Optical tools to study the subcellular organization of GPCR neuromodulation.

Modulation of neuronal circuit activity is key to information processing in the brain. G protein-coupled receptors (GPCRs), the targets of most neuromodulatory ligands, show extremely diverse expression patterns in neurons and receptors can be localized in various sub-neuronal membrane compartments. Upon activation, GPCRs promote signaling cascades that alter the level of second messengers, drive phosphorylation changes, modulate ion channel function, and influence gene expression, all of which critically impact neuron physiology. Because of its high degree of complexity, this form of interneuronal communication has remained challenging to integrate into our conceptual understanding of brain function. Recent technological advances in fluorescence microscopy and the development of optical biosensors now allow investigating neuromodulation with unprecedented resolution on the level of individual cells. In this review, we will highlight recent imaging techniques that enable determining the precise localization of GPCRs in neurons, with specific focus on the subcellular and nanoscale level. Downstream of receptors, we describe novel conformation-specific biosensors that allow for real-time monitoring of GPCR activation and of distinct signal transduction events in neurons. Applying these new tools has the potential to provide critical insights into the function and organization of GPCRs in neuronal cells and may help decipher the molecular and cellular mechanisms that underlie neuromodulation.

Notch2 pathway mediates breast cancer cellular dormancy and mobilisation in bone and contributes to haematopoietic stem cell mimicry.

BACKGROUND: Recurrence after >5-year disease-free survival affects one-fifth of breast cancer patients and is the clinical manifestation of cancer cell reactivation after persistent dormancy. METHODS: We investigated cellular dormancy in vitro and in vivo using breast cancer cell lines and cell and molecular biology techniques. RESULTS: We demonstrated cellular dormancy in breast cancer bone metastasis, associated with haematopoietic stem cell (HSC) mimicry, in vivo competition for HSC engraftment and non-random distribution of dormant cells at the endosteal niche. Notch2 signal implication was demonstrated by immunophenotyping the endosteal niche-associated cancer cells and upon co-culture with sorted endosteal niche cells, which inhibited breast cancer cell proliferation in a Notch2-dependent manner. Blocking this signal by in vivo acute administration of the γ-secretase inhibitor, dibenzazepine, induced dormant cell mobilisation from the endosteal niche and colonisation of visceral organs. Sorted Notch2HIGH breast cancer cells exhibited a unique stem phenotype similar to HSCs and in vitro tumour-initiating ability in mammosphere assay. Human samples confirmed the existence of a small Notch2HIGH cell population in primary and bone metastatic breast cancers, with a survival advantage for Notch2HIGH vs Notch2LOW patients. CONCLUSIONS: Notch2 represents a key determinant of breast cancer cellular dormancy and mobilisation in the bone microenvironment.