Directional Sensing by White Blood Cells

Speaker:      Michael Sixt (Group leader & vice president IST Austria)

Subject:       Molecular Control of Leukocyte Chemotaxis

Location:    Erasmus Medical Center Rotterdam

Date:            Monday, March 16 16:00-17:00

 Author: Jasper Veerman

While many researchers in his field study several properties of the immune system to better understand it, Michael Sixt uses the immune system of mice as a model to study his primary interest: cell movement. Leukocytes, also known as white blood cells, are very dynamic cells that move through lymphatic vessels upon activation (for example skin damage). They migrate to a location called the lymphatic node, where they present the antigen they captured. In doing so, many movement-related challenges are to be overcome. Below, I will discuss the  first challenge of the migration of the activated leukocyte into the lymph vessel.

Since cells are “blind”, they need special cues to travel to the desired location. Chemotaxis is the process where cells sense increased concentrations of a certain ligand, and therefore move towards  or away from this gradient, depending if they are attracted to, or repelled from this substance. The presumed attractor of leukocytes, CCL21, is immobilized around lymph vessels, forming a gradient with increasing concentration from  distance onwards. This proposed mechanism was tested – and proven – by destroying this CCL21 gradient, after which the leukocyte no longer moved specifically in the direction of the lymph vessels.

After analyzing the structure of CCL21, it was found to contain a rare C-terminus that could bind to, among others, polysialic acid (PSA). Removal of this recognizing tail resulted in failure of CCL21 to recognize the CCR7 receptor on the surface of the lymph vessel. A likely conclusion is that CCR7 must contain a PSA domain, to which the CCL21 binds. This conclusion was verified experimentally. Furthermore, Michael Sixt’s group is working on finding the molecular details of this binding reaction. The latest model is one in which auto-inhibition plays an important role. Normally, CCL21 has a protecting belt (the rare tail) around the binding site for CCR7. However, when bound to PSA, this belt changes position, allowing proper ligand-receptor binding.

Calcium Vessel Dynamics Upon Signal Transmission in Neurons

Speaker:      Stefan Hallerman (Carl Ludwig Institute for Physiology, University of Leipzig)

Subject:       Presynaptic Calcium Dynamics at Central Synapse

Author: Jasper Veerman

Location:    Lecture Hall 4, Erasmus Medical Center Rotterdam

Date:            Monday, March 2 17:00-18:00

 

The cerebellum is the part of the central nervous system, and is primarily responsible for the coordination of movement. Many neurons are connected to this region, whose input axons are called mossy fibers. The fast transmission of signals in synapses is important for processing information quickly. Using high frequencies to encode signals, mossy fibers can act at speeds of 800 Hz. In 3-6 week old mice the action potential is in the range of . Using artificial stimulation, the highest possible frequencies at which the signal is still properly passed on, is just above 1.6 kHz.

It is fascinating try to understand how calcium vesicles, transmitters of the signal, can fuse so synchronously in such small time intervals. In trying to do so, the vesicles were imaged using several calcium indicators. Of course, each of these indicators reduces the freedom of movement, so only a combination of several calcium indicators allows for back extrapolation of the endogenous – without indicators – situation. From this experiment, it was concluded that the cytosolic solution does not capture much of the calcium coming in.

In trying to explain this, further calcium transmission measurements had to be done at high frequency bursts. It was found that after each action potential, the calcium concentration increased, saturating after 20 action potentials. This saturation poses a problem for passing on additional signals, since after these 20 signals, an additional signal will not result in an increase of calcium concentration, making it hardly detectable. Therefore, at these high firing rates, the system approaches its limit. It can cope with the first few signals, but needs some time to recover afterwards.

Measuring forces on polypeptide chains with a simple trick

Bionanoscience Seminar, 12.03.2015

Speaker: Gunnar von Heijne (Stockholm University)

Author: Edgar Schönfeld

In some situations, the force on a polypeptide chain can be measured with a technique that does not make use of magnetic tweezers and similar stuff. This is for example the case when a polyptide migrates through the inner cell membrane in E. coli via a special channel, the ‘translocon’. In order for this to take place the migration must happen cotranslational, meaning that the ribosome is docked to the translocon while the nascent chain travels through the translocon and does not fold into a protein before it has reached the other side of the inner membrane. I learned about the existence of the translocon for the first time in this seminar, and so did some of the PHD students that attended the seminar with me.

translocon
The Leader-Peptase protein (LEP) during translation

 

One of the proteins that uses the translocon in the way I described is the protein LEP (“Leader Peptase”). It is a transmembrane protein itself and uses the translocon to reach its position in the membrane. The research team used LEP as a model system to measure the forces it is exposed to during its passage through the translocon. Their unconventional approach to measuring forces was the incooperation of so-called “arrest peptides” (AP’s) into the LEP protein, but before I get into the details of this mechanism I will explain what AP’s do.

