How to achieve cellular replication without fail: lessons from bacterial cells.

Speaker: Christine Jacobs-Wagner
Department: Microbial Sciences Institute, Yale West Campus
Subject: How to achieve cellular replication without fail: lessons from bacterial cells.
Location: TU Delft (BN Seminar)
Date: 13-10-2017     

 Author: Nemo Andrea

 The topic of today’s talk was cellular replication, which, in Christine’s opinion, is the ability that separates the living from the non-living. In order to study this process, they study bacterial replication, as bacteria both divide rapidly and do so with high accuracy. The speaker stressed how, if one stops to think about it, achieving such short (20 minutes) division times in a varying environment with in the inherently highly stochastic environment of a living cell, is a truly remarkable feat. Thus, the robustness of bacterial replication and the relative simplicity of prokaryotic systems as compared to eukaryotic model systems make bacteria the candidate of choice for her research.

As there are many topics that can be explored within cellular replication, the speaker decided to focus on a single system that her group had recently done work on. Many bacteria have plasmids, which can contain vital functions for a bacterium, thus requiring efficient segregation of plasmids upon division. While plasmids diffuse throughout the cell and are thus, on average, equally divided among the two halves of a bacterium, suggesting that in the case of cell division no problem should arise, this is not the case. If a plasmid is present in high copy number, the chance of one daughter cell having significantly fewer copies of the plasmid is very small, but if a plasmid has low copy number the probability of one daughter cell ending up with no copies due to low number noise becomes significant. It is thus that bacteria have developed methods to effectively separate the plasmids that are present in low numbers. One could argue that it is not the bacterium driving the evolution of such a system but rather the plasmid itself, as plasmids that effectively do this ensure their survival, but that is really more a matter of perspective than anything else, and beside the point argued in this talk.

The plasmid that the group focused on displayed very curious behavior. The plasmids (in elongated bacteria) are distributed equidistantly along the long axis. The plasmids do diffuse, but stay in roughly the same area over time. It should be apparent that if such a distribution is maintained, the plasmids will be divided equally among daughter cells. As it turns out, this is achieved through a particular variant of the Par system. This variant uses just two proteins: PAR A/B, which are encoded on the plasmid itself. ParB binds to the plasmid, and does this by recognizing specific sequences on the plasmid, ensuring selective binding. ParA, on the other hand, binds to ParB, after which ParB will stimulate the ATPase activity of ParA, which will then unbind from the DNA. ParA also unspecifically binds to the DNA (of the bacterium). They observed (in case of a single plasmid) an oscillation of ParA from one side of the cell to the other, with the plasmid (with ParB) following the ParA signal. They then produced a first model, to test if simple Brownian dynamics of a diffusing plasmid could reproduce such behavior. They found that this was insufficient and concluded from this that a translocating force must be present to create this behavior.

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Figure taken from: DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos. ; cropped

The key insight that was missing from the first simulation was the fact that DNA is not static, but moves around in the cell. They determined that the movement of a locus in the DNA can essentially be seen as an elastic harmonic potential, with a force of around 0.04pN. This is a really small force, even by cellular standards. They redid the simulations with this idea incorporated and found that they were able to reproduce the behavior observed in bacteria. The conceptual change is now as follows: The plasmid with ParB is pulled around through its multiple connections with multiple harmonic potentials through ParA. It will then unbind from the ParA in that area and move in a direction (slightly). In the new situation, the plasmid will ‘see’ more ParA in the direction of movement than behind it (which is the previous location, where it made ParA unbind) essentially creating a gradient of forces in the direction of movement. The speed of the plasmid seemed to correspond well with the force of the harmonic potentials. The initial symmetry breaking is caused by the stochastic nature of ATP hydrolysis.

The speaker mentioned another recent model that was developed that may explain similar phenomena, but pointed out that certain parts of this specific system are most likely not reconcilable with that model. The model presented today was also extremely elegant requiring just two components; one that binds to chromosomes, and another that modulates that first component’s lifetime. I quite enjoyed the talk, as Christine was an excellent presenter. While I am generally not easily convinced by simulations, this model seems to make sense both mechanistically and matches real world experiments. We also had a brief session afterwards to ask some questions, which was cut short due to unforeseen circumstances. I do appreciate her taking the time to sit down with us and answer some questions – even the ones not related to the talk.

