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Several advanced imaging methods including live-cell imaging, FRET imaging, optogenetics and laser ablation have been used to explore the dynamic behaviors of cellular activities!!!!

1. The dynamic of cellular events

Live-cell imaging is a powerful tool to observe the dynamic behaviors of cellular events. To visualize the protein of interest (POI) in living cells, various fluorescence proteins such as GFP, Neon, YFP, Clover, Venus, CFP, Cerulean3, BFP, tRFP, mCherry, and Ruby (kindly given by Dr. Inoue in Johns Hopkins), have been used to label the POI by genetic engineering. There are three movies showing the dynamic of microtubule growing, vesicular trafficking, and autophagosome formation are presented here.

2. The kinetic of ciliogenesis and intraflagellar transport

Primary cilium is a very tiny organelles (~1/10000 of cell body) protruding from the apical surface of almost every cells. It’s always challangeling to observe the dynamic behaviors of ciliary events due to the small size and unique architecture of primary cilium. We have overcome these technical challenges and precisely monitor the kinetic of ciliogenesis (Kobayashi et al., 2014) and intraflagellar transport. Two movies are presented here. 

3. FRET imaging

Fluorescence Resonance Energy Transfer (FRET) imaging has been widely used to real-time quantify molecular dynamics in biophysics and biochemistry including protein-protein interactions, protein-DNA interactions, and protein conformational changes. To take advantage of this powerful tool, we have used different FRET based biosensors such as Rac biosensor, HRas biosensor (Thevathasan et al., 2013), and PKA biosensor (kindly provided by Dr. Inoue in Johns Hopkins University) to measure the activities of Rac, HRas, and PKA, respectively, in living cells under different physiological conditions. One FRET imaging showing the PKA activities upon the treatment of PKA activator and inhibitor is presented here.

4. CID system

Chemically inducible dimerization (CID) system can translocate proteins to specific sites rapidly, which allows people to manipulate cellular signaling in various subcellular regions on a timescale of seconds. One of the biggest limitations of CID system at that time has been irreversibility. We have addressed the problem of reversibility and are able to activate PI3K-depenedent membrane ruffling in a reversible manner (left movie). Moreover, we have developed a novel method called “chemically inducible diffusion trap” that allows us to monitor the entry kinetic of protein of interests going into primary cilia, and translocate POIs into primary cilia for manipulation of ciliary composition or signaling locally on a second timescale (right movie).

 

5. Optogentic tools

Optogentic tools allow people to manipulate cellular signaling with high spatiotemporal resolution. We have used blue light to locally and rapidly induce either translocation or clustering of cry2 proteins in living cells. Moreover, we have also utlitzed this cry2-based system to induce membrane ruffling locally. 

 

6. Laser ablation

In order to perturb tiny structure in living cell rapidly, we used laser ablation to disrupt daughter centriole. This technique has enabled us to spatiotemporally perturb any visible structures in cells and study their physiological functions. 

 

 

7. FRAP experiment

The dynamic of POI (protein of interests) was measured by FRAP (Fluorescence recovery after photobleaching). 

 

 

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8. Life in the YCL Lab

Check out what we're doing and how we enjoy our time in the YCL Lab!!!!

 

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