Vincent T. Moy - US grants

Protein-mediated molecular adhesion: AFM studies. Numerous cellular processes including cell adhesion and cell migration are mediated by the transient interaction of adhesion molecules. Often, as in the case cell migration, there is a delicate balance between cell attachment and cell de-attachment. The strength of cell attachment is determined by the number of intermolecular bonds formed between membrane- bound receptors and their ligands on the opposing surface and the de- attachment force of the individual bonds. Whereas the number of bonds formed at the interface is closely related to the association constant of the protein-ligand interaction, the rupture force of these bonds is poorly understood and is the main focus of this proposal. The long-term objective of this proposed research is to achieve a fundamental understanding of the mechanisms involved in molecular adhesion. The current proposal examines the intermolecular forces of protein-ligand interaction in two model systems, the streptavidin-biotin pair and an antibody-antigen pair. In general, protein-ligand bonds are noncovalent and will spontaneously break by thermal agitation given sufficient time. The dissociation lifetime of the protein-ligand bond is accelerated and can be studied by applying an external force across the bond with an Atomic Force Microscope (AFM). Comprehensive measurements of force-life time relationships derived from AFM-induced separation of the streptavidin- biotin bond will be used to reveal the dissociation pathway and possible intermediate states of the complex. AFM measurements of mutagenized streptavidin will be used to identify key amino acid determinants responsible for adhesion to biotin. In contrast to the resilience of the streptavidin-biotin interaction, the binding of fluorescein to 4-4-20, an anti-fluorescyl antibody, can be thermodynamically manipulated by small changes in temperature, pH, and solvent system. The effects of these perturbations on the dissociation constant and binding enthalpy will be studied and correlated to force measurements to determine the reaction pathway of 4-4-20:fluorescein dissociation beyond the point of initial bond rupture. Together, these experiments will contribute to establishing a conceptual framework for understanding protein-mediated molecular adhesion.

DESCRIPTION (provided by applicant): The adhesive interactions between the leukocyte integrin, lymphocyte function associated antigen-1 (LFA-1) and its native ligand, intercellular adhesion molecule-1 (ICAM-1) are crucial for normal function of the immune system. It stabilizes the interactions of T lymphocytes and antigen-presenting cells during the process of T cell activation. The biophysical properties of the LFA-1/ICAM-1 interaction that are deemed important in this process include the ability of the complex to modulate between high and low affinity states and the intrinsic mechanical strength of the LFA-1/ICAM-1 complex. Until recently adhesion was examined through indirect methods that involved kinetic measurements or simple cell adhesion assays. Now, advances in atomic force microscopy (AFM) have enabled direct measurements of adhesive forces at the level of single ligand-receptor pairs. The AFM measurements, when combined with mutagensis experiments, can be used to identify the molecular determinants that are responsible for the major features in the dissociation potential of the LFA-1/ICAM-1 complex. Here, we propose to acquire AFM force measurements to investigate the mechanisms that contribute to the interaction between LFA-1 and ICAM-1 during initial T-celI/APC contact and during subsequent T-cell activation when adhesion is further strengthened. The first three objectives will measure the dynamic strength and identify the structural components of the LFA-1/ICAM-1 interaction and the last two objectives will explore mechanisms for the enhanced binding following T-cell activation. Results from these experiments will answer the following questions: 1) How does the LFA-1/ICAM-1 complex unbind? 2) How does the bond strength of the LFA-1/ICAM-1 complex change with the conformation of LFA-1? 3) What are the molecular determinants that permit the LFA-1/ICAM-1 complex to resist large pulling forces? 4) Is enhanced lymphocyte adhesion following cell activation due to receptor clustering? and 5) Does the dimeric structure of ICAM-1 strengthen its interaction with LFA-1? Ultimately, these experiments will help us achieve a better understanding of the biophysical mechanisms that determine ligand-receptor binding strength and could aid in the development of treatments for immune system related disorders.

This is a proposal to acquire an atomic force microscope (AFM) on an inverted optical microscope and to develop two AFM-related non-imaging instruments: one for measuring single-molecule force spectroscopy and inter-molecular forces; the other, for measuring elasticities of soft samples under physiological conditions at the nano-scale. Over the past 10 years, atomic force microscopy (AFM) has become an increasingly important tool in biological research. It has gained popularity in biological applications because, unlike electron microscopy, it can image samples under physiological conditions, including live cells undergoing biological processes. The AFM acquires a topographical image of the sample surface by raster scanning an atomically sharp probe over the sample. In addition to its different imaging modes, the AFM is a versatile instrument that can be applied as a nano-indenter and as a molecular force apparatus to probe the mechanical properties of the sample. As a nano-indenter, the AFM has provided direct measurements of the local viscoelastic properties of samples on the nanometer scale. As a molecular force apparatus, the AFM has been used to measure the unbinding force of individual ligand-receptor complexes and the unfolding of individual proteins. Another attractive feature of the AFM is that it can be readily combined with optical microscopy techniques such as FRET, FRAP, TIRF and confocal microscopy. By integrating optical microscopy and AFM into a single experimental platform, the optical image can be directly correlated with the AFM data, providing a powerful tool for studying biological process in situ and in real time.

The acquisition and development of these three instruments is the first step toward establishing an ultramicroscopy center at the university. The two instruments to be developed can be constructed very economically, based on the designs of existing AFMs from the principal investigator's laboratory; this will permit the commercial AFM to be dedicated to imaging applications. The commercial AFM will be the first imaging AFM in the South Florida area and will provide a much needed resource for the local research community. These instruments will provide valuable research opportunities for undergraduates and students from underrepresented groups as well as researchers from different disciplines within the university.

PROJECT SUMMARY The proposed research applies biophysical methods toward elucidating the underlying mechanism of Ca2+ triggered exocytosis of neurotransmitters during signal transmission at chemical synapses. Neurotransmitters (e.g., glutamate, GABA, catecholamine) within unstimulated neurons are sequestered within secretory vesicles docked at the plasma membrane of the presynaptic terminal. Upon the arrival of an action potential at the axon terminal, voltage-dependent calcium channels open and the resulting influx of calcium triggers a biochemical cascade that causes the neurotransmitter- containing vesicles to fuse to the plasma membrane, releasing their contents into the synaptic cleft. This process is mediated by SNARE proteins expressed on the plasma membrane and their counterparts on the vesicle's surface. Trans pairing of the neuronal v-SNARE (VAMP2) and t-SNAREs (syntaxin and SNAP-25) has been shown to form the essential molecular machinery necessary to induce vesicle fusion at the presynaptic terminal. The fusogenic function of the SNAREs is regulated by associated accessory molecules, including synaptotagmin and complexin. Although there have been considerable advances toward identifying the individual components of the fusion machinery in recent years, there is still numerous gaps in our understanding of SNARE-mediated membrane fusion and how the process is regulated. The central hypothesis of our research is that the interaction of the SNARE proteins generates a mechanical force that destabilizes the apposing membranes and thus lowers the energy requirement for membrane fusion. Calcium bound synaptotagmin promotes fusion by further lowering this energy requirement, whereas complexin inhibits fusion by preventing the SNARE complex from completely annealing. To test this hypothesis, the proposed research will employ state-of-the-art atomic force microscopy techniques to measure the force generated by the interactions of the cognate SNAREs. Results will determine if it is sufficient to bring the membranes to close proximity in order to initiate the SNARE-facilitated membrane fusion process. Moreover, we will determine by direct force measurements how the SNAREs, along with complexin and synaptotagmin, alter the energetics of the membrane fusion process, hence revealing the mechanism of SNARE- mediated membrane fusion.