Cells in the body have a remarkable ability to sense "stiffness" in their environment - that is, a cell can distinguish between a hard substrate such as bone, and a softer substrate such as brain. This ability is critical for many aspects of cell function such as migration, wound healing and the proper formation of tissues and organs. In order to sense the mechanical properties of their environment, cells adjust their internal stiffness to match that of the external surface by reorganizing their internal cytoskeleton, which is made of a dynamic network of microscopic filaments. The main component of these filaments is a protein called actin. Various actin-binding proteins crosslink the actin filaments and organize them into multifilament bundles. Palladin is a newly identified actin cross-linking protein that is important in organizing these filament networks. Previous experiments have shown that palladin plays an essential role in embryonic development: when the palladin gene is silenced in mice, it results in lethal development defects that are characterized by a failure of cells to adhere properly to a substrate and to migrate appropriately. These deficits may arise because the cell's ability to adjust its internal stiffness is compromised in the absence of palladin. This proposal will test the hypothesis that palladin, by its ability to cross-link actin and its interaction with another actin cross-linker, alpha-actinin, determines the structure and mechanical properties of actin networks and enables the cell to sense its physical environment. Two types of approaches will be used to address this question. First, the structural and mechanical properties of actin networks assembled on a glass slide will be measured to elucidate how actin cross-linking by palladin contributes to actin organization. In addition, palladin levels will be genetically manipulated in living cells to study how altered actin organization, cellular stiffness and force generation impacts cellular mechano-sensitivity.
Broader Impact This collaborative proposal will enhance the understanding of essential biological processes that underlie cell movement and tissue formation. The training of graduate and undergraduate students in interdisciplinary approaches from Physics and Cell Biology will be an integral part of the work. The cell lines that will be developed as part of this project will be made freely available to other investigators following their publication. A graduate course in Cell Mechanics will be developed based on the conceptual framework of this proposal. The PI and co-PI will also encourage minority students as well as high school students from the area to participate in research as part of the Louis Stokes Alliance for Minority Participation program at the University of Maryland and the University of North Carolina Research Apprenticeship Program. The PI will organize a one week biophysics laboratory demonstration as part of the Summer Girls Program in the Department of Physics to encourage participation of female students in science and technology fields.
Cells in the body possess exquisitely sensitive abilities to detect changes in their microenvironment, and they can respond to these changes by altering their behavior. This plays an important role in normal physiological processes such as the healing of wounds and the remodeling of injured organs. In all organs of the body, cells called myofibroblasts sense when they are near a wound, and they respond by dividing (to create more cells to help heal the wound), by migrating into the wound and secreting collagen to fill the gap, and by contracting, which serves to bring the wound-edges closer together. Myofibroblasts are able to detect changes in the mechanical properties of their microenvironment, i.e., the relative flexibility or softness of the area they reside in, and they convert these mechanical signals into chemical signals, in a process called mechanotransduction. Mechanotransduction is believed to be one of the major ways that myofibroblasts recognize and respond to injury. The process of mechanotransduction, and the precise molecular pathways that allow myofibroblasts to sense changes in their environment and respond appropriately to those changes, are not well-understood. In the current project, we used a team-science approach to explore this question. The investigators hypothesized that two specific protein molecules, palladin and alpha-actinin which bind to actin filaments in cells, play critical roles in mechanotransduction. To test this idea, Dr. Carol Otey (a cell biologist) created genetically engineered lines of myofibroblasts in which the genes for palladin and alpha-actinin were silenced, and these cells were studied in her lab, and also in the lab of Dr. Arpita Upadhyaya (a biophysicist). Dr. Upadhyaya’s lab utilized sensitive biophysical methods to measure changes in the stiffness and contractility of the genetically-engineered myofibroblasts, while Dr. Otey’s lab focused on studying changes in the chemical signaling pathways within those same cells. The studies of genetically-engineered myofibroblasts demonstrate that palladin, in particular, plays a key role in cellular force generation and mechanotransduction. These studies also identified specific chemical signaling pathways, known as Rho GTPases, that require palladin in order to function normally. In addition, both labs worked together to study the properties of palladin and alpha-actinin, as purified molecules isolated from cells. Dr. Otey’s lab purified the palladin protein, and the physical properties of actin filament networks formed by the binding of palladin, both alone and in combination with alpha-actinin, were studied by Dr. Upadhyaya’s lab. The results of studies with purified proteins show that palladin and alpha-actinin work together as molecular partners to organize the structural elements of the cell (known as the cytoskeleton). The results from this work provide new insights into the process of mechanotransduction, in particular the role of actin organization in this process, and set the stage for future studies that could explore the process of wound-healing in animal models.
