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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Targeting Epigenetic Changes in Immune Cells: Implications in Disease

Speaker: Esteban Ballestar
Department: Bellvitge Medical Research Institute (IDIBELL), Barcelona Spain
Location: Erasmus MC
Date: July 13, 2017
Author: Teun Huijben

Esteban Ballestar got his Bachelor and Master degree at the University of Valencia in Spain, followed by a PhD. Afterwards he did a Postdoc abroad and returned to start his own research group at the same university. The main interest of his group is the DNA methylation, and especially in the context of diseases involving the immune system.

The first part of Estebans talk was about mapping DNA methylation in immunological diseases to understand which proteins are involved in the disease. The group of diseases they studied were Common Variable Immunodeficiencies Diseases (CVID) in which the body has not enough primary antibodies. These diseases are mostly caused by severe deficiencies in the number of switched memory B-cells. With switched B-cells we mean activated B-cells that start producing the antibodies in high quantities after recognizing the antigen. By mapping the DNA methylations of these B-cells, they hope to find genes that are differently methylated and are mostly likely causing the disease.

Methylation of DNA means that a methyl group is added to the 5-prime end of a cytosine (5mC) nucleobase. This can only be done if the cytosine is next to a guanine (see Figure 1). DNA methylation is maintained by de-novo DNA methyltransferases (mostly DNMT1, DNMT3A and DNMT3B). DNA methylations can be actively removed by demethylation, in which the 5mC is oxidized to a 5hmC or 5caC. Adding or removing methyl group to the DNA has an effect on the gene expression of that particular gene.

zakhari01

Figure 1: DNA methylation. The cytosine of the CG-pair gets methylated by a de-novo methyltransferase (DNMT). [S. Zakhari 2015]

To identify disease causing genes, Estebans group did DNA methylation profiling of the B-cells from a CVID patient. To eliminate as much side effects as possible, thy only investigated twins of which one sibling had CVID and the other was healthy. After collecting the B-cells, they performed DNA methylation profiling and looked at genes with different methylation profiles between the two brothers. They found 230 genes that are more methylated in the CVID patient and 81 genes that are less methylated. Gene ontology analysis showed that most of the genes were related to immune responses, indicating that changing the gene expression of these genes can cause an immune related disease.

All the genes that showed different methylation profiles between healthy and CVID patient, were taken into further research. The B-cells are sorted on the fact whether they were naive (not yet switched to active) or switched. They found that in healthy persons most genes got demethylated after the transformation from naive to switched. On the other hands, the same genes in CVID patients showed no decrease in methylation, a second indication that these genes are involved in the disease.

However, the question remains whether the different methylation profile itself causes the disease, or is it a downstream effect caused by other factors. To investigate this, more research needs to be done on this subject. Also, more methylation profiles of twins are needed to draw real conclusions about the disease causing genes, since one set of results of the statistically valid enough. Overall, the talk of Esteban was interesting and he is a very good speaker. Despite using many difficult immunology term, he explained very clear the research his lab is doing.