Day 3- Single-Cell Mechanobiology

After the wonderful talk during coffee break, coming back to our last session of today!

Megan Valentine- A new model system for cellular studies of mechanobiology

Megan introduced us a new model organism that is known is our  “closest vertebrate relative”, Botryllus schlosseri (commonly known as the golden star tunicate). It is a highly dynamic organism that needs constant angiogenesis, because it has a large and transparent extracorporeal vascular network, and their vessels are constantly remodeling. What is special about them is that their vessels are inverted with respect to vertebrate, so we can have direct access to extracellular matrix via microinjection.

With the model organism Botryllus, she can directly apply physical forces and monitor the downstream responses in a living organism in real time through manipulation of the blood vessels. She found that Lysyll oxidase (LOX, responsible for crosslinking collagen) expression is stimulated by the presence of collagen, and inhibiting LOX by adding a specific inhibitor, ß-aminopropionitrile (BAPN) causes massive retraction of vessels.

This is a pretty fascinating new model system for mechanobiology studies, and this talk was ended with a nice and amusing “slurp” video (a cell swallowed by the phagocyte)!

 

Chin-lin Guo- Spontaneous Patterning of Cytoskeleton in Single Epithelial Cell Apicobasal Polarity Formation

How does mammalian cells form the specific organs? Previously, people focused on the spatial patterning and the coordination of chemical signals. Recently, ChinLin and others have found that mechanical forces also play an important role in the organization of multicellular architectures.

He has shown that long-range mechanical force enables self-assembly of epithelial tubular patterns, and the  self-organization of epithelial morphology is dependent on rigidity. Moreover, he thinks that direct cell-cell contact induces the segregation of par complex and the formation of actin belt, which is how individual cells form apicobasal polarity.

The cool part of his talk is that he uses an ECM gel to see the spontaneous single-cell partitioning of cytoskeleton on 2D platform and 3D culture. Furthermore, to differentiate between actin band and belt and the role of microtubules, he implemented the lattice light sheet microscopy (LLSM, Bi-Chang Chen), which showed the conversion of actin from band to contractile belt, and he thinks that actin band can serve as a precursor to guide cell-cell interactions.

To sum up, he thinks that single epithelial cells can form a precursor state by spontaneous partitioning of cytoskeleton to guide multicellular epithelization and apicobasal polarity formation, and there could be an intermediate state between the mesenchymal state and the epithelial state.

 

Poul Bendix- Dynamics of Filopodia: Rotation, Twisting, and Pulling

Poul is interested in the question: do filopodia rotate around its own axis? His group has surprising evidence for a new pulling mechanism originating from twisting of the actin within the filopodium. Using labeled actin, he can have 3D visualization of filopodia in different cells to find the answer.

When visualizing actin polymerization inside membrane tube, he found that filopodia exhibit buckling of their actin shaft in conjunction with pulling. In HEK cells, he found that there is twist buckling transition of filopodia that buckling releases accumulated twist, which is a strong indication of twisting of actin. Moreover, they observed retrograde flow and rotation of the actin shaft, so there could be correlation between force and actin distribution, and also correlation between coil movement and force.

They have found helical buckling and rotational behavior in the filopodial actins in various cell lines, which may facilitate the sensing and interaction of the cell with its surroundings using filopodia.

 

***

The banquet in Grand Hotel was AMAZING!! Grand hotel is one of the most famous landscapes of Taipei, with contemporary palatial architecture and delicious cuisines! After the wonderful meal, some of us went to the karaoke, and I heard it was also lots of fun!

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

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IMG_5640.JPG(Having a great meal at Grand Hotel, photo: Ivy)

 

 

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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)

 

Day 2- Cellular Processes in Single Cells II: Transcription

Good morning everyone! The sky has cleared up a bit today (with a bit drizzles). After the first day of ideas and the Shilin night market tour, we are all ready for another day of interesting sciences!

Achillefs Kapanidis- Using tracking PALM to study bacterial transcription and chromosome organization in live bacterial cells

In order to understand RNA polymerase (RNAP) behavior and its role in nucleoid organization in vivo, Achillefs’ team used photo-activated localization microscopy single-molecule tracking, and they were able to tell apart diffusing RNAP from those that are bound to DNA.

