Adapting to the Coronavirus Pandemic: Building and Incorporating a Diagnostic Pipeline in a Shared Resource Laboratory.

In March 2020, with lockdown due to the coronavirus pandemic underway, the Francis Crick Institute (the Crick) regeared its research laboratories into clinical testing facilities. Two pipelines were established, one for polymerase chain reaction and the other for Serology. This article discusses the Cricks Flow Cytometry Science Technology Platform (Flow STP) role in setting up the Serology pipeline. Pipeline here referring to the overarching processes in place to facilitate the receipt of human sera through to a SARs-CoV-2 enzyme-linked immunosorbent assay result. We examine the challenges that had to be overcome by a research laboratory to incorporate clinical diagnostics and the processes by which this was achieved. It describes the governance required to run the service, the design of the standard operating procedures (SOPs) and pipeline, the setting up of the assay, the validation required to show the robustness of the pipeline and reporting the results of the assay. Finally, as the lockdown started to ease in June 2020, it examines how this new service affects the daily running of the Flow STP. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Arkadia activates Smad3/Smad4-dependent transcription by triggering signal-induced SnoN degradation.

E3 ubiquitin ligases play important roles in regulating transforming growth factor beta (TGF-beta)/Smad signaling. Screening of an E3 ubiquitin ligase small interfering RNA library, using TGF-beta induction of a Smad3/Smad4-dependent luciferase reporter as a readout, revealed that Arkadia is an E3 ubiquitin ligase that is absolutely required for this TGF-beta response. Knockdown of Arkadia or overexpression of a dominant-negative mutant completely abolishes transcription from Smad3/Smad4-dependent reporters, but not from Smad1/Smad4-dependent reporters or from reporters driven by Smad2/Smad4/FoxH1 complexes. We show that Arkadia specifically activates transcription via Smad3/Smad4 binding sites by inducing degradation of the transcriptional repressor SnoN. Arkadia is essential for TGF-beta-induced SnoN degradation, but it has little effect on SnoN levels in the absence of signal. Arkadia interacts with SnoN and induces its ubiquitination irrespective of TGF-beta/Activin signaling, but SnoN is efficiently degraded only when it forms a complex with both Arkadia and phosphorylated Smad2 or Smad3. Finally, we describe an esophageal cancer cell line (SEG-1) that we show has lost Arkadia expression and is deficient for SnoN degradation. Reintroduction of wild-type Arkadia restores TGF-beta-induced Smad3/Smad4-dependent transcription and SnoN degradation in these cells, raising the possibility that loss of Arkadia function may be relevant in cancer.

An N-terminally truncated Smad2 protein can partially compensate for loss of full-length Smad2.

TGFbeta (transforming growth factor beta) superfamily signalling is critical both for early embryonic development and later for tissue homoeostasis in adult organisms. The use of gene-disruption techniques in mice has been essential to understanding the functional roles of the components of the pathways downstream of TGFbeta superfamily ligands, in particular, the receptors and the Smads that transduce signals from the plasma membrane to the nucleus. Smad2 functions downstream of TGFbeta, Activin and Nodal, and a number of Smad2 mutant mice have been generated by different laboratories. Although in all cases these Smad2-deficient mice were embryonic lethal, those created by deletion of the first coding exon survived longer than those generated by replacing part of the MH (Mad homology) 1 domain or deleting all or part of the MH2 domain. Moreover, they displayed a less severe phenotype, as they were capable of transiently inducing mesoderm. In the present study, we show that embryonic fibroblasts taken from the Smad2 mutant mice created by deletion of the first coding exon express a small amount of an N-terminally truncated Smad2 protein. We show this protein results from internal initiation at Met(241) and encodes the entire MH2 domain and the C-terminal part of the linker. We demonstrate that this protein is incorporated into Smad heteromeric complexes, can interact with DNA-binding transcription factors and thereby can mediate TGFbeta-induced transcriptional activation from a number of TGFbeta-responsive elements. We propose that this functional truncated Smad2 protein can partially compensate for the loss of full-length Smad2, thereby providing an explanation for the differing phenotypes of Smad2 mutant mice.

Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling.

Smad4 in partnership with R-Smads (receptor-regulated Smads) activates TGF-beta (transforming growth factor-beta)-dependent signalling pathways essential for early mouse development. Smad4 null embryos die shortly after implantation due to severe defects in cell proliferation and visceral endoderm differentiation. In the basal state, Smad4 undergoes continuous shuttling between the cytoplasm and the nucleus due to the combined activities of an N-terminal NLS (nuclear localization signal) and an NES (nuclear export signal) located in its linker region. Cell culture experiments suggest that Smad4 nucleocytoplasmic shuttling plays an important role in TGF-beta signalling. In the present study we have investigated the role of Smad4 shuttling in vivo using gene targeting to engineer two independent mutations designed to eliminate Smad4 nuclear export. As predicted this results in increased levels of Smad4 in the nucleus of homozygous ES cells (embryonic stem cells) and primary keratinocytes, in the presence or absence of ligand. Neither mutation affects Smad4 expression levels nor its ability to mediate transcriptional activation in homozygous cell lines. Remarkably mouse mutants lacking the Smad4 NES develop normally. Smad4 NES mutants carrying one copy of a Smad4 null allele also fail to display developmental defects. The present study clearly demonstrates that Smad4 nucleocytoplasmic shuttling is not required for embryonic development or tissue homoeostasis in normal, healthy adult mice.

The Dynamics of TGF-ß Signaling Are Dictated by Receptor Trafficking via the ESCRT Machinery.

Signal transduction pathways stimulated by secreted growth factors are tightly regulated at multiple levels between the cell surface and the nucleus. The trafficking of cell surface receptors is emerging as a key step for regulating appropriate cellular responses, with perturbations in this process contributing to human diseases, including cancer. For receptors recognizing ligands of the transforming growth factor ß (TGF-ß) family, little is known about how trafficking is regulated or how this shapes signaling dynamics. Here, using whole genome small interfering RNA (siRNA) screens, we have identified the ESCRT (endosomal sorting complex required for transport) machinery as a crucial determinant of signal duration. Downregulation of ESCRT components increases the outputs of TGF-ß signaling and sensitizes cells to low doses of ligand in their microenvironment. This sensitization drives an epithelial-to-mesenchymal transition (EMT) in response to low doses of ligand, and we demonstrate a link between downregulation of the ESCRT machinery and cancer survival.

TGF-ß uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition.

The best characterized signaling pathway downstream of transforming growth factor ß (TGF-ß) is through SMAD2 and SMAD3. However, TGF-ß also induces phosphorylation of SMAD1 and SMAD5, but the mechanism of this phosphorylation and its functional relevance is not known. Here, we show that TGF-ß-induced SMAD1/5 phosphorylation requires members of two classes of type I receptor, TGFBR1 and ACVR1, and establish a new paradigm for receptor activation where TGFBR1 phosphorylates and activates ACVR1, which phosphorylates SMAD1/5. We demonstrate the biological significance of this pathway by showing that approximately a quarter of the TGF-ß-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the ID genes. Finally, we show that TGF-ß-induced epithelial-to-mesenchymal transition requires signaling via both the SMAD3 and SMAD1/5 pathways, with SMAD1/5 signaling being essential to induce ID1. Therefore, combinatorial signaling via both SMAD pathways is essential for the full TGF-ß-induced transcriptional program and physiological responses.

Controlling long-term signaling: receptor dynamics determine attenuation and refractory behavior of the TGF-ß pathway.

