Nanoholes for Single-Molecule Experiments

Speaker:      Reuven Gordon (University of Victoria, Canada)

Subject:       Nano-Opto-Bio-Mechanics: Listening to Single Proteins with Light

Location:     TNW-Zuid

Date:            Friday, October 14, 16:00-17:00

Jasper Veerman

 

Having read the first four words of his presentation, the audience was none the wiser. Fortunately, Reuven Gordon started by explaining how these tie together. Studying  biology at the single-protein level, means we are dealing with nanometer-sized objects of interest. The amazing technique used to study these molecules are optical cavities, with interacting light and sound to study mechanical properties of the molecules. An example of such nanoholes is shown in the figure below. When focusing a laser on the structure, a force field is generated that can trap  polystyrene beads at the place where we see the ‘w’.

seminar-reuven-gordon

Figure 1. Schematic representation of two intersecting circular nanoholes. Holes are 120nm in diameter. Adapted from Reuven Gordon et al., Optics Express, 2015.

If a particle gets trapped inside the nanohole, a difference in the transmitted light can be observed. Effectively, a little bit more light can pass through. Interestingly, these small changes are amplified to the fourth power, making them easy to observe. As an example, the classic biological protein BSA was studied. Trapping the protein can exist in two states, which could be observed from the transmission profiles as two different ‘step heights’ as the particle was trapped inside the nanohole.

There is another practical application of this technique in pharmaceutics. Utilizing the incredible sensitivity of the method, differences in behavior of wildtype and mutant proteins can be observed. In brief, if the addition of a specific drug restores the behavior of the mutant back to the wildtype, the drug has curing potential.

In addition to the research presented above, there were many more fascinating examples of the work done in Reuven Gordon’s lab. This talk was one of the most fascinating ones I attended. Together with the other attendees, I felt highly impressed by the ingenious technique and interesting applications. I look forward to keeping track of his research in the future.

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Dissecting Back-To-Front Cell Polarity with Optogenetics

Speaker:      Mathieu Coppey (Curie Institute Paris)

Subject:       Dissecting back-to-front cell polarity with optogenetics

Location:     TNW-Zuid

Date:            Friday, September 16, 16:00-17:00

Jasper Veerman

 

The first thing that strikes the audience as Mathieu Coppey starts his talk is his enthusiasm. When he introduces himself as a physicist moving into quantitative biology, his eyes twinkle. I believe this is a characteristic commonly observed when physicists talk about their moving into more biological research.

Coppey’s lab uses optogenetic techniques to perturb and study biological systems. In such approach, one uses light to observe and control f.e. cell organization. Some specific molecules undergo a conformational change upon being irradiated by light of a certain wavelength. Such conformational changes can correlate with the (in)ability for a receptor to bind its ligand. In this way, light can be used to externally perturb a biological system. In one of the epxeriments, Coppey shows a TIRF-microscopy video of the binding of a membrane protein and its ligand in a specified region of interest that is excited by the laser. In this region, the binding, and thus prolongued stay of the fluorescently-tagged ligand, results in increasing fluorescence signal.

Polarity in biology is a different topic studied by the group, using above-mentioned and complementary techniques. Polarity is a strange phenomenon with two extremes. Firstly, breaking a pencil into two pieces will yield two different pieces. By contrast, if you take a magnet and do the same, you will be left with two magnets that have two poles each. Somehow the magnet has an intrinsic polarity in its molecules. The same can be said for some biological tissues. If part of a developing embryo is removed, we may still see the same organism emerge, only lacking a specific limb. The matter at hand is not as black and white, so current research focuses on exploring and defining the in-between region of polarity in biology.

Fraud in Science

Speaker:      Wilfred van der Wiel (University of Twente) and Derek Stein (Brown University)

Subject:       Fraud in science – not in your group?

Location:     Auditorium TU Delft

Date:            Thursday, September 8, 12:00-13:25

Jasper Veerman

 

Fraud in science is a topic that is not discussed freely and frequently among scientists. On the contrary, it is a discussion that is often avoided. With their intensions pure, no researcher can imagine their colleagues fabricating or manipulating data. Unfortunately, it is a phenomenon that does occur, as Derek Stein Wilfred van der Wiel learned the hard way. These former members of the Kavli Institute were both confronted with students committing fraud under their supervision. During the Kavli Day, they told their respective stories, shining light on a sensitive yet important subject.

