Targeting Epigenetic Changes in Immune Cells: Implications in Disease

Speaker: Esteban Ballestar
Department: Bellvitge Medical Research Institute (IDIBELL), Barcelona Spain
Location: Erasmus MC
Date: July 13, 2017
Author: Teun Huijben

Esteban Ballestar got his Bachelor and Master degree at the University of Valencia in Spain, followed by a PhD. Afterwards he did a Postdoc abroad and returned to start his own research group at the same university. The main interest of his group is the DNA methylation, and especially in the context of diseases involving the immune system.

The first part of Estebans talk was about mapping DNA methylation in immunological diseases to understand which proteins are involved in the disease. The group of diseases they studied were Common Variable Immunodeficiencies Diseases (CVID) in which the body has not enough primary antibodies. These diseases are mostly caused by severe deficiencies in the number of switched memory B-cells. With switched B-cells we mean activated B-cells that start producing the antibodies in high quantities after recognizing the antigen. By mapping the DNA methylations of these B-cells, they hope to find genes that are differently methylated and are mostly likely causing the disease.

Methylation of DNA means that a methyl group is added to the 5-prime end of a cytosine (5mC) nucleobase. This can only be done if the cytosine is next to a guanine (see Figure 1). DNA methylation is maintained by de-novo DNA methyltransferases (mostly DNMT1, DNMT3A and DNMT3B). DNA methylations can be actively removed by demethylation, in which the 5mC is oxidized to a 5hmC or 5caC. Adding or removing methyl group to the DNA has an effect on the gene expression of that particular gene.

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Figure 1: DNA methylation. The cytosine of the CG-pair gets methylated by a de-novo methyltransferase (DNMT). [S. Zakhari 2015]

To identify disease causing genes, Estebans group did DNA methylation profiling of the B-cells from a CVID patient. To eliminate as much side effects as possible, thy only investigated twins of which one sibling had CVID and the other was healthy. After collecting the B-cells, they performed DNA methylation profiling and looked at genes with different methylation profiles between the two brothers. They found 230 genes that are more methylated in the CVID patient and 81 genes that are less methylated. Gene ontology analysis showed that most of the genes were related to immune responses, indicating that changing the gene expression of these genes can cause an immune related disease.

All the genes that showed different methylation profiles between healthy and CVID patient, were taken into further research. The B-cells are sorted on the fact whether they were naive (not yet switched to active) or switched. They found that in healthy persons most genes got demethylated after the transformation from naive to switched. On the other hands, the same genes in CVID patients showed no decrease in methylation, a second indication that these genes are involved in the disease.

However, the question remains whether the different methylation profile itself causes the disease, or is it a downstream effect caused by other factors. To investigate this, more research needs to be done on this subject. Also, more methylation profiles of twins are needed to draw real conclusions about the disease causing genes, since one set of results of the statistically valid enough. Overall, the talk of Esteban was interesting and he is a very good speaker. Despite using many difficult immunology term, he explained very clear the research his lab is doing.

Modeling of the ribosome and the RNA polymerase molecular motors

Speaker: Bahareh Shakiba
Department: Institute for Advanced Studies in Basic Sciences, Zanjan, Iran
Location: TU Delft
Date: July 12, 2017
Author: Teun Huijben

Bahareh has just finished her PhD and is now doing a Postdoc at the IASBS in Iran. In this talk she explained what she has studied during her PhD, in which she was interested in the two most important molecular motors present in the Central Dogma: the RNA polymerase and the ribosome. Both proteins can be seen as molecular motors since they use energy to move along a polymer (either DNA or mRNA). She was especially interested in the dynamics of these motors, so their pausing-behaviour and force-dependence.

The first part of the talk was about modeling the RNA polymerase (RNAP). Bahareh proposed a model to simulate the transcription steps using two components of the RNAP: the bridge helix and the trigger loop, which are both located very close the active site of RNAP (see Figure 1). The bridge helix promotes opening of the double stranded DNA helix and the trigger loop binds to the DNA and drags the DNA through the polymerase. The internal movement of components in the active site of RNAP during the transcription of a single nucleotide can be divided into multiple steps, which each having a energy barrier. Using the energy barriers calculated by other people, she computed the average duration of a pause during transcription. The average length of these pauses was the same as found in experiments.

 

ÈFigure 1: The bridge helix and the trigger loop are located near the active site of RNA polymerase. [M. Thomm, University of Regensburg]

However, I have some critical notes on this finding. First of all, she didn’t explain the model very well, so it was unclear to us in what way this model was new. Especially given the fact that many people do research on this subject and multiple models already exist. Besides that she didn’t mention the reaction rates she used in the simulations and where they came from.