They found that RNAP has periphery bias that is dependent on active transcription, and RNAP clustering is a function of growth. These RNAP clusters are having a similar mobility as DNA. Interestingly, when they image the nucleoid and RNAP using 3D structural illumination, they found that RNAP will form large clusters at regions of low DNA density in rich media, leading to the suppression of other genes. The mechanism they proposed is that RNAP redistribution is due to changes in gene expression, such as stress, mutation, and overexpression. Moreover, they also found that RNAP will interact with non-specific DNA substantially.

They are also developing assays to study the non-specific interactions of DNA binding proteins with chromosomal DNA, which would definitely be a useful tool for this field!

 

Nam Ki Lee- Direct observation of transcription in a living bacterial cell

Nam Ki and his team are studying the coupling of transcription and translation, and how these two spatially separated but functionally related processes are cooperatively regulating the movement and the effective expression of genes.

They observed the movement of the actively transcribing T7 RNAP toward outside nucleoid, and this was affected by the translation by ribosome. Furthermore, they found that the movement of genes by transcription-translation coupling is seen in both E. coli RNAP and T7 RNAP. They also measured the in vivo kinetics of the T7 RNAP transcription on-rate and elongation rate, and found that deletion of the ribosomal binding site doesn’t change the elongation rate, but enhanced the transcription on-rate 1.7-fold, indicating a close relationship of transcription-translation effect.

The model that they propose is that transcription starts within the nucleoid, and then the DNA-RNAP-ribosome complex will move to the outside of the nucleoid, and the transcription initiation is enhanced!

 

David Rueda- Imaging Small Cellular RNAs with Fluorescent Mango RNA Aptamers

David and his group set out to develop fluorogenic RNA aptamers that has improved physicochemical properties (i.e., thermal stability, fluorescence brightness and ligand affinity) and better signal-to-noise ratio, and they developed “mango” I-IV (after spinach, lots of veggies and fruits *laugh*).  Interestingly, mango IV is resistant to formaldehyde fixing, which is particularly useful for cell fixation.

They shown that these aptamers could be used to image small non-coding RNAs (such as 5S rRNA and U6 snRNA) in both live and fixed human cells with improved sensitivity and resolution. In the 5S rRNA example, they showed that 5S rRNA foci are not processing bodies, but instead are associated with mitochondria!  They were also able to image U6 snRNA in live cells, and they found that there are 3 types of behavior, no moving, low mobility, and high mobility.

Interestingly, they referred to the idea of Bo Huang and developed CRISPR-mango for imaging of telomeres and specific loci! This would definitely open up a new world of RNA imaging in cells!

 

Xiaoli Weng- Using Superresolution Fluorescence Microscopy to probe the spatial organization of transcription in E. coli

Xiaoli is trying to gain insight into the regulation of gene expression at the global level by using superresolution fluorescence microscopy.

They found that RNAP forms clustered distribution under fast growth, and globally stopping transcriptions has the largest perturbations. They also probed the colocalization of the elongation factor NusA and RNAP, and found they are together in elongation complexes, with nascent rRNA. Interestingly, when they perturbed rRNA transcription with serine hydroxamate treatment or in rrn deletion strain, they found that certain RNAP independent of rRNA synthesis are retained.

Therefore, the formation of RNAP clusters and active rRNA synthesis could be independent, and genes could potentially localize with RNAP clusters to have better regulation and more efficient transcription.

 

Ivy Pei-Tzu Huang (Howard Lab & Xiong Lab, Yale University)

 

 

Day 2- Single Cell Modeling

After the coffee break and some illuminating discussions, we came back to the next session: single cell modeling!

Philip Nelson- Single-photon sensitivity in human vision

Philip started this session by giving us a really nice background on the “evolvement” of science. If we have nice night/dim light vision, we would have a better chance of survival-not falling to be a prey! However, there is this notion that the emission of light is lumpy and random, and so is the capture/absorption of light. This leads us to wonder, what is the sensitivity of human vision (perception threshold)?

He goes on to discuss the landmark experiment done by Hecht on determining the minimum number of photons required for perception of light dating back to 1942, and some considerations involved in the experiment’s design. Barlow later pointed out there could be discrepancy due to false positive reports. He also discovered the role of spontaneous isomerization in vision perception. Later on, Baylor’s single cell data showed that there are not only spontaneous isomerizations, but also some lower signal rumble. Barlow also has the insight that ” the very first synapse must discarded some genuine signals”, which Rob Smith claimed later that it is “discarding half of the real signals”.