Derek Stein has his own lab in the Brown University, with a research interest in nanofluidics. In the research, long charged molecules (like DNA), were passed through tiny channels that were also charged. The behavior of the molecules was characterized and the results were published. After questions in the field regarding the fitting of some data, discrepancies came to light. As a result, Stein tried to reproduce the analysis but kept getting different fits of the data than the student. As suspicion arose, an investigation was launched and it was indeed proven that the student had manipulated data. Having sorted out this nightmare, a next disappointment came when another long-term trusted student also committed fraud. Besides having to battle the denial of the student in question, Stein also stumbled upon unwillingness of the university to retract the diploma, presumably out of fear for bad publicity and lawsuits.

Wilfred van der Wiel had a similar experience with a student working on natural crystallites that form 1 nm wide channels. These nanochannels were loaded with a dye and studied. After publishing in Science, successive experiments by a new student did not reproduce the results. At the same time, a collaborator had moved, which delayed the process of finding the source of the discrepancy. In addition, it was very difficult to get in touch with the student, who had left the country and made up numerous excuses. The subsequent investigation uncovered that the data leading to the figures was a mix and match of acquired data taken under different experimental conditions. Also in this case the paper was retracted.

From these two stories we learn that fraud in science is something to be aware of. It is good to check and verify data and to be alert at all times. On the other hand, we must not become paranoid, as a researcher simply cannot always look over the shoulder of lab members. We need to further explore and discuss these topics if we want to find a workable solution.

3D Genome Studies

Speaker:      Wouter de Laat (Hubrecht Institute)

Subject:       From 3D genome studies that uncover genome functioning to advanced methods for prenatal diagnosis

Location:     Faculty of Applied Sciences, TU Delft

Date:            Thursday, February 11 2016, 16:00-17:00

Jasper Veerman

 

If we attach all the DNA from our chromosomes and extend it, it would span 2 meters. This comparison is often used to create an image of how wonderfully the DNA is compacted into the ‘tiny’ nucleus. A comparison that remains fascinating, regardless of how often you have heard it. In this way, Wouter de Laat introduces his talk on 3D genome studies.

Besides the amount being enormous, its function may be of even higher interest. It is estimated that about 3% of the genome is coding DNA, while the other 97% is popularly called ‘junk DNA’. However, soon after the term was coined, researchers realized that the junk DNA was immensely important. Wouter de Laan suggested that this junk may contain activating switches, up to 1 Mbp basepair (1 million basepairs)  in front of a gene. In order to find these enhancing regions, DNA sequencing may not be sufficient. It is desired to use a technique that takes into account the three dimensional structure of the DNA.

The technique used for this is 3C: chromosome conformation capture. This technique, developed by Job Dekker, consist of crosslinking nearby DNA via proteins, subsequent digestion and ligation that result in plasmid formation, connecting pieces of DNA that were spatially close. Afterwards, reverse crosslinking removes the linking proteins, making the plasmid ready for sequencing. Using thismethod, enhancer-promotor loops have been found in for example the -globin locus. Repetition over time allows tracking subsequent activation of different types of -globin throughout someone’s life.

Besides his research, Wouter de Laat set up a company called Cergentis, which focuses on application of these techniques in healthcare. For example, the plasma from a pregnant woman contains some fetal DNA, that could be used for prenatal, noninvasive screening for disease. Of course, the ethics of this are a whole different field than the scientific discovery as such.

Nanofabrication with ion-beams

Speaker:      Gaurav Nanda (Kavli Nanolab Delft)

Subject:       Nanofabrication with Helium Ion Microscope: Modification of Graphene and Growth of 3D AFM Probes

Location:     Faculty of Applied Sciences, TU Delft

Date:            Tuesday, September 15 2015, 13:00-14:00

Jasper Veerman

 

Usually speakers from outside the university come to present their work during the Technology Colloqia of the Kavli Nanolab, but now a PhD student presented to the people of his own faculty. The topic of today was the helium ion microscope and mostly its current applications. Given that this type of talk is part of the Technical Colloqia series, it focuses on the technical aspects of the research mostly, and not experimental results.