Another important aspect is that she didn’t show the distribution of pauses. Normally it is very interesting to look at a histogram of pauses, to see how they are distributed, this can be Gaussian, exponential or Gamma, for example. Seeing the distribution will give a lot of information about the underlying processes and indicates if it is fair to simple take the average pausing time and compare this with experiments.

The second part of her presentation Bahareh talked about modeling ribosome dynamics. A ribosome is a protein complex that translated the mRNA to a protein. Bahareh studied how  mRNA-hairpins influence the processivity of the ribosome, since hairpins are formed in single stranded RNA and form roadblocks in front of the ribosome.

The active site of a ribosome has three free places to bind a tRNA molecule: the E- (exit), P- (polymer) and A-(active)-site (see Figure 2). The ribosome moves forward by first displacing the large subunit, followed by movement of the small subunit. If the mRNA in front of the ribosome has internally formed a hairpin, this can either result in necessary unwinding of the hairpin or a frameshift of the ribosome. The latter means that the ribosome temporarily detaches from the mRNA, resulting in errors in the produced protein.

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Figure 2: The active site of the ribosome has three active sites, the E-, P- and A-site. A tRNA loaded with an amino acid can bind to the ribosome and transfer its amino acid to the growing peptide chain. A hairpin in front of the ribosome. [Shakiba 2016, arXiv: 1607.0719v1]

There are currently two ideas of how ribosomes solve the hairpins. One states that the moving ribosome applies a force on the hairpin forcing it to unwind. The other theory states that the ribosome itself actively uses helicase activity to make the mRNA single stranded. Both theories are simulated in the model. Then she compared the model with experimental data of ribosomes translating mRNAs having hairpins, while applying a force on the mRNA. This comparison revealed that the model with ribosomes having helicase activity did the best job in explaining the experimental data. Giving the indication that ribosomes indeed have their own helicase activity.

As already stated in this report, the talk of Bahareh was quite hard to follow, especially because she didn’t explain the models very well. Therefore, the first part of her talk didn’t really impress me, since I didn’t see what was new and surprising in her model. However, the second part was easier to understand and got a clear, well described message.

Quo Vadis: Cees Dekker

Speaker: Cees Dekker
Department: Bionanoscience, TU Delft
Location: TU Delft
Date: July 6, 2017
Author: Teun Huijben

As start of the summer Quo Vadis of the Bionanoscience Department, Cees Dekker was invited to give a talk. Literally translated from Latin, Quo Vadis means: ’Where are you going?’. In light of this title Cees decided to give a summarizing talk about covering all the exciting research happening in his lab and what he hopes to achieve in the next years.

Since Cees has the largest lab of the department, of lot of different topics are studied in his group. The different topics can be roughly divided into three categories: developing novel nanotechnologies, studies on chromosomal organization and developing the synthetic cell. Of each of these subject he highlights some interesting ongoing studies and his vision on the future.

The first topic Cees elaborates on are the novel nanotechnological techniques his group is developing. The most important technique is the solid state nanopore, which is a very small hole in a thin membrane. When a voltage is applied over the chip, an ionic current starts running and DNA can be dragged through the pore, because of its overall negative charge. While translocating the pore, the DNA blocks the ionic current partly and the decrease in current can be measured. The nanopore technique can be used in different studies. Firstly, it may enable sequencing of DNA optically, when the pore is used in combination with plasmonic structures. The gold plasmonic structures create a high-energetic field trapping the DNA in the pore. In combination with Raman spectroscopy, chemical structures of the DNA can be deduced from the emitted radiation.

Secondly, nanopores are also useful in studying nuclear pore complexes. By covering the inside of the pore with nuclear pore proteins, the transport of proteins can be mimicked through this artificial pore. The advantage is that it can be done in vitro, instead of in living cells. At last, they are also trying to sequence proteins using biological nanopores.

The second main part of Cees’ talk was about the study of chromosome organization. For multiple years, his group is interested in the higher structures of DNA and how that structure is determined. An important part of chromosome structures are supercoils, in which DNA is coiled up to store it in a compact way and suppress transcription. The main question is whether these supercoils (or plectonemes) are sequence dependent. His group developed a new technique to study the coiling, and indeed the position of the supercoils is dependent on the sequence of the DNA. Modeling of the DNA gave the insight that certain sequences have an intrinsic curvature, and the model predicted which sequence will increase the probability of having a supercoil, since the tip of the supercoils needs Experiments with these sequence indeed showed a higher probability of having a supercoil at that position. This shows that the intrinsic curvature of the DNA determines higher structures of the chromatin, so the DNA sequence not only codes for proteins, but also for its own structure.

