The role of DNA damage in cardiovascular disease

written by Raman van Wee

Seminar 4

Speaker:  Catherine Shanahan

Department: Cardiology, Kings College

Subject: The nuclear lamina: a key modulator of cardiovascular ageing         

Location: Erasmus MC         

Date: 20-12-2017   

 

Text:

The talk of Catherine Shanahan was one containing many experimental results, making it very information dense. Although it started as a relative macroscopic biological story it quickly turned very nanobiology related with subjects such as DNA damage, DNA damage repair systems and DNA damage signalling pathways. The key player in the story was prelamin-A, the inimical precursor of lamin-A, which contributed to the calcification of smooth muscle cells in various ways. The first was mechanically, indirectly, prelamin-A alters the chromosome structure, this subsequently alters mechanic bridges that connect the nucleus to the cytoplasm. Although I am not sure how this works I imagine, this makes the cell more rigid, slowly calcifying it until it is completely inflexible. At the same time entry of DNA repair machinery into the cell was prevented to, having massive DNA damage and thus failing cell activities as consequence. At last prelamin-A induced chromosome modifications, leaving epigenetic marks on the DNA in one way or another this also might result in calcification. For now it is still unclear which of the three mentioned processes contributes to ageing and in which exact manner. What is clear is that dialysis patient will benefit greatly if cardiovascular ageing could be circumvented or effectively combated. First because of reduced kidney efficiency, these patients already have elevated levels of salts in their bloodstream, moreover during dialysis these vessels are exposed to much more salts, oxidative reactants and toxic waste. This all contributes to calcification and thus ageing of the vessels which is often the cause of death for these patients, and not their weakened kidneys!

 

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Enormously elevated levels of prelamin-a contribute to the bloodvessel-related disease, often seen in dialysis patients.

 

The last thing professor Shanahan made clear was that there is still much work to be done in this field. She mentioned that issues regarding DNA damage signalling when there is in fact no real damage or the fact that many severally DNA damaged cells can still proliferate due an adaption in their cell cycle need to be surmounted in order to get a full understanding of the complexity of calcification and ageing of cells.

 

 

Conclusion:

 

Prelamin-A affects cells in (at least) three manners, contributing to calcification of smooth muscle cells. This on its turn stiffens and narrows blood vessels, posing problems for the blood flow in patients. How exactly prelamin-A works and what can be done against it is yet to be discovered.

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Biophysical properties of fibrin

Speaker: Gijsje Koenderink
Department: Systems Biophysics, AMOLF
Subject: Biophysics of fibrin clots
Location: Erasmus MC
Date: 18-12-2017     

 Author: Nemo Andrea

Gijsje Koenderink is head of the systems biophysics department at the AMOLF research institute, based in Amsterdam. She was going to discuss the topic of fibrin clots, as part of the hematology lectures at the Erasmus Medical Centre. As the more reductionist approach of AMOLF and the highly applied nature of a good fraction of research at the Erasmus MC inherently attract a different crowd, the lecture was presentedin a fashion that focused on the findings, rather than theoretical justifications.

She opened up her talk with a general introduction regarding the research carried out at AMOLF, introducing topics such as their minimal systems and the reductionist approach to understanding the interactions and behavior of these systems. Part of the focus of research at AMOLF is into the mechanical properties of cells and other biological structures. Cells display behavior that is known as strain stiffening, where the cells become stiffer as the strain on said cells increases. This holds true for many biopolymer networks too. At the same time, these networks can exhibit dynamic behavior, meaning that these structures can be both very rigid but also very flexible/dynamic, which is behavior not commonly seen in synthetic materials.