In brief, the helium ion microscope contains a tip with three atoms that are slightly apart. The tip is slightly turned to select the current from the brightest atom. Neutral gas atoms close to this tip (atom) will be ionized through the process of electron tunneling. Several accelerating ions will form a beam that is focused onto the sample. An advantages of using this technique is much deeper penetration and a 0.5nm resolution.

One of the applications of this helium ion microscope is the growth of specific 3D AFM tips. Take into your mind a surface on which we wish to create a needle that could be used to feel surfaces like an AFM. In this technique, a gas is brought around this surface. Then, the helium ion beam can be used to accurately ‘glue’ these gas molecules into a needle by constantly shining at the desired position. In this way, tips of the desired geometry can be produced with high precision. Some of the current drawbacks of this technique is fragility of the thin tip (~30 nm) and its ‘stickiness’ to some surfaces.

The Origin of Cellular Life

Speaker:      Jack Szostak (Harvard)

Subject:       The Origin of Cellular Life

Location:     Industrial Engineering, TU Delft

Date:            Friday, June 26 2015, 16:00-17:00

Jasper Veerman

 

Besides the research topic discussed below, Jack Szostak has already made brilliant contributions in the field of genetics. A major achievement was the creation of the first yeast artificial chromosome, aiding in the mapping of genes in mammals. From there, he carried on studying function of telomeres, the end of chromosomes. For these contributions, he was awarded the 2009 Nobel prize in medicine or physiology.

Currently, space explorations yield interesting pieces of evidence for potentially habitable planets somewhere else in outer space. “Are we alone?”, is a question that continuously fascinates researchers. In order to determine whether extraterrestrial life is possible, Jack Szostak aims to unravel the details on the origin of life. If it is possible to create a protocell in relatively understandable steps with reasonable probability, it is plausible that life will exist elsewhere.

Szostak starts from the justifiable assumption that from anorganic molecules and a favourable environment, certain simple lipids and amino acid progenitors can be formed. In order to build the protocell, the self assembling properties of lipids are ideal for creating a membrane-like structure. Such a compartment is required for allowing sufficient concentrations of the functional organic materials inside. These materials are likely to be a progenitor of RNA, since this polymer can both store heritable information, as well as carrying out enzymatic-like activity.

While studying these two essential ingredients, a lipid vesicle and RNA, problems in the model are overcome one at a time, leaving us optimistic that the origin of cellular life can, one day, be presented as an event that was simply bound to happen. From then, the question becomes where there is extraterrestrial life, and how we might get in touch.

Reconstructing Minimal Divisomes in the Test Tube

Speaker:      German Rivas (Systems Biochemistry of Bacterial Division, CIB Madrid)

Subject:       Reconstructing Minimal Divisomes in the test Tube

Location:     Bionanoscience, TU Delft

Date:            Tuesday, June 9 11:00-12:00

Author: Jasper Veerman

 

As bottom-up biology has become increasingly popular, scientists aim to construct the minimal cell. In trying to achieve this, a solid understanding of the splitting of a cell into two daughter cells is essential. In his research, German Rivas studies how elements of the so-called divisome are organized at the membrane.

Locating the site of division happens with tremendous precision, leaving scientists to wonder what robust mechanism underlies this process. FtsZ is an important molecule, that polymerizes around the membrane to form a ring-like structure. This structure is anchored to the cell membrane by ZipA, and can contract to split the cell contents.

Using pioneering techniques, like light scattering, ultracentrifugation and fluorescence correlation microscopy, Rivas studies the behavior of the above-mentioned molecules in lipid bilayers, vesicles and nanodisks. In ZipA containing vesicles, he was able to show that the polymerization of FtsZ resulted in cell shrinkage (division). Interestingly, this process could be controlled from outside the cell.

Some of the dynamics of the proto-ring assembly remind Rivas of his background in plaques, accumulation of molecules in a certain location. As a result, someday, Rivas wishes to unravel the effect of crowding on the assembly of the proto-ring, yielding insight in how cell-division processes can occur with such precision, despite all the busy cell contents moving about.

