Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Abstract

Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing proteinbased nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications. Limited information is derived from methods requiring fixation or fractionation of tissue (1, 2). Static analysis of metabolite composition in organs, tissues, and cellular compartments involves cell disruption. Most techniques neither measure metabolite changes in real-time nor account for likely variations in local metabolite concentration at the cellular level. Current methods have low resolution and are prone to artifacts, e.g., contamination by other cell types or subcellular compartments. Thus, little is known about the dynamic changes in concentration of metabolites such as sugars and amino acids͞neurotransmit-ters at the site of transport, i.e., in the synaptic cleft relative to the cytosol of adjacent neurons and glia or at the loading site of the phloem in plants, but also in the distribution of different sugars within cellular compartments. To better understand metabolism and compartmentation, a noninvasive technique would be of significant advantage.To generate a set of multifunctional nanosensors with specificity to a large number of different compounds, suitable binding proteins fulfilling a number of criteria are required. First, the binding proteins must undergo a conformational change on substrate binding. Ideally, they should belong to a family covering a wide spectrum of substrates. Furthermore, high-affinity binding would be advantageous, because it would provide a comparatively fast way to generate mutants with lower affinities suited for an optimal physiological detection range. Finally, for measurements in eukaryotes, ...

Molecular tools for cell and systems biology

“…Despite the relative technical ease of preparing such a sensor, the outcome is hard to predict. Accordingly, the number of reporters that are able to monitor changes in molecule counts is very limited, but reporters for cAMP (Adams et al, 1991;Nikolaev et al, 2004;Zaccolo et al, 2000), cyclic guanosine 3Ј ,5Ј-monophosphate (cGMP) (Honda et al, 2001), H 2 O 2 , myo-inositol 1,4,5-trisphosphate (Morii et al, 2002), glucose , maltose (Fehr et al, 2002), ribose , phosphate (Gu et al, 2006), glutamate (Okumoto et al, 2005), and lipopolysaccharide (LPS) (Voss et al, 2007) have been introduced. For quantification in cells, in vitro derived binding constants could be used.…”

Achieving the in Silico Plant. Systems Biology and the Future of Plant Biological Research

“…However, a similar profile of the metabolite constituents will remain challenging for the foreseeable future (Sweetlove et al, 2003). The development of novel methods for visualizing the concentration of specific metabolites in subcellular compartments of living cells (Fehr et al, 2002) provides an example of what may be possible. The challenge now is to implement these and related methods and to develop methods that will allow simultaneous imaging of many constituents in such a way that they can be integrated with parallel information about other biological processes.…”

Imaging of metabolites by using a fusion protein between a periplasmic binding protein and GFP derivatives: From a chimera to a view of reality

“…Fehr et al (4) attached two different GFP variants to the N and C termini of a PBP, which binds maltose (the maltose binding protein, MBP). Native MBP binds maltose in an effectively irreversible mode, and release requiring an interaction with the maltose transport protein.…”