The cell membrane as a point of contact

How do cells interact with their surroundings?

Receptors in the cell membrane going into action

© Maria Florencia Sánchez, Uni Münster

Specific proteins (green), called receptors, in the membrane of living cells are interacting with signal molecules (violet). We attached the signal molecules to a glass plate in a pattern of tiny dots. / Confocal fluorescence microscopy

The cell membrane is an elastic sheath that surrounds every cell in our bodies. It is the point of contact through which cells communicate with their surroundings. Via specific proteins in the cell membrane, known as receptors, the cells can sense and process a variety of information from their environment. The receptors bind to particular signal molecules that then transmit the information to the cell. The receptors assemble in groups, thereby amplifying the response in the cells. We want to gain a more precise understanding of how this clustering of receptors impacts the cells’ behaviour.

To trigger the mechanisms, we attached a signal protein to a glass plate in a pattern of tiny dots. Each dot has a diameter of three micrometres (0.003 millimetres). To achieve this, we used a special technique known as microcontact printing: we printed a biological material in a special design leaving dot-like regions empty. These were subsequently filled with signal molecules. We then placed the cells onto the glass plate. The experiment worked: where the dots in the image appear in white or paler violet, the green of the receptor overlays the violet of the signal molecule – in other words, receptors have formed clusters here. Many different signals interact at the cell membrane. The first step involves examining them immediately after the receptors have formed clusters. Several hours later, we can evaluate the effects they have on the behaviour of the cells – for example, the cells migrate, produce specific molecules or die. These processes play a role in tissue development, immune response and cancer metastasis.

Maria Florencia Sánchez
European Institute for Molecular Imaging, University of Münster
This study was conducted during my time at Goethe University Frankfurt.

Blood vessel cells seal their wounds

© Nikita Raj, Uni Münster

A view into the aorta of a mouse – the main artery leading away from the heart. The cavity inside the blood vessel is shown in black. The flow of blood can cause small wounds in the membranes of the vascular wall cells that are quickly sealed again by the cells (green areas around the hollow part of the aorta). / Confocal fluorescence microscopy

Cells in the body are exposed to constant mechanical strain. For example, muscle cells contract whenever we move, and the cells in the blood vessels are always in contact with the flowing blood, which exerts forces on them. This causes tears in the cell membrane that the cells can repair immediately. The survival of the cells depends on this because the cell membrane protects the interior of the cells and regulates the exchange of substances with their surroundings. We have discovered that tiny bubbles inside the cells, called early endosomes (blue), are involved in this repair process. When a cell is injured, the endosomes fuse with the cell membrane. They provide their own membrane, the material that surrounds them, and in doing so help reseal the wound. The emergency signal required for this to happen is triggered by calcium ions that enter the cell from the blood through the wound.

Our team has deciphered this mechanism using cell cultures of human blood vessel cells. We used a laser to artificially create wounds in their membranes and then performed high-resolution imaging to observe how the wounds were repaired. In experiments with mice, we were then able to confirm our hypothesis that wounds in blood vessel cells occur mostly at places where the blood flow, and thus the forces that act upon the blood vessel cells, change suddenly – namely at the regions where vessels branch off. One such region is shown in the image. A dye (green) has entered the injured cells together with the blood. The cells have immediately repaired the holes, which is why the dye is enclosed there, making the repaired areas visible under the microscope. We know that in atherosclerosis, a blood vessel disease, deposits in the vascular walls frequently accumulate at precisely those regions where the blood flow changes suddenly in the vessels – presumably this is also connected with the wounding of the cells. Understanding the underlying mechanisms of wound repair could potentially make it easier to identify the early stages of the disease.

