Christina Karlsson Rosenthal

Cyclin keeps dNTPs in balance

Embryonal rhabdomyosarcoma (ERMS) is an aggressive form of muscle sarcoma that arises from myoblasts or satellite cells. Langenau and colleagues now use transgenic zebrafish to image the cells responsible for sustained ERMS growth in vivo, and also define a role for non-tumour-propagating ERMS cells in supporting metastatic spread (Cancer Cell 21, 680–693; 2012). Using elegant in vivo imaging techniques, the authors visualized ERMS formation in the tail musculature of fluorescent transgenic zebrafish embryos microinjected with oncogenic transgenes, and identified distinct ERMS cell subpopulations that were fluorescently labelled based on the expression of different myogenic factors. They demonstrated that the cell subpopulation expressing myf5, a factor that marks satellite and early muscle progenitor cells, was more proliferative and harboured tumourpropagating capacity in vivo. They further showed that cells expressing myogenin, a marker of differentiated muscle cells, were not tumourpropagating, but were highly migratory and invaded normal tissue. The authors determined that the migration of myogenin-expressing cells to secondary areas of tumour growth precedes the recruitment of tumour-propagating myf5positive cells to those sites, demonstrating that differentiated non-tumour-propagating ERMS cells facilitate metastasis. Defining the mechanisms by which differentiated, migratory cells promote metastatic colonization by tumour-propagating cells will be an exciting area of future research. AIZ

EGFR probes matrix stiffness

Written by Christina Karlsson Rosenthal and Alexia-Ileana Zaromytidou EGFR probes matrix stiffness When cells migrate and spread, they mechanically test the rigidity of the extracellular matrix (ECM) by contracting it, and at certain levels of force, cells reinforce integrinmediated adhesions. Sheetz and colleagues now show that rigidity sensing is dependent on epidermal growth factor receptor (EGFR) activity, specifically on rigid surfaces (Nat. Mater. http://doi.org/b6wz; 2017). Seeding cells onto micropillars coated with fibronectin confirms the role of local contractile units in contraction testing, and show that contractile unit activity is reduced on stiff compared with soft pillar matrix. EGFR is known to act with integrins in regulating cell–ECM interactions, and the authors find that loss of EGFR, through inhibition or knockdown, decreases spreading and contractility on stiff, but not on soft substrates. Interestingly, in the absence of EGFR, overexpression of the ligand-independent receptor HER2 can rescue rigidity sensing. The authors go on to show that EGFR can be activated in a ligand-independent manner by Src and, using inhibitors and mutants of Src-dependent phosphorylation sites, they find that this is needed for mechanosensing. However, EGF also stimulates local contraction events on rigid surfaces. Finally, they demonstrate that EGF-mediated activation of motility is dependent on myosin contractility and Src kinases. These data demonstrate a previously unknown role for EGFR in rigidity sensing. CKR Formin’ a perinuclear actin cage in confined migration

Mechanical control of antigen uptake

Written by Christina Karlsson Rosenthal and Alexia-Ileana Zaromytidou EGFR probes matrix stiffness When cells migrate and spread, they mechanically test the rigidity of the extracellular matrix (ECM) by contracting it, and at certain levels of force, cells reinforce integrinmediated adhesions. Sheetz and colleagues now show that rigidity sensing is dependent on epidermal growth factor receptor (EGFR) activity, specifically on rigid surfaces (Nat. Mater. http://doi.org/b6wz; 2017). Seeding cells onto micropillars coated with fibronectin confirms the role of local contractile units in contraction testing, and show that contractile unit activity is reduced on stiff compared with soft pillar matrix. EGFR is known to act with integrins in regulating cell–ECM interactions, and the authors find that loss of EGFR, through inhibition or knockdown, decreases spreading and contractility on stiff, but not on soft substrates. Interestingly, in the absence of EGFR, overexpression of the ligand-independent receptor HER2 can rescue rigidity sensing. The authors go on to show that EGFR can be activated in a ligand-independent manner by Src and, using inhibitors and mutants of Src-dependent phosphorylation sites, they find that this is needed for mechanosensing. However, EGF also stimulates local contraction events on rigid surfaces. Finally, they demonstrate that EGF-mediated activation of motility is dependent on myosin contractility and Src kinases. These data demonstrate a previously unknown role for EGFR in rigidity sensing. CKR Formin’ a perinuclear actin cage in confined migration

