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!

 

AA

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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Dissecting the fundamentals of transcriptional regulation

Aïsha​ ​Mientjes

aishamientjes@gmail.com/a.mientjes@student.tudelft.nl

4460960

Seminar 7:​    

Speaker:​ ​Martin Howard

Department:​ John Innes Center

Subject:​ Dissecting the fundamentals of transcriptional regulation

Location:​ ​TU​ ​Delft

Date:​ 12-01-2018

 

On the 12th of January, there was a bionanoscience seminar given by Martin Howard. He was trained as a physicist, but he has since then moved into the field of biology as currently does research on epigenetic memory.

The presentation was started of by the question: when is transcription regulated in an on/off manner and when is it regulated in a continuous manner? In addition he asked whether the process is noisy/bursty or not. The rest of the seminar mainly concerned itself with the first question, because there was too little time to go into the second question.

Epigenetic inheritance means the inheritance of phenotype not based on the DNA sequence. Martin Howard studies epigenetics by zooming in on one locus. The system that he studies is called Arabidopsis, in particular flowering locus C (FLC). This locus is involved with aligning plant development with the seasons. The switch to flowering in a plant is repressed by FLC. FLC is in turn repressed by vernalization.

vernalisation

The level of FLC throughout the year.

 

Correct timing is very important for the FLC is very important because plants cannot flower too early or too late. In the graph above it is visible that the level of expression on return to the warm period depends on the length of the cold period. The Arabidopsis model is very slow, which can be difficult in experiments but also allows a very quantitative study.

 

After an explanation of the model, the speaker went on to explain about epigenetic memory. This memory can be stored in a trans way and in a cis way. Trans means that it is stored in diffusible factors and is uniformly distributed, cis means that it is stored in chromatin based histone modifications and is locally stored. There is still an active debate in biology as to how the memory is stored.

 

Systems for digital cell autonomous epigenetic memory is bi-stable. For an individual cell, it is all or nothing. With this knowledge, it can be concluded that the quantitative quality (shown in the graph above) must come from the fraction of cells. The final part of the presentation concerned an experiment with which Martin Howard showed that there is a favour for the cis memory.

 

I found this seminar very interesting because it largely concerned ecology, a subject which is not often discussed during these seminars. I found the model quite interesting and it was nice to hear that a lot of field research was done for the discussed experiments as well. The final part of the presentation was quite difficult to understand but the rest was fairly easy to follow in my opinion. In addition, I liked that Martin Howard has a very ‘physicist’ way to explore biology, he focussed on a single locus which made it quite easy to explain epigenetics.

A paradoxical human mesh

Fibrin clots at work in forming a mesh of blood cells to fill up a hole in a vessel.

(Eye of Science / Photo Researchers, Inc)

Written by Raman van Wee

Speaker:  Gijsje Koenderink

Department: FOM institute AMOLF

Subject: Biophysics of fibrin clots     

Location: Erasmus MC         

Date: 18-12-2017   

 Professor Koenderink kicked off with a great description of her as hardcore physicist meeting the biologists in the middle: mechanics of fibrin clots. This was a nice reminder of the value of Nanobiology by means of bridging this gap. Her seminar lasted for roughly half an hour and was very understandable, as Koenderink undeliberately continued perfectly on our knowledge gained in Biophysics and Thermodynamics and Transport.

 

The topic of her talk was the: “Biophysics of fibrin clots” and more specifically the mechanical characteristics of fibrin clots. Unlike many synthetic materials fibrin shows both stiffness as well as great elasticity. This results in a tissue that on the one hand doesn’t easily deform when put under stress and on the other hand doesn’t break when put under great force. This in line with the function of fibrin clots: serving as a band aid when the body is at risk of blood loss. The clots should withstand both the mechanical pulling and pushing force of moving cells and the bloodstream putting stress on it. Not only the tissue as a whole, but also individual cells showed these characteristics. By using a number of techniques including an optical tweezer she was able to stretch a single fibrin polymer, in this case by using lasers. Having measured the applied force and the stretch, the stiffness could be determined.

Another technique that was used was shear rheology, which makes use of displacement of two parallel plates with fibrin that interconnects them. By shifting the plates and measuring the resistance, the shear/stress ratio could be measured, being yet another typical characteristic for a material. A contrast worth noting between her research and most research done in Rotterdam was that at AMOLF the research was much more quantitative.

 

All in all fibrin is an extremely effective protein that supports wound healing. It is both stiff and elastic, explaining the title. Fibrin is better able to restore its original state if it stretched quickly rather than when it is stretched slowly. At last fibrin shows a stiffer behavior when first being compressed to then later be stretched.

