Cell Protrusion Dynamics on Suspended Nanofiber Networks
Protrusions are cytoplasmic extensions from the primary cell body that are used by cells to sense their fibrous surroundings. The ability of cells to extend protrusions underpins key biological processes such as cell migration and extracellular matrix (ECM) degradation. The importance of protrusions as the precursor to focal adhesion formation and subsequent migration in both normal and cancer cells has been consistently highlighted in the literature. Regarding ECM degradation, protrusions have been implicated in cleaving and reorganizing the surrounding ECM through the recruitment of matrix metalloproteinases (MMPs). Thus, elucidating the behavior of single cell protrusions is crucial towards expanding our understanding of how cells sense and interact with their surroundings, ultimately contributing to critical phenomena such as cancer metastasis, wound healing, etc.
At STEP Lab, we design suspended fiber networks with precisely tunable architecture and fiber diameter to investigate how single cells (both cancer and non-cancer) sense and interact with fibers by extending protrusions. We deposit large diameter “base fibers” orthogonal to smaller diameter “protrusive fibers”. The resulting mismatch in fiber curvature constrains bulk cell body migration along the base fiber, allowing us to investigate individual protrusions extended along the protrusive fibers. Using these suspended fiber networks we have observed the following protrusive behaviors:
Force-exerting perpendicular lateral protrusions in fibroblastic cell contraction
Aligned extracellular matrix fibers enable fibroblasts to undergo myofibroblastic activation and achieve elongated shapes. Activated fibroblasts adhere to the extracellular fibers and contract, perpetuating the alignment of these fibers. This poorly understood feedback process is critical in chronic fibrosis conditions, including cancer. Here, using fiber networks that serve as force sensors, we identify “3D perpendicular lateral protrusions” (3D-PLPs) that evolve from lateral cell extensions named twines. The specific morphology of PLPs enables them to exert force on parallel neighboring fibers, causing cells to increase their contractility. Twines originate from stratification of cyclic actin-waves traversing the cell and swing freely in 3D to engage neighboring fibers. Once engaged, a lamellum forms and extends multiple secondary twines, which fill in to form a sheet-like PLP, in a force- entailing process that transitions focal adhesions to elongated 3D-adhesions. Controlling the geometry of extracellular networks confirms that anisotropic fibrous environments support 3D-PLP formation and function, suggesting an explanation for cancer-associated desmoplastic expansion at single-cell resolution.