The continuing goal of this multifaceted project is to develop new physics-based methodologies and employ them to study complex biological structures and materials. Insights gained from studying model materials then are used in inquiries of specific phenomena of biomedical import. For instance, we have developed techniques based on fluorescence correlation spectroscopy (FCS) and quantitative microscopy that make it possible for us to examine the movement of particles--varying in size from small metabolites to viruses--through concentrated polymer solutions and dense, interconnected polymer matrices. Recently, we have focused on discerning how the swelling or shrinking a crosslinked hydrogel affects the translation of embedded nanoparticles, the purpose being to better comprehend underlying physical mechanisms. Several of these studies utilize models of non-biological origin that can be manipulated to provide insight into the behavior of somewhat more complicated biological materials. The knowledge gained ultimately should lead e.g., to better understanding of the behavior of extracellular matrix material in edematous tissue. Techniques developed in such investigations are now being employed to examine the movement of antibodies and viruses through vaginal secretions. In the latter case the goals are to understand how HIV and other viruses involved in sexually-transmitted diseases penetrate cervical mucus and other protective barriers to reach the cells which they infect. Particle tracking analysis of individual virions indicates that most are slowed down over 100-fold when compared with movement in water, and that their translocation is not driven by typical diffusion. Rather, a major factor is the relaxation of the matrix that follows mechanical perturbations such as stretching and twisting of a sample. Other methods developed in this investigation, principally fluorescence correlation spectroscopy (FCS), can be used to examine the way microbicides and other pharmaceutical agents penetrate vaginal secretions. In a related study, we have been investigating how scattering of incident and emitted light affects FCS measurements, in anticipation of using such techniques to study motions of molecules occurring within dense biopolymer matrices or optically-opaque tissues. In a different study we used novel computer-based structural modeling, combined with dynamic light scattering (DLS), static light scattering (SLS), and small angle neutron scattering (SANS), to examine conformations of clathrin triskelions in solution. Clathrin is a major protein involved in receptor-mediated endocytosis (RME), a process whereby eucaryotic cells take up growth factors and metabolites, and also regulate surface-bound receptors for those materials. As such, receptor-mediated endocytosis is an integral component of many normal and abnormal growth processes and is involved in other aspects of cell and tissue development. Clathrin, along with other proteins, is a key constituent of the coats that surround membrane vesicles participating in RME and certain other intracellular trafficking processes of eucaryotic cells. Knowledge gained from these and related studies has been used to establish a physical model of clathrin coat and network formation, and currently is being applied to investigations of the initiation of clathrin coated pit formation at the plasma membrane. The last example pertains to obtaining a basic understanding how physical boundaries influence spatial patterns that arise in concentrated ensembles of rod-shaped objects such as microtubules, amyloid plaques, and similarly-shaped biological assemblies. We constructed a biomimetic analog, composed of small hard rods confined within an enclosure of adjustable size and shape, that was subjected to mechanical shaking to mimic thermal excitation. The objective was to study the effects of steric interference between, e.g., microtubules, separately from other intermolecular interactions. We found that when the rods are confined to containers whose dimensions are of the same order of magnitude as the lengths of the rods, conditions exist where the rods self-organiize and experience a density-dependent isotropic-nematic structure transition. This model pertains at an elemental level to microtubule involvement in cell division. Recently, in order to investigate the role of cytoplasmic factors that act as molecular crowders, we added small spheres to the ensemble of objects undergoing thermal excitation. The pressure attributable to the presence of the spheres (akin to """"""""depletion forces"""""""") drives the rods to form assemblies that differ dramatically from those seen in the absence of the spheres, especially when the rods are constrained to lie near a surface. In particular, vibrofluidized rods in low density, when crowded by high densities of spheres and confined to quasi-2D planes, self-assemble into linear polymer-like structures. A paper describing this work has been submitted to the journal """"""""Soft Matter.""""""""