The last part of his talk was about making a synthetic cell, the ultimate dream of Cees. The goal is to create a vesicle (liposome) with a working division mechanism inside. One idea is to use the bacterial MinE-MinD oscillating proteins to position a FtsZ-ring in the middle of the cell and actively contract this ring to divide the cell. This idea will need some years to become reality, so right now his group is studying the different components of this idea separately and hopefully the first ’synthetic’ cell will be there is a few years.

What I found particularly nice about this talk was that Cees summarized all the research that is currently done in his large lab. In this way the department got a better idea of all the things happening within this part of Bionanoscience. Besides that, Cees is a very enthusiastic speaker what makes if nice to listen to.

Active polymer models for the 3D organization of chromosomes

Speaker: Johannes Nübler
Department: MIT
Subject: Active polymer models for the 3D organization of chromosomes
Location: TU Delft, A1.100
Date: 03-07-2017

Author: Kristian Blom

There it is, my final seminar report just before my trip to Japan starts. On the 3rd of July I visited a talk given by Johannes Nübler, postdoc at the MIT Mirny lab. Johannes works on the analysis of Hi-C and Micro-C data and modeling chromosomal organization in yeast, mouse, and human cells. His particular focus is on bridging large scale polymer models of chromatin with more fine-grained models of nucleosomes. He is interested in the role of active processes in chromatin organization, e.g. transcription and chromatin remodeling. In this talk we focused specifically on modelling Hi-C data.

The talk started with an introduction of Hi-C (High-throughput sequencing). With this method one can analyze the spatial organization of chromatin in a cell, by quantifying the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome. A shortcoming of Hi-C is that when two sites are not in close contact, no additional information is provided about how far these sites are separated from each other. Hi-C data is usually visualized by a heatmap where the number of interactions between all the different locations of a genome is shown. In order to make sense of this data, a model has been created that simulates active polymer folding and recreates the experimentally obtained heatmaps. By comparing the experimental heatmaps with the modelled heatmaps, one can understand why certain features arise under specific circumstances.

Figure 1 – Top row, the update rules used in simulations: (A) a condensin extrudes a loop by moving the two ends along the chromosome in the opposite directions, (B) collision of condensins bound to chromosomes blocks loop extrusion on the collided sides, (C) a condensin spontaneously dissociates and the loop disassembles; (D) a condensin associates at randomly chosen site and starts extruding a loop. Bottom row, (E) we use polymer simulations to study how combined action of many loop extruding condensins changes the conformation of a long chromosome. Image from 10.7554/eLife.14864

After the introduction we looked at interaction heatmaps of entire chromosomes, characterized by repeating blocks of high/low interaction intensity in the vertical and horizontal direction. The blocks with high interaction intensity are called topologically contacted domains. A typical feature in these interaction heatmaps are the dense squares along the diagonal, which indicates that neighboring areas on the chromosome are relatively often in contact with each other. To simulate those features passive polymer folding was not enough, and therefore a loop extrusion model was introduced (Figure 1). In this model DNA folding arises due to an extrusion complex containing two subunits that attaches to the DNA, forming a small loop in the process. Thereafter the two subunits slide along the DNA in opposite directions, making the loop bigger. This mechanism actively folds the DNA, allowing far located parts on the sequence to come in closed contact with each other. During sliding the extrusion complex searches for a specific motif, which causes a protein called CTCF to bind to the DNA. When a subunit encounters a CTCF that is pointed towards the subunit, it will stop sliding. If the CTCF motif is pointed in the sliding direction of the subunit, it won’t be recognized and the subunit keeps sliding. The result of this behavior is that the pair of CTCF motifs at the end of a loop are pointing towards one another. One of the exciting results of the extrusion model is that loops formed by extrusion will be unknotted, allowing for easy access to the genetic information. Another very nice and cool result is that a collection of chromosomes that are intertwined will naturally segregate when extrusion takes place.

Although you had to have a bit of background knowledge in Hi-C to understand everything, I really liked the theoretical aspect of the talk. The fact that the model is very simple but produces very interesting results makes it a very powerful model. For the future it should be experimentally validated whether DNA looping is an active process involving some sort of extrusion enzyme, or a passive process that arises due to DNA-DNA interactions. For now it seems that the former comes closest to reality, but we have to wait for evidence to know the truth. Now it is time to go to Japan, cheers!

