Improved sensors for fructose-1,6-bisphosphate enable in vivo imaging of glycolysis

The authors developed HYlight2, an improved genetically encoded sensor for fructose-1,6-bisphosphate that offers significantly enhanced sensitivity and enables in vivo imaging of glycolytic flux across diverse tissues and organisms, including neurons, pancreatic islets, and the liver.

The Gist

Imagine your body's cells are like bustling factories that need fuel to keep running. The most common fuel they burn is sugar, and the process of turning that sugar into energy is called glycolysis.

Inside these factories, there's a critical moment early in the process where a specific molecule, called Fructose-1,6-bisphosphate (or FBP), is created. Think of FBP as the "green light" on a traffic signal. When you see a lot of FBP, it means the factory is working hard and burning fuel quickly. When you see little FBP, the factory is idling.

The problem is that scientists have struggled to see this "green light" clearly inside living things. The old tools (sensors) were like foggy glasses; they could tell you the light was on, but they were dim and hard to read, especially in complex environments like a living brain or liver.

The Breakthrough: HYlight2
The researchers in this paper created a new, super-powered pair of glasses called HYlight2. They built this sensor by taking a natural protein and running it through a "training camp" inside a test tube filled with bacteria parts. They made thousands of random changes to the protein's DNA, screened them, and picked the winners over four rounds.

The result is a sensor that is much sharper and brighter than the old ones:

• In the lab: It shines about three times brighter than previous versions when it spots FBP.
• In living cells: It shines about two times brighter when detecting changes in energy use, such as when neurons (brain cells) get excited.

Seeing the Invisible in Action
The team didn't just stop at the lab bench. They used HYlight2 to peek inside living creatures and watch their energy factories in real-time. They successfully used this new sensor to:

1. Watch how energy use changes in the neurons of tiny worms (C. elegans).
2. Observe sugar processing in the pancreas of zebrafish.
3. See the unique patterns of energy use in the mouse liver.

In short, this paper introduces a much brighter, clearer tool that lets scientists finally watch the "green light" of sugar burning flicker and flash inside living animals, revealing how different tissues manage their energy on the fly.

Technical Summary

Technical Summary: Improved Sensors for Fructose-1,6-bisphosphate Enable In Vivo Imaging of Glycolysis

1. Problem Statement

Glycolysis is a fundamental metabolic pathway, and its flux varies significantly across different tissues and cell types. While some tissues (e.g., the liver) exhibit spatial heterogeneity, others (e.g., the brain) dynamically regulate glycolysis in response to physiological demand. A critical bottleneck in studying these dynamics is the lack of high-performance biosensors capable of monitoring Fructose-1,6-bisphosphate (FBP) in vivo. FBP is the product of the first committed step of glycolysis, making its concentration a direct and tightly correlated proxy for glycolytic flux. Existing sensors lack the necessary sensitivity and dynamic range to effectively capture rapid metabolic changes in complex living systems.

2. Methodology

To address the limitations of current tools, the researchers developed HYlight2, an improved genetically encoded FBP sensor. The development process involved the following key steps:

• Mutagenesis Strategy: The team employed random whole-gene mutagenesis within an E. coli lysate system to generate a diverse library of sensor variants.
• Screening Process: They conducted four rigorous rounds of screening to isolate variants with superior performance characteristics.
• Validation Models: The selected sensor (HYlight2) was validated across multiple biological scales:
  • In vitro biochemical assays.
  • Mammalian cell cultures.
  • Stimulated neuronal activity models.
  • In vivo imaging in three distinct organisms: Caenorhabditis elegans (nematode) neurons, zebrafish pancreatic islets, and mouse liver.

3. Key Contributions

The primary contribution of this work is the creation and validation of HYlight2, a next-generation FBP biosensor that significantly outperforms its predecessors. The study provides:

• A robust engineering pipeline for optimizing metabolite sensors using E. coli lysate mutagenesis.
• A validated tool capable of high-resolution, real-time imaging of glycolytic flux in diverse physiological contexts.
• Demonstration of the sensor's utility in bridging the gap between in vitro biochemical properties and complex in vivo metabolic dynamics.

4. Key Results

The HYlight2 sensor demonstrated substantial improvements in performance metrics compared to previous iterations:

• Dynamic Range: In in vitro assays, HYlight2 achieved a relative change in fluorescence ($\Delta R/R$) of approximately 9, representing a significant enhancement in signal magnitude.
• Mammalian Cell Performance: The sensor showed a three-fold improvement in signal response when expressed in mammalian cells.
• Neuronal Activity: During stimulated neuronal activity, HYlight2 provided a two-fold improvement in detecting glycolytic responses, allowing for the capture of rapid metabolic shifts.
• In Vivo Versatility: The sensor successfully detected altered glycolytic activity in:
  • C. elegans neurons.
  • Zebrafish pancreatic islets.
  • Mouse liver tissue, confirming its applicability across different organ systems and species.

5. Significance

This research represents a major advancement in metabolic imaging. By providing a sensor with a high dynamic range and improved sensitivity, HYlight2 enables researchers to:

• Visualize Metabolic Heterogeneity: Directly observe spatial variations in glycolysis within tissues like the liver.
• Study Dynamic Regulation: Monitor real-time glycolytic responses in the brain during neuronal activation, offering new insights into the metabolic demands of neural activity.
• Broaden Research Scope: Facilitate comparative metabolic studies across diverse model organisms (from nematodes to mammals), potentially accelerating the understanding of metabolic diseases such as diabetes and neurodegenerative disorders where glycolytic dysregulation is a key factor.

In summary, HYlight2 overcomes previous technical limitations in FBP detection, serving as a powerful tool for dissecting the spatiotemporal dynamics of glycolysis in living systems.