Lipid spirals in Bacillus subtilis and their role in cell division

The fluid mosaic model of membrane structure has been revised in recent years as it has become evident that domains of different lipid composition are present in eukaryotic and prokaryotic cells. Using membrane binding fluorescent dyes, we demonstrate the presence of lipid spirals extending along the long axis of cells of the rod-shaped bacterium Bacillus subtilis. These spiral structures are absent from cells in which the synthesis of phosphatidylglycerol is disrupted, suggesting an enrichment in anionic phospholipids. Green fluorescent protein fusions of the cell division protein MinD also form spiral structures and these were shown by fluorescence resonance energy transfer to be coincident with the lipid spirals. These data indicate a higher level of membrane lipid organization than previously observed and a primary role for lipid spirals in determining the site of cell division in bacterial cells

Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

Super-resolution light microscopy, correlative light and electron microscopy, and volume electron microscopy are revolutionising the way in which biological samples are examined and understood. Here, we combine these approaches to deliver super-accurate correlation of fluorescent proteins to cellular structures. We show that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy. In conventional section-based correlative microscopy experiments, the specimen must be moved between imaging systems and/or further manipulated for optimal viewing. These steps can introduce undesirable alterations in the specimen, and complicate correlation between imaging modalities. We avoided these issues by using a scanning electron microscope with integrated optical microscope to acquire both localisation and electron microscopy images, which could then be precisely correlated. Collecting data from ultrathin sections also improved the axial resolution and signal-to-noise ratio of the raw localisation microscopy data. Expanding data collection across an array of sections will allow 3-dimensional correlation over unprecedented volumes. The performance of this technique is demonstrated on vaccinia virus (with YFP) and diacylglycerol in cellular membranes (with GFP)

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure

A New Monte Carlo Algorithm for Protein Folding

We demonstrate that the recently proposed pruned-enriched Rosenbluth method (P. Grassberger, Phys. Rev. E 56 (1997) 3682) leads to extremely efficient algorithms for the folding of simple model proteins. We test them on several models for lattice heteropolymers, and compare to published Monte Carlo studies. In all cases our algorithms are faster than all previous ones, and in several cases we find new minimal energy states. In addition to ground states, our algorithms give estimates for the partition sum at finite temperatures.Comment: 4 pages, Latex incl. 3 eps-figs., submitted to Phys. Rev. Lett., revised version with changes in the tex

Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families

Somatic mutations in salivary duct carcinoma and potential therapeutic targets

Background: Salivary duct carcinomas (SDCa) are rare highly aggressive malignancies. Most patients die from distant metastatic disease within three years of diagnosis. There are limited therapeutic options for disseminated disease. Results: 11 cases showed androgen receptor expression and 6 cases showed HER2 amplification. 6 Somatic mutations with additional available targeted therapies were identified: EGFR (p. G721A: Gefitinib), PDGFRA (p. H845Y: Imatinib and Crenolanib), PIK3CA (p. H1047R: Everolimus), ERBB2 (p. V842I: Lapatinib), HRAS (p. Q61R: Selumetinib) and KIT (p. T670I: Sorafenib). Furthermore, alterations in PTEN, PIK3CA and HRAS that alter response to androgen deprivation therapy and HER2 inhibition were also seen. Materials and Methods: Somatic mutation analysis was performed on DNA extracted from 15 archival cases of SDCa using the targeted Illumina TruSeq Amplicon Cancer Panel. Potential targetable genetic alterations were identified using extensive literature and international somatic mutation database (COSMIC, KEGG) search. Immunohistochemistry for androgen receptor and immunohistochemistry and fluorescent in situ hybridization for HER2 were also performed. Conclusions: SDCa show multiple somatic mutations, some that are amenable to pharmacologic manipulation and others that confer resistance to treatments currently under investigation. These findings emphasize the need to develop testing and treatment strategies for SDCa. © Khoo et al

Somatic mutations in salivary duct carcinoma and potential therapeutic targets

Background: Salivary duct carcinomas (SDCa) are rare highly aggressive malignancies. Most patients die from distant metastatic disease within three years of diagnosis. There are limited therapeutic options for disseminated disease. Results: 11 cases showed androgen receptor expression and 6 cases showed HER2 amplification. 6 Somatic mutations with additional available targeted therapies were identified: EGFR (p. G721A: Gefitinib), PDGFRA (p. H845Y: Imatinib and Crenolanib), PIK3CA (p. H1047R: Everolimus), ERBB2 (p. V842I: Lapatinib), HRAS (p. Q61R: Selumetinib) and KIT (p. T670I: Sorafenib). Furthermore, alterations in PTEN, PIK3CA and HRAS that alter response to androgen deprivation therapy and HER2 inhibition were also seen. Materials and Methods: Somatic mutation analysis was performed on DNA extracted from 15 archival cases of SDCa using the targeted Illumina TruSeq Amplicon Cancer Panel. Potential targetable genetic alterations were identified using extensive literature and international somatic mutation database (COSMIC, KEGG) search. Immunohistochemistry for androgen receptor and immunohistochemistry and fluorescent in situ hybridization for HER2 were also performed. Conclusions: SDCa show multiple somatic mutations, some that are amenable to pharmacologic manipulation and others that confer resistance to treatments currently under investigation. These findings emphasize the need to develop testing and treatment strategies for SDCa. © Khoo et al

Endosomal cargo recycling mediated by Gpa1 and phosphatidylinositol 3-kinase is inhibited by glucose starvation

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalized protein and lipid cargoes recycle back to the surface efficiently in glucose-replete conditions, but this trafficking is attenuated following glucose starvation. We find that cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for endosome to surface recycling. Furthermore, we find that the yeast Gα subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct Gα subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose that this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and overexpression of Gpa2 alter PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation

Enhanced RAD21 cohesin expression confers poor prognosis and resistance to chemotherapy in high grade luminal, basal and HER2 breast cancers

Introduction: RAD21 is a component of the cohesin complex, which is essential for chromosome segregation and error-free DNA repair. We assessed its prognostic and predictive power in a cohort of in situ and invasive breast cancers, and its effect on chemosensitivity in vitro.Methods: RAD21 immunohistochemistry was performed on 345 invasive and 60 pure in situ carcinomas. Integrated genomic and transcriptomic analyses were performed on a further 48 grade 3 invasive cancers. Chemosensitivity was assessed in breast cancer cell lines with an engineered spectrum of RAD21 expression.Results: RAD21 expression correlated with early relapse in all patients (hazard ratio (HR) 1.74, 95% confidence interval (CI) 1.06 to 2.86, P = 0.029). This was due to the effect of grade 3 tumors (but not grade 1 or 2) in which RAD21 expression correlated with early relapse in luminal (P = 0.040), basal (P = 0.018) and HER2 (P = 0.039) groups. In patients treated with chemotherapy, RAD21 expression was associated with shorter overall survival (P = 0.020). RAD21 mRNA expression correlated with DNA copy number, with amplification present in 32% (7/22) of luminal, 31% (4/13) of basal and 22% (2/9) of HER2 grade 3 cancers. Variations in RAD21 mRNA expression in the clinical samples were reflected in the gene expression data from 36 breast cancer cell lines. Knockdown of RAD21 in the MDA-MB-231 breast cancer cell line significantly enhanced sensitivity to cyclophosphamide, 5-fluorouracil and etoposide. The findings for the former two drugs recapitulated the clinical findings.Conclusions: RAD21 expression confers poor prognosis and resistance to chemotherapy in high grade luminal, basal and HER2 breast cancers. RAD21 may be a novel therapeutic target