Disrupting ultrashort nucleic acid duplex with mechanical force

Speaker: Kevin Whitley

Department: University of Illionois at Urbana -Champain

Subject: Disrupting ultrashort nucleic acid duplex with mechanical force

Location: Bionanoscience Department, TU Delft

Date: 30-06-2017

Author: Mirte Golverdingen

Kevin Whitley did research to the hybridization kinetics of DNA under force. He tried to understand the transition state of nucleic acid hybridization. Hybridization is used for gene manipulation and targeting for DNA nanomachines, tweezers and bipadal walkers. Short strands (about 10 bp) show a ‘all of nothing’ behavior when hybridizing. Fist one or two base pairs bind and then all base pairs bind without stable intermediates. This describes a two-state reaction in a schematic energy landscape. The kinetics of any of these states is determined by the transition state between them. Whitley searched for the transition state between the bound and the unbound DNA.

To research the hybridization kinetics of the DNA the end-to-end extension of the DNA was researched. This is the length of the top of the energy to the bottom of the energy where the DNA is hybridized. Bell’s equation describes the energy barrier between the end-to-end extension. So, by measuring the rates of the Bell’s function you can calculate the end-to-end distance.

Whitley measured the hybridization rates by using optical beads that are bound to the DNA by double stranded handles and with a 9 nucleotides long middle strand. This short part is the binding site for short DNA. The distance between the two beads was approximately one. By using a flow chamber Whitley was able to look at oligo that were free in solution. He was able to measure the koff and kon rate under a constant amount of force. In this way, he could measure the moment a ss DNA oligomer binds to the middle strand by measuring the position of the bead. The ds DNA is stiffer than ss DNA, and therefore the position of the bead changes. By using this method, Whitley could measure the difference between ss and ds DNA up until a force of 12 pN.

By using Fleezers, which is a combination between fluorescence and optical tweezers, Whitley was able to label the oligomer with a fluorophore (see figure 1). In this way, also forces lower than 12 pN can be measured. Again, they calculated the binding and unbinding rates of the DNA. While the unbinding rate of the DNA goes up linearly when the force increases, the binding rate also goes up, however, not linearly. When the oligomer becomes longer, the unbinding rate goes down. For the binding rate no dependence on the length of the oligomer can be found.

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Figure 1: Measurement of single-oligonucleotide hybridization kinetics under force. (A) Schematic of the hybridization assay (not to scale). An engineered DNA molecule (red) containing a short, central ssDNA region flanked by long double-stranded DNA (dsDNA) handles is held under constant force by polystyrene beads (grey spheres) held in optical traps (orange cones). A fluorescence excitation laser (green cone) is focused on the central ssDNA region. Short oligonucleotides (blue) labeled with a Cy3 fluorophore at the 3΄ end (green disk) bind and unbind to the complementary ssDNA sequence in the center of the tethered DNA. The binding and unbinding is observed by the fluorescence emitted from the attached fluorophores.

Adapted from: Kevin D. Whitley, Matthew J. Comstock, Yann R. Chemla; Elasticity of the transition state for oligonucleotide hybridization. Nucleic Acids Res 2017; 45 (2): 547-555. doi: 10.1093/nar/gkw1173

When looking at Bell’s equation, the binding rate should be an exponential force-dependence, however, non-exponential behavior is reproducible across other conditions. The end-to-end distance is therefore not constant, and the Bells’ equation changes so an integral is used instead.  By calculating the unbound and bound extension by using the worm like chain model for double stranded DNA they were able to calculate the end-to-end extension. In this way, they also obtained the persistence length and contour length of the end-to-end extensions. He repeated these calculation for all different lengths of oligomers. The transitions state persistence length is comparable to ssDNA, however it is a bit stiffer.

The transition state is probably no random process, as simple polymer estimations predict a low probability of spontaneous alignments. The single strand that is a bit stiffer, as measured in the transition state, this could correspond to prearranging of the strands, prior to nucleation. Some enzymes can pre-organize mRNA so the transition state energy barrier becomes lower. Moreover, fast target finding is possible when there are preorganized bases by enzymes. In this way, they can speed hybridization up by pre-paying the entropy penalty.

Whitley told a clear story on obtaining the end-to-end distance of the DNA hybridization translation state. The combination of Optical Tweezers and fluorescence was very cool. Moreover, using the mathematics in combination with the biology inspired me as Nanobiology student.

Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Speaker:  Clara Belzer

Department: Laboratory of Microbiology, Wageningen University, The Netherlands

Subject: Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Location: BioTechnology department TU Delft

Date: 29-06-2017

Author: Mirte Golverdingen

To broaden my view of the studies done in the Applied Science building I visited a lunch lecture in the BioTechnology department. The microbiologist Clara Belzer from Wageningen University was the speaker and she spoke of the newly discovered Akkermansia muciniphila bacteria. Belzer and her group are interested in anaerobic micro-organisms that can degrade ligases and mucus in the gut. Verrucomicrobia is one of the phyla that lives in the gut, the bacteria Akkermansia muciniphila is one of the Verrucomicrobia and isolated by the group for a quest of new bacteria.

