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.

The integrin-adhesome network, which contains >150 proteins, is mechano-transducing and located at discreet positions along the cell-cell and cell-extracellular matrix interface. A small subset of the integrin-adhesome is known to maintain normal muscle morphology. However, the importance of the entire adhesome for muscle structure and function is unknown. We used RNA interference to knock down 113 putative Caenorhabditis elegans homologs constituting most of the mammalian adhesome and 48 proteins known to localize to attachment sites in C. elegans muscle. In both cases, we found >90% of components were required for normal muscle mitochondrial structure and/or proteostasis vs. empty vector controls. Approximately half of these, mainly proteins that physically interact with each other, were also required for normal sarcomere and/or adhesome structure. Next we confirmed that the dystrophy observed in adhesome mutants associates with impaired maximal mitochondrial ATP production (P < 0.01), as well as reduced probability distribution of muscle movement forces compared with wild-type animals. Our results show that the integrin-adhesome network as a whole is required for maintaining both muscle structure and function and extend the current understanding of the full complexities of the functional adhesome in vivo.—Etheridge, T., Rahman, M., Gaffney, C. J., Shaw, D., Shephard, F., Magudia, J., Solomon, D. E., Milne, T., Blawzdziewicz, J., Constantin-Teodosiu, D., Greenhaff, P. L., Vanapalli, S. A., Szewczyk, N. J. The integrin-adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces in Caenorhabditis elegans.

In this study, we report a microfluidic device for the whole-life culture of the nematode Caenorhabditis elegans that allows the scoring of animal survival and health measures. This device referred to as the NemaLife chip features: (1) an optimized micropillar arena in which animals can crawl, (2) sieve channels that separate progeny and prevent the loss of adults from the arena during culture maintenance, and (3) ports that allow rapid accessibility for feeding the adult-only population and introducing reagents as needed. The pillar arena geometry was optimized to accommodate the growing body size during culture and emulate the body gait and locomotion of animals reared on agar. Likewise, feeding protocols were optimized to recapitulate longevity outcomes typical of standard plate growth. Key benefits of the NemaLife Chip include eliminating the need to perform repeated manual transfers of adults during survival assays, negating the need for progeny-blocking chemical interventions, and avoiding the swim-induced stress across lifespan in animals reared in liquid. We also show that the culture of animals in pillar-less microfluidic chambers reduces lifespan and introduces physiological stress by increasing the occurrence of age-related vulval integrity disorder. We validated our pillar-based device with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated reversals can be scored across the lifespan in the NemaLife chip. Overall, the capacity to generate reliable lifespan and physiological data underscores the potential of the NemaLife chip to accelerate healthspan and lifespan investigations in C. elegans.

Summary Mechanotransduction channels have been proposed as force sensors in various physiological processes, such as hearing and touch. In particular, TMC1 has been shown to constitute the pore of hair cell mechanotransduction channels, but little is known about how force is sensed by TMC channels. Here, we identify UNC-44/ankyrin as an essential component of the TMC-1 mechanotransduction channel complex in the sensory cilia of Caenorhabditis elegans mechanoreceptor neurons. Ankyrin binds indirectly to TMC-1 via evolutionarily conserved CIB proteins, which are required for TMC-1-mediated mechanosensation in C. elegans OLQ neurons and body wall muscles. Mechanosensory activity conferred by ectopically expressed TMCs in mechanoinsensitive neurons depends on both ankyrin and CIB proteins, indicating that the ankyrin-CIB subcomplex is required for TMC mechanosensitivity. Our work indicates that ankyrin is a long-sought intracellular tether that transmits force to TMC mechanotransduction channels.

Locomotion of the nematode is a key observable used in investigations ranging from behavior to neuroscience to aging. However, while the natural environment of this model organism is 3D, quantitative investigations of its locomotion have been mostly limited to 2D motion. Here, we present a quantitative analysis of how the nematode reorients itself in 3D media. We identify a unique behavioral state of-a roll maneuver-which is an essential component of 3D locomotion in burrowing and swimming. The rolls, associated with nonzero torsion of the nematode body, result in rotation of the plane of dorsoventral body undulations about the symmetry axis of the trajectory. When combined with planar turns in a new undulation plane, the rolls allow the nematode to reorient its body in any direction, thus enabling complete exploration of 3D space. The rolls observed in swimming are much faster than the ones in burrowing; we show that this difference stems from a purely hydrodynamic enhancement mechanism and not from a gait change or an increase in the body torsion. This result demonstrates that hydrodynamic viscous forces can enhance 3D reorientation in undulatory locomotion, in contrast to known hydrodynamic hindrance of both forward motion and planar turns.