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Agrobacterium-mediated transformation, pathogenesis by trans-kingdom conjugation

Speaker: Paul van Hooykaas
Department: Molecular Genetics, Leiden University
Subject: Agrobacterium-mediated transformation, pathogenesis by trans-kingdom conjugation
Location: Gorlaeus Laboratories, Leiden Bioscience Campus
Date: 28-09-2017

Author: Nemo Andrea

As is convention, Paul opened up his talk by outlining what the issues were that his research may be able to contribute to, and how these issues affect society. The genus of bacteria is the primary focus of his research, and thus he explained how and where these bacteria function. These bacteria are known to cause tumours in plants, which waste the plant’s resources. These tumours cell are plant cells with atypical morphology and will grow in growth factor deficient media. As plants are central to the food supply at all levels and as food supply for the increasing global population becomes an increasing challenge, research into such pathogens will be of importance. While fighting the reduction in crop yield due to this bacteria is a worthwhile exploration, the primary aim of this research was studying the means through which the bacterium induces this effects in plants – with the aim of generating molecular tools.

Agrobacterium will attach itself on plants and subsequently induce changes inside the plant cell. One of the effects of this is the induced production of opines, which are a type of chemical believed to aid the bacteria grow. This would explain the motivation of these bacteria to infect these plants. Relatively early on it was realized that a large plasmid was essential for the pathogenic effect. This plasmid, when present in non-pathogenic bacteria, was sufficient to make these bacteria cause tumors in plants. All the virulence ‘genes’ are therefore on the plasmid. A piece of this plasmid will end up in the plant cell, on which genes encoding opines and auxins and cytokinins are found. Not all genes on this section are fully understood yet, but many are believed to be related to cell growth – which would explain the tumour like behavior in the plant cells.

They found that this plasmid was quite resistant to random mutations, indicating that it is a very robust mechanism. This suggested it may a great method to inject specific genes into plants, by modifying the plasmid and inserting genes of interest. The method of infection used by agrobacteria works on many plant types. The monocots clade of plants do not form tumours like other clades, but it was found that they do produce opines, indicating that the plasmid section is successfully inserted in the plant genome. This means this method could be applied to a wide range of plant types. The speaker briefly highlighted the societal concern about GMO crops, but was of the opinion GMO crops are essential in solving food problems in the future, a notion I share. Another interesting discovery made was that certain plants (e.g. sweet potato) appear to still carry T-DNA (which is the DNA inserted by the bacterium) in their DNA outside of infection. While the speaker did not expand on this, it would be interesting to investigate why these genes have been retained (as they are detrimental to the plant in the context of bacterial infection).
They set out to identify the specific genes that mediate the transfer of the plasmid segment into the plant cells. They discovered that two genes, Vir D1 and D2, release the Vir genes from the plasmid. They induce nicks in the circular plasmid, which then releases the Vir strand as a linear piece of DNA. This single stranded DNA can then reform into a plasmid in the plant cell or be integrated in the genome. They noticed that this process resembles conjugation in bacteria, where bacteria can exchange genetic information by exchanging plasmids. They had also previously discovered that Agrobacterium can also transfer the T-DNA into fungi and even yeast, which is a remarkable fact. However, it has been known for a while that bacteria can conjugate DNA into yeast cells. These two things combined made them look at the conjugation machinery in greater detail.

Other discoveries made them believe that not only DNA is inserted in the plant cell, but also proteins. This would be unheard of and a significant discovery. They used a clever method of splitting up a GFP protein and leaving one part attached to the protein (in the virulent plasmid) and the other part expressed in the plant cell. If the protein from the bacterium would somehow be inserted in the plant cells, the GFP would recombine and become fluorescent. They had to use this method as GFP would not fit through the nanomachinery that is responsible for the transfer. By this approach they were able to confirm that even proteins were able to be transported into the plant cell.