They discovered that the bacteria are very dominant along the mucus gut and an important player in human microbiota composition. The bacteria are a potential maker for healthy intestine and has an extraordinary mucus degrading capacity. The importance of the bacteria is shown in the fact that after a month after birth the bacteria can already be detected in the gut system. This is certainly not the case for all gut bacteria.

The human microbiota changes during life stages and perturbations, this is partly caused by the place of the bacteria on the body. However, over a life time the microbiota can also change due to physiological changes of the body by diet changes. Therefore, the change in microbiota can contribute to or starting diseases. Moreover, some microbiotia do not depend on food, they depend on mucus and mother milk.

Bacteria influence the host cell by a difference in glycal level. This has a microbial benefit; mucosal glycan foraging can solve the problem of the competition for glycans in the gut. For the host, the mucosal microbes can provide resistance against the colonization of pathogens. It directly inhibits the pathogens by production of antimicrobial compounds. Moreover, the depletion of nutrients modulates the immune response of the host.

In the gut, there are 5 major microbiological fila, some are mucus degraders and some are mucus binders. Mucus can be used as the source of growth because mucus glycans can be created by the bacteria. However, there are not only glucose molecules in these mucus glycans, there are also other more ‘awkward’ sugars which are hard to break down. These sugars need to be broken down by enzymes. The Akkermansia muciniphila is adapted to digest mucin, it contains a high number of genes that encodes for enzymes which can degrade mucin. None of all Verrucomicrobia have the same number of enzymes that are able to digest mucin as the A. muciniphila.  So, the A. muciniphila is really adapted to the work of mucin digestion.

So, to find out if A. muciniphila is also present in other animals, Belzer et al. went to the zoo to find the bacteria in mucus of mammals and non-mammals (see figure 1). The A. muciniphila found in these animals differ as the animals have different digestive physiologies, diets and mucus structure. Still, the bacteria were not very different, which is very remarkable. The only difference between the animals A. muciniphila was found in the antibiotic resistance and CRISPR Cas system, as the animals differ in defending to bacteria and viruses in their environment.

So, why are the bacteria so similar? This could be due to a spontaneous infection very recently, so the bacteria were not able to evolve since. The bacteria could also have a very stable genome that does not evolve. Or the bacteria are not specific on the type of mucus.  One genome, however, was different from the rest. The python strain of the A. muciniphila looks really different. Moreover, only a small number of all animals are tested on the A. muciniphila. Therefore, more different genomes could be discovered.

Belzer saw a similarity between organisms and the A. muciniphila system: As soon as the host was on a diet or starving, the A. muciniphila levels are going up. This is similar for all organisms and systems. This could indicate that A. muciniphila could be an obese related bacterium. Belzer moreover, showed that the A. muciniphila works as a growth support of beneficial microbes in the gut. Moreover, when the A. muciniphila levels rises in the gut, organisms were metabolic healthier.

It was very interesting to visit the BioTechonolgy department for this seminar. Some metabolic details were hard to follow for me; however, I was still able to follow her talk. I was surprised by the complex microbiota in the gut. The similarity of the A. muciniphila is very interesting and research for this bacteria in other animals can contribute to the understanding of this bacteria.

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Figure 1: Akkermansia muciniphila is universally distributed in intestinal tracts all over the animal kingdom. (a) Phylogenetic tree indicating the position of A. muciniphila among selected full-length 16S rRNA clones from mammalian gut samples. Red colored samples derive from human sources. Thermotoga thermarum is used as an outgroup. The tree was generated using the neighbor joining method. Full details and high-resolution information are provided in Supplementary Figure S1. (b) Schematic representation of the tree in (a) with the five different clades their position and similarity to A. muciniphila. (c) Taxonomic tree of mammals generated using iTol webtool from tree of life project using all available sequences from NCBI (Letunic and Bork). Animal silhouettes indicate single species as a representative of that order. When an animal species from the mammalian orders was positive for Akkermansia-like sequences the animal logo belonging to that order is colored red, when it was negative the animal logo is colored gray. No Akkermansia sequences have been reported yet in any of the animals belonging to the mammalian orders depicted in black.

Adapted from: Belzer, C., & De Vos, W. M. (2012). Microbes inside—from diversity to function: the case of Akkermansia. The ISME journal, 6(8), 1449-1458.

