Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.

The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes implicated in both the complete demolition and fine size control of microtubule-based arrays are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here, we use atomic force microscopy (AFM) to image depolymerizing arrays at single-microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-scale cytoskeletal structures.

Irreversible synthesis of an ultrastrong two-dimensional polymeric material.

Polymers that extend covalently in two dimensions have attracted recent attention1,2 as a means of combining the mechanical strength and in-plane energy conduction of conventional two-dimensional (2D) materials3,4 with the low densities, synthetic processability and organic composition of their one-dimensional counterparts. Efforts so far have proven successful in forms that do not allow full realization of these properties, such as polymerization at flat interfaces5,6 or fixation of monomers in immobilized lattices7-9. Another frequently employed synthetic approach is to introduce microscopic reversibility, at the cost of bond stability, to achieve 2D crystals after extensive error correction10,11. Here we demonstrate a homogenous 2D irreversible polycondensation that results in a covalently bonded 2D polymeric material that is chemically stable and highly processable. Further processing yields highly oriented, free-standing films that have a 2D elastic modulus and yield strength of 12.7 ± 3.8 gigapascals and 488 ± 57 megapascals, respectively. This synthetic route provides opportunities for 2D materials in applications ranging from composite structures to barrier coating materials.

Single-Particle Studies Reveal a Nanoscale Mechanism for Elastic, Bright, and Repeatable ZnS:Mn Mechanoluminescence in a Low-Pressure Regime.

Mechanoluminescent materials, which emit light in response to elastic deformation, are demanded for use as in situ stress sensors. ZnS doped with Mn is known to exhibit one of the lowest reported thresholds for appearance of mechanoluminescence, with repeatable light emission under contact pressure <10 MPa. The physical basis for such behavior remains as yet unclear. Here, reliable microscopic detection of mechanoluminescence of single ZnS:Mn microparticles, in combination with nanoscale structural characterization, provides evidence that the mechanoluminescent properties of these particles result from interplay between a non-centrosymmetric crystal lattice and its defects, viz., dislocations and stacking faults. Statistical analysis of the distributions of mechanoluminescence energy release trajectories reveals two distinct mechanisms of excitation: one attributable to a piezo-phototronic effect and the other due to dislocation motion. At pressures below 8.1 MPa, both mechanisms contribute to mechanoluminescent output, with a dominant contribution from the piezo-phototronic mechanism. In contrast, above 8.1 MPa, dislocation motion is the primary excitation source. For the piezo-phototronic mechanism, we propose a specific model that accounts for elastic ZnS:Mn mechanoluminescence under very low pressure. The charged interfaces in stacking faults lead to the presence of filled traps, which otherwise would be empty in the absence of the built-in electric field. Upon application of external stress, local enhancement of the piezoelectric field at the stacking faults' interfaces facilitates release of the trapped carriers and subsequent luminescence. This field enhancement explains how <10 MPa pressure produces thousands of photons.

Cell derived extracellular matrix-rich biomimetic substrate supports podocyte proliferation, differentiation and maintenance of native phenotype.

Current technologies and available scaffold materials do not support long-term cell viability, differentiation and maintenance of podocytes, the ultra-specialized kidney resident cells that are responsible for the filtration of the blood. We developed a new platform which imitates the native kidney microenvironment by decellularizing fibroblasts grown on surfaces with macromolecular crowding. Human immortalized podocytes cultured on this platform displayed superior viability and metabolic activity up to 28 days compared to podocytes cultured on tissue culture plastic surfaces. The new platform displayed a softer surface and an abundance of growth factors and associated molecules. More importantly it enabled podocytes to display molecules responsible for their structure and function and a superior development of intercellular connections/interdigitations, consistent with maturation. The new platform can be used to study podocyte biology, test drug toxicity and determine whether sera from patients with podocytopathies are involved in the expression of glomerular pathology.

Biomimetic engineering of conductive curli protein films.