The nanomachine in question is the Type 4 bacterial secretion system. Interesting to note is that this machinery is found in many bacteria and is used for conjugation, effector translation and more. Different types of bacteria have slightly altered versions of this nanomachine with different functions. The fact that this secretion system is responsible for the protein transport was surprising, but this does explain the parallels between conjugation and the mechanism of infection of Agrobacterium. The next question was how this machine could transport both DNA and proteins. As DNA and proteins are radically different structures, one would expect that only one of the two would be transported. They determined that VirD2 (nicking protein mentioned earlier) remains attached to the linear DNA and its C terminal domain is recognized by the secretion system, explaining DNA recognition and transport.


Structure of the type IV secretion system. Taken from: Waksman et al. 2010

When looking at the injected proteins in detail, they identified various proteins, of which they found one named virD5 to be of particular interest. Recent results have found that it inhibits growth in plants and yeast. Mechanistically, they found that it functions by binding to kinetochore associated proteins Spt4 and Ndc10. There, it destabilizes chromosomes and leads to chromosome missegregation. By overexpressing a gene that is known to counteract the pathway VirD5 deregulates, they were able to rescue the chromosome destabilization effect. The speaker postulated that the destabilization may buy time for the virulent T-DNA to be inserted in the DNA, as the cell cycle checks will be delayed. Additionally, the author described how this plasmid, provided the VirD5 protein is removed (as to not cause genetic instability), may prove to be very useful for industrial modification of plants.

When I was planning this seminar, I was worried it would have a heavy chemistry focus. Luckily, the chemistry aspects were kept to a minimum, making the talk quite accessible. I always enjoy hearing about plant biology, as this topic isn’t covered anywhere in the nanobiology course (understandably). The main attraction for me was the Type 4 Secretion system. Just like the bacterial flagella or ATP-synthase it is a wonderful (and versatile) nanomachine. There are many interesting directions to take in research regarding it, from the evolutionary reason for a particular adaption of the motor to the biophysics of unfolding and pulling a protein through it. I wonder what the reason is that the system is able to transport proteins into yeast but not animal cells. Perhaps modification of such a system may be an efficient way to inject proteins (for example medication) into cells.

Mechanotransduction in collective cell migration and its synthetic mimic

Speaker: Joachim Spatz
Department Max Planck institute for medical research department of cellular biophysics
Subject: mechanotransduction in collective cell migration and its synthetic mimic
Location: TU Delft (BN Seminar)
Date: 08-09-2017
Author: Nemo Andrea

Joachim Spatz is a researcher from Germany who is working on a range of topics ranging from more traditional cell migration to microfluidics. We were informed that he was interested in the TU Delft’s BaSyC project. His talk today was a lecture in two parts with the first part focusing on cell migration and the latter on a new microfluidics-based approach to sequentially add components to a bilipid membrane.

 Part 1 – Cell migration

The talk started by introducing the motivation for his current work on migration: collective movement. Collective movement, as seen on the macro scale in flocks of birds and fish, is also seen in biology. Retinal cells are the textbook example of a collective form of cell migration during the formation of the eye. The two behaviors (on the macro and microscale) are very much the same behavior and are worth exploring from a physics point of view. Elucidating the principles that lead to collective motion is therefore of strong interest.

 To introduce his work on migration, we first had to be introduced to a few techniques, namely Traction Force Microscopy (TFM). This technique allows one to determine the forces exerted on the ECM by cells migrating on or through the material. From this, one can then also infer the stress between cells themselves (versus cells vs ECM). This can be done because the epithelial cells that they used exhibit a form of collective migration, where the cells are linked through various proteins. From these tractions and stresses, they were able to infer that each cell has a force correlation length that corresponds to about 10 cell lengths. This is then the maximum distance that cells can ‘’affect’’ other cells by exerting force/traction.