CRISPR Systems: Nature’s Toolbox for Genome Protection

Speaker: Prof. Dr. Jennifer Doudna

Department: UC Berkely

Subject: CRISPR Systems: Nature’s Toolbox for Genome Protection

Location: Wageningen University

Date: 30-9-2016

Author: Mirte Golverdingen

 

On Friday September 30th, I travelled to Wageningen to join the Lecture of prof. dr. Jennifer Doudna. She is one of the pioneers on the very promising CRISPR field. She started her lecture by explaining what CRISPR is and how it was discovered. CRISPR, Clustered regularly interspaced short palindromic repeats, are hallmarks of acquired immunity in bacteria.

The discovery of CRISPR started with curiosity on how bacteria defend themselves from viruses. 10 years ago, it emerged that many bacteria have an array of repetitive arrays in their genome, CRISPRs. In 2005 it was shown that the arrays contained virus RNA’s, this showed that CRISPR indeed could be a viral immune system. Over the next several years they did research to the way bacteria adapted to the viral DNA. It turned out that bacteria use a mechanism that adapts the viral DNA, then, crRNA is transcribed from this DNA and these CRISPR-RNAs can target complexes of virus DNA.

The Cas9 gene is the only gene that is necessary to fight of viruses by using CRISPR. From CRISPR to CRISPR systems there is much variation in how the DNA guides the DNA cutting work. These variations are classified into two classes, Class 1 and Class 2 systems.

Doudna did research to Class 2, consisting of CRISPR systems that contain one single enzyme responsible for detecting and destroying DNA. Cas 9 is a dual RNA guided DNA endonuclease. It is programmed by the crRNA, tracrRNA duplex and holds on to an unwound double stranded DNA helix. (See Figure 1) TracrRNA is important for creating crRNA but it also works in the complex with the crRNA. It trims away short molecules of RNA that are still functional. You can simplify this into a single guide instead of a dual crRNA-tracrRNA chimera. This Cas9 protein still worked.

honours-6

Figure 1: Cas9 can be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features. (A) (Top) In type II CRISPR/Cas systems, Cas9 is guided by a two-RNA structure formed by activating tracrRNA and targeting crRNA to cleave site-specifically–targeted dsDNA (see fig. S1). (Bottom) A chimeric RNA generated by fusing the 3′ end of crRNA to the 5′ end of tracrRNA. Jinek & Chylinski et al. Science 337, 816 (2012).

Cas9 and its guide RNA act like a molecular scalpel to cut DNA. It searches on all the DNA in the cell to look if it can find DNA that match. When a match occurs the DNA is opened and cut with a double stranded break.

For the biotechnology, this is very interesting. Genome editing begins with dsDNA cleavage and results in non-homogenous end joining or Homologous recombination with a donor DNA between the two ends. Normally it is very hard to fix the double stranded break. However, the Cas9 is simple and cheap, it uses the DNA as donor so you can easily build in new genes. The CRISPR-Cas9 genome editing technology is useful because of the power of base pairing. It is a very convenient way to recognize DNA and to reprogram the specificity of the systems. All CRIPSR systems are based on this simple idea. Secondly, there are many applications of this technique in animal and plants. Moreover, it is a programmable DNA manipulation program with all the possibilities occurring from this idea.

The DNA target recognition is driven by pam motifs. Cas9 works by unwinding the DNA, however it does not hydrolize ATP. Cas9 first binds to PAM motives in the theta DNA curtains of the phage. PAM contact triggers the DNA unwinding, Cas9 is therefore a catalysator. In this way, the DNA can be unwound. The conformational change when Cas9 binds to the DNA is partly responsible for this DNA unwinding.

Cas9 searching mechanism is based on finding PAM motives, that are only seen in phage DNA. When there is recognition, unwinding finds place. The DNA will be cut and the virus is killed. Cas9 therefore does not slide along the DNA strand, instead it binds and searches very quickly and then leaves to another spot on the DNA.

I was very excited about this talk, it showed how the CRIPSR-Cas9 system worked. And it also showed something from research work done behind this revolutionary discovery. I hope there will be very renewing applications of this technique. The decade in this field will be even more exciting compared to the first decade.