13Electronic Supplementary Information (ESI) available: Movies demonstrating progeny washing. Worm 14 development and arena optimization information. Protocol for lifespan assay in a microfluidic device. Abstract 34Caenorhabditis elegans is a powerful animal model in aging research. Standard longevity assays 35 on agar plates involve the tedious task of picking and transferring animals to prevent younger 36 progeny from contaminating age-synchronized adult populations. Large-scale studies employ 37 progeny-blocking drugs or sterile mutants to avoid progeny contamination, but such 38 manipulations change adult physiology and alter the influence of reproduction on normal aging. 39Moreover, for some agar growth-based technology platforms, such as automated lifespan 40 machines, reagents such as food or drugs cannot be readily added/removed after initiation of 41 the study. Current microfluidic approaches are well-suited to address these limitations, but in 42 their liquid-based environments animals swim rather than crawl, introducing swim-induced 43 stress in the lifespan analysis. Here we report a simple microfluidic device that we call NemaLife 44 that features: 1) an optimized micropillar arena in which animals can crawl, 2) sieve channels 45 that separate progeny and prevent the loss of adults from the arena during culture 46 maintenance, and 3) ports which allow rapid accessibility to feed the adult-only population and 47 emulate the body gait, locomotion, and lifespan of animals reared on agar. We validated our 51 approach with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) 52 and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also 53 showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated 54 reversals can be scored across the lifespan. Overall, the capacity to generate reliable lifespan 55 and physiological data from the NemaLife chip underscores the potential of this device to 56 accelerate healthspan and lifespan investigations in C. elegans. 57 58 59 60 61 62 63 64 65Aging is a significant risk factor for a broad range of diseases including neurodegenerative 66 disorders, diabetes and cancer 1-5 . With the growing aging population, the socioeconomic 67 burden attributed with age-associated diseases is staggering and development of therapies that 68 promote healthy aging is imperative. C. elegans is a powerful model organism for aging 69 investigations with a short lifespan (3-5 weeks), remarkable genetic similarity with humans (~ 70 38 % orthologs 6 ) and conserved signaling pathways 7 . Additionally, a fully mapped genome 8 and 71 incredible genetic plasticity 9,10 makes C. elegans an attractive tool for aging studies. Advances in 72 fluorescent microscopy 11 and genomic technology (RNAi, CRISPR) 12,13 have further expanded 73 the number of possible ways in which C. elegans can be used to study healthy aging. 74Lifespan analysis has become a classic method for evaluating the effects of a wide variety of 75 gene...

Intervening in aging processes is hypothesized to extend healthy years of life and treat age-related disease, thereby providing great benefit to society. However, the ability to measure the biological aging process in individuals, which is necessary to test for efficacy of these interventions, remains largely inaccessible to the general public. Here we used NHANES physical activity accelerometer data from a wearable device and machine-learning algorithms to derive biological age predictions for individuals based on their movement patterns. We found that accelerated biological aging from our “MoveAge” predictor is associated with higher all-cause mortality. We further searched for nutritional or pharmacological compounds that associate with decelerated aging according to our model. A number of nutritional components peak in their association to decelerated aging later in life, including fiber, magnesium, and vitamin E. We additionally identified one FDA-approved drug associated with decelerated biological aging: the alpha-blocker doxazosin. We show that doxazosin extends healthspan and lifespan in C. elegans. Our work demonstrates how a biological aging score based on relative mobility can be accessible to the wider public and can potentially be used to identify and determine efficacy of geroprotective interventions.

Transcriptome‐based drug screening is emerging as a powerful tool to identify geroprotective compounds to intervene in age‐related disease. We hypothesized that, by mimicking the transcriptional signature of the highly conserved longevity intervention of FOXO3 (daf‐16 in worms) overexpression, we could identify and repurpose compounds with similar downstream effects to increase longevity. Our in silico screen, utilizing the LINCS transcriptome database of genetic and compound interventions, identified several FDA‐approved compounds that activate FOXO downstream targets in mammalian cells. These included the neuromuscular blocker atracurium, which also robustly extends both lifespan and healthspan in Caenorhabditis elegans. This longevity is dependent on both daf‐16 signaling and inhibition of the neuromuscular acetylcholine receptor subunit unc‐38. We found unc‐38 RNAi to improve healthspan, lifespan, and stimulate DAF‐16 nuclear localization, similar to atracurium treatment. Finally, using RNA‐seq transcriptomics, we identify atracurium activation of DAF‐16 downstream effectors. Together, these data demonstrate the capacity to mimic genetic lifespan interventions with drugs, and in doing so, reveal that the neuromuscular acetylcholine receptor regulates the highly conserved FOXO/DAF‐16 longevity pathway.