 The behavior they were interested in is related to wound healing. They spatially constrained the cells in a rectangular cutout, which could then be removed after which the cells would spread. Interestingly, they observed that cells do not spread out homogenously (equal dilation everywhere), but rather that certain cells would move outwards first and that these cells would drag others with them. These cells, dubbed ‘’leader cells’’, and their dynamics where studied. They wondered what the rules where for this system and how this leader cell first emerges. To do this, they observed the stress and force exerted by cells before the leader cell appears/can be identified. They found that the mean traction in the regions where a leader cell will appear is significantly higher than in cells that will not produce a leader cell, and similarly for stress. They then wished to find the rules for the spacing of leader cells, as they observed the spacing between leader cells (along the cell boundary) had a minimum value. This was not just an artifact, as when the aritificially patterned the cells in a way where the leader cells were spaced closer together, they would return to the natural spacing distance.

They found that the spacing of the leader cells depended on the ECM stiffness (which affects the traction exerted by each cell). This was verified by adding factors that increase or decrease internal actomyosin contraction, which similarly affects cell traction. An increase in traction was accompanied by an increase in leader cell spacing. They postulate is because with greater traction a leader cell can affect larger number of cells due to increased force correlation length.  

 Part 2 – Microfluidics

 In line with their work on migration, they wished to make bilipid membranes that had integrins in the membrane. Such vesicles could then adhere to the environment and could work as an artificial model for some aspects of cell migration. In their attempts to create these they ran into problems with the low mechanical strength and the low yield of the fabrication process. They then developed a new system: water droplets in oil with the water droplets being stabilized by a polymer shell. Through  this strong polymer shell (which can be moved using standard microfluidics platforms) they can inject many different proteins using a series of picoinjectors. As there is no limit to how many picoinjects can be used, and each picoinjector is placed after the other, one has the advantage of being able to sequentially add elements to the vesicle. This is a massive advantage, as various systems will not properly self-assemble if they are all ‘’’thrown in at once’’. Not only does this allow for precise control, the system also had very high throughput with the picoinjectors being able to handle roughly 500 cells per second.

microfluidics

image taken from a presentation by Marian Weiss titled ‘‘Droplet-Based Microfluidics for Sequential Bottom-Up Assembly of Functional Cell-Like Compartments

 Joachim showed us some beautiful demonstrations, with three differently labeled fluorescent actin types being injected into a single cell, and even adding actin and myosin into a vesicle. This then allowed the myosin to contract the actin, making the vesicles active in a real sense. As they did this they observed that the active actin vesicles were slowly rotating. They were able to turn this rotation into motion across a line using another bead attached to the outside of the vesicle, making essentially a very crude form of a migrating cell. (although mechanically completely different from natural cell migration).

 After this, they also added lipids to the vesicle, which, in the right concentration, allowed for the formation of a lipid bilayer in the polymer vesicle. This a remarkable feat, as making a membrane isn’t easy with traditional methods. Next to this, they also demonstrated ester formation and dynamics inside these vesicles in some truly breathtaking video fragments. Importantly, they also demonstrated that is was very easy to remove the polymer shell and just leave the bilipid membrane. To get back to their original goal, they managed to add integrins into this membrane using this technique. Depending on the concentration, they could modulate the extent to which cells adhered to fibrinogen. They are currently working on making artificial mitochondria, and were able to get ATPase and bacterial rhodopsin to work in this artificial context, which I consider to be a remarkable achievement.

 I found this to be the most excited seminar I have attended to date. The first part was interesting to me because I knew a fair amount about cell migration due to my Honours Programme Project, and the second part was extremely fascinating due to the wonderful things that they were able to do. I am very excited for work on the BaSyC project of the TU, so seeing a new promising method like this makes me very, very excited about what is to come.

Whole genome sequencing of spermatocytic tumours

Speaker: Anne Goriely
Department: Josephine Nefkens Institute
Subject: Whole genome sequencing of spermatocytic tumours
Location: Erasmus MC
Date: 28-06-2017
Author: Renée van der Winden

Anne Goriely came to talk to us about her work on spermatocytic tumours. In her talk she first gave a lot of background information before going into detail about her latest findings. She started by giving us some information on spermatocytic tumours. These testicular tumours are very rare and mostly occur in older men. They are slow growing, but can become extremely large (3-30 cm in diameter). Luckily, the prognosis is usually very good. The cell of origin for these tumours is an adult spermatogonia. This is interesting, since the germline usually does not mutate because that is evolutionarily very disadvantageous. These mutations occur through a copy error during stem cell division and the mutation rate increases with age, which explains why these tumours are usually found in older men.