 

 

 

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.

“Biophysical properties of fibrin”

 

Speaker:             Gijsje Koenderink  
Department:     Biological Soft Matter
Subject:              Biophysical properties of fibrin
Location:           Rotterdam, Wytemaweg 80 
Date:                    18-12-2017
Author:               Maricke Angenent

 

Gijsje Koenderink is the group leader of the research group ‘Biological Soft Matter’ at AMOLF, one of the Dutch research laboratories. Her group is mostly interested in the physical mechanisms behind self-organisation and the mechanical properties of the cytoskeleton. She explained that research is performed in two main areas. One of those is the cellular approach, in which a minimal cell is constituted with the help of cytoskeleton proteins that show a high level of self-assembly. This so-called minimal cell can then be used to examine all sorts of physical properties individually. The other approach is on the level of tissues. Living cells are brought into extracellular matrices that resemble the biological tissue environment. As a result you can analyse how cells respond to specific factors in the environment and thereby determine sensory mechanisms of the cell. In this presentation Gijsje used the subject of fibrin fibres and their biophysical properties to exemplify the current methods and approaches used in her lab.

Fibrin is a protein that is involved in blood clotting. You can imagine that this particular protein must be mechanically very stable, as you do not want any premature rupture of the fibrin complexes resulting in unwanted bleeding. Mechanical stability requires that a protein can withstand rather large forces without its overall structure being changed. In case of the fibrin proteins this stability was tested by using a polymerized fibrinogen protein (fibrinogen circulates in the blood stream and is enzymatically turned into fibrin fibres in case of injury, causing blood clotting), which was glued to one of two plates. Subsequently the other plate would be moved in such a way that the fibrinogen protein was stretched to several times its own length. From this experiment it was observed that a fibrin network is initially very random, however once stretched the various fibrin fibres are aligned into a more ordered structure. Gijsje even hypothesized that the lengthening of the fibres not only happens due to the straight alignment, but that the proteins itself might be stretched as well!

A different method used to determine the degree of stretching, was shear rheology. This technique utilizes a round plate on which the material to be examined can be deposited. A second plate is put on top of this round plate such that the protein you are analysing is enclosed between the two plates. Thereafter you can move the plates in order to apply (known) forces to the protein. From the results of this experiment and corresponding graphs, the elastic modulus of the fibrin fibres was determined. The elastic modulus can be understood as the relation between the deformation and stretch of a material. The conclusion was that the more fibrin is stretched (=more deformation) the stiffer the fibrin clots become. One explanation for this was that a relaxed fibrin fibre can be assumed to be a “floppy protein”. Once this floppy protein is stretched further it gets increasingly difficult to increase the stretch even more, thus leading to more stiffness.

Logically, the next step in the experiment was to question why fibrin is stretchy. Somehow the internal structure of fibrin fibres must be stretched, however with the previous assays you do not see what is going on internally. Therefore yet another approach had to be taken. The optical tweezers were introduced, which allow you to perform measurements on one single fibre at the time. Optical tweezers make it possible to hold very tiny particles, like single molecules, between two beads and then move them around by applying forces in any direction. In Gijsjes experiment, a single fibrin fibre was glued to both of the beads of the tweezer and then pulled apart. So now you are actually stretching a single fibre and thus you can determine the elastic modulus of a single fibrin instead of the modulus from the entire network, as was done before.

optical tweezers
Figure 1:  Optical tweezers. Protein stuck to two beads. One is kept in the same position, the other is moved to introduce stretch.

It is clear now that fibrins stretch on the single cell level in addition to the stretching caused by the alignment of cells. Most probably this molecular stretching originates from molecular unfolding of the protein. When coiled coil regions unfold due to induced conformational changes, you can expect lengthening. This lengthening can explain why a single fibre can stretch under certain conditions.

A final remark was that fibrin clots get stronger after they have been compressed. During the process of compression the clots are actually softened, however once you bring the material back to the decompressed state you see that the network has become stiffer than before. The tested hypothesis was that during compression water is pushed out of the fibrous network. Consequently the fibres are brought closer together which allows them to make new (irreversible) bonds. Once the fibres are then decompressed you actually stretch the new bonds which stiffens the fibre network. Double optical tweezer were used to verify this hypothesis.

So with the help of various laboratory set-ups, more and more is known about the biophysical properties of fibrin. Once the stretching and mechanical strength of fibrin clots is fully understood we can start to think of various clinical applications. For example, the gained knowledge can be used in thrombosis treatments or the biophysical properties can be used to increase mechanical stability of mutated fibrin variants. I think Gijsje presented us with an interesting seminar. I had not expected that the topic would be very relevant to us as nanobiologists, but it turned out that there was actually quite some overlap with topics from our curriculum.

