Bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), have high rates of antimicrobial tolerance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome antimicrobial tolerance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a nanoscale drug carrier for antimicrobial codelivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs outperforming negatively charged formulations. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic codelivery to treat recalcitrant bacterial infections.

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.

To control their shape and movement, cells leverage nucleation promoting factors (NPFs) to regulate when and where they polymerize actin. Here we investigate the role of the immune-specific NPF WASP during neutrophil migration. Endogenously-tagged WASP localizes to substrate-induced plasma membrane deformations. Super-resolution imaging of live cells reveals that WASP preferentially enriches to the necks of these substrate-induced membrane invaginations, a distribution that could support substrate pinching. Unlike other curvature-sensitive proteins, WASP only enriches to membrane deformations at the cell front, where it controls Arp2/3 complex recruitment and actin polymerization. Despite relatively normal migration on flat substrates, WASP depletion causes defects in topology sensing and directed migration on textured substrates. WASP therefore both responds to and reinforces cell polarity during migration. Surprisingly, front-biased WASP puncta continue to form in the absence of Cdc42. We propose that WASP integrates substrate topology with cell polarity for 3D guidance by selectively polymerizing actin around substrate-induced membrane deformations at the leading edge. A misregulation of WASP-mediated contact guidance could provide insight into the immune disorder Wiskott-Aldrich syndrome.

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.