Nikita Raj and Volker Gerke
Institute of Medical Biochemistry, University of Münster
The experiments with mice were conducted in cooperation with Oliver Söhnlein, Institute of Experimental Pathology at the University of Münster

Gateways for the transport of substances

© Thea Jacobs, Uni Münster

A layer of cells surrounding the maturing egg of a fruit fly. At the point where the membranes (pink) of three cells meet, the cells loosen their connections in a controlled manner and open the intercellular spaces (green). / Confocal fluorescence microscopy

Within multicellular organisms, cells build connections with one another forming layers of cells known as epithelia that cover the surfaces of organs and separate them from other tissues. Special connections between the cells ensure on the one hand that a cell layer is stable and will protect the body and organs against invading pathogens, and on the other that it remains permeable to certain substances or migrating cells. This allows, for instance, dissolved nutrients to be transported across epithelia, or immune cells to migrate from the blood vessels to sites of inflammation in the tissue. We investigate a related process in the developing eggs of the fruit fly.

The oocytes (egg cells) are enclosed by a layer of epithelial cells through which they absorb yolk proteins. Adhesion proteins on the surface of the cells hold the cells together and seal the spaces between the cells. Our team discovered that the cells gradually rearrange and break down specific adhesion proteins on their membranes. This process occurs where three cells meet – the points of contact between two cells are preserved. Over the next 16 hours, yolk proteins are absorbed into the egg cell. The process subsequently reverses, causing the intercellular spaces to close. This controlled regulation is crucial for maintaining overall tissue stability and enabling the rapid and efficient absorption of yolk into the egg. Furthermore, controlling these openings is vital because unregulated gaps in the tissue could potentially allow pathogens to enter.

Thea Jacobs and Stefan Luschnig
Institute of Integrative Cell Biology and Physiology, University of Münster

A fine-mesh sieve made of proteins

© Sarah Weischer, Sonja Bauer, Uni Münster

Two proteins (multicoloured and grey) in the cell membrane of a nephrocyte – a type of cell in fruit flies that filters substances. With the aid of super-resolution microscopy, we were able to precisely localise individual molecules (multicoloured) of one of the two proteins to the nearest five nanometres (0.000005 millimetres). / TIRF microscopy and DNA-PAINT

Video: For this microscopy technique we label the molecules such that they flash up under the microscope. As can be seen in the video, we take a large number of individual images and combine them to reconstruct an overall image. We then analyse which of the detected signals are so close to one another that they probably each originate from one particular molecule.

Research groups around the world have achieved rapid advances in light microscopy in recent decades. The technology uses laser light to make fluorescently labelled molecules shine. Particularly high-resolution techniques can even be employed to distinguish between individual molecules in cells. In this experiment we examined how proteins are arranged in the membrane of nephrocytes in fruit flies. Much like cells in the human kidney, nephrocytes filter waste substances out of the haemolymph – the “blood” in flies. Their cell membrane consists of many small invaginations over which a slit diaphragm comprising different proteins extends, acting like a fine-mesh sieve. Studying these filtration mechanisms in fruit flies can provide insights into the function of the human kidney and the mechanisms of kidney diseases.

The trick with many super-resolution microscopy methods is to make sure that the molecules being studied light up briefly one at a time under the microscope rather than all fluorescing at the same time. This is the only way we can distinguish between closely spaced molecules in the images. For the microscopy method we used here (DNA-PAINT), the proteins to be investigated are tagged with DNA strands. Though DNA is normally double-stranded, in this case we use only single strands. Their counterparts float in a liquid in which the tissue sample lies. They are fluorescently labelled and repeatedly dock to and then separate from their target strands. Because they remain briefly in place while bound to the target strands, they emit sufficient light signals in that instant for them to flash up under the microscope. As the range of such labelling and imaging techniques in light microscopy is huge, highly specific skills are required. Because we want to share our knowledge and microscopes, scientists at our university have joined forces to form the Münster Imaging Network – Microscopy. The network is coordinated from the Multiscale Imaging Centre, which is also home to some of the microscopes that can be used by research groups university-wide.

Sarah Weischer
Münster Imaging Network – Microscopy, University of Münster
The image was created during a research project pursued by Sonja Bauer and Michael Krahn, Medical Clinic D – Internal Medicine at Münster University Hospital. The expertise in DNA-PAINT was contributed to our Imaging Network by the research group led by Carsten Grashoff, Institute of Integrative Cell Biology and Physiology at the University of Münster.