Alina Hategan

Cross-correlated TIRF/AFM reveals asymmetric distribution of force-generating heads along self-assembled, "synthetic" myosin filaments.

Myosin-IIs rod-like tail drives filament assembly with a head arrangement that is often considered to be a symmetric bipole that generates equal and opposite contractile forces on actin. Self-assembled myosin filaments are shown here to be asymmetric in physiological buffer based on cross-correlated images from both atomic force microscopy and total internal reflection fluorescence. Quantitative cross-correlation of these orthogonal methods produces structural information unavailable to either method alone in showing that fluorescence intensity along the filament length is proportional to height. This implies that myosin heads form a shell around the filament axis, consistent with F-actin binding. A motor density of approximately 50-100 heads/micrometer is further estimated but with an average of 32% more motors on one half of any given filament compared to the other, regardless of length. A purely entropic pyramidal lattice model is developed and mapped onto the Dyck paths problem that qualitatively captures this lack of length dependence and the distribution of filament asymmetries. Such strongly asymmetric bipoles are likely to produce an unbalanced contractile force in cells and in actin-myosin gels and thereby contribute to motility as well as cytoskeletal tension.

Visualization of the dynamics of fibrin clot growth 1 molecule at a time by total internal reflection fluorescence microscopy

Individual fluorescently labeled fibrin(ogen) molecules and their assembly to make a clot were observed by total internal reflection fluorescence microscopy (TIRFM). We used the bleaching of the fluorescent labels to determine the number of active fluorophores attached nonspecifically to each molecule. From the total intensity of bleaching steps, as single-molecule signature events, and the distribution of active labeling, we developed a new single-molecule intensity calibration, which accounts for all molecules, including those “not seen.” Live observation of fibrin polymerization in TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics of individual fibrin fibers quantitatively at the molecular level. Some fibers thickened in time to thousands of molecules across, equivalent to hundreds of nanometers in diameter, whereas others reached an early stationary state at smaller diameters. This new approach to determine the molecular dynamics of fiber growth provides information important for understanding clotting mechanisms and the associated clinical implications.

Cells on gels: smooth and skeletal muscle cell responses to substrate compliance

Cell adhesion has been recognized as being regulated by receptor-ligand interactions, but substrate compliance is emerging as an equally important determinant of adhesion, structure, and ultimately cell state. For contractile cells, such as the Smooth Muscle Cell line A7r5, spreading was investigated as a function of both adsorbed collagen and substrate compliance. Collagen levels of 10/sup 3/ng/cm/sup 2/ produce a significant short term increase in cell spreading on rigid substrates. Longer-term spreading was dependent, however, on substrate compliance (controlled by a variably crosslinked polyacrylamide gel), in which cell area increased two-fold on stiff substrates (8 kPa) versus soft gels (1 kPa). To investigate their influence on cytoskeletal function, cells transfected with GFP-actin and - paxillin were controllably peeled using a translating micropipette and a shear fluid force. F-actin remodeling on stiff surfaces caused fracturing of the cytoskeleton during peeling, with the residual actin co-localized with paxillin. Preliminary results of fluorescent AFM on F-actin also show a change in assembly that marginally translates to a change in rigidity on stiff substrates. Results were also obtained with the skeletal muscle line C2C12 and indicated only a short term dependence on substrate compliance. This study suggests that the coupling of short-term chemical signaling and long-term mechanical signaling contributes to contractile cell mechanotransduction, ultimately affecting cell remodeling.

HIV-Tat Protein Enhances Amyloid Beta Peptide Aggregation

We show that amyloid beta 1-40 peptide aggregation under physiological conditions in the presence of HIV-Tat protein results in significant structural modifications. Atomic force microscopy imaging shows that the predominant typical singular uniform amyloid fibrils that formed at a 200 micromolar concentration of amyloid, turned into a population with more double twisted fibers when 0.08 micromolar HIV-Tat was present and at higher Tat concentrations (0.4 to 1.8 micromolar) it turned into populations with predominantly thick unstructured filaments and nonspecifically aggregated large patches. At a 1/10 molar ratio of HIV-Tat to amyloid beta peptide, the fibrils were much larger and irregular along the length, their dimensions being similar to those of amyloid fibrils formed at extreme concentrations (>1 mM), but without the uniformly striated structure. The rupture length under air flow of filaments with similar dimensions increased significantly with the presence of HIV-tat protein at polymerization, suggesting internal structural changes within the filament backbone that lead to bigger mechanical resistance. Importantly, the presence of Tat in the amyloid fibrils significantly increases the neurotoxicity of the fibrils, as shown in neuronal cell culture experiments. Future studies will involve localization of Tat molecules throughout the amyloid filaments and aggregates, for a better understanding of the interaction between HIV-Tat protein and amyloid beta peptide.