On this page you can find a brief description of projects that are currently undertaken. For a background of these project, please refer to the page "Our interest".
The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, also known as ErbB proteins, are single-pass membrane proteins that signal through a kinase cascade. Under physiological conditions, their stimulation is initiated by ligand binding that induces dimerization and consequent activation of the intracellular kinase domain followed by tyrosine phosphorylation of their C-terminal domain. This very simple view is complicated by the fact that receptors are organized into larger-order assemblies both in the absence and presence of stimulation, and their activation involves rearrangement of receptors within these mesoscale clusters.
Currently undertaken projects are aimed at the following major problems.
A. Biological tissues are much more flexible than conventional cell culture surfaces implying that cells are usually studied under conditions that do not mimic the human body. Furthermore, malignant transformation alters the stiffness of tissues. For these reasons, we are interested in how culturing on surfaces of different stiffness affects clustering, mobility and signaling of ErbB proteins.
B. While the basic principle of receptor clustering and its alteration by ligand binding is widely accepted, several lingering questions remain. It is not fully understood what kind of forces generate mesoscale receptor clusters, and how mutations and receptor isoforms affect dimerization and the size/composition of larger, mesoscale clusters. To shed light on these principles, we are studying- how mutations in EGFR family members give rise to receptor activation despite being present is different domains of the protein- how different isoforms of ErbB4 differ in their biophysical and signaling properties- how intrinsically disordered domains contribute to the formation of signaling-competent receptor dimers and larger-order clusters.
C. Ligand binding can be easily studied if it is labeled by a fluorophore. Most ligands binding to ErbB family members are not available fluorescently conjugated. In order not to impair their binding properties, it is essential to ensure that the fluorophore is conjugated to the peptide at a defined, non-random location. We are devising ways to label these ligands fluorescently while preserving their binding properties.
Cell-penetrating peptides are short peptides, typically made of about 5–30 amino acids, that are able to enter cells by crossing the plasma membrane. Many of them contain several positively charged amino acids, such as arginine or lysine. Because the outer surface of the cell membrane and many membrane-associated molecules carry negative charges, these positively charged peptides can interact strongly with the cell surface. Importantly, they can often enter cells without causing obvious membrane damage. Some well-known examples of cell-penetrating peptides include TAT, penetratin, and polyarginine peptides.
A particularly useful property of these peptides is that they can carry other molecules into cells. They may be chemically linked to a cargo molecule or mixed with it through electrostatic interactions. The cargo can be very diverse, including DNA, RNA, siRNA, proteins, peptides, nanoparticles, or small-molecule drugs. This is important because many biologically useful molecules cannot cross the cell membrane efficiently on their own, especially if they are large, highly charged, or hydrophilic.
They may enter cells in different ways. In some cases, they can interact directly with the plasma membrane and pass through it directly without the involvement of any active, biological process. In many other cases, especially in living cells exhibiting very active membrane turnover, they are taken up by endocytosis. The exact mechanism depends on the peptide sequence, concentration, cargo, cell type, and experimental conditions.
Independent of which uptake pathway is used, cell-penetrating peptides have to cross a bilayer once so that they reach the cytoplasm. After endocytic uptake, they cross the endosomal membrane in a process called endo-lysosomal escape, while when taken up by direct membrane crossing, they traverse the plasma membrane.
However, there are still challenges, such as improving cell-type specificity, avoiding degradation, reducing toxicity, escaping from endosomes after uptake, and ensuring that the cargo reaches the correct intracellular target.
The positive charge of cell-penetrating peptides comes into interaction with the dipole potential of the membrane, a several hundred-millivolt, positive potential present inside the lipid bilayer. The dipole potential is due to the preferential orientation of carbonyl and water dipoles on the surface of the membrane. We have shown previously (paper 1, paper 2) that reduction of the dipole potential can enhance the cellular uptake of cell-penetrating peptides.
In our current projects, we are trying to optimize the structure of cell-penetrating peptides to deliver biologically relevant cargo to cells more efficiently. We are conjugating cell-penetrating peptides with sequences equipping them with cell specificity as well as extracellular matrix-penetrating potential.
The plasma membrane is a system in which lipid molecules are arranged in an ordered fashion. However, order is characterized by several different parameters that report on distinct physical or biological properties of the membrane.
A. The dipole potential is due to the non-random arrangement of dipoles of surface-attached water and the carbonyl groups of lipids.
B. Although the membrane interior is hydrophobic, water penetrates into the membrane, even into the hydrophobic layer to a certain extent. This is characterized by membrane hydration.
C. The rotational mobility of fatty acids or fluorophores incorporated into the membrane reports on fluidity that can be measured by the fluorescence anisotropy of membrane-embedded dyes.
D. The membrane surface is exposed to tension, which is partially due to principles very similar to the surface tension of water, but also partially the result of biological factors, such as the cytoskeleton or membrane turnover.
Currently, we are looking at
Experimental investigation of purinergic signaling is complicated by the complexity of the system generated by the expression of several receptor classes as well as the release and degradation of ATP by cells.
Therefore, responses to exogenous ATP are confounded by the expression of multiple receptor subclasses, as well as by ATP production and the degradation of both endogenously produced and exogenously applied ATP.
We are currently
Fluorescently-labeled antibodies are widely used in cell biology due to their ease of application and affordability. Although it has been known for quite some time that the labeling procedure negatively impacts the fluorescence properties of dyes and the affinity of the antibody, these shortcomings are mainly important for those only who would like to evaluate fluorescence intensities quantitatively. Since we belong to this group of researchers, we have been studying this phenomenon in the past years.
Our current efforts are aimed at elucidating what kind of local properties govern which sites are labeled in the antibodies.