High resolution imaging using scanning ion conductance microscopy with improved distance feedback control

Microscopy is an essential technique for observation on living cells. There is currently great interest in apply scanning probe microscopy to image living biological cells in their natural environment at the nanometer scale. Scanning ion conductance microscopy is a new form of scanning probe microscopy, which enables non-contact high resolution imaging of living biological cells. Based on a scanned nanopipette in physiological buffer, the distance feedback control uses the ion current to control the distance between the pipette tip and the sample surface. However, this feedback control has difficulties over slopes on convoluted cell surfaces, which limits its resolution. In this study, we present an improved form of feedback control that removes the contribution of up to the third order slope from the ion current signal, hence providing a more accurate signal for controlling the distance. We show that this allows faster and lower noise topographic high resolution imaging.     

Differential Notch signalling distinguishes neural stem cells from intermediate progenitors

During brain development, neurons and glia are generated from a germinal zone containing both neural stem cells (NSCs) and more limited intermediate neural progenitors (INPs). The signalling events that distinguish between these two proliferative neural cell types remain poorly understood. The Notch signalling pathway is known to maintain NSC character and to inhibit neurogenesis, although little is known about the role of Notch signalling in INPs. Here we show that both NSCs and INPs respond to Notch receptor activation, but that NSCs signal through the canonical Notch effector C-promoter binding factor 1 (CBF1), whereas INPs have attenuated CBF1 signalling. Furthermore, whereas knockdown of CBF1 promotes the conversion of NSCs to INPs, activation of CBF1 is insufficient to convert INPs back to NSCs. Using both transgenic and transient in vivo reporter assays we show that NSCs and INPs coexist in the telencephalic ventricular zone and that they can be prospectively separated on the basis of CBF1 activity. Furthermore, using in vivo transplantation we show that whereas NSCs generate neurons, astrocytes and oligodendrocytes at similar frequencies, INPs are predominantly neurogenic. Together with previous work on haematopoietic stem cells, this study suggests that the use or blockade of the CBF1 cascade downstream of Notch is a general feature distinguishing stem cells from more limited progenitors in a variety of tissues.     

RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels

Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.     

BluB cannibalizes flavin to form the lower ligand of vitamin B12

Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms. The biosynthesis of the lower ligand of vitamin B(12), 5,6-dimethylbenzimidazole (DMB), is poorly understood. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and D-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease.     

The translational landscape of mTOR signalling steers cancer initiation and metastasis

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the 'cancerous' translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.     

A novel retinoblastoma therapy from genomic and epigenetic analyses

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.     

Sustained translational repression by eIF2α-P mediates prion neurodegeneration

The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.     

Genetic dissection of an amygdala microcircuit that gates conditioned fear

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ(+) neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ(-) neurons in CEl. Electrical silencing of PKC-δ(+) neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called CEl(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.