An arrest peptide is a sequence of amino acids that induces translational stalling. An example is the sequence HAPIRGSP from the SecM protein of E.Coli. Whenever this sequence is translated by the ribosome the Proline (P) at the end of the sequence will not be incooperated well, which results in a blockage of further elongation of the nascent chain. However, the cell seems to have ways to overcome this blockage. It is believed that it does so by applying a ‘pulling force’ on the nascent chain, in the exact moment when the elongation stalls. In the case of LEP this force is provided by another protein that is translated parallely, and the research team of G.v Heijne measured this force.

The fractions of arrested and fully translated LEP protein
The fractions of arrested and fully translated LEP protein (pulse-chase technique)

In order to prepare LEP for their experiment, they genetically engineerd a 19 amino acid long hydrophobic sequence H and an AP near the C-terminus of the LEP polypeptide. H and AP are thereby a distance L apart. The researchers had reason to speculate that the force depends on L and on the hydrophobity of H. Thus they conducted a large number of experiments in which they varied the distance L and the hydrophibity of H. The engineerd LEP sequences were cloned into a plasmid and introduced into E.coli. Then the proteins where radioactively labelled, the cells were lysed and the protein products were made visible on a gel. Everytime two bands were seen, one representing fully translated LEP and the other incompletely (arrested) LEP. The intensity of these bands varied with different distances L and hydrophobities. At a distance L of circa 30 and 40 aminoacids the intensity of the band representing the fully translated LEP was at a maximum, which indicates strong pulling forces. As we can see, the ratio of fully-translated and arrested LEP fractions gives a good benchmark of the force. Despite the high precision of the force measurements the translation into Newton is not very accurate yet. The forces we are talking about are in the range of circa 10 to 40 pN.

The power of this method of force measurement is that it is relatively simple and precise and can be applied in many other situations. The team of G.v Heijne used it for instance in a later experiment in which they measured the electrostatic force that a peptide chain has to overcome to migrate through a membrane.

Special Thanks to Gunnar von Heijne, who sent me the ppt of his presentation to share it with you.

 

Presentation: presentation_g_v_heijne

Papers:

(Nature, 2012) http://casimir.researchschool.nl/upload/files/nat_struct_mol_biol_2012_ismail.pdf

(Nature, 2015) http://casimir.researchschool.nl/upload/files/nat_struct_mol_biol_2015_ismail.pdf

When DNA replication runs into a problem

Hematology Seminar by Puck Knipscheer

Erasmus Medical Center, Department of Hematology 19-1-2015

At the moment, Puck Knipscheer is the group leader of the Knipscheer research group at the Hubrechts Institute Utrecht. This group is currently trying to get a better understanding of the so called Fanconi Anemia pathway (FA pathway).  This pathway plays an important role in the DNA repair mechanism that repairs interstrand crosslinkings (ICLs). As can be seen in figure 1, interstrand crosslinking (ICL) is the phenomenon that the two strands of DNA are linked together and can be induced by exo- and endogenous agents such as carmustine and nitrogen mustard, which are agents used in chemotherapy. This means that understanding interstrand crosslinking and its repair mechanisms could be and already is very useful in treatment of cancer.

puckhematology
Your body, and that of many other animals, has a repair mechanism to repair interstrand cross links and as said, the FA pathway plays an important role in this. What the FA pathway basically does is coordinating and initiating several DNA repair mechanisms. This is done by the ubiquitination of the proteins FANCI and FANCD2, who in their turn activate other repair mechanisms like homologous recombination, nucleotide excision repair and mutagenic translesion synthesis. People who have a mutation in their DNA that prevents the pathway from being executed suffer from the genetic disease Fanconi Anemia (FA). These people have several symptoms, like developmental defects, bone marrow failure, increased cancer risks and cellular sensitivity to ICLs. Understanding the pathway, like Puck Knipscheer aims to do, can therefore contribute to both cancer treatments as well as understanding and perhaps healing of FA. DNA replication and repair is studied Xenopus Laevis (African clawed frog) egg extracts, because they are known to have unique form eukaryotic DNA replication and are fully soluble.

Kasper Spoelstra – wkspoelstra@hotmail.nl