Extra centrosomes provide route to invasion

Written by Nathalie Le Bot, Christina Karlsson Rosenthal and Alexia-Ileana Zaromytidou Mechanotransduction in collective cell migration The Drosophila melanogaster ovary egg chambers contain nurse cells and an oocyte surrounded by epithelial follicle cells. During oogenesis, polar cells stimulate a group of epithelial cells, termed border cells, to move between nurse cells towards the oocyte. Successful border cell migration requires E-cadherin expression in both border and nurse cells. Montell and colleagues now provide an elegant characterization of the role of E-cadherin in this process (Cell 157, 1146–1159; 2014). Using Drosophila strains with E-cadherin depleted in border, nurse or polar cells, they demonstrated that E-cadherin-based adhesions between border and nurse cells are necessary for directional migration of border cells, whereas E-cadherin depletion in polar cells disrupts border cell clusters. The authors generated transgenic flies expressing E-cadherin modified to include a Förster resonance energy transfer (FRET)-based tension sensor, to register mechanical force transduction across E-cadherin molecules. They observed a higher level of tension at the front of the border cell cluster, indicating higher adhesive strength and/ or actomyosin contractility at that site. Morphodynamic profiling of protrusion and retraction behaviour of border cells expressing dominant-negative forms of PVR (PDGFand VEGF-receptor related) and EGFR (epidermal growth factor receptor) confirmed the known role of these receptors in guiding border cells. Comparing these profiles with those from expression of dominant-negative Rac and depletion of E-cadherin indicated that PVR and EGFR stimulate Rac activity and function upstream of E-cadherin in border cell guidance. Further tension sensor experiments revealed that the guidance receptors and Rac promote tension on E-cadherin at the front of the cluster. Experiments with a Rac FRET probe and Rac photoinhibition combined with depletion of adherens junction proteins showed that E-cadherin is involved in a positive feedback amplification of Rac signalling at the front of the border cell cluster to promote forward protrusion stabilization and to allow directional migration through E-cadherin-based adhesion between border cells. AIZ Fasting promotes HSC function

Opposing enzymes cooperate in ERAD

Lung epithelial repair depends on progenitor cell populations located in specific niches, but little is known about the signalling crosstalk occurring between the progenitors and their niches in homeostasis and post-injury repair. Kim and colleagues have set up a 3D co-culture system of bronchioalveolar stem cells (BASCs) and lung-derived endothelial cells to obtain colonies growing from single BASC clones (Cell 156, 440–455; 2014). These clones expand to differentiate into three types of colonies: bronchiolar, bronchioalveolar or alveolar structures. They find that the multipotentiality of the bronchioalveolar structures obtained was conserved following multiple passages or subcutaneous transplantations. They noticed that secretion of thrombospondin-1 (Tsp1) from the endothelial cells increased after injury, and that the lung endothelial cells defective for Tsp1 lost their capacity to induce alveolar differentiation. They also observed impaired alveolar repair in Tsp1-null mice. Further analysis led the authors to delineate that the growth factor BMP4 produced after injury activates NFAT-dependent Tsp1 production in endothelial cells to drive alveolar differentiation and repair. This 3D co-culture system will help in the analysis of maintenance and repair of lung stem cells in homeostasis and repair. NLB Myosin forces in haematopoiesis