Seminar 7
Mutations found in relation to age (Goriely et al., 2017)

Next, Goriely brought up Apert syndrome. This disorder only has a paternal origin and the mutation causing it is spontaneous, which means it has to have occurred during spermatogenesis. It was found that the higher the age of the father, the more likely it was that the offspring would have the disorder. This is called the paternal age effect. Apert syndrome shares this feature with other disorders. It also turns out that only gain-of-function mutations are enriched with age. This can be beneficial for the current generation, but harmful for the next. That is why they are called ‘selfish mutations’.

Goriely’s lab searched for these selfish mutations in testis. To do so they made slices of testis and stained them. They found that there was a higher level of staining in some testicular tubes, which indicated mutated spermatogonia. These selfish clones can spread over large areas of the testis and different clones can be found in the same testis. It is even possible for mutated and non-mutated clones to be side by side in a tube. Some of these mutations are strong and thus lead to impaired spermatogenesis, but this is not the case for all mutations. It turns out that these selfish clones are more numerous with increasing age and that all men have them. In that sense they can be compared to moles on the skin.

Lastly, Goriely pointed out that when a distribution was made of the occurrence of spermatocytic tumours versus age, it turned out to be bimodal. Moreover, spermatocytic tumours are rare, while selfish mutations are common. This led to the belief that there might be a second source for these tumours. At this point whole genome sequencing was used to determine that aneuploidy occurs in the testis. Hypotheses connected to this finding are that aneuploidy comes before the selfish mutation and thus they might be passenger mutations. Thus aneuploidy might drive tumorigenesis. Moreover, aneuploidy can cause a gene imbalance, causing meiosis to fail so that the cell re-enters mitosis. This can lead to the giant tumours observed.

I thought this seminar was very easy to follow due to all the background information given. I liked the topic of the talk, but I do feel that for a large part of the talk we kept coming back to the same conclusion: that mutation rate goes up with age. I would be interested to see if the hypotheses given are true and what might then be done to help treatment of these tumours.

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!

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

 

Light harvesting and photo-protection in photosynthesis

Speaker: Lijin Tian
Department:
 Biophysics of photosynthesis
Subject:
Light harvesting and photo-protection in photosynthesis
Location: TU Delft
Date: 24-05-2017

Author: Kristian Blom

At the 24th of June I visited a talk given by dr. Lijin Tian, a postdoc candidate for the Nynke Dekker lab who is currently working in the lab of Prof. dr. Roberta Croce in the department of biophysics of photosynthesis at the VU. The main research goal of Prof. dr. Croce is to get an understanding of the light reactions of photosynthesis at the molecular level, with particular emphasis on light absorption, excitation energy transfer and photo-protection.

Dr. Lijin Tian started the talk by introducing photosynthesis and the main actors involved in this process. Photosynthesis can be divided in two separate processes: light-dependent and light-independent. In the light-dependent process the solar energy is harvested by chlorophylls and thereafter converted into chemical energy. In the light-independent reactions, called the Calvin cycle, carbohydrate molecules are assembled from carbon dioxide using the chemical energy harvested during the light-dependent process. For the light-dependent process there are two multiprotein complexes, called Photosystem I and II, who catalyze the process of light harvesting and conversion of light energy into chemical energy. Both these complexes can on their turn be divided into two parts: an antenna system that harvest the light energy and is responsible for the energy transfer to the reaction center, and a core complex in which charge separation and electron transfer takes place.