New Frontiers in Nanofluidics Research

 

Speaker: Derek Stein

Department: Department of Physics, Brown University

Subject:

Location: TU Delft, Bionanoscience, Kavli Institute of Nanoscience

Date: 9-9-2016

Author: Mirte Golverdingen

Derek Stein gave his lecture to show what the new frontiers are in the Nanofluidics research.  He started with the research to use nanoscience for protein sequencing. The general idea was to combine mass spectrometry and nanotubes. The techniques are combined so they overcome the limitations of each technique. The nanotubes were already known by the public. Mass spectrometry, however, was a more obscure technique for us. Derek therefore gave some background to give more understanding of this technique.

Mass Spectrometry is a powerful tool; it can tell the weight of every single molecule. Therefore, it is an extremely well established technique. The tool separates ions by their mass to charge ratio (m/z) in vacuum. Nowadays it has such a high resolution that it can resolve ions differing in mass by a single Dalton.

Another new development is electrospray ionization. This is a soft ionization technique that can be used for proteins without destroying the conformation of the molecule. It works as follows; you take a little tube with a diameter of several dozens of nanometers. Through the tube you pass a fluid with the analyzing molecules. Then you put a strong voltage between the liquid and a metal part at the end of the evaporation chamber in the vacuum chamber. Small nanodroplets that leave the tip of the tube come into an evaporation chamber where there is a gas that dries them out. This results in forces that become stronger than the forces in the liquid droplets, so the droplets blow up and this results in smaller droplets. At the end of this process the droplets are shrinking into ions.

Stein used both techniques to take a mass spectrum of adenosine monophosphate. The resolution of this technique is very precise and it showed a peak at 346,4 FW. For Single-Ion Detection Stein used an electron multiplier, the Channeltron. In a process called secondary emission, a single ion can induce the emission of roughly 1 to 3 electrons. In the Channeltron a chain reaction of emissions is created, so one ion sprays out a lot of electrons. In this way, the signal of one ion is amplified. Therefore, Stein could detect the single ions that resulted from the electrospray ionization.

However, if you use mass spectrometry for sequencing proteins, peptide fragmentation is necessary. The polypeptide will break on the backbone, between the O and the NH atoms. This is ideal because the peptides will break into amino acids, however, you do not know how the protein breaks apart. More tests are therefore needed to discover the sequence of the protein, and, how longer the protein, how more tests are needed.

Stein’s lab wants to combine mass spectrometry with a source of single ions detection. If you can start your measurement by passing a single polymer across a nanotube, then you will break the polymer apart and use the mass spectrometer to measure which amino acid passes. The order of arrival will tell the exact sequence of the protein, without using further tests. Because this will take place in a vacuum chamber, no random motion will occur. In his lab, they have built a system to start test this idea. (See figure 1)

schematic-representation-of-the-setup

Figure 1: Schematic representation of the setup Stein has proposed. The protein gains access to the vacuum chamber by passing a nanotube. At the end of the tube fragmentation takes place. The amino acids then are recognized by mass spectrometry. The sequence of the amino acids is determined by the sequence of the protein that entered the nanotube. Liszweski, K. (2016). The Next Next Thing in Sequencing. GEN. 36.Retrieved from: http://www.genengnews.com/gen-articles/the-next-next-thing-in sequencing/5653?kwrd=Oxford%20Nanopore

Stein showed me a very exciting application in biology of nanotechnology. He also showed how important knowledge of biology, chemistry and physics is in the nanobiology field. I am very excited about new ways of thinking about protein sequencing and adapting nanotechnology in the biochemistry field. This is also something I want to learn more about. By adapting the protein fragmentation into the mass spectroscopy, you can determine the sequence of the protein more easily. This idea uses nanotechnology in the form of nanotubes and mass spectroscopy. The idea of Stein is very exciting and could be a revolution in the protein sequence world.

Dissecting back-to-front cell polarity with optogenetics

speaker: Mathieu Coppey
position: Physics and chemistry departments Curie Institute Paris
date: 16-sept-2016
time: 16:00-17:00
author: Teun Huijben

Mathieu Coppey works in the Physics and Chemistry Departments Curie Institute in Paris. He is interested in cell polarity and biophysical methods to manipulate this polarity. In his talk he enlightens us about the CRY2/CIBN technique; what it is and how it is used to study cell polarity.