In line with the reductionist approach, research done at AMOLF makes use of purified fibrin with only a few of the commonly associated factors such as thrombin and Factor XIII to serve as a model system for fibrin behavior. Fibrinogen is a glycoprotein with long unstructured tails that, upon cleavage by thrombin, can self-assemble into polymers. After cleavage, fibrinogen is known as fibrin. These polymers form dense networks that form, with the aid of platelets, blood clots that are essential for healing of wounds. Problems in this mechanism of wound healing, which could be with the system being too active (spontaneous clot formation in the absence of wounds – thrombosis) or too inactive (lack of clot formation – e.g. von Willebrand disease) are important areas of active research. Recently, the long unstructured regions of fibrin have been visualized by AFM.

fibrin_afm.pngHeight mode AFM images of a single fibrin molecule in top left and growing fibril in the other images. Taken from [1].

These fibrin networks are not passive structures, but are under stress from various forces, as would be expected from the biological role of clotting. Examples of forces are the shear flow induced by the flow of blood, forces from migrating cells and platelets. These networks also have to be degradable, and are thus also subject to enzymatic degradation, which differs per region and condition. Fibrin networks naturally form in networks where the fibers are randomly aligned, but will line up when the network is stretched. To quantify mechanical property of polymer networks, rheological measurements are indispensable. Using a rheometer, it was shown that, as one would expect, fibrin networks also exhibit strain stiffening. This matches the behavior of another common ECM protein: collagen.

Naturally, predicting strain stiffening is not a new challenge in the field of polymer physics, and, like with collagen, existing theory can explain a good portion of the measured strain stiffening of fibrin. When the strain on the networks get exceedingly large however, the theoretical model fails to accurately predict the stress. Thus, an extension of the model was required for the high strain regime. One of the hypotheses as to why the model fails to predict all regions of strain was that fibrin fibers are themselves elastic. In the theory used for the strain stiffening, the assumption is made that the polymers are inelastic. In order to test whether fibrin is itself elastic they turned to optical tweezers. They combined optical tweezers with a multi-channel microfluidic flow device in order to efficiently capture single fibrin molecules and measure the behavior of such a molecule under forces.

From these measurements that fibrin exhibits elastic behavior, in that it will return to its original length and elastic modulus when it is released from a stretched position (i.e. it is not deformed, no hysteresis). Naturally, then the question arises: what part of the molecule mediates this reversible stretching. Atomic force measurements then determined that the alpha coiled coil regions of the protein are highly stretchable. To test whether they could see this conformational change in protein structure leading to lengthening happen, they turned to X-ray diffraction. Using a synchrotron x-ray beam, they were able to view changes to the protein as it unfolds. Traditionally, you need a crystal of your protein in order to determine the structure using x-ray diffraction technique, but fibrins natural periodic spacing in polymer network was sufficient to determine spacing of the protein structural elements. In low strain regimes, the X ray diffraction pattern clearly showed the known spacing between the subunits of fibrin, but when large strain was applied to the network (in the region where polymer theory failed to predict the behavior, and stretching of the proteins was expected) these specific patterns disappeared, confirming that the protein indeed undergoes conformational changes under high strain.

Remarkably, they found that while extension of a fibrin network is quite reversible, compression is not. After having been compressed and decompressed, the networks become significantly stiffer than in their pre-compression state. They hypothesized that this was because upon compression, the fibrin molecules become more closely spaced and can undergo more interactions and form new connections, which would strengthen the network. This would naturally require the protein to be able to form more interactions than the known binding patterns. In order to test this they used an optical trap setup where they held 2 fibrin molecules in 2 traps and lined them up in a cross pattern. It was then observed that these molecules ‘stuck’ to each other and required significant force to be separated again. Thus, it seems feasible that this is the mechanism that explains the stiffening after compression.

It was apparent that this lecture was meant for the researchers at the Erasmus Medical Centre, rather than at more biophysically oriented researchers, as no formulas or theories were discussed on the slides. It was interesting to see that techniques like optical tweezers are relatively unknown among the researchers at the EMC, seeing as they are prominently featured in the nanobiology programme. Curiously, when I signed up for this lecture, I did not know the speaker, but as part of my bachelor end project I am speaking with people at AMOLF, so it was interesting to see one of the people I met there back at the EMC. A small world.

