ATM phosphorylates PP2A subunit A resulting in nuclear export and spatiotemporal regulation of the DNA damage response.

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

Silk Reconstitution Disrupts Fibroin Self-Assembly.

Using atomic force microscopy, we present the first molecular-scale comparison of two of the most important silk dopes, native (NSF) and reconstituted (RSF) silkworm fibroin. We found that both systems depended on shear to show self-assembly. Significant differences in the nature of self-assembly between NSF and RSF were shown. In the highest studied concentration of 1000 mg/L, NSF exhibited assembly into 20-30 nm-wide nanofibrils closely resembling the surface structures found in natural silk fibers. RSF, in contrast, showed no self-assembly whatsoever at the same concentration, which suggests that the reconstitution process significantly disrupts silk's inherent self-assembly capability. At lower concentrations, both RSF and NSF formed fibrils under shear, apparently denatured by the substrate. Using image analysis, we quantified the properties of these self-assembled fibrils as a function of concentration and found low-concentration fibrils of NSF to form larger continuous structures than those of RSF, further supporting NSF's superior self-assembly capabilities.

Self-strengthening tape junctions inspired by recluse spider webs.

Adhesive tapes are versatile and widely used yet lack adhesion strength due to their tendency to fail via peeling, a weak failure mode. A tape with surprising adhesive properties is the recluse spider's 50 nm-thin silk ribbon with a 1 : 150 aspect ratio. Junctions of these microscopic sticky tapes can withstand the material's tensile failure stress of ≈1 GPa. We modeled these natural tape-tape junctions and revealed a bi-modal failure behavior, critically dependent on the two tapes' intersection angle. One mode leads to regular, low-strength peeling failure, while the other causes the junction to self-strengthen, eliminating the inherent weakness in peeling. This self-strengthening mechanism locks the two tapes together, increasing the junction strength by 550% and allowing some junctions to remain intact after tensile failure. This impressive adhesive strength of tapes has never before been observed or predicted. We found that recluse spiders make tape junctions with pre-stress to force the locked, high-strength failure mode. We used this approach to make junctions with synthetic adhesive tapes that overcame the weak peeling failure.

Digital Polymerase Chain Reaction Paired with High-Speed Atomic Force Microscopy for Quantitation and Length Analysis of DNA Length Polymorphisms.

DNA length polymorphisms are found in many serious diseases, and assessment of their length and abundance is often critical for accurate diagnosis. However, measuring their length and frequency in a mostly wild-type background, as occurs in many situations, remains challenging due to their variable and repetitive nature. To overcome these hurdles, we combined two powerful techniques, digital polymerase chain reaction (dPCR) and high-speed atomic force microscopy (HSAFM), to create a simple, rapid, and flexible method for quantifying both the size and proportion of DNA length polymorphisms. In our approach, individual amplicons from each dPCR partition are imaged and sized directly. We focused on internal tandem duplications (ITDs) located within the FLT3 gene, which are associated with acute myeloid leukemia and often indicative of a poor prognosis. In an analysis of over 1.5 million HSAFM-imaged amplicons from cell line and clinical samples containing FLT3-ITDs, dPCR-HSAFM returned the expected variant length and variant allele frequency, down to 5% variant samples. As a high-throughput method with single-molecule resolution, dPCR-HSAFM thus represents an advance in HSAFM analysis and a powerful tool for the diagnosis of length polymorphisms.

AFM-based mechanical characterization of single nanofibres.

Nanofibres are found in a broad variety of hierarchical biological systems as fundamental structural units, and nanofibrillar components are playing an increasing role in the development of advanced functional materials. Accurate determination of the mechanical properties of single nanofibres is thus of great interest, yet measurement of these properties is challenging due to the intricate specimen handling and the exceptional force and deformation resolution that is required. The atomic force microscope (AFM) has emerged as an effective, reliable tool in the investigation of nanofibrillar mechanics, with the three most popular approaches-AFM-based tensile testing, three-point deformation testing, and nanoindentation-proving preferable to conventional tensile testing in many (but not all) cases. Here, we review the capabilities and limitations of each of these methods and give a comprehensive overview of the recent advances in this field.

Brown recluse spider's nanometer scale ribbons of stiff extensible silk.

The silk of the recluse spider features a ribbon-like morphology unlike any other spider silk or synthetically spun polymer fiber. These protein ribbons represent free-standing polymer films with a thickness of about 50 nm. Stress-strain characterization of individual fibers via atomic force microscopy reveals that these ribbons, only a few molecular layers of protein thin, rival the mechanical performance of the best silks.