Bioelectronic systems derived from peptides and proteins are of particular interest for fabricating novel flexible, biocompatible and bioactive devices. These synthetic or recombinant systems designed for mediating electron transport often mimic the proteinaceous appendages of naturally occurring electroactive bacteria. Drawing inspiration from such conductive proteins with a high content of aromatic residues, we have engineered a fibrous protein scaffold, curli fibers produced by Escherichia coli bacteria, to enable long-range electron transport. We report the genetic engineering and characterization of curli fibers containing aromatic residues of different nature, with defined spatial positioning, and with varying content on single self-assembling CsgA curli subunits. Our results demonstrate the impressive versatility of the CsgA protein for genetically engineering protein-based materials with new functions. Through a scalable purification process, we show that macroscopic gels and films can be produced, with engineered thin films exhibiting a greater conductivity compared with wild-type curli films. We anticipate that this engineered conductive scaffold, and our approach that combines computational modeling, protein engineering, and biosynthetic manufacture will contribute to the improvement of a range of useful bio-hybrid technologies.

Nanoparticle Wettability Influences Nanoparticle-Phospholipid Interactions.

We explored the influence of nanoparticle (NP) surface charge and hydrophobicity on NP-biomolecule interactions by measuring the composition of adsorbed phospholipids on four NPs, namely, positively charged CeO2 and ZnO and negatively charged BaSO4 and silica-coated CeO2, after exposure to bronchoalveolar lavage fluid (BALf) obtained from rats, and to a mixture of neutral dipalmitoyl phosphatidylcholine (DPPC) and negatively charged dipalmitoyl phosphatidic acid (DPPA). The resulting NP-lipid interactions were examined by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). Our data show that the amount of adsorbed lipids on NPs after incubation in BALf and the DPPC/DPPA mixture was higher in CeO2 than in the other NPs, qualitatively consistent with their relative hydrophobicity. The relative concentrations of specific adsorbed phospholipids on NP surfaces were different from their relative concentrations in the BALf. Sphingomyelin was not detected in the extracted lipids from the NPs despite its >20% concentration in the BALf. AFM showed that the more hydrophobic CeO2 NPs tended to be located inside lipid vesicles, whereas less hydrophobic BaSO4 NPs appeared to be outside. In addition, cryo-TEM analysis showed that CeO2 NPs were associated with the formation of multilamellar lipid bilayers, whereas BaSO4 NPs with unilamellar lipid bilayers. These data suggest that the NP surface hydrophobicity predominantly controls the amounts and types of lipids adsorbed, as well as the nature of their interaction with phospholipids.

Automated fabrication of photopatterned gelatin hydrogels for organ-on-chips applications.

Organ-on-chip platforms aim to improve preclinical models for organ-level responses to novel drug compounds. Heart-on-a-chip assays in particular require tissue engineering techniques that rely on labor-intensive photolithographic fabrication or resolution-limited 3D printing of micropatterned substrates, which limits turnover and flexibility of prototyping. We present a rapid and automated method for large scale on-demand micropatterning of gelatin hydrogels for organ-on-chip applications using a novel biocompatible laser-etching approach. Fast and automated micropatterning is achieved via photosensitization of gelatin using riboflavin-5'phosphate followed by UV laser-mediated photoablation of the gel surface in user-defined patterns only limited by the resolution of the 15 µm wide laser focal point. Using this photopatterning approach, we generated microscale surface groove and pillar structures with feature dimensions on the order of 10-30 µm. The standard deviation of feature height was 0.3 µm, demonstrating robustness and reproducibility. Importantly, the UV-patterning process is non-destructive and does not alter gelatin micromechanical properties. Furthermore, as a quality control step, UV-patterned heart chip substrates were seeded with rat or human cardiac myocytes, and we verified that the resulting cardiac tissues achieved structural organization, contractile function, and long-term viability comparable to manually patterned gelatin substrates. Start-to-finish, UV-patterning shortened the time required to design and manufacture micropatterned gelatin substrates for heart-on-chip applications by up to 60% compared to traditional lithography-based approaches, providing an important technological advance enroute to automated and continuous manufacturing of organ-on-chips.

Patterning of sub-50 nm perpendicular CoFeB/MgO-based magnetic tunnel junctions.

Perpendicular magnetic tunnel junctions (p-MTJs) were patterned into nanopillars using electron-beam lithography to study their scaling and switching behaviour. Magnetoresistance measurements of annealed and unannealed p-MTJ films using scanning probe microscopy showed good agreement with Monte Carlo modeling. p-MTJ pillars demonstrated clear parallel magnetic states, both 'up' or both 'down' following AC-demagnetization. Significant variability in the resistance of p-MTJ pillars was observed and attributed to edge features generated during patterning or local inhomogeneity in the MgO layer.