Protein assembly in healthy and pathological tissues by Atomic Force Microscopy (AFM) – AFM-2-BioMed

-Atomic force microscopy imaging of native membranes
-Atomic force microscopy imaging of reconstituted membrane proteins
-Atomic force microscopy imaging of supported lipid bilayers
-Lens laser micro-dissection
-PCR

-Atomic force microscopy imaging of the +cAMP conformation of Mlok1
-Atomic force microscopy imaging of clathrin (preliminary)
-High-speed Atomic force microscopy imaging rod outer segment disks (preliminary)
-Laser micro-dissection of lenses
-AQP0 mRNA analysis by PCR (preliminary)

-High-resolution and high-speed AFM imaging the membrane systems under varying conditions (pathological, addition of mediators)
-Mouse cataract model setup

None, so far, on the project topics.

Membrane proteins carry out many vital functions such as transport, energy transduction, signalling, and communication, etc. Notably ~70% of nowadays used drugs target membrane proteins, underlying the medical importance of this class of proteins. At present, the number of solved membrane protein structures is constantly increasing; however, the way these proteins are organized and work together in native membranes is usually unclear. At the same time it has been known that often in biomembranes the proteins assemble and work cooperatively as parts of complex supramolecular machineries. Thus, the lack of knowledge on supramolecular organization of membrane proteins creates certain bottlenecks for elaboration of complete synthetic views of membrane-related biological phenomena and therefore limits development of molecular-based approaches in medicine.

Atomic force microscopy (AFM) is a surface-sensitive technique employing a sharp nanometric tip apex to analyse the surface features. This technique has been proved to study membrane proteins in their native environment – the biomembranes. The resolution of AFM, and especially its signal-to-noise ratio is high enough to allow direct visualization of both the submolecular features and oligomeric organization of membrane proteins, and therefore AFM can describe the overall architecture of biomembranes.

Given the importance of membrane proteins in both physiological and medical contexts, we are proposing a comparative study of biomembranes from healthy and pathology-affected tissues. The AFM technique will be applied mainly as an imaging tool to depict how the membrane proteins are organized in the membranes, how a pathology alters this organization, and subsequently how drugs affect the structure of biomembranes.

We are planning to focus our research on the three following biological systems:

1). Membranes of eye lens fiber cells. Here, the membrane proteins assemble in characteristic junctional microdomains linking the neighbouring cells and thus providing the microcirculation flow in the tissue. It has been shown that malformation of these protein microdomains is related with cataract development. Animal models with artificially (drug) induced cataracts will be used to study the molecular mechanisms of cataract formation.

2). Disk membranes are located in the outer segments of retina’s rod cells. These membranes carry the rhodopsin molecules – the protein responsible for the initial step of vision, i.e. light absorption. It has been shown that the disk membranes have a distinctive spatial architecture: the membrane proteins are organized in protein domains separated by a lipid girdle. With use of animal models we will study the molecular bases of induced retinopathies.

3). Membranes rich in nicotinic Acetylcholine Receptor (nAChR), an important agent of the neuronal transmission. We will perform high-resolution AFM studies of this receptor molecule in its native environment - the membrane. Our aim will be to describe the organization of AchR molecules and show how binding of ligands/drugs can influence both conformation and supramolecular organization of the AChRs.

We believe that our studies will provide valuable data on fine structure of biomembranes involved in important physiological processes and will shed light on molecular aspects of related pathologies. In perspective, AFM has a potential to be used in clinical diagnostics, where in combination with other conventional analytical tools it will help making decisions on the medical treatment and thus contribute to the development of personalized molecular medicine.