Mitotic suppression of repair prevents telomere fusions

By Nathalie Le Bot, Emily J. Chenette, Christina Karlsson Rosenthal and Alexia-Ileana Zaromytidou Mitotic suppression of repair prevents telomere fusions Repair of DNA double-strand breaks (DSBs) requires accumulation of the ubiquitin ligase RNF8 followed by the repair factors 53BP1 and BRCA1 at break sites. Recruitment of these proteins and subsequent repair is inhibited in mitotic cells, but it has been unclear how and why. Durocher and colleagues reveal that RNF8 recruitment is controlled by the mitotic kinases CDK1 and PLK1, and that this is important for the maintenance of genomic stability (Science http://doi.org/r8g; 2014). RNF8 is normally recruited through binding to MDC1 but the authors found that CDK1mediated phosphorylation of RNF8 on Thr 198 disrupts its interaction with RNF8. In RNF8-depleted cells, restoration of a non-phosphorylatable RNF8 mutant restored irradiation-induced RNF8 foci in mitosis, whereas a phospho-mimicking version prevented foci formation in interphase. Reversing this phosphorylation event was sufficient to restore the recruitment of BRCA, but not that of 53BP1, to mitotic DSB sites. The authors then identified PLK1 and CDK1 phosphorylation sites in 53BP1, and demonstrated by mutagenesis that PLK1and CDK1-mediated phosphorylation of 53BP1 prevents its recruitment to DSBs. Cells expressing non-phosphorylatable mutants of both RNF8 and 53BP1 were extremely sensitive to ionizing radiation because of impaired whole-chromosome segregation associated with telomere fusions, a process mediated by the repair machinery. Thus mitotic chromosomes may be sensitive to telomere fusions, explaining why DSB repair must be suppressed in mitosis. CKR Reprogramming goes through a primitive-streaklike state

RIPK3 kinase activity determines death pathway

Brown adipose tissue (BAT) undergoes mitochondrial uncoupling and expends energy in response to adrenergic stimulation. Mechanistically, norepinephrine promotes the release of free fatty acids (FFAs), which activate Ucp1 to mediate thermogenesis. Shirihai and colleagues report that norepinephrine also induces mitochondrial fragmentation in BAT to promote thermogenesis (EMBO J. 33, 418–436; 2014). Norephinephrine and FFA treatment in mouse primary brown adipocytes synergistically induced energy expenditure, indicating that adrenergic signalling promotes thermogenesis through FFA-independent pathways. Indeed, norephinephrine, but not FFA alone, induced mitochondrial fragmentation. Norepinephrine promoted protein kinase A (PKA)-mediated phosphorylation of the pro-fission protein Drp1, and its localization with the mitochondria. Intriguingly, inhibiting FFA release by treating cells with Orlistat blocked depolarization but did not affect mitochondrial fragmentation, suggesting that Drp1-mediated fragmentation is stimulated by norephinephrine but not FFA. Overexpressing a dominant-negative Drp1 mutant blocked norephinephrine-stimulated energy expenditure and mitochondrial depolarization, revealing the importance of mitochondrial fission in thermogenesis. Consistent with this observation, knocking down the pro-fusion protein Mitofusin 2 (Mfn2) caused mitochondrial fragmentation and depolarization, and increased oxygen respiration. Mfn2 knockdown also synergized with FFAs to promote energy expenditure in brown adipocytes. Thus, adrenergic signalling promotes thermogenesis by promoting FFA release and mitochondrial uncoupling, and by activating Drp1 and stimulating mitochondrial fission. EJC Evading the defence against brain metastasis

