NemaFlex: a microfluidics-based technology for standardized measurement of muscular strength of C. elegans

Muscle strength is a functional measure of quality of life in humans. Declines in muscle strength are manifested in diseases as well as during inactivity, aging, and space travel. With conserved muscle biology, the simple genetic model C. elegans is a high throughput platform in which to identify molecular mechanisms causing muscle strength loss and to develop interventions based on diet, exercise, and drugs. In the clinic, standardized strength measures are essential to quantitate changes in patients; however, analogous standards have not been recapitulated in the C. elegans model since force generation fluctuates based on animal behavior and locomotion. Here, we report a microfluidics-based system for strength measurement that we call ‘NemaFlex’, based on pillar deflection as the nematode crawls through a forest of pillars. We have optimized the micropillar forest design and identified robust measurement conditions that yield a measure of strength that is independent of behavior and gait. Validation studies using a muscle contracting agent and mutants confirm that NemaFlex can reliably score muscular strength in C. elegans. Additionally, we report a scaling factor to account for animal size that is consistent with a biomechanics model and enables comparative strength studies of mutants. Taken together, our findings anchor NemaFlex for applications in genetic and drug screens, for defining molecular and cellular circuits of neuromuscular function, and for dissection of degenerative processes in disuse, aging, and disease

Characterization of the unfolded state of bovine α-lactalbumin and comparison with unfolded states of homologous proteins

The unfolded states of three homologous proteins with a very similar fold have been investigated by heteronuclear NMR spectroscopy. Secondary structure propensities as derived from interpretation of chemical shifts and motional restrictions as evidenced by heteronuclear 15N relaxation rates have been analyzed in the reduced unfolded states of hen lysozyme and the calcium-binding proteins bovine α-lactalbumin and human α-lactalbumin. For all three proteins, significant deviations from random-coil predictions can be identified; in addition, the unfolded states also differ from each other, despite the fact that they possess very similar structures in their native states. Deviations from random-coil motional properties are observed in the α- and the β-domain in bovine α-lactalbumin and lysozyme, while only regions within the α-domain deviate in human α-lactalbumin. The motional restrictions and residual secondary structure are determined both by the amino acid sequence of the protein and by residual long-range interactions. Even a conservative single point mutation from I to L in a highly conserved region between the two α-lactalbumins results in considerable differences in the motional properties. Given the differences in oxidative folding between hen lysozyme and α-lactalbumin, the results obtained on the unfolded states suggest that residual long-range interactions, i.e., those between the α- and the β-domain of lysozyme, may act as nucleation sites for protein folding, while this property of residual structure is replaced by the calcium-binding site between the domains in α-lactalbumin