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)

 

Nanotechnology in Single Cell Biology & Cellular Processes in Single Cell: Cell Cycle

It’s been raining nonstop since I landed in Taipei – not storming but a constant pitter patterning of rain drops. I don’t mind it. It’s actually kind of nice and soothing. Especially with the poster session taking place in a covered, outdoor courtyard, the rain adds an almost zen element, contributing to a very relaxing environment to discuss Science!

 

Going back to this morning, I got lost trying to find the lecture hall on campus. Luckily, a very nice campus security guard took pity on me and my poor attempt at speaking Mandarin and was able to point me in the right direction. I made it just in time for the start of the morning session, which kick started with the Advanced Microscopy for Single Cells and was followed by Single-Cell Mechanobiology I (Michael will be covering these sessions).

 

Lunch followed, and I couldn’t help but overflow my plate with food to the point where it was almost embarrassing (the key word is almost). This boost of energy sustained me through the poster session (take that jetlag!). The Nanotechnology in Single Cell Biology session followed immediately afterwards, where Dr. Bianxiao Cui started by posing the key biological question of “How do cells sense surface topography?”. To help answer this overarching question, the Cui group etches tunable nanopillar features into glass and has discovered that certain proteins preferentially accumulate on the nanopillars. One important class of proteins are those that contribute to clathirin-mediated endocytosis (i.e. FCHo1, TfnR, Epsin1, Amphiphysin1, CLTA, DNM2, and AP2). Moreover, when cells are cultured on nanobar features that possess high curvature at the end and low curvature along its length, these endocytic-related proteins display distinct curvature preferences by accumulating at the ends of the nanobars. The resulting curvature induced by the features was found to also affect endocytosis kinetics. To read more about this work, please refer to Dr. Cui’s recently published paper. Another important class of proteins that the Cui group discovered to be sensitive to curvature were those responsible for forming branched actin, such as FBP17, N-WASP, and Cortectin.

 

Dr. Haw Yang then proceeded to share his key biological question of “How do molecular interactions give rise to larger-scale processes?” or, more specifically, “How do rare events frame the mechanisms and kinetics of biophysical interactions?”. In order to address these types of questions, Dr. Yang presented a multi-resolution imaging approach that combines two-photon laser scanning microscopy and 3D single-particle tracking to investigate nano-bio interactions in living single cells. One application that Dr. Yang demonstrated is the ability to provide mechanistic insights for cellular trafficking of nanoparticles. For example, his group discovered that TAT-coated nanoparticles move on the membrane surface rather than inside of the endosome in addition to there being a faster-than-expected slowdown of these nanoparticles when approaching the cell membrane.

 

Scott Thourson ended the nanotechnology session by presenting tunable, conductive PEDOT:PSS polymer microwires. He started with a demonstration that these microwires were capable of stimulating a single heart cell, which could be visualized by the “beating” or contraction of the cell. Thourson then proceeded to motivate the use of these polymer microwires in combination with the patch clamp method in order to obtain more quantitative results about membrane potential. Since these wires are comprised of a soft conductor (Young’s modulus around 2 Pa, similar to brain tissue) with high charge density, they are ideal for use in single neuron stimulation, which Thourson highlighted is sufficient to affect behavior. Moreover, these polymer wires can be grown with tunable properties that can influence stimulation parameters, making them a promising interfacial material to interact electrically with single cells.

 

After a brief coffee break, the afternoon continued with the Cellular Processes in Single Cell: Cell Cycle session. Dr. Paul Wiggins kick-started the session discussing the “wrestling match” that occurs between replication and transcription and his group’s interest in understanding how frequent these replication conflicts are as well as what are the structural consequences of such conflicts. Using Single-Molecule Fluorescence Microscopy, the Wiggins group is able to visualize the replication process in single cells and characterize the replication complexes with single-molecule resolution, revealing that 1) replication is inherently discontinuous with pervasive disassembly/assembly and 2) transcription-induced conflicts are a key contributing factor for replisome disassembly. Furthermore, it appears that replication conflicts are actually quite frequent (5 conflicts per cell cycle). For more information regarding this work, please refer to their recently published work.

 

Dr. Sheng-Hong Chen started his talk by motivating the importance of p53 and MDMX in cancer, stating that the tumor supressor p53 is mutated in over 50% of cancer patients and that the oncogene, MDMX, is often overexpressed. Therefore, current therapeutic approaches to combating cancer often involve the dual approach of activating p53 while inhibiting MDMX. The success of this approach, however, hinges on the timing of the activation/inactivation. In order to obtain a better understanding of the optimal temporal parameters, Dr. Chen began by dissecting the dynamics of p53 in single cells. Upon exposure to gamma irradiation, Dr. Chen described an oscillatory behavior of p53 that followed with cell-cycle arrest. On the other hand, with UV irradiation, there was a single pulse of p53 that triggered apoptosis. The Chen group is interested if it is possible to re-direct p53 dynamics by modulating MDMX. MDMX suppression was found to trigger biphasic p53 dynamics in single cells: 1) an initial post-mitotic pulse and 2) an oscillation phase. Furthermore, cell sensitivity to DNA damage is also different in each of the phases. In the first, MDMX works in synergy with DNA damage to cause cell death while, in the second, MDMX inhibited cell death by re-directing UV-induced apoptosis to cell cycle arrest — highlighting the key take away that timing is important!

 

Olivia J. Scheideler (University of California – Berkeley)

Frozen Single Cell under Raman Spectroscopy

BPJ_112_12.c1.inddCryopreservation is the technology used to stabilize cells for a variety of applications, including diagnosis and treatment of disease. Because we don’t completely understand the mechanisms of freezing damage, poor or inadequate methods of preservation have limited our ability to use cells for cell therapy. Also, observations of cell responses could not be correlated to viability on a cell-by-cell basis using conventional low-temperature microscopy techniques. This study establishes our ability to measure the viability of individual frozen cells based on the correlation of cytochrome c distribution, a signal that can be detected using Raman spectroscopy, with trypan blue staining. With Raman spectroscopy, we are able to observe cells during freezing, and identify specific chemical and morphological changes inside the cell that result in life or death.

The cover image for the June 20 issue of the Biophysical Journal is an artistic rendering of frozen cells surrounded by extracellular ice and unfrozen solution. The background image is Lake Michigan in cold winter. Floating ice is separated by unfrozen water. The distribution pattern of the floating ice and unfrozen water is just like a frozen sample: ice crystals are separated by unfrozen concentrated solution. Schematic diagrams of frozen cells were imbedded in the background image to mimic a real frozen cell sample. In the diagram, the blue area represents unfrozen solution, the white area represents extracellular ice, the red line represents a region of cell membrane in close proximity to extracellular ice, and the black line represents a region of cell membrane in unfrozen solution between adjacent extracellular ice crystals. The schematic diagrams were precisely positioned in the background image such that the unfrozen solution in the diagram was co-located with unfrozen water in the background image and the extracellular ice crystals in the diagram were co-located with the floating ice in the background image. Our studies found that interactions between the cell membrane and extracellular ice resulted in intracellular ice formation (IIF), and increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF.

Raman spectroscopy has enhanced our understanding of freezing damage. These studies can enable the development of new and improved cell preservation protocols and eventually improve the growth of cellular therapies and our ability to treat patients.

– Guanglin Yu, Yan Rou Yap, Katie Pollock, Allison Hubel

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)