Structurally distinct periodic and dynamic chromatin domains revealed by super-resolution fluorescence microscopy

Speaker: Kirti Prakash

Department: Institute of BMolecular Biology, Mainz, Heidelberg University Heidelberg. Carnergie institution for science, Baltimore usa, current affiliation institute paris

Subject: Structurally distinct periodic and dynamic chromatin domains revealed by super-resolution fluorescence microscopy

Location: Bionanoscience Department, TU Delft

Date: 26-06-2017

Author: Mirte Golverdingen

A large spectrum of chromatin domains between DNA and the chromosome territory level remains unexplored.  We do understand the morphology of the DNA helix since the 1950’s and a decade ago Bolzer et al. showed the morphology of intra-chromatic departments of chromatins (see Figure 1).  However, the morphology and behavior of the region between single DNA morphology and intra-chromatic departments is still fairly unknown.  Genomic methods have already found a large spectrum of chromatin domains between the DNA and the chromosomal territory level, however their regulation is not known yet. To learn more of the regulation of gene expression, we have to obtain a better understanding of the unknown region.

 

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Figure 1: False color representation of all CTs visible in this mid-section after classification with the program goldFISH.

Adapted from: Bolzer, A., et al.  (2005). Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS biology, 3(5), e157.

Kirti Prakash studied these intermediate chromatin features by using single-molecule localization methods (SMLM). Classical microscopy methods, such as conventional light and electronic microscopy, are not able to identify intermediate chromatin features because of specificity and a poor resolution. Therefore, nanoscale imaging is required to describe chromatin complexity.

SMLM is the best method to study chromatin organization because it has the best resolution in a small area. From all SMLM methods, PALM/STROM has the best resolution and distance in xyz area. When asked if the time resolution had not to be taken into account, Prakash mentioned that time resolution is not important for the research he is doing. Moreover, he mentioned that the PALM/STORM microscope set-up technique is very simple, because no much alignment is needed.

However, the preparation of the buffers and fluorophores is very complicated, because the combination of buffer and fluorophores need to have a high photon expression number, prevents photo-bleaching and it has to blink for a long time. The search to a combination that has all three characteristics is time consuming and hard. Therefore, I think that PALM/STORM is not that better than other SMLM methods as Prakash suggested in his talk.

Dyes as Hoechst and DAPI undergo phot-conversion when binding to the minor DNA groove, therefore they are a good tool of chromatin studies for SMLM. The red shift of the fluorophore makes that you can exclusively excite the dyes that are bound to the DNA. This makes that the dyes do not bleach and that there are still a lot of signals. This high labeling density is required to study unknown structures.

According to Prakash, this method of direct DNA staining is better than previous methods based on antibody staining o histone proteins.  This is because you can have a 5 times higher labeling density. However, can we really compare the DNA-staining with histone labeling? Histones behave different from DNA itself, so are you not comparing apples with oranges?

By using the technique Prakash was able to study the hierarchical structure of the chromatin in a single experiment. He made 2D images of a 3D domain, so different in z-direction could not be measured. He showed that there were clusters of chromatin in the cell. However, is this really a fair conclusion if you have no idea of the clustering of chromatin in the z-direction.

By studying the clusters visualized, Prakash could research the chromatin complex morphology. He observed ring and rod like chromatin domains and displacement of active histone modification during stress. In mammalian cells, the chromatin complexes rearrange after stress. So, according to Prakash SMLM contributes to describe the spectrum of unknown chromatin domains between the nucleosome and chromosome level.

Prakash, however, did not convince me of the value of SMLM to the study of unknown chromatin domains. He compared the results of Histone labeling with his DNA staining assay, which was, I think, not fair. Moreover, his visualization of the chromatin complexes was in 2D without a verification that this 2D representation was valid. I think that a more validated study to chromatin complexes with this assay would have a better contribution to research on these complexes.

Quantitative single molecule imaging in living cells and animals

Speaker: Christof Gebhardt

Department: Institute of Biophysics, Universität Ulm

Subject: Quantitative single molecule imaging in living cells and animals

Location: Bionanoscience Department, TU Delft

Date: 07-04-2017

Author: Mirte Golverdingen

Life is an interplay of inanimate biomolecules, Christof Gebhardt tries to understand how these biomolecules function in isolation, interaction and what happens with them when we are ill. To understand this the research to reaction kinetics, their spatial distribution, stoichiometry and forces can help. Moreover, biomolecules can be better understood if we manipulate single biomolecules in vitro and in vivo.

