Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SMSC (Single-Molecule Studies on Condensin)

Teaser

This project aimed at understanding the molecular mechanism of condensin protein using various single-molecule biophysical tools. Condensin is the key protein to organize the chromosome structures in every living cell. It belongs to SMC protein family which are crucial for...

Summary

This project aimed at understanding the molecular mechanism of condensin protein using various single-molecule biophysical tools. Condensin is the key protein to organize the chromosome structures in every living cell. It belongs to SMC protein family which are crucial for chromosome organization and segregation. How these SMC proteins organize the chromosome is still a mystery. Recently, in 2018, our group showed DNA loop extrusion mediated by condensin using single-molecule fluorescence assay for the first time. I the current project, I reconstituted DNA loop extrusion using AFM microscopy to understand the mechanism of loop extrusion mediated by condensin. The study is under preparation of a paper submission in Molecular Cell.

Objective 1 (RO1): HS AFM video imaging to watch the structural change of condensin at high spatiotemporal resolution (~ 1 nm, 50 ms).
-. I imaged condensin holocomplex using HS AFM imaging technique with a high spatiotemporal resolution (down to 1 nm and up to 10 frames per second) (Fig. 2 and 3). I observed that the SMC arms of the holocomplex are very flexible (Fig. 3), and there was a transient interaction between hinge region and head domains. Also, we imaged the condensin with/without ATP molecules, and we found that the SMC arms movement occurs with an ATP independent manner. However, ATP binding increased the binding of hinge domain to the head domains (Fig. 4). This suggests that ATP binding induce DNA pulling from hinge domain to the head domains.

Objective 2 (RO2): Real-time visualization of condensin-shaping chromosome using HS AFM.
-. I succeeded in co-imaging condensin and DNA. I reconstituted condensing-mediated DNA loop extrusion on the AFM microscopy (Fig. 1). Also, it shows ATP hydrolysis dependent behaviour. Using both high speed AFM and dry AFM, I could obtain the intermediates of loop extrusion mediated by condensin. I observed that only single condensin localized at the stem of DNA loop. The conformations of condensin at the stem varying, also and the neck size of DNA loop was highly correlated to the width of condensing at the stem. This suggests that the conformational changes of condensin are directly connected to the DNA loop extrusion.

Objective 3 (RO3): Understanding force-generating mechanism of condensin using smFRET-combined magnetic tweezers.
-. Using single-molecule fluorescence assay, I observed single-cohesin’s migration action along the DNA. Labeled cohesin showed that cohesin compacts DNA with an ATP independent manner using clustered cohesin molecules. Also, there was no evidence of ATP dependent behaviour of cohesin, but we observed that cohesin compacts DNA in an ATP independent manner.

Work performed

1 : HS AFM training
I was trained by Dr. Allard Katan to use HS AFM independently. I succeeded to handle the HS AFM by myself. Technically, I obtained important skills for HS AFM imaging such as sample preparation, surface functionalization, handling HS AFM cantilevers, and imaging.

2 : HS AFM imaging
Using HS AFM, I imaged condensin holocomplex and DNA loop extrusion mediated by condensin. Also, I imaged not only condensin but also cohesin and BsSMC to see whether the structures are also conserved. The three proteins showed similar results that they all have flexible SMC arms and the conformational changes were very dynamic. This results will be submitted in a high profile journal such as Nature Structural Molecular Biology.

3: HS AFM imaging for DNA-condensin interaction
I obtained the DNA loop structures induced by condensin and BsSMC. We could observe that single condensin is enough to pull the DNA loop. In addition, the protein width at the stem of DNA loop was highly correlated with the neck size of DNA loop, indicating that there is a certain conformational changes of condensin at the stem of DNA loop.

4: Single-molecule fluorescence microscopy combined magnetic tweezers.
Our group succeeded to develop loop extrusion assay to clearly show DNA loop extrusion mediated by condensin. I applied this platform to cohesin and BsSMC, and we found that cohesin compacts DNA with an ATP independent manner. Also, using this assay, I observed ATP dependent translocational movement of cohesin.

Final results

My plan was to enhance the potential and future career prospects using this fellowship. My goal was to obtain skilled and professional research PI, creating new research direction, and having professional collaborations. Because of this projects, I could be skilled for the HS AFM imaging skills and single-molecule fluorescence skills to visualize DNA compaction mediated by SMC proteins. In addition, potential many future directions could be formed such as various SMC proteins or SMC and the interaction between SMC proteins and others. Moreover, the collaborations with the World-leading groups such as Prof. Christian Hearing, Prof. Frank Uhlmann, and Prof. Byung Ha Oh helped me increase the understanding about SMC protein fields and important biochemical skills.
The meetings with Prof. Dekker made me learn his long-term vision how to create new research directions and how to find the most important problems in my research field. Also, how to manage the research efficiently. Also, discussion with other group members inspired me how to make a connection between SMC proteins and synthetic cells. Also, my finding is important to understand some diseases related to SMC proteins such as cancers and genetic diseases.
Moreover, HS AFM imaging of SMC proteins show a potential to contribute to “liquid-phase structure biology”. My results were different from other structural studies. I observed more flexible structures of SMC proteins instead of rigid structures, indicating that the structure results can be dependent on which technique we use. Also, the most important structural results should be based on liquid-phase imaging.

Quality of the proposed measures to communicate the action activities to different target audiences
When my results are published, I will upload it on “youtube” because I have nice movies such as SMC’s dynamic conformational changes and DNA compaction images. Already, I uploaded my HS AFM movie on our web page to advertise this result to students (http://ceesdekkerlab.net). I used my movies and results when Bachelor or Master students visited our department or our lab

Website & more info

More info: https://ceesdekkerlab.nl.