New probes, singularities, and a reason for RNAi mismatches in vivo (Part 2)

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Me, Michael Shannon, in rainy Taipei

Takeharu Nagai (part 2/2): Singularities in cells: leaders, followers and citizens

Takeharu describes a familiar situation. Our results, whether its molecular behaviour or cellular behaviour are generally an averaged description of phenomena. We get an idea of what’s going on, but ignore the minority phenomena: the singularities that lead to group behaviours.

After a brief rundown of things he considers singularities: the big bang, the benign to malignant switch, formation of iPS stem cells with only 4 genes altered, populist fascism under Trump etc, he sets out to investigate this idea armed with a plethora of new fluorescent probes, and some cool model systems.

Here, he focuses on cAMP signalling in social amoeba, which transition from single celled entities to an intercommunicating mass, a multicellular being. To initiate this switch, all you have to do is starve them of nutrients.

By using two markers, Flamindo2 (Odaka) and R-FlincA (Horikawa, in press) he derives a ratiometric measurement for cAMP activity, known to be associated with this switch in amoeba behaviour. Combining this with a high speed microscope tiling technique, he is able to look at the behaviour of both single cells and the whole population of cells.

What he finds is amazing – single amoeba cells become leaders, displaying a burst of cAMP, before setting off their neighbours. “Early followers” then signal to “late followers”, and within a matter of hours, to “citizens”, setting off a continuous spiral of cAMP signalling which causes the amoeba to group together and become multicellular. The fluorescence videos of this are quite amazing – and while the paper isn’t out yet, you can view some similar behaviours online at Take’s website.

Interestingly, several leaders seem to be selected, but only one gains ultimate dominance as the seed of the spiral of signalling. One of the goals of the Nagai lab now is to find out how this leader is selected.

Okay, next up, Sua Myong

Sua Myong – How does RNAi actually work?

Sua employs FISH, a super resolution technique, to view RNA interference in single cells. What she finds is the first insight into the function of RNAi with reference to biologically relevant miRNAs since Fire and Mello won the nobel prize for the work and revolutionised the field.

The investigative technique works by targeting particular mRNAs with search RNAi strands loaded with 30 to 40 fluorophores each. By watching the transient binding of these RNAs in TIRF, the PSFs can be localised and the relative number of RNAs between conditions can be quantified.

The group tried to figure out which parts of shRNA were important in terms of its structure and its interactions with DICER and RISC, the proteins that bind it to genes of interest and cut the genes, respectively. To do this they altered the shRNA supplied to the cell by first changing the size of the hairpin loop, before measuring silencing using the FISH technique described above.

Longer loop size improved silencing, and it was found that this was dependent on better association with the DICER protein.

Introducing mismatches in the nucleotides was also trialled, as this is common in biological settings – many endogenous microRNAs have these mismatches which prevent them from binding the target gene perfectly.

What they found was that the altered shRNA had not problem binding DICER, but was inhibited in its handover to the RISC complex.

This is important, because it may be a way for cells to control the power of microRNAs, in cases where protein translation must be fine tuned.

Thanks for reading – that’s me over and out for this meeting.

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A local bicycle repair shack in Taipei

Michael Shannon (Dylan Owen lab, KCL)

Sensor and probe development, day 4 of the BPS conference in Taipei

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Michael Lawson, Amy Palmer, and Julie Biteen

 

Here are the talks on Sensor and Probe development, all on the last day of the conference (Part 1)

Amy Palmer probes the cellular ionosome

 

First up today, Amy Palmer develops new probes to investigate the ‘ionosome’* – the complex but under-recognised flow of metal ions within living cells. This is a relatively untapped field – 30 % of proteins in cells require a metal cofactor to function, yet only really Calcium has been addressed with fluorescent probes, or even recognised as a regulator of cell function.

Zinc ions are particularly interesting, and that’s the focus for today’s talk. It’s level is sensed by cells, which adjust their metabolism in response. 10% of proteins require Zinc, so it does a lot of different jobs.

OK so first of all the probe made by Amy and her team. This is a FRET probe, composed of two Zinc binding domains between the donor and acceptor. When Zinc binds, conformational change occurs and the probes come within the magic distance, facilitating energy transfer which can be detected. In addition, a signal sequence can be added to direct the construct to somewhere specific eg – membrane, nucleus, cytoplasm.

To prove the probe works, the group carried out in situ calibrations – within the same cells they will be testing. To soak up all the Zinc for a low signal readout, they used a chelator (in this case TPEN) and to get a high Zinc signal they used a Zinc carrier (Zinc Pyrithione). This works pretty nicely, and their yellow FRET signal is ratiometric with reference to the green donor signal alone. There also doesn’t seem to be any perturbation of endogenous Zinc concentration due to the probe itself, which is nice.

Next they looked at the kD values for binding of Zinc to some of the proteins it regulates, and it turns out that they are not fully occupied under physiological conditions. This is important as it points towards Zinc indeed being a regulator, functioning by binding on and off to the protein of interest.

One example of this is CDK2 in the cell cycle. Zn fluctuations accompany high CDK2 cytoplasmic recruitment/activity, when the cell has just exited mitosis. The group found that these zinc fluctuations are required for the decision to translocate CDK2 from nucleus (inactive) to cytoplasm (active).

One of the next steps for the group might be to target the probes to particular organelles, to investigate Zinc’s role there. Very interesting stuff.