For the harvesting of light energy there is an critical value regarding the amount of energy influx into the reaction centers. Above this value irreversible damage to the photosynthetic system can be caused. To circumvent this problem, photo-protective mechanisms in the antenna system can reduce the energy influx by quenching excess excitation energy as heat in a process known as nonphotochemical quenching (NPQ). In their latest research paper, Prof. dr. Roberta Croce and her lab show that LHCII, the main light harvesting complex of algae, can only switch to a quenched conformation as a response to a pH change when LHCSR1 (light-harvesting complex stress related 1) is present in low concentration.

Figure 1: Fluorescence traces of LCHII-only and LCHII+LHCSR1 cells. (A) LHCII-only, (B) LHCII+LHCSR1 cells with (red)/without (black) nigericin. The signal was collected at 680 nm. Nigericin (100 μM) addition and pH changes are indicated by arrows.
Image from: E Dinc, L Tian, LM Roy, R Roth, U Goodenough, R Croce (2016) “LHCSR1 induces a fast and reversible pH-dependent fluorescence quenching in LHCII in Chlamydomonas reinhardtii cells”

Regarding the talk itself, I didn’t found it that interesting. For me it was hard to follow the story, since the speaker his English wasn’t that good. Also, and this is not the first time that I notice this, the slides were overcrowded with images and data. It was quite surprising for me that there isn’t any research lab in BN that focusses on photosynthetic systems, since it is such a fundamental field in cell biology. At the end of the talk one of the PI’s of BN asked a lot of questions regarding the status of the research field in photosynthetic systems. From my point of view it almost looked like he/she was planning to start a new lab with a specialization in the biophysics of photosynthesis. Perhaps that within a few years from now we have a new lab in the BN department.

 

EMT controlled phenotype switching drives malignant progression

Speaker: Geert Berx
Department: Molecular and Genetic Oncology Lab, Ghent University, Belgium
Location: Erasmuc MC Rotterdam
Date: May 24, 2017
Author: Teun Huijben

Geert Berx is introduced by one of our teachers Riccardo Fodde as one of the pioneers in the field of the epithelial-mesenchymal transition (EMT). After this introduction he starts to give us an introduction on EMT.

As the name implies, EMT is the transition of an epithelial cell to a mesenchymal cell. The epithelial cells are polar and very tightly connected to there surrounding cells, thereby forming a clear boundary between the underlying tissue and the outside world. To become a mesenchymal cell, the mesenchyme is the connective tissue lying underneath the epithelium, the cell has to loose its polarity and connections to other cells. This is done by losing the cell-cell interaction protein E-Cadherin (epithelial Cadherin). E-Cadherin is down-regulated as a result of binding of transcription factors to specific E-boxes near the promotor.

The most important transcription factor doing this, and discovered by Geert Berx himself, is ZEB2. If ZEB2 is high in expression the E-cadherin is down-regulated, resulting in less cell-cell interactions enabling EMT. Control experiments in different mouse models and human cell lines showed that knocking-out ZEB2 resulted in more E-cadherin and no EMT, proving this theory.

The reason why EMT is widely studied is because of its importance in cancer. When malignant epithelial cells undergo EMT, they can travel through the mesenchyme to the blood and travel then to new places to form metastases. For a long time, people thought of EMT in a very binary way; a cell is either epithelial or mesenchymal. However, Geert and his colleagues proved that there are also multiple transitional states between epithelial and mesenchymal cells, and they showed at least 8 different metastable intermediates. The distinct states differ in levels of amongst other things E-cadherin, EpCAM and ZEB2. In both normal tissue as tumors, a wide variety of these states is found, indicating that the EMT system is way more difficult than thought.

Further research into the importance of ZEB2 in EMT and tumor formation resulted in many new insights. ZEB2 appeared also important in the maintenance of stem cells, spontaneous tumor formation and the p53 pathway. However, in the study of ZEB2 importance in human melanoma cell lines they found something interesting. When ZEB2 was knocked-out the tissue didn’t differentiate anymore, and high levels of its counterpart ZEB1 were measured. Indicating that ZEB2 is important in differentiation and proliferation. Further studies showed that ZEB1 is important in stem cell maintenance and tumor invasion. This resulted in a clear model where either ZEB1 of ZEB2 is present, supported by experimental data.

