Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.DOI: http://dx.doi.org/10.7554/eLife.12727.001
BRCA1 and BRCA2 account for most cases of familial, early onset breast and/or ovarian cancer and encode products that each interact with hRAD51. Results presented here show that BRCA1 and BRCA2 coexist in a biochemical complex and colocalize in subnuclear foci in somatic cells and on the axial elements of developing synaptonemal complexes. Like BRCA1 and RAD51, BRCA2 relocates to PCNA+ replication sites following exposure of S phase cells to hydroxyurea or UV irradiation. Thus, BRCA1 and BRCA2 participate, together, in a pathway(s) associated with the activation of double-strand break repair and/or homologous recombination. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary breast and/or ovarian cancer.
Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that “paints the cell” with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.
Restoring functional β-cell mass is an important therapeutic goal for both type 1 and type 2 diabetes (1). While proliferation of existing β-cells is the primary means of β-cell replacement in rodents (2), it is unclear whether a similar principle applies to humans, as human β-cells are remarkably resistant to stimulation of division (3,4). Here, we show that 5-iodotubercidin (5-IT), an annotated adenosine kinase inhibitor previously reported to increase proliferation in rodent and porcine islets (5), strongly and selectively increases human β-cell proliferation in vitro and in vivo. Remarkably, 5-IT also increased glucose-dependent insulin secretion after prolonged treatment. Kinome profiling revealed 5-IT to be a potent and selective inhibitor of the dual-specificity tyrosine phosphorylation–regulated kinase (DYRK) and cell division cycle–like kinase families. Induction of β-cell proliferation by either 5-IT or harmine, another natural product DYRK1A inhibitor, was suppressed by coincubation with the calcineurin inhibitor FK506, suggesting involvement of DYRK1A and nuclear factor of activated T cells signaling. Gene expression profiling in whole islets treated with 5-IT revealed induction of proliferation- and cell cycle–related genes, suggesting that true proliferation is induced by 5-IT. Furthermore, 5-IT promotes β-cell proliferation in human islets grafted under the kidney capsule of NOD-scid IL2Rgnull mice. These results point to inhibition of DYRK1A as a therapeutic strategy to increase human β-cell proliferation.
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