Crocker Lab	University of Pennsylvania
	Department of Chemical and Biomolecular Engineering

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Research Overview

Our research interests are in soft matter physics and its borders with cell biology. Our expertise lies in figuring out how to make careful mechanical measurements on very small objects, from cells and colloidal particles all the way down to single macromolecules. On the one hand, we use novel methods originally developed for studying soft matter to probe interesting biological systems, such as the cytoskeleton of eukaryotic cells. Alternatively, we can use biochemistry to construct novel and interesting soft-matter experiments and materials and perhaps ultimately to engineer useful devices.

Cell Mechanics

It is becoming increasingly clear that mechanical stress and deformation play a significant role in the behavior of eukaryotic cells. It also appears likely that cells are mechanically sophisticated, having an array of mechanical senses whose outputs are integrated together and used during differentiation decisions, tissue remodeling and, of course, motility. Understanding these aspects of cell mechanics would seem to be a prerequisite both for understanding tissue growth and morphology at the cellular scale and realizing the ultimate potential of tissue engineering. Despite this potential impact, the mechanical response (e.g. rheology) of cells and its physical and biochemical determinants remain largely mysterious. The challenge of understanding cell mechanics is a consequence of the mechanical complexity of cells; it seems likely that different cell compartments and discrete structural elements will each have their own distinct mechanical response. Different measurement techniques might measure different components, or different combinations of components, all of which might depend on cell type. Our approach to dealing with this complexity is to apply a suite of four or five different techniques to a single cell type in our laboratory, and to seek results that are mutually consistent between two or more techniques. In a recent study, we found that the results of our four methods, along with a substantial portion of literature findings, could be collapsed onto two ‘master curves’. These curves, we concluded, corresponded to the different mechanical responses within two spatially distinct compartments, one close to the membrane and the other deeper in the interior. Much of our current effort is focused on using pharmacological interventions to determine the sub-cellular structures and molecular species responsible for these two observed mechanical responses, in other words, to dissect their molecular determinants. We have already determined, surprisingly, that actin and myosin appear to play no significant role in the mechanics of the deep interior. Ultimately, identifying the molecular players and their spatial arrangement is a must if we are to make contact with minimal in vitro cytoskeleton models and soft matter theory. In a second line of research, with our collaborators Andy Lau and Tom Lubensky we have developed a unique tool for quantifying the stress fluctuations within a soft material, which we call stress fluctuation spectroscopy. A major current area of soft-matter research relates to understanding materials containing active elements that cause internal deformation, sliding or stress, for example, molecular motors. While cells are obviously an example of such materials, how quantities such as motor activity and intracellular stress relate in general to the mechanical response and function remain unclear. Given the array of different motors and stress generation mechanisms in cells, it seems likely that the observed basal stress and stress fluctuations will be a superposition of many different sources, some of which may be intimately related to the mechanics and others that are not. That is, in addition to dissecting the determinants of cell rheology using techniques such as two-point microrheology, we are also currently using motor inhibitors and stress fluctuation spectroscopy to dissect the molecular determinants of intracellular stress generation.

Self-Assembly by Molecular Recognition

The term self-assembly describes the spontaneous organization of component parts, for example, atoms arranging themselves into an ordered crystal. Much attention has been placed on the idea of using self-assembly to ‘grow’ useful devices from nanoscopic synthetic parts (also called ‘bottom up’ assembly) rather than current nanofabrication techniques based on photolithography (‘top down’ assembly). Ideally, we would like to have a technology platform that allows us to self-assemble arbitrary, complicated structures from a large number of different parts, and to do so rapidly and with a low probability of forming defective assemblies. In principle, this would seem to require parts that exclusively stick to each other in a user specified way. In practice, one of the most successful approaches is due originally to Ned Seeman. This approach, called DNA nanotechnology, uses DNA to stick structural components together by exploiting the tendency of single stranded DNA to form a double helix preferentially with strands having a complementary base sequence. While this method has been used by a number of researchers to form an ever increasing array of complicated structures, the details of why some designed structures form while others do not remain poorly understood. In our lab, we are trying to piggyback on DNA nanotechnology to self-assemble novel structures from micron-sized objects, specifically micron-sized polymer spheres called colloids. We chose colloids because the formation of ordered colloidal crystals is a comparatively well understood and prototypical example of self-assembly, and colloids are large enough to be imaged using an optical microscope, making it relatively easy both to watch the assembly process as it occurs, and to see what you made in the end. We were the first group to successfully form colloidal crystals using interactions due to DNA (or any molecular recognition system). In the end, we found issues of colloidal stability, reversible binding, equilibrium growth and annealing to be critically important. Along the way we developed a novel method for modifying the surface of colloids with a polymer brush and functional groups. Our current efforts are directed at forming novel two and three dimensional alloy structures. This exploratory research receives input from detailed simulations by Talid Sinno’s group.

Binding in the Single- and Few-Molecule Limit

As a spin-off of our self-assembly work, we performed direct measurements of the effective pair potential of mean force prevailing between two colloidal particles bearing complimentary DNA. This effort required the refinement of an extended optical tweezer measurement system to achieve resolution at the few nanometer level. Direct measurements of the DNA induced interaction were well described by a semi-analytic model we developed based on polymer physics, chemical equilibrium and a generalized mass action principle. While our first measurements were at high DNA density (up to ~100 molecules could sterically reach between the colloids), we soon realized that our instrument was equally well suited to studying binding in the single or few molecule limit as well. That work confirmed what we had begun to suspect—that the kinetics of DNA hybridization near the melting temperature are non-exponential. We are currently investigating the possibility of making more biologically relevant studies of ligand-receptor binding. Many of the existing techniques for performing single molecule mechanics measurements are poorly suited for various technical reasons for studying binding at small forces, while our instrument is optimal for that condition. Unlike many surface techniques that yield single-molecule binding affinities and kinetics, our instrument can also study the more complicated and realistic case of multi-valent or cooperative binding processes in the few molecule limit.