 

 

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

Speaker:                            Mikael Simons
Department:                    MPI Experimental Medicine, TU Munich/DZNE Munich
Subject:                             Cell Biology of myelin in the healthy, diseased and aging brain
Location:                           Rotterdam, Wytemaweg 80
Date:                                   02-11-2017
Author:                              Maricke Angenent

 

Myelin is one of the most abundant structures found in the nervous system. It has a sheath-like structure that seemingly envelopes nerve fibers, or so called axions, thereby providing an insulating and protecting coating to the axion. One of the well-known functions of myelin is to maximize the conduction velocity of axions. However that is not all, myelin also plays an important role in axonal transport and preventing axonal degeneration. The latter is what Mikael Simons is most interested in. His main goal is to understand the process of myelin formation and how myelin structures change during specific diseases. Once this understanding is established, you can start to think of applications such as introducing remyelation as a treatment for degenerative nerve diseases. The talk had a well-defined structure with three main topics. First up was the formation of myelin, followed by a theory of how myelin maintains axonal functioning and ending with myelin in the diseased and aging brain.

myelination

Myelin formation mainly occurs in the first year postnatally, but continues until the age of 20 or when the brain has reached the adult stage.  Oligodendrocytes are the cells which drive the formation of the axon wrapping. The precursors of those cells move to the correct location where they differentiate in the mature oligodendrocytes. Those mature cells can then interact with the central part of axons. The question posed was how the multilayered membrane structure of myelin is created, when it all starts from this one oligodendrocyte (a point source). To study this M. Simons and his colleagues used high pressure freezing to conserve the myelin structure in the best possible way. Previously, due to the compact structure with a high level of lipids, myelin samples were often damaged and thus difficult to analyze correctly. In addition, high resolution volume microscopy by ion beam milling was put into practice, to directly image the growing myelin sheaths. With these two advanced techniques, it was easy to identify growth profiles and convert this to models of how myelin structures are created. The conclusion was that there are actually two distinct growth movements. Firstly an inner loop wraps myelin sheaths around the axon, and secondly all of the layers slide latterly along the axon to the sides. Surprisingly, it was the inner layer where growth seems to be happening, despite the fact that actually membrane growth is initiated by the oligodendrocyte, which is located on the edge of the axon. This means that the newly produced membrane must be transported all the way to the inner loop. An explanation for this would be that the membrane is transported through cytoplasmic channels and then deposited on the inner layer of the myelin sheaths.

These cytoplasmic channels have another function as well. They are involved in maintaining axonal functioning. Since axons are isolated by myelin wrapping, their metabolism thus depends on the permeability of myelin sheaths. Energy sources needed for axonal processes, such as metabolites, are provided through the sheaths. This happens with the help of cytoplasmic channels within myelin, which connect the cell body and the inner most membranes to the environment. This is considered to be an important aspect of axonal maintenance.

In the next part of the seminar, Simons continued with an explanation of how the myelin membranes are compacted and what the effect of this compacting is on the functioning of axons. One of the proteins involved in this process is called myelin basic protein, ‘PLP’, which by self-interaction and polymerisation compacts the membranes. In the normal dispersed state, charges of opposite membranes repel. However in the presence of the polymerized protein, the charges are neutralized and the membranes can be compacted from the outer layer towards the inner layers. The second involved protein is CNP, which localizes cytoplasmic channels and keeps the cytosol within the membrane at this position. As a result CNP prevents that all the cytosol is being squeezed out and thus avoids over compaction of the membranes. In the experiments it became clear that CNP knockouts result in severe axonal degeneration. This indicates that if CNP is not present in the cell, the coupling of the myelin sheath to the axon (and thereby metabolite provision) is lost. So if CNP is not present, there are no cytoplasmic channels which means that no energy sources can be provided, resulting in axonal degeneration.

Finally, a few diseases were addressed involving axonal degeneration. The underlying mechanisms were discussed in some detail, but I found this part rather difficult to understand. The main point made was that macrophages and outfoldings of the myelin membranes (and accumulations of those in lysosomes) are among the main contributions to axonal degeneration. For me the last part of the presentation was a bit to detailed, but overall it was an interesting topic with a new take on myelin functioning.

