Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle.

Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology.

Photoconvertible Behavior of LSSmOrange Applicable for Single Emission Band Optical Highlighting.

Photoswitchable fluorescent proteins are capable of changing their spectral properties upon light irradiation, thus allowing one to follow a chosen subpopulation of molecules in a biological system. Recently, we revealed a photoinduced absorption band shift of LSSmOrange, which was originally engineered to have a large energy gap between excitation and emission bands. Here, we evaluated the performance of LSSmOrange as a fluorescent tracer in living cells.

Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell.

Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell.

A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.

We sought to develop a sensitive and quantitative technique capable of monitoring the entire flux of autophagy involving fusion of lysosomal membranes. We observed the accumulation inside lysosomal compartments of Keima, a coral-derived acid-stable fluorescent protein that emits different-colored signals at acidic and neutral pHs. The cumulative fluorescent readout can be used to quantify autophagy at a single time point.

Simultaneous single cell stable expression of 2-4 cDNAs in HeLaS3 using psiC31 integrase system.

An important consideration in the design of multigene delivery technology is the availability of suitable vectors to introduce multiple genes stably and stoichiometrically into living cells and co-express these genes efficiently. As a promising system for this purpose, we developed multi-cDNA expression constructs harboring two to three tandemly situated cDNAs in a single plasmid. The utility of this vector system is amplified by combining it with the psiC31 recombinase system which mediates site-specific integration of the genes into naturally occurring chromosomal sequences.

Fluorescence imaging using a fluorescent protein with a large Stokes shift.

Keima is a far-red fluorescent protein endowed with a large Stokes shift. It absorbs light maximally at around 440nm and emits maximally at around 620nm. While the original Keima is obligately tetrameric (tKeima), the dimeric and monomeric versions (mKeima and dKeima, respectively) have been generated.

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex.

Lighting up cells: labelling proteins with fluorophores.

During the past decade, rapid improvements have been made in the tools available for labelling proteins within cells, which has increased our ability to unravel the finer details of cellular events. One significant reason for these advances has been the development of fluorescent proteins that can be incorporated into proteins by genetic fusion to produce a fluorescent label. In addition, new techniques have made it possible to label proteins with small organic fluorophores and semiconductor nanocrystals.