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.

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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.

Mechanotransduction in collective cell migration and its synthetic mimic

Speaker:             Joachim P. Spatz
Department:     Max Planck Institute for Medical Research, department of Biophysical      Chemistry, Heidelberg, Germany
Subject:              Sequential bottom-up assembly of synthetic cells
Location:            TU Delft, BN-seminar
Date:                    08-09-2017
Author:               Maricke Angenent

 

Joachim Spatz was the invited speaker of today’s seminar. He presented a talk which was organized in two main topics, starting with an introductory part on the self-organization of biological systems. This organization is also observed on cellular level and by making simulations of this process, the physical laws of collective cell migration can be studied in more detail. Subsequently, Spatz moved on to his most recent subject of research, which involves the field of synthetic biology. He elaborated on the progress his department has made on the creation of a synthetic cell and how this cell could be brought into a biological environment.

 

Collective cell migration
When comparing different forms of collective movement, whether it be on a macro or micro scale, you will find similarities no matter what the object is. One of those is that each system will find hierarchy at some point in the process. Since the similarities in collective migration were so striking, it was nothing but logical to set out for overlapping ‘rules’ involved in the behavior. This is what lead Spatz to his conducted experiments, which were mostly focused on the upper layer of skin, the epidermis. When you look from the top you can clearly see the dynamics of individual skin cells and how they are all mechanically interconnected. Consequently simulations allow for in depth analysis of how the movement is actually established. With special measurement techniques, forces on the various cells were measured, which were then used to calculate stresses between individual cells. The result obtained was that the force correlation length corresponds to the length of approximately 10 cells, meaning that the force exerted by one cell can influence the migration of other cells separated by maximally 9 others.

seminar report 1Fig 1. The forces involved in collective cell migration can be compared to rope pulling. The force is constantly passed to the adjacent cell, up to 10 cells further
Source: http://www.cell.com/trends/cell-biology/fulltext/S0962-8924(11)00127-9

The next experiment involved a confinement of cells. During the assay the cells were (at time t=0 ) allowed to spread to an area without any other cells. Interestingly, the result showed that even though cells are genetically identical, they do not move in the same direction nor at the similar speed. Now by combining the knowledge of before and by again computing the forces present, a so-called leader cell could be identified. A cell which is the first to move from the confined space and seemingly ‘pulls’ the other cells with him. Subsequently it was studied what the requirements were to become such a leader. To do so, the field looked at leader cells which had already appeared and then went back in time to study the prior forces and their particular location.

What I found surprising is that the system of cells always regulates back to the essential spacious organization with respect to the leader cells, even after being disrupted. Showing that it is indeed a very mechanically regulated system, including cells that collectively integrate forces together.

The synthetic cell
Overall I thought this part of the lecture was more difficult to understand than the first part, mostly because of quite some difficult terminology which I am not very familiar with, yet. Despite that it was very interesting to listen to, as it appeared to me as a promising approach to making an actual fully function cell. The main idea was that Spatz and his research group are now making synthetic cells by first stabilizing water droplets and then sequentially adding various essential cell compartments. To do so the water particle, which is a mechanically very strong template, is brought into an electric field causing the cell membrane to become more porous. Subsequently, pico-injection was utilized to inject quantified volumes into the ‘synthetic cell’.  What I picked up as most important message was that the sequential addition was of real importance for it allows to reach complexities which you could not reach if you try to make the entire cell in one go.

On a personal note, I thought the seminar was very fascinating and I learned a lot of new information. I was not acquainted with all the theories behind collective cell migration, and to be honest had never acknowledged it before. However it turns out to be a very regulated process with many physical laws to be discovered. For me the second part about making a synthetic cell was what really sparked my interest. I found it amazing to find out that the basic molecule they are using to make a synthetic cell is just an ordinary water molecule! I look forward to hearing more about this kind of recent innovations in the biological field and all of the possible applications.

Mechanotransduction in Collective Cell Migration and its Synthetic Mimic

Written by: Raman van Wee

Speaker:  Joachim P. Spatz

Department: Biophysical Chemistry

Subject: Mechanotransduction         

Location: Applied Sciences  

Date: 08-09-2017   

 

Text:

Spatz kicked off by showing us several videos of collective movement, both at the population level and at the cellular level. The latter included wound healing and formation of lateral line in zebrafish. Next a video of the motion of an epithelial monolayer of upper skin came by, it was very chaotic and movingly, which actually surprised me, I was expecting a rather static situation. Quantitively speaking groups of 10 cells up to 200 micrometer showed to behave as a collective, tuning the direction of the force to the group. In contrast there are leader cells, which go into a space on their own, followed up by the rest of the group. By reversing videos, behavior of leader cells could be investigated before it became apparent that the cell would become a leader cells. These cells seem to be predetermined, as they are at least 1000 micrometers apart of each other. The system regulates itself as shown by putting several leader cell too close to each other leading to the system eliminating those that are unwanted and thus leading to a stable, well distanced situation of leader cells. Remarkably if a group of followers, following a leader cell, threatens to exceed 10 followers, a new leader is brought forward from the group. I would say this requires extensive communication and coordination to execute well.

