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

Abstract

Vitamin B12 (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 B12 is associated with negative consequences on the developing brain. Vitamin B12 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 B12 concentration in intact living cells. Initially, a construct was designed by using the vitamin B12 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 B12. This sensor is stable to pH changes, and measures the vitamin B12 in a concentration-dependent manner with an apparent affinity, Kd of ∼157μM. In case of E. coli cells, an increase in the emission intensity ratio was specifically detected after exposure to vitamin B12. In both in vitro and in vivo measurements, FRET ratio increases after the addition of vitamin B12. Furthermore, the results show that the SenVitAL can measure the vitamin B12 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 B12 import and metabolism and, would help to elucidate their complex roles in biology.