Background
Serine serves as a metabolic nexus in tumors, coordinating one-carbon metabolism, nucleotide synthesis, and redox regulation. In recent years, targeting the serine metabolic pathway, particularly the rate-limiting enzyme PHGDH, has attracted considerable attention as a promising anticancer strategy. Nevertheless, tumor cells preserve serine homeostasis through the coordinated regulation of endogenous synthesis and exogenous uptake, which often limits the efficacy of single-enzyme inhibition approaches. Notably, the function of serine transporters (SerTs) is intimately linked to their nanoscale spatial organization on the plasma membrane. However, owing to the lack of highly specific probes and ultra-resolution imaging techniques,
the mechanisms underlying their assembly and functional relevance remain largely unexplored.

Research Highlights
Researchers from Wuhan University of Science and Technology and the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences have jointly published a paper in Research entitled “Mechanistic Insight into Serine Flux Regulation through Nanoscale Organization of Glucose and Serine Transporters by Substrate Probe-based dSTORM Imaging.” The study developed a small-molecule substrate-based fluorescent probe (Ser-probe) derived from the structure of serine. This chemical probe not only exhibits high binding specificity and a one-to-one binding ratio with serine transporters (SerTs), but also possesses an extremely small molecular size. Compared with traditional biological probes such as antibodies, it offers significant advantages in terms of compactness, enabling highly efficient labeling of densely distributed target proteins on the cell surface. Leveraging the superior labeling performance of this probe combined with direct stochastic optical reconstruction microscopy (dSTORM), the researchers achieved super-resolution imaging and quantitative analysis of multiple serine transporters (SerTs). The work systematically revealed the nanoscale clustering behavior and regulatory mechanisms of SerTs in breast cancer cells.
1. Probe Synthesis and Specificity Validation
Through precise chemical modification of the serine molecule, the research team preserved its transporter-binding moiety and conjugated it with a fluorophore, thereby successfully synthesizing Ser-probe. This probe exhibits high affinity and specificity. Competitive experiment demonstrated that its binding can be completely inhibited by free serine, which confirms its ability to specifically label SerTs under physiological conditions. The development of this probe provides a novel tool for super-resolution imaging of membrane transporters.
2. Enhanced SerT Clustering in Malignant Tumors Correlates Positively with Function
Using dSTORM imaging, it was observed that from normal breast epithelial cells (MCF10A) to low-malignant MCF7 cells and highly malignant MDA-MB-231 cells, the point density on the cell membrane, average cluster area, and cluster coverage of SerTs all increased significantly. In MDA-MB-231 cells, the average cluster area of SerTs reached twice that of MCF10A cells, and the cluster coverage increased by 4.6-fold. Furthermore, flow cytometry analysis revealed that cells with stronger SerT clustering exhibited higher uptake capacity of fluorescently labeled serine, indicating a positive correlation between the clustering tendency of these transporters and their transport function.
3. SerT/GluT Clustering Cooperativity Sustains Cellular Serine Homeostasis
By employing dual-color dSTORM imaging, the study revealed significant colocalization of SerTs with glucose transporters (GluTs) on the plasma membrane, which was particularly pronounced in MCF7 cells with high PHGDH expression. The colocalization coverage reached 4.23%, markedly higher than the 1.41% observed in MDA-MB-231 cells. These findings suggest that cells with strong endogenous serine synthesis capacity are more prefer to forming SerT/GluT functional microdomains, thereby coordinately regulating glucose uptake and serine metabolism.
4. Lipid Rafts and Glycosylation as Key Mechanisms of Cluster Assembly
Cholesterol depletion (MβCD treatment) or glycosidase digestion (PNGase F treatment) significantly disrupted the clustered assembly and colocalization of SerTs and GluTs, indicating that lipid raft microdomains and glycoprotein cross-linking networks constitute the physical basis for maintaining the stability of these transporter nanoclusters. This finding provides potential targets for the intervention of membrane protein assembly.
5. Metabolic and Pharmacological Interventions Induce Spatial Reorganization of SerTs and GluTs
Glucose deprivation markedly weakened the clustering capacity of GluTs, reducing the SerT/GluT colocalization coverage from 4.23% to 0.71%. The PHGDH inhibitor CBR-5884 induced enhanced clustering at low concentrations, whereas at high concentrations its cytotoxic effects led to cluster disassembly. Combined interventions (inhibitor in combination with low glucose or free sialic acid) synergistically disrupted the protein cluster structures and significantly enhanced the anticancer effect. Cell viability decreased from 92.55% under single-drug treatment to 87.71% with inhibitor in combination with low glucose, or 78.84% with inhibitor in combination with free sialic acid.

Significance and Perspectives
This study is the first to reveal a direct connection between the spatial organization of SerTs and their function at the nanoscale. It breaks the conventional paradigm of metabolic research, which has been largely limited to enzyme activity and expression levels of key proteins, and offers a new perspective for investigating the role of nutrient transporters in the regulation of tumor metabolism.

Academic Value
Developed the substrate probe applicable to super-resolution imaging of SerTs;
Revealed the regulatory axis of “transporter nanocluster distribution-metabolic function-drug response”;
Established a model of the synergistic role of lipid raft and glycosylation in membrane protein assembly.

Translational Prospects
The clustering pattern of SerTs may serve as a biomarker of tumor metabolic state;
Combined targeting of metabolic enzymes and transporter assembly (e.g., PHGDH inhibitors with sialic acid) can enhance therapeutic efficacy;
Provides a new strategy for developing therapies aimed at regulating the spatial organization of membrane proteins.

The complete study is accessible via DOI: 10.34133/research.0805