By using the “high-tech” instruments available now, scientists are able to confirm directly that rod cells impose no threshold, and test out the models proposed. Take home message here is that even with challenging technical details, we could still try to have the model which in Philip’s words: “The best fitting model is the one that maximizes a likelihood function defined by the rather limited experimental dataset.”

 

Po-Yi Ho- Interrogating the Bacterial Cell Cycle by Cell Dimension Perturbations and Stochastic Modeling

Po-Yi is testing the growth law of bacteria by perturbing the cell dimensions and looking at the bacteria cell cycle, using E.coli as a model system.

Single bacteria cells grow exponentially. Particularly, average E. coli cell volume scales exponentially with growth rate, with a scaling exponent equal to the time from initiation of a round of DNA replication to the cell division at which the corresponding sister chromosomes segregate, this is known as the Schaechter’s growth law. So how are cell division and DNA replication coupled and regulated? They found that cells initiate replication at a constant size delta per origin of replication. So they proposed this “adder-per-origin” model, which states that cells add a constant volume delta per origin between initiations, then divide after a constant C+D minutes.

When they try to perturb the bacterial cell dimensions by varying the expression levels of mreB and ftsZ (bacterial homologue of actin and tubulin, respectively), they found that decreased mreB levels resulted in increased cell width (with little change in cell length), whereas decreased ftsZ levels resulted in increased cell length. Moreover, they also found that the growth law remained valid over a range of growth conditions and dimension perturbations, which is kind of impressive! In conclusion, the timing of replication initiation governs that of cell division, and cell volume is the key.

 

Rosanna Smith- Gene reporters and the problem of measurement in live cells

Rosanna showed that even though fluorescence reporters are a convenient tool for us to measure protein expression in live cells, there could be some “side-effects” (that are not so loved obviously) that could perturb the results and affect the interpretation of data.

They showed that some reporters that are used to assess gene expression may have noise that impacts the single cell reporting. As an example, they used the pluripotency factor Nanog, and looked at the stochastic transcription from two alleles. They illustrated that different reporters can have different Nanog expression patterns, meaning that the reporters will perturb the dynamics that we are originally trying to measure.

This talk prompts me to think more about the experimental design and the intrinsic technical difficulties associated!

***

After a delicious and satiating lunch along with nice relaxing small talks, we are ready for the cultural tour to National Palace Museum, and the highly-anticipated Taipei 101 tour! The National Palace Museum has all the art treasures dating back to thousands of years ago! Next, we went up the observatory deck of Taipei 101. The elevator is pretty impressive (even for people afraid of height), it only took us 37 sec to get to floor 89 (382 meters above the ground)! The view of the whole Taipei city is amazing, although there was some fog and the visibility was not the best. The wind damper used to stabilize this skyscraper is also pretty impressive, it helped this tall building survive through different typhoons that hit Taiwan!

-Ivy

Getting ready for the Biophysical Society Thematic Meeting on Single-Cell Biophysics!

(IAMS building, photo credit: IAMS)

I am very grateful to have the opportunity to attend my first Biophysical Society Meeting! This year, the meeting “Single-Cell Biophysics: Measurement, Modulation, and Modeling” will be held June 17-20 at Dr. Poe Lecture Hall Foyer of the Institute of Atomic and Molecular Science (IAMS) building, National Taiwan University (NTU). As its name implies, the meeting this year is focused on single-cell biophysics, and it is bringing together people from different fields around the world! I can’t wait to have nice discussions on exciting sciences, meeting new friends, and becoming part of this wonderful community!

(National Taiwan University Library, photo credit: Office of International Affairs, NTU)

As a local here in Taiwan, I have pursued my undergrad and master degree at National Taiwan University (NTU). I remember those good old days of working till midnight in the lab, and then go across the street from our lab to grab some delicious Taiwanese street snacks (chicken fillet/popcorn chicken & bubble milk tea …and so many more)! (Good science and delicious food go together really well! Guess I am lucky to be schooled here in Taipei!)

(Taipei, photo credit: Travel Taipei)

We will be covering this thematic meeting over these days, hope you enjoy reading our articles! I believe it would be a rewarding experience!

🌸Ivy