A cell has polarity when one end of the cell is different from the other end. This can be a different shape, for example in a neuron, different protein concentrations or organel distributions. Mathieu Coppey and his group are interested in this polarity and questions they ask are like, how is this polarity established, maintained, and most important, can we manipulate or create this polarity by using biophysical methods like optogenetics? Using this optogenetics they try to develop a quantitative biophycisal approach to manipulate protein distributions at the subcellular scale.

The optogenetics method Coppey uses is the CRY2/CIBN dimerizer. CIBN is together with GFP and CAAX bound to the membrane as CIBN-GFP-CAAX, using CAAX as anchor that is embedded in the membrane. Cytoplasmic CRY2-mCherry undergoes a conformational change when illuminated with blue light. This conformational change enables CRY2 to bind CIBN (see figure 1A below). By binding to CIBN CRY2 moves from the cytoplasm to the plasma membrane, resulting in a higher concentration near the membrane and a lower concentration in the cytosol. This can be seen in figure 1B, before illumination CRY2 is equally distributed over the cell, but after illumination with blue light CRY2 gathers near the plasma membrane.

crycibfig1

figure 1: (A) in this optogenetic technique CIBN-GFP is bound to a CAAX-anchor that is embedded in the membrane and cytoplasmatic CRY2 is bound to mCherry. When illuminated with blue light, CRY2 undergoes a conformational change so it can bind CIBN. (B) before illumination all the CRY2-mCherry is equally distributed throughout the cytoplasm, (C) but after blue illumination it is localized against the plasma membrane.

Now we have seen that it is possible to translocate CRY2 to the plasma membrane, we can use this technique to manipulate local subcellular protein concentrations. One way Coppey and his colleagues did this, is by only illuminating a small part of the cell. As can be seen in figure 2 below, when the area indicated with the red square is illuminated with blue light, after some time the CRY2-mCherry concentration in that area increases noticable. And when the blue illumination is moved to the green square, also the CRY2-mCherry moved to this area. These images are made with a TIRF microscopy, so only the membrane bound CRY2-mCherry is visible.

schermafbeelding-2016-09-21-om-09-10-47

figure 2: TIRF images. (A) When only the area indicated with the red square is illuminated with blue light the CRY2-mCherry concentration increases in this area (see B). When later the area indicated with the green square is illuminated the mCherry level on the membrane on the other side of the cell  increases (see C)

With localizing the illumination on only a small part of the cell, it is possible to get a high concentration on CRY2-mCherry at that location at the plasma membrane. When they combined this with Rho GTPases it gave them the possibility to activate other proteins only at this location, and this is exactly what Coppey and his colleagues did. They fused the catalytic domain (DHPH) of the Intersectin (ITSN) guanine exchange factor (GEF) to CRY2-mCherry. This catalytic domain triggers the RHO GTPase Cdc42. This is done by exchanging the GDP for a GTP and Cdc42 is inactive when GDP is bound and active when GTP is bound. So when a part of the cell is illuminated with blue light, ITSN-DHPH-CRY2-mCherry binds to membrane bound CIBN and Cdc42 is only activated in this region. Cdc42 has an influence on the cell movement, as can be seen in figure 3 below. Because after 2 minutes of illumination the part of the cell that was illuminated starts expanding, and a little later, the other side of the cell starts retracting. The local activation of Cdc42 is causing this. What once was a cell without polarity had changed into a cell with a clear polarity between front and back.

schermafbeelding-2016-09-27-om-18-38-38

figure 3: (A) Scheme of the cell movement after illumination with blue light, the dashed blue rectangle indicates the area that is illuminated, the grey area (front of the cell) responds by expanding and the blue area (rear) responds by retracting. (B and C) The displacement and area of the front (black), rear (blue) and illuminated region (green) over time after illumination.

I think in the future this invention will make it possible to study intracellular signaling pathways better than we can now. By only activating a specific protein in a small part of the cell, the local effect of that activation will be become clear. Also the time it takes before the intracellular signaling or diffusion to transfer the signal to the other side of the cell can be studied.