Understanding the complex dynamics of growth factor signaling requires both mechanistic and kinetic information. Although signaling dynamics have been studied for pathways downstream of receptor tyrosine kinases and G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors, they have not been investigated for the transforming growth factor-ß (TGF-ß) superfamily pathways. Using an integrative experimental and mathematical modeling approach, we dissected the dynamic behavior of the TGF-ß to Smad pathway, which is mediated by type I and type II receptor serine/threonine kinases, in response to acute, chronic, and repeated ligand stimulations. TGF-ß exposure produced a transient response that attenuated over time, resulting in desensitized cells that were refractory to further acute stimulation. This loss of signaling competence depended on ligand binding, but not on receptor activity, and was restored only after the ligand had been depleted. Furthermore, TGF-ß binding triggered the rapid depletion of signaling-competent receptors from the cell surface, with the type I and type II receptors exhibiting different degradation and trafficking kinetics. A computational model of TGF-ß signal transduction from the membrane to the nucleus that incorporates our experimental findings predicts that autocrine signaling, such as that associated with tumorigenesis, severely compromises the TGF-ß response, which we confirmed experimentally. Thus, we have shown that the long-term signaling behavior of the TGF-ß pathway is determined by receptor dynamics, does not require TGF-ß-induced gene expression, and influences context-dependent responses in vivo.

Arkadia regulates tumor metastasis by modulation of the TGF-ß pathway.

TGF-ß can act as a tumor suppressor at early stages of cancer progression and as a tumor promoter at later stages. The E3 ubiquitin ligase Arkadia (RNF111) is a critical component of the TGF-ß signaling pathway, being required for a subset of responses, those mediated by Smad3-Smad4 complexes. It acts by mediating ligand-induced degradation of Ski and SnoN (SKIL), which are 2 potent transcriptional repressors. Here, we investigate the role of Arkadia in cancer using model systems to address both potential tumor-suppressive and tumor-promoting roles. Stable reexpression of Arkadia in lung carcinoma NCI-H460 cells, which we show contain a hemizygous nonsense mutation in the Arkadia/RNF111 gene, efficiently restored TGF-ß-induced Smad3-dependent transcription, and substantially decreased the ability of these cells to grow in soft agar in vitro. However, it had no effect on tumor growth in vivo in mouse models. Moreover, loss of Arkadia in cancer cell lines and human tumors is rare, arguing against a prominent tumor-suppressive role. In contrast, we have uncovered a potent tumor-promoting function for Arkadia. Using 3 different cancer cell lines whose tumorigenic properties are driven by TGF-ß signaling, we show that loss of Arkadia function, either by overexpression of dominant negative Arkadia or by siRNA-induced knockdown, substantially inhibited lung colonization in tail vein injection experiments in immunodeficient mice. Our findings indicate that Arkadia is not critical for regulating tumor growth per se, but is required for the early stages of cancer cell colonization at the sites of metastasis.

DT40 cells lacking the Ca2+-binding protein annexin 5 are resistant to Ca2+-dependent apoptosis.

Annexins are widely expressed Ca(2+)-dependent phospholipid-binding proteins with poorly understood physiological roles. Proposed functions include Ca(2+) channel activity and vesicle trafficking, but neither have been proven in vivo. Here we used targeted gene disruption to generate B-lymphocytes lacking annexin 5 (Anx5) expression and show that this results in reduced susceptibility to a range of apoptotic stimuli. By comparison B-lymphocytes lacking annexin 2 (Anx2) showed no such resistance, providing evidence that this effect is specific to loss of Anx5. The defect in the ANX5(-/-) cells occurs early in the apoptotic program before nuclear condensation, caspase 3 activation, and cell shrinkage, but downstream of an initial Ca(2+) influx. Only UVA/B irradiation induced similar levels of apoptosis in wild-type and ANX5(-/-) cells. Unexpectedly, ANX5(-/-) cells permeabilized in vitro also failed to release mitochondrial cytochrome C, suggesting a possible mechanism for their resistance to apoptosis. These findings demonstrate a role for Anx5 in determining the susceptibility of B-lymphocytes to apoptosis.