The conventional co-immunoprecipitation (co-IP or pulldown) commonly used for assessing protein-protein interactions (PPIs) needs a large number of cells and therefore is unsuitable for studying the PPIs in rare cells such as sensory hair cells, circulating tumor cells, and embryonic stem cells. To overcome this problem, a single-molecule pull-down assay (SiMPull) has been proposed by Jain et al. in 2011(Jain et al., 2011a). However, the technique requires meticulous multistep cleaning, surface-passivation, and functionalization of quartz slides, and these procedures together are extremely time-consuming and have high operational barriers.

Here, we report a highly innovative microbead-based SiMPull for cell population and a nanobead-based approach for SiMPull designed for cell populatio...[ Read more ]

The conventional co-immunoprecipitation (co-IP or pulldown) commonly used for assessing protein-protein interactions (PPIs) needs a large number of cells and therefore is unsuitable for studying the PPIs in rare cells such as sensory hair cells, circulating tumor cells, and embryonic stem cells. To overcome this problem, a single-molecule pull-down assay (SiMPull) has been proposed by Jain et al. in 2011(Jain et al., 2011a). However, the technique requires meticulous multistep cleaning, surface-passivation, and functionalization of quartz slides, and these procedures together are extremely time-consuming and have high operational barriers.

Here, we report a highly innovative microbead-based SiMPull for cell population and a nanobead-based approach for SiMPull designed for cell populations and, more importantly, single cells. We used commercially available, pre-surface-functionalized agarose and magnetic beads to replace complicated surface modification on glass coverslip to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions by fused fluorescent protein or immunofluorescent labeling under microscope. Our agarose beads-based SiMPull has been applied to detect TMC1(Transmembrane Channel Like 1)-FLAG, confirm the interaction between multiple components of the MET (mechanoelectrical transduction) complex in hair cells for the first time. Besides hair cells, we also applied this method to other rare cells - γδ T cells.

For single-cell SiMPull by magnetic nanobeads, we used a microwell array chip to trap single cells and applied surface modified magnetic nanobeads to microwells. After cell lysis, the magnetic beads could capture target proteins and pull them down to glass surface on the bottom of the microwell by adding magnetic field to the device. Then proteins of interest could also be observed under microscope. We could successfully pull down overexpressed GFP molecules and PKA (Protein Kinase A) complexes from a single HEK 293 cell and detect TMC1-FLAG molecules in a single hair cell.

We believe that our methods are substantially simpler and faster than existing single-molecule pull-down methods and are considerably more widely applicable to all cell types. These two crucial features would enable universal application of our method in common biological and clinical laboratories.

We proposed a new dye uptake-based drug screening method targeting Transient receptor potential vanilloid 1 (TRPV1) channel in the second part of the thesis. TRPV1 is a nonselective cation channel with high calcium permeability and wildly distributed in a diverse range of tissues. TRPV1 is involved in the detection and transduction of chemical or physical nociceptive stimuli, such as high temperature and and painful stimuli in the dermal and epidermal layers of the skin(Riera et al., 2014). TRPV1 has also been reported to be linked with inflammation(Bertin et al., 2014), chronic pain(Gouin et al., 2017), nerve damage(Bai et al., 2018), etc. and be considered as a therapeutic target for those related disease. Therefore, finding modulators of TRPV1 channels is potentially useful in the treatment of those disease.

Calcium uptake and patch clamp are commonly used in the measurement of TRPV1’s channel activity and identification of TRPV1’s antagonists (Fenwick et al., 2017; Gavva et al., 2007). However, patch clamp is more complicated and not suitable for high throughput screening. And calcium imaging also required multistep pretreatment including calcium indicator permeation and wash steps. Here, we provide an alternative way to screen TRPV1 modulators which is based on FM (Fei-Mao) styryl dyes (FM dye) uptake though TRPV1, and is simpler, easier to operate and cheaper compared to using calcium indicators and patch clamp.