Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.

No abstract

The transient receptor potential (TRP) channels are implicated in various cellular processes, including sensory signal transduction and electrolyte homeostasis. We show here that the GTL-1 and GON-2 TRPM channels regulate electrolyte homeostasis in the C. elegans intestine. GON-2 is responsible for a large outwardly rectifying current of intestinal cells, and its activity is tightly regulated by intracellular Mg(2+) levels, while GTL-1 mainly contributes to appropriate Mg(2+) responsiveness of the outwardly rectifying current. We also used nickel cytotoxicity to study the function of these channels. Both GON-2 and GTL-1 are necessary for intestinal uptake of nickel, but GTL-1 is continuously active while GON-2 is inactivated at higher Mg(2+) levels. This type of differential regulation of intestinal electrolyte absorption ensures a constant supply of electrolytes through GTL-1, while occasional bursts of GON-2 activity allow rapid return to normal electrolyte concentrations following physiological perturbations.

No abstract

To measure the activity of neurons using whole-brain activity imaging, precise detection of each neuron or its nucleus is required. In the head region of the nematode C. elegans, the neuronal cell bodies are distributed densely in three-dimensional (3D) space. However, no existing computational methods of image analysis can separate them with sufficient accuracy. Here we propose a highly accurate segmentation method based on the curvatures of the iso-intensity surfaces. To obtain accurate positions of nuclei, we also developed a new procedure for least squares fitting with a Gaussian mixture model. Combining these methods enables accurate detection of densely distributed cell nuclei in a 3D space. The proposed method was implemented as a graphical user interface program that allows visualization and correction of the results of automatic detection. Additionally, the proposed method was applied to time-lapse 3D calcium imaging data, and most of the nuclei in the images were successfully tracked and measured.