*me and Amy agreed that this was a cooler name for this network than metallosome – what do you think?

Takeharu Nagai (part 1/2) – Acid resistant fluorescent protein for super resolution

Takeharu’s group looks in strange places for new fluorophores.

First up is the work of Hajime Shinoda, a “very handsome and cool student” in Take’s words. He has developed a new fluorescent protein, called Gamillus isolated from a flower hat jellyfish, which survives at low pH, where EGFP and others lose their signal.

That’s because it has a trans-isomeric conformation, instead of a cis one. It can’t gain H+ ions in a way that would disrupt the aromatic rings in the chromophores of the rest of the green cis proteins, so it is compatible for imaging low pH environments, like the inside of lysosomes. A nice control is shown: use GFP, you can’t see lysosomes, use Gamillus, and suddenly little polkadots appear in strategic cellular locations. It works!

Next, handsome Hajime altered the protein, so that it might be compatible for super resolution. He used the very problem that Gamillus solves, transition to cis isomerism, to achieve photoswitchability. Reversibly switchable Gamillus blinks on exposure to UV light, by switching between the inactive cis and active trans form. (Shinoda, unpublished data).

Michael Shannon

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)

 

 

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)

 

 

Pre-Meeting Introduction: Single-Cell biophysics: measurement, modulation and modelling

Hi everyone. I’m Michael and I’ll be one of three people covering the meeting. I’m a single cell biophysicist from London, currently working in Dylan Owen’s lab at King’s College (KCL).

The lab and I work on ways to quantitate biological phenomena on the nanoscale, particularly molecular clustering. I personally have been looking into the nanoscale goings on of integrin adhesions in migrating T cells. It’s a fun system to work in, because the adhesions are so much smaller than what are found in most other cells. What I’ve found appears to be a specific system of membrane nanoclustering, that is altered to tune the speed of a migrating T cell.

We think that this might be pretty important, especially considering a speed change experienced by cells with a mutation in PTPN22 – an integrin signal modulating phosphatase that is associated with autoimmune disease predisposition.

As well as this, we are very interesting in the dynamics and ultrastructure of the actin cytoskeleton, and mechanisms of membrane protein nanoclustering – how this might relate to the picket-fence model, lipid rafts and scaffold type protein regulation.

Some very relevant topics await: on the first day I’m very interested to hear Suliana Manley for live super resolution microscopy and Pakorn Kanchanawong on his cadherin adhesome work. I hope you are too!

As light rain continues to fall over Taipei, I think I can hear some kind of horn (of Gondor?), which I’m going to take it as my signal to start exploring the city. I always enjoy the sounds you get in such a place – city sounds are my favourite as they are so complex and multi-layered – familiar somehow and yet so different to what I’m used to in London.

So, with that, I’ll see you all tomorrow, for an engaging first day full of imaging techniques, mechanobiology, nanotechnology and the cell cycle!

Michael Shannon (Dylan Owen lab, KCL)

A Heart Attack After Lunch (Thankfully in a Dish)

The session kicked off with a heart attack (I hope the food did not have too much cholesterol!). However in this case,  Adam Engler at UCSD, has developed assays to cause heart attack in a dish in his laboratory. They designed a material, which can increase in stiffness in a step wise fashion. This helps them to assay how dynamically changing matrix affects heart development and may lead to a heart attack in older hearts. They then looked at the 9p21, a large non-coding RNA. Single nucleotide polymorphisms in this RNA increases the risk of familial heart attack. They show that indeed this ncRNA can regulate connexin expression levels. Alteration in these levels leads to an increased risk of heart attack. This is such a fantastic model to study heart attacks in vitro. Heart being a mechanical tissue, these materials now provide amazing ways to look at those aspects in a dynamically changing mechanical landscape.

Next Krystyn Van Vliet from MIT attempted to address an important issue — why  mesenchymal stem cells (MSCs) are not used for any FDA approved therapy.  She postulates that this is because of heterogeneity in the stem cell population. To make the cells segregate reliably into distinct populations based on cell diameter, cell stiffness and nuclear fluctuations, they build a microfluidics based device. They show that based on these three features, they can separate the cells that are still stem-like and have not exited the cell cycle. Moreover, this device is now in clinical trials in Singapore and provides a promising way to use MSCs for reliable therapeutic purposes with the major ill effects of the same.

This was followed by a talk by Yunn Hwen Gan from NUS. Her lab studies Burkholderia pseudomallei, a pathogen that causes melioidosis. This bacterium infects mammalian cells and, using a typeIII/IV secretion system, it leads to cell fusion and form large multinucleate cells. This induces interferon 6 and cytosolic DNA, which helps mount an immune response. The interesting part of their discovery was that, in this bacterium, unlike other bacteria, it uses the secretion system once it has infected the host and is inside the host cell. This provides interesting ways to think about therapy.

The next talk of the session was by Samuel Safran from Weizman Institute. He educated me about some very important theories on how holes close. This becomes particularly relevant when cells are challenged to squeeze through narrow pores and this may create holes in the nuclear membrane. The hole closing would depend on line tension (leading to shrinkage) and lateral stress (growth). Outflow of liquid from the hole in a 3D system would also contribute to this. The flow of fluid – chromatin in the case of nucleus, that is very viscous- would change the dynamics of hole closure. This is an important problem, as inability to close the holes, would be detrimental to the cell.