The second part of the seminar was about making a synthetic cell. This begins by making droplets having bilayer of membrane. Using sequential pico-injection microfluids, proteins and lipids could be inserted into the future cells. This sequential addition is very important, blending it all together at once doesn’t do it. Although once in the synthetic cell, the different components can not be separated with the eye, they do actually collaborate into larger structures. Interestingly, including myosine leads to the cell rotating around its axis. The need/strength for this rotating became clear when rotation was blocked by attaching a bead. In that case tension would build up until the cell would lose contact with the glass.

Afterwards we had the opportunity to speak to Spatz directly and ask him a few questions. We came to realization that he thinks in one year from now his synthetic cells will have some sort of mitochondria like energy factories. For the duplication part of life he thinks other research previously done can be very beneficial, drastically lowering the time required to integrate it in his cell. Besides, what actually surprised me was that his seminar was primarly based on 2 papers, whereas I expected more.

 

Conclusion:

Collective migration is mechanically regulated by the length up to which cells collectively integrate forces together. The cells follow this rule to select and follow new leaders. In synthetic cells, components have to be inserted one by one although they eventually entangle. Forces in a cell can lead to the cell rotating around its axis.

Information of the structure of the universe

written by: Raman van Wee

Speaker:      Robbert Dijkgraaf

Department: Physics dept. Princeton

Subject:       The relation between the extremely large & the tiny world

Location:     Applied Sciences

Date: 07-09-2017   

Professor Dijkgraaf told us about the various scales scientists work at. We concluded that life (& Nanobiology) is centered at the middle of this sizescale.
According to Dijkgraaf this makes studying life extra challenging. In the outer sizes a different set of rules and equations hold, but in the middle, where life happens to be, the collide, making it extra hard. I found this both surprising and intriguing. Surprising because it posed quite a contradiction with my
instinctual beliefs, intuitionally I felt the center of scale is the relatively easy part and going out into space or into the world of quarks would be more challenging. It intrigued me as well, because as a Nanobiologist I am working right around this center!
The concepts in the introduction were well imaginable and thus comprehensible, as the seminar continued, it became less and less logical for me. A lot of concepts were new for me and were touched on only briefly, hence I lost parts of the seminar. Just like Dijkgraaf said one could easily get lost in H space while I didn’t even know what is comprised. Despite me not following all the content, it still widened my horizon, as it became apparent how much there is around I still don’t know anything about. A lot of those concepts and theories are also more or less related to my field of study and are therefore worth taking a closer look at some time. Although slightly off topic, when Cees Dekker introduced Dijkgraaf, he told us about some of the many committees, boards or councils Dijkgraaf has working in. I found this interesting as well, besides his own research he is also looking at science from different levels and angles, which I believe is anything but boring.

Conclusion:
All in all life is at the center of the sizescales. Beyond the cellular and organelle level there is yet so much to be discovered. In the future a beautiful goal
would be to implement concepts in the universe scale into the atomic scale and vice versa. Personally I am most interested in some of the quantum related theories and concepts.

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.

What you can learn from computer modelling

Speaker: Christine Jacobs-Wagner
Department: Casimir Research School
Subject: How to achieve cellular replication without fail: lessons from bacterial cells
Location: Delft
Date: October 13, 2017

Although Christine Jacobs-Wagner studied biochemistry in college, she would describe herself more as a cell biologist. According to her, one is not defined by education, but rather by the questions they pose themselves. That’s why her lab is very multidisciplinary, which made her seminar really interesting. I liked the way she didn’t just come to Delft to lecture us about what’s she’s done, but rather to get feedback from the scientists here, who might have a stronger modelling background than hers. This is how science should work, and it’s a great feeling to be a part of it.

She spoke about the importance of distributing plasmids evenly during cell division. If one of the daughter cells doesn’t inherit an essential plasmid for some reason, it will not survive. That’s why it is crucial to have a method in place to guarantee the survival of both daughter cells. The ParA and ParB proteins make up such a system. This was the focus of her research.

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Plasmids should be distributed like these green dots

The ParA proteins bind to the nucleoid of a bacterium, and the ParB associates with plasmid DNA. When they interact, ParB induces a reaction which breaks the ParA-DNA bond. This is all well understood, but how would this have the desired effect of distributing all plasmids evenly? Well, it might be easier to observe what happens when there’s only one plasmid in the cell. It starts to oscillate along the long axis, but why?

 

 

Then she proceeded with an in silico model, a computer simulation. Unfortunately, the first model didn’t show the desired behaviour. The plasmid made a random walk, so the Par proteins didn’t do anything. At least, not until she adapted the model to make the DNA non-static. She could model ParA molecules as tiny springs, which oscillate around an equilibrium position. This was a very small change, but it had a great impact. It was all she needed to accurately model the oscillatory behaviour.

It was really cool to see such a small change make such a big difference. For further research and exploration of the ParA-B system, an analytical model would be useful, but that will also be a big challenge. I’m excited for the future!