Day 3- Chromosome Dynamics

After lunch and poster session, we are getting back to have some more exciting sciences!

Johan Elf- Single Molecule Studies of Cas9 Search Kinetics in Living Cells

Using single fluorescent microscopy, Johan is trying to investigate how long it takes for Cas9 to locate to a single target sequence in living cells.

He compared the searching time of Cas9 with the lac repressor lacI on target DNA in cells. LacI slides on the major groove of DNA, and it takes about 3 minutes for a lacI to find a operator. On the other hand, Cas9 has guide RNA that needs base pairing with its targeting sequence, so it needs to unfold the DNA and compare if the DNA matches with its sequence. With their smart method “dCas9 (deactivated Cas9) single molecule binding assay”, they discovered that it takes around 6 hr for an individual Cas9 to bind its target! That was a pretty long time! But it only takes around 30 ms for its searching time per PAM, indicating that there could be many short interactions. The dissociation rate of Cas9 is pretty long, matching the generation time of the cell.

It amazes me that it takes 6hr for Cas9 to bind a single dsDNA target which is 100X longer than lacI!

 

Melike Lakadamyali- Decoding Chromatin Organization with Superresolution Microscopy

Melike first gave us some background on chromatin organization over different length scale, and the long term goal is to visualize the chromosome fiber. She is interested in how nucleosomes are arranged in vivo.

She is using a model system that has asymmetric cell division, the neuroblast cell, and they are looking into interphase compaction and mitotic compaction. She found that interphase chromatin compaction scales with cell size in small cells but not large cells. Next, she found that mitotic compaction rates scale with the nuclear volume and interphase chromatin compaction. This is interesting because the fact that it is dependent on nuclear volume indicates that there may be a factor that compacts chromatin, and the concentration as well as the accessibility of this factor is important.

She will be trying to look into the histone modifications and also cohesin and condensin as the next step.

 

Yujie Sun- Labeling and imaging of the chromosome & superresolution techniques for study of 3D genomic questions

The first part of Yujie‘s talk is about multicolor labeling and long term imaging of chromosome loci. Besides fusing GFP to dCas9, putting fluorescence tags on sgRNA is a favored alternative. He demonstrated that one can have more fluorescence tags on sgRNA, and sgRNA better stands photobleaching. The reason for the bleach resistant of fluorescent sgRNA is attributed to fast exchange rate of MCP on MS2, so it recovers faster with better recovery magnitude. He also demonstrated with an example which involves labeling of single, non-repetitive locus, MUC4 & HER2 gene labeling in a single cell. He goes on to talk about a brilliant idea of  all-in-one sgRNA expression plasmid, which can express multiple sgRNAs in one plasmid! This would be useful for reliable activation/repression of genes simultaneous in a single cell.

In the second part of the talk, Yujie focuses on the superresolution techniques for study of 3D genomic questions. Excitingly, he showed they were able to direct dynamic observation of Pol II clustering in live cells for the first time! To study if and how serum stimulation enhances Pol II clustering,  they used actin mutant/spatial depletion along with serum stimulation with tcPALM, immuno-FISH, and two-color superresolution imaging. They found that nuclear actin is required for the establishment of the serum induced transcription program, and also required for enhanced level Pol II clustering upon IFN-gamma treatment. Moreover, upon serum stimulation, serum response genes are localized within Pol II clusters, and nuclear actin filaments and Pol II clusters colocalize.

 

Sangyoon Han- Emerging Role of Differential Molecular Association in Force-transmitting Nascent Adhesions

Sangyoon is interested in the factors that are affecting the decision process of nascent adhesions, which could be coming from early molecular assembly (talin, vincullin, and paxillin), and/or affected by mechanical forces. He also addresses the three challenges faced: how to measure forces from small adhesions, heterogeneous molecular movement, and how to link molecular movement to the force.

For measuring forces from small adhesions, he uses the TFM (high resolution traction force microscopy), and by using sparsity force reconstruction, he was able to suppress noise without underestimating the force. He found that nascent adhesions of a living cell transmit significant amount of forces. Next, he wanted to know if all the nascent adhesions are the same? He uses a very neat method – machine learning! By machine learning of adhesion tracks, he was able to characterize different types of nascent adhesions.

For those interested in his technical methods, he has TFM package, adhesion tracking and classification packages available!

 

Ivy (Howard Lab / Howard Lab facebook & Xiong Lab, Yale University)

 

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