Imaging nanoscale nuclear structures with expansion microscopy.

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, limiting the numbers of three-dimensional (3D) nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy (ExM) solves many of these limitations, but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here, we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120-130 nm (pre-expansion). Using the DNA damage response proteins BRCA1, 53BP1 (also known as TP53BP1) and RAD51 as exemplars, we quantitatively describe the 3D nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin-regulated events and show the simultaneous assessment of four elements. This study thus demonstrates how ExM can contribute to the investigation of nanoscale nuclear processes.

FrenchFISH: Poisson Models for Quantifying DNA Copy Number From Fluorescence In Situ Hybridization of Tissue Sections.

PURPOSE: Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. The gold standard for detecting copy number changes in tumor cells is fluorescence in situ hybridization (FISH) using locus-specific probes that are imaged as fluorescent spots. However, spot counting often does not perform well on solid tumor tissue sections due to partially represented or overlapping nuclei. MATERIALS AND METHODS: To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes or a homogeneous Poisson point process model for automated spot counting. RESULTS: We benchmarked the performance of FrenchFISH against previous approaches using a controlled simulation scenario and tested it experimentally in 12 ovarian carcinoma FFPE-tissue sections for copy number alterations at three loci (c-Myc, hTERC, and SE7). FrenchFISH outperformed standard spot counting with 74% of the automated counts having < 1 copy number difference from the manual counts and 17% having < 2 copy number differences, while taking less than one third of the time of manual counting. CONCLUSION: FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue by both speeding up and improving the accuracy of spot count estimates.

Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events.

BACKGROUND: The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. OBJECTIVE: This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. METHODS: Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. RESULTS: NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. CONCLUSION: NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.

Platelet glycoprotein VI and C-type lectin-like receptor 2 deficiency accelerates wound healing by impairing vascular integrity in mice.

Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.

Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule.

Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of multiple signaling pathways. A novel approach to the treatment of PDAC is described, involving the targeting of cancer genes in PDAC pathways having over-representation of G-quadruplexes, using the trisubstituted naphthalene diimide quadruplex-binding compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[ lmn][3,8]phenanthroline-1,3,6,8(2 H,7 H)-tetraone (CM03). This compound has been designed by computer modeling, is a potent inhibitor of cell growth in PDAC cell lines, and has anticancer activity in PDAC models, with a superior profile compared to gemcitabine, a commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to analyze the effects of this quadruplex-binding small molecule on global gene expression. This has revealed the down-regulation of a large number of genes, rich in putative quadruplex elements and involved in essential pathways of PDAC survival, metastasis, and drug resistance. The changes produced by CM03 represent a global response to the complexity of human PDAC and may be applicable to other currently hard-to-treat cancers.

CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution.

Single molecule imaging approaches like dSTORM and PALM resolve structures at 10-20 nm, and allow for unique insights into protein stoichiometry and spatial relationships. However, key obstacles remain in developing highly accurate quantitative single molecule approaches. The genomic tagging of PALM fluorophores through CRISPR-Cas9 offers an excellent opportunity for generating stable cell lines expressing a defined single molecule probe at endogenous levels, without the biological disruption and variability inherent to transfection. A fundamental question is whether these comparatively low levels of expression can successfully satisfy the stringent labelling demands of super-resolution SMLM. Here we apply CRISPR-Cas9 gene editing to tag a cytoskeletal protein (α-tubulin) and demonstrate a relationship between expression level and the subsequent quality of PALM imaging, and that spatial resolutions comparable to dSTORM can be achieved with CRISPR-PALM. Our approach shows a relationship between choice of tag and the total expression of labelled protein, which has important implications for the development of future PALM tags. CRISPR-PALM allows for nanoscopic spatial resolution and the unique quantitative benefits of single molecule localization microscopy through endogenous expression, as well as the capacity for super-resolved live cell imaging.

Quantifying receptor trafficking and colocalization with confocal microscopy.

Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocytosis. Unbiased and robust image analysis workflows are required for the identification, and study, of aberrant trafficking. After a brief review of related strategies, identifying both good and bad practice, custom workflows for the analysis of live cell 3D time-lapse data are presented. Strategies for data pre-processing, including denoising and background subtraction are considered. We use a 3D level set protocol to accurately segment cells using only the signal from fluorescently labelled receptor. A protocol for the quantification of changes to subcellular receptor distribution over time is then presented. As an example, ligand stimulated trafficking of epidermal growth factor receptor (EGFR) is shown to be significantly reduced in both AG1478 and Dynasore treated cells. Protocols for the quantitative analysis of colocalization between receptor and endosomes are also introduced, including strategies for signal isolation and statistical testing. By calculating the Manders and Pearson coefficients, both co-occurrence and correlation can be assessed. A statistically significant decrease in the level of ligand induced co-occurrence between EGFR and rab5 positive endosomes is demonstrated for both the AG1478 and Dynasore treated cells relative to a control. Finally, a strategy for the visualisation of co-occurrence is presented, which provides an unbiased alternative to colour overlays.