[1] Protopopova ADBarinov NAZavyalova EGKopylov AMSergienko VIKlinov DVVisualization of fibrinogen αC regions and their arrangement during fibrin network formation by high-resolution AFMJ Thromb Haemost 2015135709.

How do endosomal vesicles move? A surprising interaction network in and between organelles

Speaker: Sjaak Neefjes
Department: Bionanoscience
Subject: How do endosomal vesicles move? A surprising interaction network in and between organelles
Location: TU Delft
Date: 24-11-2017
Author: Renée van der Winden

Sjaak Neefjes came to talk to us about his research on the dynamics of certain endosomes that are active in the immune response of the body. He first explained very briefly what this immune response looks like to get an overview of what he was discussing. Then he focused on the endosomes themselves, how they are formed, how dynamic they are and how they are transported.

When the body is invaded by pathogens, certain macrophages engulf and digest these pathogens and present the pathogen’s antigens on the outside of their own membrane for other cells to see. These macrophages are appropriately called antigen-presenting cells (APCs). Now let’s dive into these APCs. In the Golgi body of the APCs class II MHC proteins are fabricated. It is these proteins that are involved with the presenting of the foreign antigens. These proteins are engulfed in an endosome, which eventually exits the cell, thus presenting the antigens to other cells. These endosomes can have two forms: multivesicular and multilamellar. For now, only multivesicular endosomes were discussed.

One of the questions Sjaak Neefjes discussed is how dynamic these endosomes are. Can proteins leave the endosome after they have been put into it? To test this, green fluorescent protein (GFP) was attached to the MHC II proteins and could thus be seen in the endosomes. Next, protease was added to the GFP expressing cells, so that the proteins were broken up. This resulted in GFP moving from the endosomes to the nucleus. This indicates that, indeed, particles can leave the endosome once they are inside it.

Another subject of this seminar was the movement of these endosomes. They are made somewhere in the center of the cell and have to be moved to the membrane if they are going to be ejected. It appeared this movement happened only in small bursts and was bidirectional. The endosomes can be moved to the outside of the cell, but also back to the center. It was already known that so-called motor proteins exist that bind to endosomes and can transport them across the cell. The questions Sjaak Neefjes discussed was ‘Why do the motor proteins bind the endosome?’ and ‘Why only for a few steps?’.  It turns out this is a rather complex interaction of multiple molecules in the cell, which I will not go into right now. In the end, the take-home message was that cholesterol is a key factor in this process. A certain protein in the cell acts as a cholesterol sensor, resulting in endosomes being transported further into the cell when cholesterol binds and endosomes moving to the periphery of the cell when cholesterol does not bind.

Seminar 10
Schematic difference between high and low cholesterol conditions (van der Kant & Neefjes, 2014)

I found this seminar quite difficult to follow, because Sjaak Neefjes named a lot of proteins and substances by name. Usually, these names consist simply of letters and numbers, which make a lot of sense if you have been working with them for a long time, but make a talk sound like gibberish at times if you are not familiar with them. I did appreciate that the seminar started with a general overview of the process we were discussing. This places the research into a context that you can wrap your head around as an audience and provides you with a sense of purpose for this research from the start.

Cell biology of myelin in the healthy, diseased and aging brain

Speaker: Mikael Simons
Department: Clinical Genetics
Subject: Cell biology of myelin in the healthy, diseased and aging brain
Location: Erasmus MC
Date: 02-11-2017
Author: Renée van der Winden

Today Mikael Simons came to talk to us about his research on myelin, the sheath that covers axons in nerve cells. He first explained a bit about the general structure and workings of myelin and then he moved on to discuss what happens to myelin during disease and aging.

A critical part of Simons’ research was to start fixating the axons by high pressure freezing instead of using chemical fixation. Chemical fixation does not work well on myelin, because it contains fatty molecules. Using this new method of fixating allowed Simons’ group to see details not seen before in myelin.

