threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. Drosophila melanogaster OGT/supersex combs (sxc) is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved, and the underlying mechanisms linking these phenotypes to embryonic development, are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, amongst others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development.Wellcome Trust Investigator Award (110061); MRC grant (MC_UU_12016/5); and Royal Society Research Grant

Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding.

Clathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.Supported by NIH R01 GM106963 to L.M.T. and Wellcome Trust grants 090909/Z to D.J.O., 097040 to A.G.W., and 100140 to CIMR.This is the final version of the article. It first appeared from Cell Press / Elsevier via https://doi.org/10.1016/j.devcel.2016.05.00

Hotspot ESR1 mutations are multimodal and contextual modulators of breast cancer metastasis

Constitutively active estrogen receptor α (ER/ESR1) mutations have been identified in approximately one-third of ER+ metastatic breast cancers. Although these mutations are known as mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant but not local recurrences in five independent breast cancer cohorts. In concordance with transcriptomic profiling of ESR1-mutant tumors, genome-edited ESR1 Y537S and D538G-mutant cell models exhibited a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally conferred enhanced cell–cell contacts while decreasing cell-extracellular matrix adhesion. In vivo studies showed ESR1-mutant cells were associated with larger multicellular circulating tumor cell (CTC) clusters with increased compactness compared with ESR1 wild-type CTCs. These preclinical findings translated to clinical observations, where CTC clusters were enriched in patients with ESR1-mutated metastatic breast cancer. Conversely, context-dependent migratory phenotypes revealed cotargeting of Wnt and ER as a vulnerability in a D538G cell model. Mechanistically, mutant ESR1 exhibited noncanonical regulation of several metastatic pathways, including secondary transcriptional regulation and de novo FOXA1-driven chromatin remodeling. Collectively, these data provide evidence for ESR1 mutation–modulated metastasis and suggest future therapeutic strategies for targeting ESR1-mutant breast cancer

Determination of the pK(a) of the N-terminal amino group of ubiquitin by NMR

This work was supported by the Medical Research Council (grants U117533887 and grant U117565398 until March 2015) and by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001142, FC10029), the UK Medical Research Council (FC001142, FC10029), and the Wellcome Trust (FC001142, FC10029)

Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in sickle cell disease

Photograph of a scene at Turner Falls

The protein phosphatase 4 complex promotes the Notch pathway and wingless transcription

The Wnt/Wingless (Wg) pathway controls cell fate specification, tissue differentiation and organ development across organisms. Using an in vivo RNAi screen to identify novel kinase and phosphatase regulators of the Wg pathway, we identified subunits of the serine threonine phosphatase Protein Phosphatase 4 (PP4). Knockdown of the catalytic and regulatory subunits of PP4 cause reductions in the Wg pathway targets Senseless and Distal-less. We find that PP4 regulates the Wg pathway by controlling Notch-driven wg transcription. Genetic interaction experiments identified that PP4 likely promotes Notch signaling within the nucleus of the Notch-receiving cell. Although the PP4 complex is implicated in various cellular processes, its role in the regulation of Wg and Notch pathways was previously uncharacterized. Our study identifies a novel role of PP4 in regulating Notch pathway, resulting in aberrations in Notch-mediated transcriptional regulation of the Wingless ligand. Furthermore, we show that PP4 regulates proliferation independent of its interaction with Notch