Matrix Organization and Dimensionality

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications, trials & patents

Abstract

We are currently exploring the cell-matrix adhesions formed to 2D compared to 3D fibrillar extracellular matrix under dynamic conditions. An extracellular matrix molecule that has been studied in considerable depth in regular 2D cell culture is fibronectin. We and others previously described two distinct mechanisms for assembly of fibrillar fibronectin, one involving lateral translocation of the alpha5-beta1 integrin along flat tissue culture substrates, and a second mechanism involving the translocation of entire focal adhesions along a basement membrane substrate driven by an actomyosin "contractile winch" system. We are continuing these studies of the mechanisms of fibronectin matrix assembly. We are also exploring in depth our previously published observation that there is an unusually high level of integrin activation in a 3D fibrillar collagen environment to determine the role of actomyosin contractility in this activation state using NAMs (New Approach Methodologies), in particular by using synthetic and cell-derived 3D extracellular matrix models using human cells. Our previous research had provided an in-depth characterization of the effects of temperature on the formation of collagen type I fibrils, including striking changes in local fibril stiffness using atomic force microscopy (AFM) with a tiny probe tip to clarify that local fibril thickness and stiffness could be augmented by polymerization at low temperature (e.g., 12 degrees centigrade). Although originally established using in-house highly purified rat-tail collagen, we collaborated to first confirm that this phenomenon could be documented for commercial rat-tail collagen using AFM. These fibrillar collagen preparations mimic normal versus more fibrotic tissues that have thicker, stiffer fibrils. These preparations were used in a collaborative study to establish that perivascular cells can serve as key sensors and mediators of vascular capillary structural/mechanical changes. Cancer invasion through basement membranes is the initial step of tumor dissemination and metastasis, and crossing of this flat, two-dimensional tissue barrier is a key pathological sign of invasion. We used a three-dimensional in vitro invasion model consisting of human cancer cell spheroids encapsulated by a basement membrane and embedded in 3D type I collagen gels to visualize early events of cancer invasion using confocal microscopy. Outward motility of a long, slender protrusion of the cancer cell body is followed by attachment to collagen using the alpha2-beta1 integrin, followed by inward three-dimensional translocation of collagen inward toward the expanding spheroid. This process occurs without the help of accessory cells such as cancer-associated fibroblasts or macrophages. Contractility of the long protrusion attached to collagen fibrils helps the cancer cell to squeeze through even small basement membrane perforations for subsequent migratory invasion in a 3D collagen matrix. We are continuing to characterize these novel, unusually long and prehensile protrusions in cancer cell function. These studies are providing insights into the roles of dimensionality and specific genes in the interactions of cells with the extracellular matrix. They can range from a "1D" interaction as a cell interacts with a single bundle of collagen, to 2D on basement membranes and flat substrates, to 3D in native interstitial extracellular matrices. Understanding the differing nature of these interactions may provide insights useful for rational tissue engineering. In all our research projects, our Section emphasizes rigorous and responsible conduct of research to conduct investigations that are reproducible and transparent, e.g., by describing in detail our specific methods, reagents, and the instrument settings we use, to be able to ensure that our work can be replicated. We perform at least 3 independent biological repeats (vs. technical repeats) for each experiment we report, as well as using orthogonal approaches with skepticism to test any important conclusion. A key component of rigorous and reproducible research, we feel, is the continuous conscientious use of electronic lab notebooks that provide forensic tracking and backups (we use the Federal version of LabArchives), as well as retaining two backups of all primary data, of which one copy is retained at NIH after a lab member leaves. We evaluate and communicate error and uncertainty by using quantification, unbiased data analysis approaches, and appropriate statistical tests. In our publications, we discuss reservations, caveats, and limitations of our work and the assumptions on which it is based. We aim to construct experimental tests to obtain yes/no and/or quantitative answers in order to rigorously test our hypotheses. We welcome negative results as opening new avenues of exploration into areas that had not been predicted. We avoid conflicts of interest in our research, as well as in our reviewing for journals and grant funding agencies, and we take care to submit our research to journals with unbiased peer review, as well as avoiding “predatory” journals. We encourage collaboration and interdisciplinary research, which enriches and broadens our approaches. This approach includes sharing our underlying primary data as much as practical, e.g., by deposition in public repositories after publication of our research. Public availability of the raw data underlying research allows others to evaluate and replicate our findings. We also continue to emphasize the training and mentoring of the next generation of biomedical researchers.