Special projects between Bavaria and Florida

Prof. Dr. W. Goldmann

Biomedizinische Physik
Friedrich-Alexander-Universität Erlangen-Nürnberg

Dr. F. Rico

Miller School of Medicine
University of Miami

Mechanical activation of beta2 integrin

Beta2-integrins were chosen as a model system to study the structure-function relationship of integrins because of their high significance in immunological, inflammatory and neoplastic diseases. We will measure the magnitude of forces exerted on integrins in response to altered internal mechanical stresses. Magnetometry will be done on antibody-bound beads (precoated with monoclonal antibodies IB4, 24, 107) at sizes between 0.1 and 4.5 µm diameter in the presence of various divalent cations (Mn++, Ca++ und Mg++) to determine the activation of integrins in K562 stably transfected cells. Biochemical events of the mechanically-stimulated beta2 integrin signaling will be monitored by Western Blots using antibodies against specific phosphotyrosine residues on src and src-kinase substrate, CAS (Crk-associated substrate).

Final report

Experiments carried out using magnetometry and beads of 4.5 µm ∅ precoated with the monoclonal antibody IB4 show that bead binding occurred on transfected K562 cells expressing the CD11b/CD18 receptor. This was tested under non-activating (1 mM Ca++/Mg++ ) and activating (0.5 mM Mn++ ) conditions, which both showed bead binding. Thereafter the method of nanoscale particle tracking was used in which IB4-coated beads served as fiducial markers of CD11b/CD18 receptors and the actin cytoskeleton. Results from bead motion experiments indicate: i) persistent bead motion under all conditions which is dependent on the actin cytoskeleton, and that ii) ligated beads with activated (Mn++-treated) integrins move less diffusively (i.e. more directed) than on inactivated cells (Mg++ and Ca++-treated). In future experiments, we will determine how integrin-bead-binding motion translates into cellular biochemical signals under the above conditions using Western Blot analysis.


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