Myelin is made by cells called oligodendrocytes. When myelin is being formed, it grows in two directions. The inner layer that is directly on top of the axon drives growth and moves in a spiral-like manner, so that the myelin sheath thickens. The outer layer of myelin moves sideways, so that the entire length of the axon is covered. One of the research questions that Simons had, was what the driving force of the inner layer (also called the inner tongue) is. It turns out this is actin, which is only present at this site during growth. Another question was how myelin maintains axonal function. At least one of the reasons for this turned out to be that oligodendrocytes convert glucose into lactate, which is used as an energy source by axons. However, this leads us to another question: How can lactate get through the myelin to reach the axons? Simons found out that this happens through so-called cytoplasmic channels. The layers of myelin are held together by a protein called myelin base protein (MBP). At certain locations another protein, CNP, intervenes to keep the layers open, thus forming channels through the myelin.

image descriptionFigure 1: Schematic overview of myelin growth and cytoplasmic channels (Simons et al., 2014)

To research what happens to myelin during disease, Simons’ group mimicked two diseases, MS and NMO. In these disease myelin is damaged. Simons was particularly interested to see what myelin looks like when it is broken down. They found out that during NMO the inner tongue vesiculates first and the vesiculation then spreads through the myelin. In MS there is usually more splitting of the myelin sheath and also bulging out of the sheath. The hypothesis for the breakdown mechanism is that MBP dissociates, causing the myelin to fall apart. This turns out to be true in culture, but has yet to be shown to be true in vivo.

Lastly, Simons talked briefly about myelin in relation to aging. It turns out that there are more microglia and more lysosomes with age. The hypothesis was therefore that microglia clear myelin during aging. This hypothesis is supported by the fact that it was sometimes possible to observe myelin fragments in microglia.

All in all I thought this seminar was easy to follow and I thought it was quite interesting. I am interested in neuroscience as a whole, so I always like to follow seminars that are related to it.

How microglia/macrophages coordinate myelin repair

Speaker: Mikael Simons
Department: Institute of Neuronal Cell Biology, Technical University Munich
Subject: How microglia/macrophages coordinate myelin repair
Location: Erasmus MC
Date: 02-11-2017     

Author: Nemo Andrea

 Mikael Simons gave a talk on recent developments in the area of myelin formation and repair. He is an active researcher in this area and presented some of his recent findings. Myelin is a fatty substance (high lipid content) that is found surrounding axons that aids in signal transduction and provides nutrients. These myelin sheaths are formed (in the central nervous system) by oligodendrocytes. These oligodendrocytes are rather distinct looking cells, with many branches coming out of the main body that protrude and reach other axons where they form myelin sheaths around said axons. Oligodendrocytes have been observed to have extensions to up to 50 axons. Said protrusions do not cover the whole axon, but rather a section of around 1uM. Mikael presented work where the process of myelin formation was studied in unprecedented detail and covered various other topics related to these oligodendrocytes.

The traditional function of myelin is the isolation of nerves and subsequent improvement in signal transduction. The speaker stressed that myelin is not just a simple lipid layer around the axons; they are a an area of active communication between the oligodendrocytes and the axons. The inner fold of the myelin sheath (which consists of many wrappings) contains a functional cytoplasm where the axon and oligodendrocytes can exchange factors. Mikael showed us new work on the formation of this sheath and, how it is wrapped around the axon temporally. As the myelin sheath has high lipid content, traditional fixation methods would introduce too many artifacts for close study. Instead, they opted to study the formation process by high pressure freezing EM. This avoids the artifacts, and allows for a high-resolution 3D reconstruction by block etching a frozen neuron. (imaging slices of neuron to get a 3D reconstruction of said neuron). By comparing many neurons at different timepoints in myelin formation they came up with a model. They observed that the inner layer (called the inner tongue) wraps itself around the axon while also extending out laterally (somewhat akin to the shape of a croissant).

