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.

 

 

 

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Biophysical properties of fibrin

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

 Author: Nemo Andrea

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

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

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

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

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

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

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

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

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

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

“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.

 

 

Disrupting ultrashort nucleic acid duplex with mechanical force

Speaker: Kevin Whitley

Department: University of Illionois at Urbana -Champain

Subject: Disrupting ultrashort nucleic acid duplex with mechanical force

Location: Bionanoscience Department, TU Delft

Date: 30-06-2017

Author: Mirte Golverdingen

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

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

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

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

Honours12

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

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

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

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

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

Optical Tweezers: gene regulation, studied one molecule at a time

Speaker:  Steven M. Block
Institute Speaker: Applied Physics and Biological Sciences, Stanford University
Organising Department: Kavli Institute
Subject:  Optical Tweezers: gene regulation, studied one molecule at a time    
Location: Faculty of Industrial Design, TU Delft
Date: 01-12-16

Author: Nemo Andrea

Professor Block is a well-known figure in the bionanoscience department. He is considered to be one of the founders of the single molecule biophysics field. He currently holds the Ascherman Chair in the Departments of Applied Physics and Biology at Stanford University. One of his former students is Elio Abbondanzieri, who is currently one of the members of the bionanoscience department at the TU Delft. He gave an intriguing seminar on Optical tweezers and their applications and discussed riboswitches.

In the introductory section, Professor Block discussed the importance of the single molecule biophysics field, and how bulk analysis can have it shortcomings. This was followed by a short section on one of the workhorses of this field: the optical trap. Having been introduced to the working concept and applications of these optical traps in our course, it was interesting to see someone who pioneered the use of optical tweezers.

The more contemporary discussion started with the versatility of RNA polymerase (RNAP), which coincidentally was also the focus of the preceding week’s BN seminar. This RNAP molecule, as he explained is a very well document and very versatile protein complex which is regulated in part by RNA hairpins. To demonstrate both the applications of optical tweezers and the properties of DNA he explained how they could determine how many DNA molecules were tethered in their optical trap by measuring the force-distance curves and comparing it to the mechanical properties of DNA. This is not a new experiment by any stretch of the imagination, but it is key in illustrating the use of the optical trap.

The second part of his seminar revolved around the research done in part by Elio, namely researching the manner in which RNAP moves along the DNA, in particular focusing on testing the validity of the inchworm model. He explained the particular circumstances that made that experiment particularly challenging. To hear about the complexity of environmental noise and finding new ways to minimise noise in experiments was refreshing and educational, as we often solely see elegant and low noise data but never hear about what problem solving steps were taken in the actual experiment that ultimately lead to said results.

The Final and main focus of his seminar revolved around RNA hairpins and riboswitches. He explained how various theoretical formulas can be used to determine many properties of various RNA hairpins. They studied riboswitches, with a particular focus on a riboswitch that as adenine as its substrate. The idea behind this particular type of riboswitch is that when the substrate binds to the RNA molecule, it will affect the transcription of the gene itself. This way we have a way in which RNA molecules can become part of the complex gene regulation networks. This concepts also ties in nicely with the RNA world hypothesis.

While this seems feasible in theory, it is not enough to just describe a model qualitatively in order for it to be accepted. It must be mentioned that this is still an area of active research and only three riboswitches have currently been studied in any detail. Steven Block and his lab and others set out to characterise the mechanical properties and kinetic behaviour of the adenine riboswitch, with the goal of being able to describe it more quantitatively.

They studied force extension curves of the specific riboswitch (which ultimately is just RNA being transcribed) by using optical tweezers. They used a framework (Dudko 2006) [3] to find the free energy of activation and characterise various other aspects of the conformations of this RNA molecule as it would be transcribed. This ultimately lead to the creation of a 5 state system as described in a paper by Greenleaf et al. (2008). This group created an energy landscape of this system which elegantly describes the behaviour of this system and the effect of adenine on the energy levels.

Left: the adenine riboswitch as depicted in [1], right: the TPP riboswitch as seen in [2]

They also ran experiments on a different riboswitch, one that is found in eukaryotes and functions with TPP as a substrate. Structural analysis of this molecule had led to the proposal of a model wherein two hairpins would (blue and red in the image above) would come together in order for the riboswitch to be switched on. In order to verify this hypothesis, they combined the optical trap with FRET. They labeled each arm and observed the intensity and wavelengths of light emitted. This allowed them to make a model which suggests that there are various conformations in which this riboswitch can reside. More can be read about this in reference [2].

It was intriguing to see the results and applications of optical tweezers, a topic we have covered in physics courses and are heralded as one of the prime example of the nanobiology field and the new technologies that are created when the worlds of molecular biology and physics meet. Additionally, I had never explored riboswitches in any great detail, making this topic a completely new area for me. While they may only be a uncommon way to regulate gene expression, the mechanisms through which they regulate the genome are very unique and certainly require further study. Even with the things above, the thing I enjoyed most was seeing how theory led to experiments which then led to new theories and models.

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692365/
[2]https://elifesciences.org/content/4/e12362
[3]http://journals.aps.org/prl/abstract/10.1103/PhysRevLett.96.108101