THESIS
2021
1 online resource (xv, 123 pages) : illustrations (some color)
Abstract
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...[
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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.
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