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, dept. of molecular cellular and developmental biology
Subject:              How to achieve cellular replication without fail: lessons from bacterial cells
Location:            TU Delft, BN-seminar
Date:                    13-10-2017
Author:               Maricke Angenent

 A cell partitioning mechanism which does not involve any of the complicated processes such as cytoskeleton filament assembly, motor protein functioning and the participation of the actin proteins. That is a system we were introduced to by Christine Jacobs-Wagner, who is currently the principle investigator in the Jacobs-Wagner lab at the university of Yale. She started her seminar by giving a short outline on cell replication in general. Hereafter the presentation would consist of two distinct parts. Firstly the focus was on how to ensure that partition of cell compartments happens equally among daughter cells. The second part was supposed to be about how different processes are integrated, especially how the cell cycle function integrates cell metabolism. The latter topic would have been rather innovative as the cell cycle and metabolism are usually viewed separately. Unfortunately an hour proved too short to cover both of the subjects, so instead Christine set on answering some additional questions on the first subject.

 

Christine started by emphasizing the fact that replication is a truly remarkable process. As she said “the ability to replicate distinguishes the non-living organisms from the living”, which is the source of her fascination for the process. In her lab, bacterial replication in particular is analyzed, since replication is fast and it is an incredibly robust processes. Replication is a stochastic process, meaning that it is submissive to internal variations, yet the process seems to never fail. One of the proposed reasons for this observation is that various processes are highly integrated and often rely on intracellular organization. But how exactly is this spatial organization achieved?

 

Though diffusion is an effective means of molecular transport in bacteria, due to travel distances being small, this only leads to random distributions. This in  itself is not a problem, however when you have a low copy number, the probability of finding all copies of a cellular compartment on one side of the cell considerably increases. Consequently, the chance to obtain unequal daughter cells also rises. Diffusion in bacteria is thus ineffective for partitioning cellular compartments in low count. A different separation machinery must be present in bacteria to partition single components. As Christine explained, this mechanism makes use of stochastic interactions that are transformed into active transport, which results in the spatial patterning of low count cell compartments. Based on experiments, it was concluded that when you have more than one copy in the cell, they are equidistantly distributed among the axis of the cell ensuring their propagation to the daughter cells. This distribution was analyzed for the plasmid system, as this system is best understood compared to others.

In this experiment, the mechanism of just a single plasmid was taken into account rather than of a cluster of plasmids, which you would normally encounter in real situations. Two proteins are of central importance in the partitioning mechanism: parA and par B. The former mediates active transport and binds to the chromosomal DNA  as a dimer. The latter binds to a specific sequence of a plasmid, resulting in a plasmid rich region (or so called cargo). Bound parA interacts with the parB cargo, resulting in the oscillation of a single plasmid cluster over the nucleoid. What is interesting, is that parA does not form any cytoskeleton filaments, nor does it use any motor proteins, yet the oscillations point to a form of active transport. In modeling this process it is important to assume that chromosomal DNA is not static. Instead the DNA (and thus also when parA is bound) experiences intrinsic fluctuations, which is observed as “wiggling”. Now the plasmid cargo (=parB) diffuses until it encounters parA, where it activates hydrolyzation. Consequently the parA is released from the DNA and parB can go on to interact with a different parA-DNA complex. When plotting the movement of parB over time, you obtain evidence for oscillatory behavior, where the plasmid goes from one end of the nucleoid to the other end of the nucleoid. This movement usually follows the direction of the parA gradient as the chance for interactions is higher in an environment with a higher concentration of bound parA.

seminar report 2The above figure shows the interaction of two parB plasmids, resulting in an opposite spatial organization.

In practice not just one, but multiple plasmid cargo’s are present in the cell. All of them undergoing interactions with the DNA-bound parA. These cargo’s influence each other’s direction because as they get closer the surrounding will be depleted of parA making sure that the cargo’s will turn around to go to a more favorable surrounding (where more parA is present). This influence happens continuously and as a result a regular pattern arises in which the cargoes are aligned along the axis of the bacteria. The regular patterning leads to correct partitioning of low counts during replication.

 

So in a nutshell, Christine introduced us to a replication mechanism, simpler and easier to generate than the conventional system. A mechanism in which there is no need for cytoskeletal structures or motor proteins since the DNA fulfills a mechanical function instead. Only two factors are needed, factor A binding the DNA and factor B ensuring the lifetime of binding of A. This simple mechanism relies on random fluctuations in the chromosomal DNA and stochastic interactions between various plasmids, which leads to a perfectly organized, spatial pattern. Overall I thought the seminar was fascinating, especially because I had never heard of this alternative partitioning machinery before. In addition, Christine incorporated many expressions which we have just covered in our lectures on physical biology of the cell, showing me how the theoretical information is also really applied nowadays in the laboratories.