However, when ZEB2 was over-expressed, they found more metastases, which contradicted the current model. Further investigation resulted in the finding that TNF (tumor necrose factor) down-regulates the ZEB2 protein, resulting in higher ZEB1 levels and thereby creating more metastases. All of this knowledge together resulted in an oscillating model of ZEB1 and ZEB2 levels during tumor progression (see Figure 1).

Schermafbeelding 2017-05-24 om 13.43.33Figure 1. The levels of ZEB1 and ZEB2 oscillate during the progression of cancer. In the primary tumor ZEB2 is highly expressed, resulting in a high proliferation. During the transient state, ZEB2 is down-regulated paving the way for ZEB1 to be active and facilitate invasion. In the metastases again ZEB2 is present to stimulate proliferation and tumor outgrowth.  

After all, I found the talk by Geert Berx very interesting. Although it made very clear how many players are important in the progression of cancer and how difficult it is to do research on it.

The Pathways Traveled: Structural Studies of Virus Assembly

Speaker: Dr. Elizabeth Wright
Department:
Department of Pediatrics, Emory University
Subject: The Pathways Traveled: Structural Studies of Virus Assembly
Location: A1.100 TU Delft
Date: 19-05-2017

Author: Kristian Blom

On the 19th of May I visited a BN colloquium given by Elizabeth Wright, principal investigator at Emory University. The Wright lab is interested in the use of cryo-electron microscopy (cryo-EM) and molecular biology approaches to explore the three dimensional structures of viruses and cells. The goal is to use this information to aid in the development of novel antimicrobials, therapeutics, and vaccines.

Dr. Wright started by mentioning the benefits and methods in cryo-EM. One of the benefits is that samples stay in their ‘native’ state because all the molecules within the sample are frozen and thus do not move over time. The other benefit, which I think is even better than the first, is that with conventional cryo-EM specimen preparation artifacts are eliminated. While I’m writing I now realize that the cause of this benefit wasn’t mentioned during the talk, but I think it has to do with the cooling of the sample.

Within the realm of cryo-EM, there are different methods one can use to analyze your sample. The most extensively used methods are single particle analysis, electron crystallography, helical reconstruction and tomography. The latter method is imaging by sections. From these sections it is possible to make a 3D image by stacking the individual 2D images. During the talk dr. Wright showed us one example of a 3D image constructed by cryo-EM tomography.

After a short review of the different methods we moved to the recent advances in cryo-EM. These advances can be separated in three different areas: Sample preparation, data collection and data processing. Especially the data collection part has made some big improvement in 2008, when Direct-Electron introduced the large-format Direct Detection Device (DDD®). In traditional transmission electron microscopy (cry-TEM) cameras use a so-called scintillator. This is a material that produces a flash of light by the passage of a particle through it. For cryo-TEM this particle is an electron that causes a photon to be emitted by the scintillator to the CCD sensor. In contrast, the DDD directly detects image-forming electrons in the microscope without the use of a scintillator. This direct electron sensing results in better resolution, signal-to-noise ratio and sensitivity. The data processing improvements do mainly come from faster computing, better algorithms and auto-segmentation.

Figure 1: The difference between traditional transmission electron data collection and DDD data collection. Image from: http://www.directelectron.com/products/de-series

The second part of the talk was devoted to the current studies of the Wright lab. Besides the research, there is also a big interest to the technological developments of cryo-EM. One of the recent innovations is the correlation of fluorescence microscopy with electron microscopy. This allows one to improve the resolution and identify certain parts of the sample by staining them with a specific fluorophore.

To be honest, I didn’t found the talk that interesting. The slides that dr. Wright used during her talk were overcrowded with text and sometimes lacking important information. Therefore it was quite hard for me to keep my focus. Every time I was halfway through reading one slide, dr. Wright already went to the next slide.  Also I couldn’t found any structure in her presentation, and that is even more annoying for me because I always need some structure if I want to understand the complete picture of the talk. What I do like about here presentation is that she knew a lot about her field of research. All the questions she got from the audience were answered in a very nice way.

The Pathways Traveled, Structural Studies of Mononegales Virus Assembly

Speaker: Elizabeth R Wright
Department: Department of Pediatrics, Emory University
Subject: The Pathways Traveled, Structural Studies of Mononegales Virus Assembly
Location:
Delft University of Technology
Date: 19 May 2017
Author: Romano van Genderen

Professor Wright started by giving us an overview of different size scales and the techniques used when studying life on that scale. One thing she pointed at is that electron microscopy, her field of interest, is improving on two different terrains. It can now both make pictures of smaller and smaller structures, but also on a bigger scale.

Afterwards, she discusssed the advantages of Cryo-EM. She told us that since it does not use any staining methods, it can measure a specimen in its native state, unlike techniques such as crystallography. Also, there are few to no artifacts visible from the preparation of the specimen.

Next, she showed us the recent advances in the field of Cryo-EM. We now can not only prepare better samples due to techniques like active substrates, but also gather better data thanks to phase plates and process the data more efficiently, not only because of more processing power of contemporary computers, but also because of advantages in auto-segmentation algorithms. But according to her, the most important improvement was the invention of direct electron detectors. These DDCs are far more useful than the previously used CCD cameras, which turn electrons into photons that are then counted. This conversion leads to loss of signal. These new cameras therefore give better signals, and can also be used to record videos in real-time with framerates up to fifty frames per second.

Her lab is currently using these new tools to their best abilities, leading to research on correlative microscopy, overlaying EM and fluorescence images to better locate compounds in the cell. They also study enveloped viruses, the topic of the rest of her talk.

She started by explaining the structure of such a virus, a completely new topic to me. To be specific, she studied paramyxo and pneumoviruses. These viruses have glycoproteins on their surface, a lipid envelope, matrix proteins and a nucleocapsid protein that surrounds and protects their RNA genome. These viruses are very common, for example the common measles belongs to this class.

Paramyxo_virion.jpg

Figure 1: The structure of a myoxivirus

This class has commonly been regarded as hard to purify. Firstly, people are unsure whether or not it still looks like its native configuration after purification. Also, there are a lot of artifacts and damage introduced by current purification methods. And to finish things off, you also only get very small numbers of virus particles back from it.

This is why professor Wright wanted to improve this method. She used a method currently used in protein purification, using nickel-NTA along with histadine tags binding to it. But in her case she incorporated the nickel-NTA into the cryo-EM grid and added HIS tags to the surface glycoproteins. This makes the grid attract the viruses and leads to far better yields and also removes a lot of artifacts from the sample.

Next, she wanted to study the interior of these viruses during virus assembly and release. There were two common hypotheses on the location of the membrane protein during these processes. One says that the matrix protein covers the inside of the capsid. The other says that it forms a protective layer around the nucleocapsid protein for even more protection. Using cryo-EM, she was able to directly see the matrix protein, and that it does not cover the nucleocapsid protein.

Also, she saw a mesh of fusion proteins, proteins that play a role in binding to the host. These proteins form a two-layered lattice, but there is something weird about this lattice. It seems to have a hole in it. This hole does seem to be the same size as the protein on the host’s surface, suggesting that this a binding pocket.

The final part of her talk made the topic a bit more practical. She talked about how her techniques were used to discover more about the virus known as RSV. This virus causes asthma in newborns and there is currently no vaccine known against it. Her research found that this virus is filamentous when secreted. Also, that its structure can be discovered in large detail by using cryo-EM. One peculiar fact she found was that the RSV-F fusion protein forms a hexamer-of-trimers when in its pre-fusion form. This knowledge can be used to develop a vaccine for this virus.

I did really enjoy the first half of the talk, where the techniques and their advantages were discussed. I did notice a lack of disadvantages, a fact that I find very suspicious to say the least…

On the other hand, the second half was not that interesting, because the main points got drowned in all the details about the virus and its shape. Also, the images were not understandable, even when she told what we were supposed to see. But perhaps that was the fault of the lighting or the screen in the room.