Model of Myelin Biogenesis in the CNS, Related to Figure 7(A) Model of how ...

Structure of the ”inner tongue” wrapping around an axon as depicted in Snaidero et al.

Intrigued by this finding they decided to take a closer look at the inner tongue structure. They identified the presence of cytoplasmic channels that had many MT present. They hypothesize that this is for transport of various factors. They sought to identify the force generation structure that is responsible for the wrapping behavior. Naturally, they considered actin as a prime candidate. To test whether actin is the force generating component, they disrupted the actin network by adding cofilin, an actin depolymerizing agent. In these cells, they still observed some wrapping around the axon, but the wrapping was far from complete, suggesting that indeed actin is responsible for the wrapping motion. As some forms of cell migration use active cell-ECM or cell-cell adhesions in order for the force generated by actin to be turned into motion, no factors mediating adhesion have been found. The speaker hypothesized that the friction between the outer membrane and axon are enough for motility; a reasonable assumption, as amoeboid cells also move in this fashion.

They then turned to other mechanisms unique to myelin. They worked with a protein called Myelin Basic Protein (MBP) and studied its function. This protein is highly positively charged, and the speaker found that these proteins interact with the negatively charged proteins in the membrane. In the absence of negative charge, the MBP proteins repel and fail to interact. By changing the pH of a solution (past the isoelectric point of the protein) in vitro they were able to induce spontaneous polymerization of the protein.  If negative charges are present to cancel out the charge, they start to polymerize. In this way, they zipper the two membranes together (myelin consists of various layers of lipid membranes). The comparison is really quite accurate, as the zippering only occurs in one site at a time and moves along the membrane.

Representation of MBP zippering membranes together. Taken from: Weil et al.

Next, the speaker turned to pathologies related to oligodendrocytes and myelin. Two primary examples of diseases are MS (multiple sclerosis) and NMO (Neuromyelitis optica). In NMO, the astrocytes are destroyed in the central nervous system, which is believed to damage and destroy the oligodendrocytes in the process resulting in the indirect destruction of myelin around the axons. In MS, it is believed that the myelin is damaged directly, although the exact antibody has yet to be determined. They tried to model these diseases and study the mechanistic consequences of each pathology. For NMO they used the known antibody to model the disease. For MS, they used a antibody that results in the destruction of myelin directly (as a coarse model for MS). In the NMO case they observed that the myelin sheath started to vesiculate (turn into vesicles) from the inside out. After the entire sheath has been vesiculated, the microglia remove the damaged myelin. In the MS model, they observed that the outer membranes started splitting or bulging. They dubbed these outfoldings of the membrane ‘myelinosomes’. These outfoldings are then cleared by microglia. They compared these structures to MS biopsies and found that MS lesions have significantly greater number of these myelinosomes than other tissues.

This lecture was quite interesting, as cells in the central nervous system tend to have rather fascinating MT and Actin structures; which are topics I find particularly interesting. Oligodendrocytes are very fascinating cells, due to their unique shape and function. It was also good to see EM find applications in a biological process such as this, which is hard to resolve conventionally. It would be an interesting challenge to resolve the MT and actin structure in greater detail in these cells during myelination, but the cells are hard to culture in practice, making it easier to use mice for research. This is probably a limiting factor for their study (in labs such as the ones we have in Delft). I asked the speaker whether he had any plans on studying the effects of disrupting the MT cytoskeleton during myelination and he indicated that this was what they are currently working on. I will be following this with great interest.

[edit] 02-12-17
The youtube channel iBiology has posted 2 videos on this topic, one featuring the speaker of this seminar. I have included the links below:
Bench to Bedside: Myelination 1 – Myelination, Remyelination and Multiple Sclerosis – Mikael Simons
Bench to Bedside: Myelination 2 – Neuropathology of Multiple Sclerosis – Christine Stadelmann

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.

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!