Engineering Diffusion Barriers in the Plasma Membrane

Each cell is enveloped by a plasma membrane. This membrane fulfills a large number of tasks that are essential for the cell, including protection against the undesired influx of dangerous substances, but also the specific uptake of desired substances. A lipid bilayer represents the central structure of most plasma membranes. It is an extremely thin layer that is impenetrable for many substances, but which is still surprisingly dynamic and fluid. Hundreds of different types of proteins are embedded in this lipid layer, for example those proteins that transport certain substances from the outside to the inside in a highly selective manner. In our project we are trying to better understand one such transporter protein, namely the transporter for serotonin. Serotonin is a neurotransmitter, i.e. a messenger substance that is important for the transmission of signals between nerve cells. After the serotonin has been released into the vicinity of the nerve cell, a signal is triggered on nearby nerve cells. To end the signaling process, it is therefore necessary to remove serotonin from the vicinity of the nerve cell and make it available for further releases. In order to operate this uptake process efficiently, it seems important not to distribute the serotonin transporter evenly across the nerve cells, but to place it in those places where neurotransmitter uptake makes sense. In our project we therefore address two questions: What is the spatial arrangement of the serotonin transporter in the plasma membrane of nerve cells? To answer this question, we will use novel microscopy methods that make it possible to determine the distribution of the protein by means of single-molecule localization far below the classic optical resolution limit. Does the size of the protein play a role in the spatial arrangement? This question is based on a result from our joint preliminary work, which showed that the serotonin transporter is usually a structure made up of several proteins and moves as a so-called serotonin transporter oligomer in the plasma membrane. The mobility changes depending on the size of this oligomer, so that large protein complexes move more slowly than small ones. This difference is massively amplified as soon as immobile obstacles are built into the plasma membrane. Our assumption is now that nerve cells build immobile obstacles into the plasma membrane at certain points in order to virtually freeze the mobility of the transporter there. To test this, we will establish a semi-synthetic model system that will allow us to specifically build immobile obstacles in the plasma membrane and determine the effect on the movement of the serotonin transporter.

Transporter proteins are a class of proteins that facilitate the movement of an ion and/or molecule across the cell membrane. There are several different subclasses of transporters. Neurotransmitter transporters for monoamines, including serotonin, belong to the solute carrier 6 transporter family, which uses the electrochemical gradient of sodium for the transport of the monoamine. These transporters are primarily located perisynaptically in the pre-synaptic neuron. In this project we specifically studied the behavior of the serotonin transporter. This protein regulates the function of serotonin, which is colloquially known as the hormone of happiness. The serotonin transporter serves a crucial function within our nervous system: it is responsible for the reuptake of serotonin and thereby the termination of its action. Dysregulation of the serotonin transporter has been linked to multiple mental disorders such as depression and obsessive-compulsive disorder. The serotonin transporter is also the target of many medically approved and recreational drugs. Despite its clinical significance little is known about the mechanisms governing serotonin transporter assembly and function. Understanding its underlying molecular behavior, including movement and organization, will potentially allow for developing better treatment approaches. So far, there were limited options to selectively visualize the transporter in microscopy. Most modes of labelling were not exclusive to the serotonin transporter or posed other challenges with advanced microscopy techniques. Hence, we developed a version of the serotonin transporter with a short tag-sequence, which allows for specific labelling with custom fluorophores. This makes it ideal for a plethora of microscopy methods. We found that the developed transporter version retains its functionality and high-order oligomerization behavior. To assess the organization of the transporter in more detail, we performed superresolution experiments which allow for visualisation on a nanoscale. In particular, we were interested in the relation to the underlying cellular structures, which is known to arrange in a periodic pattern. We hypothesized that the serotonin transporter might be affected in its movement by those structures. It is a rather large molecule that has been shown to form high-order structures. First findings indicate that serotonin transporter arranges in a periodic fashion that may relate to the underlying cell structure.