Multi-range ERK responses shape the proliferative trajectory of single cells following oncogene induction.

Oncogene-induced senescence is a phenomenon in which aberrant oncogene expression causes non-transformed cells to enter a non-proliferative state. Cells undergoing oncogenic induction display phenotypic heterogeneity, with some cells senescing and others remaining proliferative. The causes of heterogeneity remain unclear.

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage.

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels.

A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium.

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein.

Fast protocol for the visualization and quantitative high-throughput analysis of sprouting angiogenesis by confocal microscopy.

We describe an optimized, cost-effective, reproducible, and robust protocol to study sprouting angiogenesis in glass-bottom 96-well plates by confocal microscopy, ideal for screening of drug or shRNA libraries. Effective and stable knockdown of gene expression in primary endothelial cells is achieved by lentiviral transduction. Dynamic behavior of individual cells and fluorescent proteins is analyzed by time-lapse imaging, while competitive advantages in tip cell formation are assessed using mixtures of differentially labeled cell populations.

Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy.

Phosphatidylinositol-3 kinase signaling controls survival and stemness of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling.

A Biosensor for the Mitotic Kinase MPS1 Reveals Spatiotemporal Activity Dynamics and Regulation.

Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1-3]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3, 4]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity.

High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs.

Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids.

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors.

An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids.

PLK1 plays dual roles in centralspindlin regulation during cytokinesis.

Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. Here, we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells.

Live imaging of cell division in 3D stem-cell organoid cultures.

Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells.

Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening.

Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers.

Sequential cancer mutations in cultured human intestinal stem cells.

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells.

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE).

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization.

A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space.

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency.

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting.

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells.

Spatial regulation of cyclic AMP-Epac1 signaling in cell adhesion by ERM proteins.

Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins.

Spatiotemporal regulation of chloride intracellular channel protein CLIC4 by RhoA.

Chloride intracellular channel (CLIC) 4 is a soluble protein structurally related to omega-type glutathione-S-transferases (GSTs) and implicated in various biological processes, ranging from chloride channel formation to vascular tubulogenesis. However, its function(s) and regulation remain unclear. Here, we show that cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of G(13)-coupled, RhoA-activating receptors, such as those for lysophosphatidic acid, thrombin, and sphingosine-1-phosphate.

Direct spatial control of Epac1 by cyclic AMP.

Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane.

A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.

We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker.