Spatiotemporal Mapping of Signaling Events via Hybrid Proximity Ligation

Abstract

The study of proteins plays a crucial role in understanding complex biological processes, as they are key to cellular function and communication. However, traditional technologies face significant challenges in studying specific issues, such as post-translational modifications (PTMs) at the single-cell level and the dynamic tracking of secreted proteins. These limitations hinder the ability to explore the intricacies of protein behavior and interaction. To address these bottlenecks, our work introduces innovative approaches that overcome these technical obstacles, paving the way for deeper insights into cellular mechanisms and advancing life sciences research.

At the single-cell scale, PTMs (such as phosphorylation and glycosylation) are extremely low in abundance, and traditional enrichment strategies (such as antibody immunoprecipitation) are incompatible with the micro-scale preprocessing of single-cell samples, leading to insufficient capture efficiency of modification sites. Furthermore, conventional mass spectrometry workflows are not optimized for the nano-liter-scale preprocessing required for single-cell analysis, resulting in inefficiencies in sample preparation and data acquisition. These drawbacks highlight the need for advancements in single-cell proteomics to overcome current technical barriers and achieve more comprehensive PTM profiling. To address these issues, we developed single-cell chip digital PLA (sc-cdPLA), an innovative method based on proximity ligation assay (PLA) technology. sc-cdPLA combines flow cytometry and digital PCR, first sorting single cells into 96-well plates using flow cytometry, then using a dual-probe PLA system with anti-p-MEK and total MEK antibodies to achieve absolute quantification of phosphorylation levels, enabling detection at the femtomolar level. In the FUCCI cell cycle model, we observed significant differences in MEK2 and phospho-MEK activity between the G1 and S/G2/M phases. Quantitative analysis revealed that the number of MEK2 protein molecules in the G1 phase was approximately 1.4-fold higher compared to the S/G2/M phase, while the number of phospho-MEK molecules was approximately 2.5-fold higher. These findings indicate heightened activity and sensitivity in the G1 phase, suggesting that drug treatments targeting phosphorylation pathways may be more effective during this stage. The sc-cdPLA method enables precise quantification of phosphorylation changes at the single-cell level, offering valuable insights into dynamic cellular processes.

The secretome, encompassing the proteins secreted by cells, plays a vital role in intercellular communication, immune response, and tissue repair. Investigating these aspects provides insights into fundamental cellular mechanisms and has significant implications for advancing disease diagnostics and therapeutic development. However, tracking secreted proteins faces even more complex challenges: first, secreted proteins undergo multiple PTMs to become functional, and in vitro recombinant expression systems cannot simulate the diversity of modifications under physiological conditions; second, the cross-organ transport of secreted proteins (such as leptin secreted by adipose tissue crossing the blood-brain barrier) involves dynamic concentration gradient changes, and in situ detection is limited by background noise from body fluids (such as serum protein interference) and low abundance target signals (leptin concentration in cerebrospinal fluid is only at pg/mL level). Traditional antibody methods also cannot distinguish the source of secretion pathways (such as vesicle transport or direct secretion).

To improve secretome investigation, we developed bio-PLA, which integrates BioID (proximity-dependent biotin identification) technology for in situ detection of adipokines secreted by the adipose tissue. By mating Adipoq-Cre female mice with BioID-flox male mice, we achieved adipose tissue-specific expression of BioID, enabling proteins produced by adipose tissues to bind with biotin. Bio-PLA is mainly composed of two probes. Probe A is connected with biotin, which can detect biotin through biotin-streptavidin-biotin bridge. Probe B is connected with antibody, which can detect a specific adipokine. We optimized the probe design and length, finding that the 93 nt Probe-A had the strongest signal. Additionally, we designed trans and cis probes, with the trans probe showing a significantly stronger signal due to reduced spatial hindrance.

In an obese mouse model, we applied bio-PLA to analyze the spatio-temporal distribution and blood content of adipose tissue-secreted protein (leptin). This revealed that obese mice have higher levels of biotinylated leptin in both blood and brain. Additionally, in situ PLA detection in the mouse brain revealed significant amounts of adipose-derived leptin in the Pir and Rh/Re. Through further comparison of obese and lean mice, we found that obese mice exhibited stronger fluorescence signals in the Pir and Rh/Re regions, indicating higher levels of adipose-derived leptin in obese mice. This may be attributed to the increased adipose tissue in obese mice, which produces more leptin, enabling these leptin molecules to more easily cross the blood-brain barrier, thereby generating stronger signals within the brains of obese mice. Lastly, using LCM-seq, we identified multiple genes and pathways within the nuclei associated with leptin and obesity, such as the PI3K/AKT pathway, ERK1/ERK2, and JNK pathway. These genes and pathways play crucial roles in regulating appetite, energy metabolism, inflammatory responses, and signal transduction. All of these results demonstrated bio-PLA's excellent performance in in situ detection, blood detection, and distal detection of secreted proteins.

This study developed sc-cdPLA and bio-PLA technologies, achieving dynamic detection of PTMs at the single-cell level and in situ tracking of cross-organ transport of secreted proteins, filling gaps in sensitivity, spatiotemporal resolution, and physiological simulation capabilities of existing technologies. Experimental validation showed that sc-cdPLA has a detection limit at the femtomolar level in single-cell phosphorylation quantification, revealing the cell cycle dependency of MEK2 phosphorylation activity, providing a precise molecular dynamics basis for targeted therapy; bio-PLA captured adipose-derived biotinylated leptin in the blood, cortex, and thalamus of obese model mice, expanding the current understanding of leptin's role and opening new perspectives for metabolic disease research.

Date of Award	3 Aug 2026
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Liang ZHANG (Supervisor)