The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore-size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and STED imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1–30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size, or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

Diffusion-controlled water permeation across bilayers of polyunsaturated phospholipids was measured by 17O nuclear magnetic resonance. In 100-nm extruded liposomes containing 50 mM MnCl2, water exchange between internal and external solutions was monitored via changes in the linewidth of the 17O water resonance of external water. Liposome size and shape were characterized by light scattering methods and determination of liposome trapped volume. At 25 degrees C, the following water permeability coefficients were determined: 18:0-18:1n-9 PC, 155 +/- 24 microns/s; 18:0-18:3n-3 PC, 330 +/- 88 microns/s; and 18:0-22:6n-3 PC, 412 +/- 91 microns/s. The addition of 1 M ethanol reduced permeability coefficients to 66 +/- 15 microns/s for 18:0-18:1n-9 PC and to 239 +/- 67 microns/s for 18:0-22:6n-3 PC. Furthermore, the addition of 50 mol% 18:1n-9-18:1n-9 PE reduced the water permeability from 122 +/- 21 microns/s for pure 18:1n-9-18:1n-9 PC to 74 +/- 15 microns/s for the mixture. The significant increase in water permeation for membranes with polyunsaturated hydrocarbon chains correlates with looser packing of polyunsaturated lipids at the lipid-water interface and the suggested deeper penetration of water into these bilayers. Ethanol may block water diffusion pathways by occupying points of water entry into bilayers at the interface. The addition of dioleoylphosphatidylethanolamine increases lipid packing density and, consequently, reduces permeation rates.

Subunit vaccines have been investigated in over 1000 clinical trials of cancer immunotherapy, but have shown limited efficacy. Nanovaccines may improve efficacy but have rarely been clinically translated. By conjugating molecular vaccines with Evans blue (EB) into albumin-binding vaccines (AlbiVax), here we develop clinically promising albumin/AlbiVax nanocomplexes that self-assemble in vivo from AlbiVax and endogenous albumin for efficient vaccine delivery and potent cancer immunotherapy. PET pharmacoimaging, super-resolution microscopies, and flow cytometry reveal almost 100-fold more efficient co-delivery of CpG and antigens (Ags) to lymph nodes (LNs) by albumin/AlbiVax than benchmark incomplete Freund’s adjuvant (IFA). Albumin/AlbiVax elicits ~10 times more frequent peripheral antigen-specific CD8+ cytotoxic T lymphocytes with immune memory than IFA-emulsifying vaccines. Albumin/AlbiVax specifically inhibits progression of established primary or metastatic EG7.OVA, B16F10, and MC38 tumors; combination with anti-PD-1 and/or Abraxane further potentiates immunotherapy and eradicates most MC38 tumors. Albumin/AlbiVax nanocomplexes are thus a robust platform for combination cancer immunotherapy.

Multimodal imaging‐guided photothermal therapy (PTT), for the therapy of cancer, based on a ferritin (FRT) nanocage loaded with the near‐infrared dye IR820 (designated DFRT) is demonstrated. The dual roles of DFRT (in imaging and PTT) are successfully balanced by using two different excitation wavelengths: 550 nm for high quantum‐yield fluorescence imaging on the one hand and 808 nm for photoacoustic imaging and PTT with high photothermal conversion efficiency on the other.

Many fundamental cell processes, such as angiogenesis, neurogenesis and cancer metastasis, are thought to be modulated by extracellular matrix stiffness. Thus, the availability of matrix substrates having well-defined stiffness profiles can be of great importance in biophysical studies of cell-substrate interaction. Here, we present a method to fabricate biocompatible hydrogels with a well defined and linear stiffness gradient. This method, involving the photopolymerization of films by progressively uncovering an acrylamide/bis-acrylamide solution initially covered with an opaque mask, can be easily implemented with common lab equipment. It produces linear stiffness gradients of at least 115 kPa/mm, extending from ∼1 kPa to 240 kPa (in units of Young's modulus). Hydrogels with less steep gradients and narrower stiffness ranges can easily be produced. The hydrogels can be covalently functionalized with uniform coatings of proteins that promote cell adhesion. Cell spreading on these hydrogels linearly correlates with hydrogel stiffness, indicating that this technique effectively modifies the mechanical environment of living cells. This technique provides a simple approach that produces steeper gradients, wider rigidity ranges, and more accurate profiles than current methods.

Chirality can be used as a design tool to control the mechanical rigidity of hydrogels formed from self-assembling peptides. Hydrogels prepared from enantiomeric mixtures of self-assembling β-hairpins show non-additive, synergistic, enhancement in material rigidity compared to gels prepared from either pure enantiomer, with the racemic hydrogel showing the greatest effect. CD spectroscopy, TEM, and AFM indicate that this enhancement is defined by nanoscale interactions between enantiomers in the self-assembled state.

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.