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 those changes by altering their behavior. This response 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. All of these changes in behavior are triggered by chemical signaling pathways within the cells. Myofibroblasts are able to detect changes in the mechanical properties of their environment, i.e., the relative flexibility or softness of the area that they reside in, and they convert these mechanical signals into chemical signals in the cytoplasm, though a process called mechanotransduction. The complex cellular process of mechanotransduction, and the precise molecular pathways that allow myofibroblasts to sense changes in their microenvironment and respond appropriately to those changes, are not well understood, but this process is believed to involve the structural elements of the cell referred to as the actin cytoskeleton. In the current project, we used a team-science approach to invertigate these processes using completementary approaches from the fields of biology and biophysics. The investigators hypothesized that two specific protein molecules, called palladin and alpha-actinin, play critical roles in mechanotransduction, as both of these proteins had been shown to be important components of the actin cytoskeleton in many types of cells. To test this idea, Dr. Carol Otey (a cell biologist) created genetically engineered cultures of myofibroblasts in which the genes for palladin and alpha-actinin were silenced, and these cells were studied in her lab, and also shared with Dr. Arpita Upadhyaya, a biophysicist. Dr. Upadhyaya used sensitive biophysical methods to measure changed 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. These studies demonstrated that palladin, in particular, plays a key role in mechanotransduction. The studies also identified specific signaling pathways, known as the Rho GTPase pathways, 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 living cells. Dr. Otey's lab purified the palladin protein from insect cells, and the physical properties of actin filament networks formed by the binding of palladin, either alone or in combination with alpha-actinin, were studied by Dr. Upadhyaya's lab. The results of these experiments show that palladin and alpha-actinin work together as molecular partners to organize the structural elements of the cells. These results provide new insights into the precise molecular pathways that underlie the process of mechanotransduction, and set the stage for future studies that could explore, and perhaps improve upon, the process of wound-healing in animal models.