Super resolution methods in combination with fluorescent proteins as markers can help us to understand biomolecules better. Gebhardt uses super resolution in combination with GFP. Next to green colours, other colours of the fluorescent protein are developed. However, single GFP expression for single molecule detection is hard to visualize using fluorescent microscopy because of the fluorescent background.

HILO illumination is an imaging method that only measures a small part of the cell, in this way the background can be reduced.  By adding a mirror to the HILO set-up, the side of the object can be imaged and in this way, you have more space to alter the cell at the top (See Figure 1). Moreover, imaging from the side reduces the contrast. The 3D image of the cell can be imaged by moving the objective up and down. By using other tags than GFP, Gebhardt was able to use HILO for in vitro, in cellula, in vivo and in silico studies.

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Figure 1: Scheme of the reflected light-sheet principle (not drawn to scale). A laser beam is focused by an objective to form a vertical light sheet that is reflected by 90° off an AFM cantilever next to a cell in a Petri dish. Fluorescence is detected by a second high-NA objective. Three-dimensional optical sectioning is achieved through vertical displacement of the sample. Adapted from: Gebhardt, J. C. M., et. al (2013). Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nature methods, 10(5), 421-426.

Gebhardt for instance, studied the transcription factor and DNA (TF-DNA) interaction times in chromatin organisation and transcription regulation. The transcriptional repressor CTCF interacts with the chromatin, however, it is not known how CtCF interacts with the chromatin during the cell cycle. Moreover, the influence of the regulator role of TF-DNA interaction time on transcription repression is a research topic of the research group of Gebhardt.

Kinetic information from the data can be obtained by researching video images of with an HoloAR labelled nuclear receptors the diffusion coefficient, the DNA bound tag and the DNA residence time of molecules can be obtained. To overcome the problem of photobleaching that occurs after continuous illumination, dark times were used. By measuring the DNA residence time of different TFs, Gebhardt showed that every transcription factor has another DNA residence time. There can be made a differentiation between specific (1/10/100 s) and unspecific (<1) DNA binding residence times.

CtCF chromatin interaction kinetics

The 3D organization of chromatin exists of nucleosomes, looped gene loci and chromosome territories. The chromatin architecture influences gene expression, differentiation, development, DNA replication and DNA repair. However, the control of the DNA loops is still unclear.

Chromatin topology is mediated by architectural proteins. Gebhardt was able to calculate the topologically associating domain, this shows how likely it is in a loop if chromatins interact with each other. There are certain TFs that is able to interact with the architectural proteins. CtCF is one of these transcription factors, therefore, it is seen as a potential ‘master weaver’. However, it is not clear if CtCF is activating, repressing or insulating the architectural proteins. CtCF contains 11 zinc finger motives that are able to dimerize the DNA. It has more than 100000 target sites on the DNA and it recruites cohesion. Gebhardt his study focusses on the kinetics of the CtCF TF, how long does it bind to the chromatin? And is the TF stable or mobile?

He was able to design a method by using fluorescent proteins that shows a wide spread binding kinetics. By developing a method based on the measured fluorescence, Gebhardt showed that CtCF shows three dissociation states. The residence times of these states are: <1s, <10s and ~1000s. The last two are specific, while the first one is specific and occurring in G1 and G2 phase of the cell cycle.

CtCF has low- and high-affinity sites on the DNA and its dynamic interactions (~4s) are changing the loops within topological associating domains (TADs) or initiate abortive loop formation. CtCF chromatin interaction depends on the current phase of the cell-cycle. In the S-phase, much less high (stable) resistance times are measures, so in the S-phase an overexpression of CtCF blocks cells. It could be that replication dissolves CTCF-chromatin interactions during S-phase. In the M-phase: CtCF is excluded from chromatin, this may be caused by phosphorylation.

By studying the interaction of CtCF with the DNA, Gebhardt et al. showed that CtCF-chromatin interaction is cell-cycle dependent. Kinetic information on the interaction of biomolecules with DNA can help us understand the complex interactions in the cell better. I liked that he was able to adapt the traditional way of imaging with HILO to a more advanced and better adapted method. Moreover, the discovery of the tree dissociation states of CtCF with their corresponding residence times invites us to research the dissociation states of other transcription factors, giving more insight to the complex cell dynamics.

Dynamics of translation of single mRNA molecules in vivo

Speaker: Marvin Tanenbaum

Department: Hubrecht Institute, Utrecht

Subject: Dynamics of translation of single mRNA molecules in vivo

Location: Bionanoscience Department, TU Delft

Date: 24-03-2017

Author: Mirte Golverdingen

Marvin Tanenbaum is interested in the control and principle of gene expression. Gene expression is not static, it is, instead, very dynamic, as seen during the cell cycle. It has a role in the regulation of all biology and this is visualized when looking at the influence of gene expression on the central dogma. Gene expression regulates transcription and translation in the cells. However, because the cell cytoplasma is so complex and chaotic, single molecule visualization of this process is hard. So, how do we get from the chaos to the pattern each cell has?

