UC Berkeley

The actin cytoskeleton plays a major role in driving cell migration. As cells navigate the surrounding matrix, the actin cytoskeleton dynamically adapts and transduces the geometrical cues into phenotype. The resulting cytoskeletal reorganization drives activation of mechanotransductive signals and has been shown to govern many aspects of cell biology. Studies using 2D platforms have produced many insights into the mechanisms of actin dynamics during cell migration, but how these mechanisms and broadly how the actin cytoskeleton contributes to motility through 3D matrices is not well understood. To increase understanding of 3D motility, we utilized an in vitro model of glioblastoma (GBM) invasion in engineered hyaluronic acid (HA) matrices to investigate the contributions of actin turnover proteins.

We employed a CRISPR knock-out (KO) screen to examine the contributions of core actin turnover proteins to 2D versus 3D motility. We first quantified cell speed and persistence in random single cell migration on 2D HA and then fold area change and farthest cell traveled of cell spheroids in 3D HA. Surprisingly, we found that only cofilin-1 (CFL) KO had differing effects on 2D versus 3D motility, and that CFL KO enhanced 3D motility. Further investigation revealed that CFL KO uniquely increased both the length and number of actin-based protrusions. With this data, we hypothesized that CFL KO stabilized actin filaments within the protrusions, allowing for better penetration of the surrounding matrix and thus increasing 3D motility.

Through targeted shRNA-induced knock-down (KD) of CFL, we validated the 2D and 3D motility results from the screen. Additional studies with Boyden chamber inserts, PDMS microchannels, and 2D scatter assays indicated that CFL suppression only enhanced protrusion stability and did not improve other mechanisms of 3D migration. Invasion assays in 3D collagen gels revealed that this effect is seemly HA-specific, as CFL KD cells did not have significantly increased fold area change or farthest cell traveled compared to NT control. Immunofluorescence staining demonstrated that CFL suppression did not alter protrusion architecture and that the protrusions expressed markers reminiscent of microtentacles and filopodia. Collectively, this work highlights the differing contributions of CFL to 2D and 3D motility and points to actin stabilization as a driver of GBM cell motility through HA matrices.