PTEN prevents junction instability

Written by Nathalie Le Bot, Christina Karlsson Rosenthal and Maria Trajkovska Macropinocytosis supports cancer cell proliferation Cancer cells have a high and unique metabolic demand, and thus it is of interest to gain insights into the mechanisms that govern nutrient uptake. Overexpression of oncogenic Ras is known to stimulate macropinocytosis, an endocytic process whereby cells internalize extracellular fluid and its contents. Bar-Sagi and colleagues (Nature 497, 633–637; 2013) have now demonstrated the functional importance of this process in Ras-transformed cancer cells. By studying these cells both in vitro and in vivo, the authors showed that they displayed increased levels of macropinocytosis, which enhanced albumin internalization and subsequently increased intracellular levels of glutamine and its downstream metabolite α-ketoglutarate. Furthermore, catabolized proteins entered the central metabolism, and protein-derived amino acids were metabolized through several pathways. The increased level of macropinocytosis translated into an increased proliferation of cells deprived of glutamine but supplemented with albumin. Increased protein uptake and proliferation could also be generalized to cells with enhanced levels of macropinocytosis regardless of Ras status. Interestingly, in mice, inhibition of macropinocytosis using a specific inhibitor of macropinosome formation resulted in attenuation or regression of tumour growth. This shows that in cancer cells, increased levels of macropinocytosis can be a beneficial nutrient uptake mechanism to support proliferation. Therefore, inhibiting macropinocytosis could have potential therapeutic benefits. MT PTEN prevents junction instability

Replication origin regulation

Written by Nathalie Le Bot, Christina Karlsson Rosenthal and Maria Trajkovska Macropinocytosis supports cancer cell proliferation Cancer cells have a high and unique metabolic demand, and thus it is of interest to gain insights into the mechanisms that govern nutrient uptake. Overexpression of oncogenic Ras is known to stimulate macropinocytosis, an endocytic process whereby cells internalize extracellular fluid and its contents. Bar-Sagi and colleagues (Nature 497, 633–637; 2013) have now demonstrated the functional importance of this process in Ras-transformed cancer cells. By studying these cells both in vitro and in vivo, the authors showed that they displayed increased levels of macropinocytosis, which enhanced albumin internalization and subsequently increased intracellular levels of glutamine and its downstream metabolite α-ketoglutarate. Furthermore, catabolized proteins entered the central metabolism, and protein-derived amino acids were metabolized through several pathways. The increased level of macropinocytosis translated into an increased proliferation of cells deprived of glutamine but supplemented with albumin. Increased protein uptake and proliferation could also be generalized to cells with enhanced levels of macropinocytosis regardless of Ras status. Interestingly, in mice, inhibition of macropinocytosis using a specific inhibitor of macropinosome formation resulted in attenuation or regression of tumour growth. This shows that in cancer cells, increased levels of macropinocytosis can be a beneficial nutrient uptake mechanism to support proliferation. Therefore, inhibiting macropinocytosis could have potential therapeutic benefits. MT PTEN prevents junction instability

Mad1 doubles as nuclear import regulator

Glucose metabolism is often altered in cancer to promote cell survival under stress. SAICAR (succinylaminoimidazolecarboxamide ribose5’-phosphate) is an intermediate of de novo purine nucleotide synthesis. Lee and colleagues report that this metabolite stimulates pyruvate kinase isoform M2 (PKM2), a glycolytic enzyme crucial in the metabolic reprogramming of cancer cells towards growth and survival when glucose is scarce (Science 338, 1069–1072; 2012). The authors tested metabolites from lung cancer cells for their ability to interact with PKM2 and identified SAICAR as a compound binding to PKM2 under low glucose conditions. Synthetic SAICAR was shown to stimulate the pyruvate kinase activity of PKM2, and this metabolite was elevated and able to activate PKM2 in glucose-deprived human cancer cell lines. Depletion of PAICS and ADSL, the SAICAR synthesizing and cleaving enzymes, respectively, showed that SAICAR levels affected glucose metabolism as well as cancer cell proliferation and survival in glucoselimiting situations. To demonstrate the functional relevance of the SAICAR–PKM2 interaction, the authors designed a PKM2 point mutant that was refractory to the effects of SAICAR. They showed that, in contrast to wild-type PKM2, expression of this mutant was not able to sustain the survival of cancer cells proficient in SAICAR production but depleted of endogenous PKM2, demonstrating that the SAICAR–PKM2 interaction was necessary for cancer cell survival in glucose-depleted settings. These findings reveal an additional layer in the regulation of the metabolic reprogramming of cancer cells. AIZ