During transcription, translation and the path of mRNA to the cytoplasm, gene expression is regulated by a large number of molecules.  These processes are most of the time studied by using GFP tracking. Twenty years ago, Singer et al. tracked mRNA for the first time by using fluorescent proteins that were attached to hairpin loops. However, for real-time visualization of translation, GFP tagging does not work because of the very weak signal of single molecules. Moreover, GFP has a ‘maturation’ time that takes very long in comparison with the translation time.

Tanenbaum started to label the ribosomes indirectly by using a GFP -Antibody-Small-peptide.  In this way, more fluorescent proteins can bind to the peptide, creating a stronger signal. However, this method was very hard to implicate. Therefore, they used a chain of GFP proteins attached to a SunTag protein that can attach to a protein label. This sequence of the protein label was translated and the resulting mRNA’s were labeled with a red mCherry dye that was attached to the hairpins of the mRNA (see Figure 1A). This system is 15~20 fold brighter than a single GFP protein, and has a good affinity for the targets.

After developing this imaging system, they applied it to obtain direct observation of translation by using the developed SunTag. When the mCherry red dots also shows green dots, translation is happening.To test if there was really translation going on, puromycin (a translation inhibitor) was added. After this addition, no green dots were seen anymore. Therefore, the combination of red and green dots really shows real-time translation(see Figure 1C). However, because translation is a very dynamic process, tracking a single mRNA was very hard. Therefore, they attached the mRNA to the plasma membrane. In this way, a single mRNA could be tracked during his whole life time (see Figure 1D and 1E).

Now real-life imaging of translation was possible, the quantitative nature of the translation could be measured. They counted the number of the ribosomes on the translation site, resulting in 20 ribosomes on each translation site. Moreover, to calculate the speed of the ribosomes, they added the translation initiation inhibitor harringtonine. In this way, you can measure the rate of the disappearing ribosomes. They calculated that ribosomes have a translation speed of 3-5 codons/s.

By adding an untranslated region (UTR), they could see a single ribosome decode a mRNA, because by using an UTR only once in a while a ribosome will bind to the mRNA. They observed only small flashes of green light after adding UTR. So, in this way they could observe single ribosomes that read RNA’s. Moreover, they observed a mRNA cycle between a translating and non-translating state. However, they have no idea yet why this cycle is there.

In conclusion, Tanenbaum visualized real-time translation of single mRNA’s for the first time by using the SunTag system. They could quantify the number of ribosomes on the translation site and the ribosome translation speed by using this method. Moreover, they were able to visualize a single ribosome decoding a mRNA. I think that this technique is an important contribution to the research of gene expression. Moreover, wide applications of the assay can contribute to the understanding of the complex process of translation. Being able to see the ‘abstract’ process of translation in real time in a cell is overall very cool.

Honours 8

Figure 1: Fluorescence Labeling of Nascent Chains to Visualize Translation of Single mRNA Molecules.

(A) Schematic of nascent polypeptide labeling using the SunTag system and mRNA labeling (A) and membrane tethering (D) using the PP7 system.

(B) A mCherry-SunTag24x reporter gene was co-transfected with either GFP or scFv-GFP, and the expression of the SunTag24x-mCherry reporter was determined by FACS (Experimental Procedures). Binding of the scFv-GFP to the SunTag nascent chain did not detectably alter protein expression.

(C) A representative U2OS cell is shown expressing scFv-GFP, PP7-3xmCherry, and the translation reporter (SunTag24x-Kif18b-PP724x). Cytosolic translation sites (scFv-GFP) co-localize with mRNAs (PP7-3xmCherry). Ribosomes were dissociated from mRNA by addition of puromycin (right panel). Note that translation sites and mRNA do not perfectly overlap because of the brief time difference in acquiring GFP and mCherry images.

(D) Schematic of nascent polypeptide labeling and membrane tethering of the mRNA using the PP7 system.

(E) U2OS cells expressing scFv-GFP (green), PP7-2xmCherry-CAAX (red), and the translation reporter (SunTag24x-Kif18b-PP724x). A single time point of the cell (top panel) and a zoomed-in view from the white-boxed area containing a few mRNAs (lower) are shown.

Adapted from: Yan, Xiaowei et al. (2016) Dynamics of Translation of Single mRNA Molecules In Vivo. Cell , Volume 165 , Issue 4 , 976 – 989