Reconstitution of cadherin function

Embryonal morphogenesis is based on the progressive differentiation and reorganizaion of the embryonal cells, and the same applies to maintenance and reorganization of adult tissue and organs. Direct cell-cell-contacts via "cadherins" (calcium-dependent adhesion proteins) play a pre-eminent role in theses processes. The 2 nm thick cadherin filaments protrude 20 nm from the cell membrane and specifically bind to cadherins of the adjacent cell. While nearly 20 classic types of cadherins are known, only cadherins of the same type result in tight trans-adhesion between cells. As an example, the E-cadherins of an epithelial cell firmly connect it to the rest of the tissue, thus an epithelial tumor cell can metastasize only after it has successfully switched off E-cadherin function. Many other pathologic processes are also related to defects in cadherin function. Unfortunately the molecular mechanism of cadherin assembly is still unknown. The adhesive structures formed between two cells are too small for detailed analysis by electron microscopy, while the cadherin molecules themselves are too large for crystal structure analysis. The present project aims at reconstitution of cadherin trans-adhesion in an atomic force microscope. 1, 2, 4, or 8 cadherin fingers will be linked to a flexible polymeric fork, the stem of which is attached to a nonadsorptive gold surface. An equal/similar fork with 1, 2, 4, or 8 cadherins is flexibly linked to the measuring tip. Analysis of force- distance cycles is expected to show whether trans-adhesion is tip-to-tip or involves extensive overlap of antiparallel cadherins, whether interdigitated zippers are formed or not - and which parts of the cadherins come into direct contact with each other during trans-adhesion. In the second stage, the arrangements and lateral diffusion of the cadherins on the gold surface will closely resemble that on a natural cell membrane and the binding of defined cadherin oligomers will be measured optically. In the third stage, the binding of defined cadherin oligomers to native cell membranes will be monitored via fluorescence microscopy. The goal is a detailed understanding of cadherin function, thereby closing an important gap in the field of cell biology and providing a rational basis for therapeutic strategies.

The architecture and processes of living cells rely on specific interactions between particular biomolecules. Structures are built by long-lasting interactions while rapid processes are mediated by groups of molecules which transiently interact with defined forces. Measurement of these forces under different biochemical conditions help to elucidate mechanisms of pairwise/groupwise interactions and how these interactions are regulated. Such measurements can be done with a single pair of interacting molecules, yielding the discrete forces and distances necessary for separating complementary molecules under various conditions. In practice, one of the two interacting molecules is "chained" to the tip of a force microscope, using a thin, flexible polymer chain of similar length as biomolecule`s diameter. The complementary molecule is tethered to a flat surface in a similar way and the interaction forces are measured during repeated contact and separation of the complementary molecules. In one part of this project, the linking of proteins to microscope tips was greatly simplified, protein consumption was reduced from 0.2 to 0.005 mg, and the mechanism of coupling was unraveled, leading to general insights on protein coupling to biosensors and biochips. In particular, the speed of protein coupling to the tip was compared to the same linking reactions in aqueous solution, i.e. far away from the surface. The surprising result was that protein coupling on the tip was up to 104 -fold faster than anticipated from reaction in liquid solution. Pre-adsorption of protein was identified as the hitherto unrecognized "helper" that served to enrich the protein concentration from <0.01% in solution to nearly 100% on the surface, thereby reducing the coupling time from several months in liquid solution to 1 hour on the tip surface. In the second part of this project, the presence/absence of pre-adsorption was also found to be the predominant parameter of protein coupling to biosensor surfaces, in which cases anti-adsorptive surfaces are much preferred. The difficulty of sensor molecule coupling to anti-adsorptive surfaces was overcome with exceptionally fast coupling reaction that do not require help by pre-adsorption. The studies on solid surfaces were made possible by a new set of short linear molecules that possess a sulfur atom at one end which binds to gold, giving a dense lawn of the thin molecules on gold. The top portion of each molecule has the ability to oppose protein adsorption. In addition, a defined fraction of the top ends carry a robust but slow coupling function (aldehyde) which can be converted into any other (fast) coupling function within minutes while the chip is already mounted in the biosensor. The design of these new linker molecules was filed for patent (A1469/2006).