Biology subjects

Ding, E. A.

Publications and source records attributed to Ding, E. A..

4 recordsLinked to original sources

bioRxiv2025

Druggable genome CRISPRi screen in 3D hydrogels reveals regulators of cortactin-driven actin remodeling in invading glioblastoma cells

To identify new therapeutic targets that limit glioblastoma (GBM) invasion, we applied druggable-genome CRISPR screens to patient-derived GBM cells in micro-dissectible biomimetic 3D hydrogel platforms that permit separation and independent analysis of core vs. invasive fractions. We identified 12 targets whose suppression limited invasion, of which ACP1 (LMW-PTP) and Aurora Kinase B (AURKB) were validated in neurosphere assays. Proximity labeling analysis identified cortactin as an ACP1- AURKB link, as cortactin undergoes serine phosphorylation by AURKB and tyrosine dephosphorylation by ACP1. Suppression of ACP1 or AURKB in culture and in vivo shifted the balance of cortactin phosphorylation in GBM and reduced actin polymerization and actin-cortactin co-localization. Additional biophysical analysis implicated AURKB in GBM cell adhesion and cortical stiffness, and ACP1 in resistance to mechanical stress and shape plasticity needed for 3D migration. These findings reveal a novel targetable axis that balances kinase and phosphatase activities to regulate actin polymerization during GBM invasion.

cancer biology↗

bioRxiv2024

Dissecting neurofilament tail sequence-phosphorylation-structure relationships with multicomponent reconstituted protein brushes

Neurofilaments (NFs) are multi-subunit, bottlebrush-shaped intermediate filaments abundant in the axonal cytoskeleton, with "bristles" composed of the subunits disordered tail domains. Precisely how the tails variable charge patterns and repetitive phosphorylation sites mediate their conformation within the brush remains an open question in axonal biology. We address this problem by grafting recombinant NF tail protein constructs (NFL, NFM, and NFH) to functionalized substrates, forming phosphorylatable brushes of defined stoichiometry. Atomic force microscopy reveals that NFM-based brushes are highly extended, while brushes incorporating the much larger NFH are surprisingly compact even after multisite phosphorylation. A self-consistent field theory predicts multilayered brush morphologies for NFM and phosphorylated NFH brushes. Further experiments with designed mutants reveal that N-terminal negative charges in NFH repel phosphorylated residues to generate the multilayer morphology and that charge segregation in NFM promotes collapsed conformations, lending new insight into how NF tail sequence features determine protein brush conformation.

biophysics↗

bioRxiv2024

Viscoelastic high-molecular-weight hyaluronic acid hydrogels support rapid glioblastoma cell invasion with leader-follower dynamics

Hyaluronic acid (HA), the primary component of brain extracellular matrix, is increasingly used to model neuropathological processes, including glioblastoma (GBM) tumor invasion. While elastic hydrogels based on crosslinked low-molecular-weight (LMW) HA are widely exploited for this purpose and have proven valuable for discovery and screening, brain tissue is both viscoelastic and rich in high-MW (HMW) HA, and it remains unclear how these differences influence invasion. To address this question, hydrogels comprised of either HMW (1.5 MDa) or LMW (60 kDa) HA are introduced, characterized, and applied in GBM invasion studies. Unlike LMW HA hydrogels, HMW HA hydrogels relax stresses quickly, to a similar extent as brain tissue, and to a greater extent than many conventional HA-based scaffolds. GBM cells implanted within HMW HA hydrogels invade much more rapidly than in their LMW HA counterparts and exhibit distinct leader-follower dynamics. Leader cells adopt dendritic morphologies, similar to invasive GBM cells observed in vivo. Transcriptomic, pharmacologic, and imaging studies suggest that leader cells exploit hyaluronidase, an enzyme strongly enriched in human GBMs, to prime a path for followers. This study offers new insight into how HA viscoelastic properties drive invasion and argues for the use of highly stress-relaxing materials to model GBM.

bioengineering↗

bioRxiv2023

Glioma Cells Secrete Collagen VI to Facilitate Invasion

While glioblastoma (GBM) progression is associated with extensive extracellular matrix (ECM) secretion, the causal contributions of ECM secretion to invasion remain unclear. Here we investigate these contributions by combining engineered materials, proteomics, analysis of patient data, and a model of bevacizumab-resistant GBM. We find that GBM cells cultured in engineered 3D hyaluronic acid hydrogels secrete ECM prior to invasion, particularly in the absence of exogenous ECM ligands. Proteomic measurements reveal extensive secretion of collagen VI, and collagen VI-associated transcripts are correspondingly enriched in microvascular proliferation regions of human GBMs. We further show that bevacizumab-resistant GBM cells deposit more collagen VI than their responsive counterparts, which is associated with marked cell-ECM stiffening. COL6A3 deletion in GBM cells reduces invasion, {beta}-catenin signaling, and expression of mesenchymal markers, and these effects are amplified in hypoxia. Our studies strongly implicate GBM cell-derived collagen VI in microenvironmental remodeling to facilitate invasion.

bioengineering↗