Live cell imaging of vitamin B12 dynamics by genetically encoded fluorescent nanosensor

Vitamin B-12 (cobalamin) is a co-factor of various enzymes and involved in the metabolism of every cell of human body. During infancy, low level of vitamin B-12 is associated with negative consequences on the developing brain. Vitamin B-12 deficiency causes various neurological abnormalities and pernicious anemia. Different methods such as, isotopic labeling, mass spectrocopy and Nuclear Magnetic Resonance spectroscopy have been used for measuring the level of cellular metabolites or signaling molecules, but these methods require the disruption and fractionation of tissues which suffer from contamination. Fluorescence Resonance Energy Transfer (FRET)-based genetically encoded nanosensors have been evolved as an ideal mean to determine the metabolite concentration in live cells. Here, we report the designing of FRET-based nanosensor for direct visualization of changes in vitamin B-12 concentration in intact living cells. Initially, a construct was designed by using the vitamin B-12 binding protein (BtuF), cyan (CFP) and yellow (YFP) variants of green fluorescent protein. This construct was then shuttled in different expression vectors. The constructed FRET sensor was termed as SenVitAL (Sensor for Vitamin Anemia Linked) which is found to be very specific for vitamin B-12. This sensor is stable to pH changes, and measures the vitamin B-12 in a concentration-dependent manner with an apparent affinity, K-d of similar to 157 mu M. In case of E. coli cells, an increase in the emission intensity ratio was specifically detected after exposure to vitamin B-12. In both in vitro and in vivo measurements, FRET ratio increases after the addition of vitamin B-12. Furthermore, the results show that the SenVitAL can measure the vitamin B-12 concentration in the cytosol of yeast and mammalian cells which proves its potential in eukaryotic system. Thus, the sensor can serve as novel indicator to investigate the vitamin B-12 import and metabolism and, would help to elucidate their complex roles in biology. (c) 2017 Elsevier B.V. All rights reserved.