Engineered cell line for inducible protein expression for single-molecule imaging

This technology is cell line for detecting and tracking transmembrane protein interactions, including GPCR dimerization, within the plasma membrane of living cells to study protein dynamics and receptor interactions.

Unmet Need: Real-time, high-resolution GPCR interaction monitoring

Current methods for studying GPCR interactions in live cells, including ensemble fluorescence techniques and single-particle tracking (SPT), struggle with low resolution and indirect interaction detection, often producing ambiguous results. These approaches cannot effectively differentiate between physical interactions and random proximity due to technical limitations in spatial and temporal resolution. Therefore, it is crucial to develop tools to regulate protein expression in a model amendable to single cell imaging to characterize GPCR dynamics.

The Technology: Cell line for precise GPCR interaction tracking

This technology is a Chinese hamster ovary (CHO) cell line to precisely monitor and track single molecule interactions using single-molecule FRET (smFRET) imaging. This cell line uses inducible expression of self-labeling tags for site-specific covalent labeling. By combing bright, organic fluorophores this technology achieves high-resolution and real-time tracking without the need for potentially toxic photostabilizers. The platform addresses the limitations of conventional methods by providing direct measurements of receptor dynamics and interactions at a molecular scale, essential for advancing our understanding of GPCR functions. Validation testing has demonstrated its effectiveness in accurately detecting and analyzing receptor dimerization and conformational changes.

The technology has been validated using Chinese Hamster Ovary (CHO) cell lines.

Applications:

• Identifying and optimizing GPCR-targeting drugs
• Enhancing therapeutic design and testing
• Developing advanced diagnostic tools for GPCR-related diseases
• Studying GPCR dynamics and interactions at high resolution
• Investigating protein conformational changes in live cells
• Exploring cell signaling pathways and receptor behaviors

Advantages:

• Provides molecular-scale resolution, surpassing the limitations of conventional fluorescence techniques
• Enables real-time monitoring of GPCR interactions and dynamics in living cells
• Uses site-specific covalent labeling for accurate detection of receptor interactions
• Employs bright, self-healing fluorophores, eliminating the need for toxic photo-stabilizers and reducing photobleaching
• Applicable to a wide range of transmembrane proteins, not limited to GPCRs

Lead Inventors:

Jonathan A Javitch, MD, PhD

Wesley B. Asher, PhD

Related Publications:

• Zheng Q, Jockusch S, Zhou Z, Altman RB, Zhao H, Asher W, Holsey M, Mathiasen S, Geggier P, Javitch JA, Blanchard SC. “Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging.” Chem Sci. 2017 Jan 1; 8(1): 755-762.

• Asher WB, Geggier P, Holsey MD, Gilmore GT, Pati AK, Meszaros J, Terry DS, Mathiasen S, Kaliszewski MJ, McCauley MD, Govindaraju A, Zhou Z, Harikumar KG, Jaqaman K, Miller LJ, Smith AW, Blanchard SC, Javitch JA. “An automated single-molecule FRET platform for high-content screening of GPCR interactions.” Nat Methods. 2021 Apr; 18(4): 397-405.

Tech Ventures Reference:

• IR CU24352

• Licensing Contact: Jerry Kokoshka