Multicolor subunit counting method for deciphering membrane receptor stoichiometry

I worked on improving the subunit counting method, a robust single-molecule imaging method that can decipher membrane protein stoichiometry in the native environment. The method utilizes the step-wise photobleaching property of the fluorophores to count the number of subunits of a protein. Until now, this method was restricted to one color only (GFP is widely used in subunit counting method as it is quite photostable). In this project, I aimed to screen potential photostable fluorophores for bleaching step counting and use them together with GFP for subunit counting. I established principles of two multicolor subunit counting methods using SNAP-tag and YFP. YFP is a photostable fluorescent protein but shares overlapping emission spectra with GFP. I established a principle to unmix GFP and YFP emission spectra for single-molecule imaging to count both GFP and YFP bleaching steps. Besides, I also used mEGFP nanobody, and bioconjugation-based systems (e.g., Spytag/Spycatcher, FimGt/DSF, etc.). I validated the methods using NMDA receptor (an ion channel and obligatory dimer of dimer) stoichiometry. I used Xenopus laevis oocytes to study the stoichiometry of the ion channels. I also studied the AMPA receptor stoichiometry using the GFP/SNAP-tag multicolor subunit counting method. GluA1/GluA2 heteromers of AMPA receptors prefer to assemble in 2:2 stoichiometry in the brain. The
molecular mechanism behind this preference is not well understood. I used different structural domainswapped chimera structures of GluA1 and GluA2 subunits to study their assembly pattern. During my Ph.D. I learned about TIRF microscopy, animal handling, different programming languages (e.g., Mathematica, MATLAB) and further improved my molecular biology skills.

Related publications:

1. Yu, C., Runge, H. F. P., Mukhopadhyay, A., Zolles, G., & Ulbrich, M. H. (2023). γ-2 and GSG1L bind with
comparable affinities to the tetrameric GluA1 core. Cellular & molecular biology letters, 28(1), 54.
https://doi.org/10.1186/s11658-023-00470-9

2. Mukhopadhyay, A., Ulbrich, M. H. (2022). Dual color subunit counting method to decipher membrane
receptor stoichiometry. (German Biophysical Society Meeting-2022).

3.Yu, C., Mukhopadhyay, A., Ulbrich, M. H. (2021). Stoichiometry of GluA1/GluA2 heteromer of AMPA
Receptor. DOI: 10.1016/j.bpj.2020.11.580 (Biophysical Society Annual Meeting-2021).